Advances in Cancer Research Volume 78

Advances in

CANCER RESEARCH Volume 78

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Advances in

CANCER RESEARCH Volume 78

Edited by

George F. Vande Woude ABL-Basic Research Program National Cancer Institute Frederick Cancer Research and Development Center Frederick, Maryland

George Klein Microbiology and Tumor Biology Center Karolinska lnstitutet Stockholm, Sweden

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Contents

Contributors to Volume 78 vii

Cell Transformation by the E7 Oncoprotein of Human Papillomavirus Type 16: Interactions with Nuclear and Cytoplasmic Target Proteins Werner Zwerschke a n d Pidder Jansen-Durr I. II. III. IV.

Background: The Role of Papillomaviruses in Human Cancer 2 Cellular E7-Binding Proteins 6 Modulation of Cellular Functions by E7 9 The Role of HPV-16 E7 in Cell Proliferation and Immortalization 17 References 22

Tumor Invasion: Role of Growth FactoFlnduced Cell Motility Alan Wells I. Introduction 32 II. Tumor Invasiveness 34 III. Cell Motility 40 IV. Motility in Tumor Invasion 76 V. Therapeutic Interventions 83 VI. Summary and Future Directions 89 References 90

Nonenzymatic Interactions between Proteinases and the Cell Surface: Novel Roles in Normal and Malignant Cell Physiology Paolo Mignatti a n d Daniel B. Rifkin I. Introduction 103 II. Extracellular Matrix-Degrading Proteinases: Classification and Structural Features 104 III. Proteolysis-Independent Roles of Extracellular Matrix-Degrading Proteinases 124 IV. Conclusions and Perspectives 143 References 146

V

vi

Contents

Molecular Pathogenesis of AIDS-Associated Kaposi's Sarcoma: Growth and Apoptosis Kaoru Murakami-Mori, Shunsuke Mori, and Benjamin Bohavida I. Introduction 160 11. Histopathogenesis 161 111. Clinical Features 163 N. In Vitro and in Vivo Models 165 V. Molecular Mechanisms of Kaposi's Sarcoma Cell Growth 168 VI. Roles of Virus Infections in Kaposi's Sarcoma Development 179 VII. Apoptosis in Kaposi's Sarcoma Cells 181 VIII. Concluding Remarks and Therapeutic Implications 188 References 190

Perspectives on Cancer Chemoprevention Research and Drug Development Gary 1. Kelloff I. 11. 111. N. V. VI.

Introduction 200 Nature of Carcinogenesis 202 Definition of Chemoprevention and Chemoprevention Agent Discovery 214 Chemopreventive Agent Development 272 Cancer Chemoprevention at Major Cancer Target Sites 286 Surrogate End Points in Defining Chemopreventive Efficacy-Importance of Evaluating Both Phenotypic and Genotypic Effects 312 VII. Major Issues and Challenges for Cancer Chemoprevention 314 References 321

.

Index 335

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Benjamin Bonavida, Department of Microbiology and Immunology, UCLA School of Medicine, University of California, Los Angeles, Los Angeles, California 90095 (159) Pidder Jansen Diirr, Institut fiir BiomedizinischeAlternsforschung der h e r reichischen Akademie der Wissenschaften, A-6020 Innsbruck, Austria (1) Gary J. Kelloff, Chemoprevention Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 (199) Paolo Mignatti, Department of Surgery, S. A. Localio General Surgery Research Laboratory and Cardiovascular Surgery Research Laboratory, New York University School of Medicine, New York, New York 10016 (103) Shunsuke Mori, Department of Microbiology and Immunology, UCLA School of Medicine, University of California, Los Angeles, Los Angeles, California 90095 (159) Kaoru Murakami-Mori, Department of Microbiology and Immunology, UCLA School of Medicine, University of California, Los Angeles, Los Angeles, California 90095 (159) Daniel B. Rifkin, Department of Cell Biology and The Kaplan Cancer Center, New York University School of Medicine, New York, New York 10016 (103) Alan Wells,* Departments of Pathology and Cell Biology, University of Alabama at Birmingham and Birmingham Veterans Administration Medical Center, Birmingham, Alabama 35294 (31) Werner Zwerschke, Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, D-69120 Heidelberg, Germany ( 1)

*Current address: Department of Pathology, University of Pittsburgh and Pittsburgh Veterans Administration Medical Center, Pittsburgh, Pennsylvania 15661.

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Cell Transformation by the E7 Oncoprotein of Human Papillomavirus Type 16: Interactions with Nuclear and Cytoplasmic Target Proteins Werner Zwerschke’ and Pidder lansen-DU&**

* Deutsches Krebsforschungszentrum Forschungsschwerpunkt Angewandte Tumowirologie 0-69120 Heidelberg, Germany ZInstitut fiir Biomedizinische Alternsforschung der Osterreichischen Akademie der Wissenschafien A-6020 Innsbruck, Austria

I. Background: The Role of Papillomaviruses in Human Cancer A. Papillomavirus Oncogenes and Their Role in Tumorigenesis B. Oncogenic Proteins Encoded by Human Papillomaviruses II. Cellular E7-Binding Proteins A. Domain Structure of E7 B. Subcellular Localization of HPV-16 E7 C. cd2-Interacting Proteins D. Proteins Interacting with the C-Terminus of E7 III. Modulation of Cellular Functions by E7 A. Cell Cycle Deregulation by E7 B. Regulation of Other Genes by HPV-16 E7 C. Modulation of Cellular Carbohydrate Metabolism by HPV-16 E7 D. Modulation of Apoptosis by HPV-16 E7 IV. The Role of HPV-16 E7 in Cell Proliferation and Immortalization A. Model for the Induction of Cell Proliferation by E7 B. The Role of E7 in Immortalization of Human Cells References

The E7 oncoprotein of human papillomavirustype 16 (HF’V-16) has long been known potent immortalizingand transforming agent. However, the molecular mechanisms underlying cell transformation and immortalization by E7 remain largely unknown. It is

as a

“Address correspondence ro P. Jansen-Diirr, Institut fur Biomedizinische Alternsforschung, Osterreichische Akademie der Wissenschaften, Rennweg 10, A-6020 Innsbruck, Austria. Advances in CANCER RESEARCH 0065-23OWOO$30.00

Copyright 8 2000 by Academic Press. All rights of reproduction in any form ICXNC~.

2

Werner Zwerschke and Pidder jansen-Diirr believed that E7 exerts its oncogenic function at least in part by modulating cellular growth regulatory pathways. Increasing experimental evidence suggests that cell transformation by E7 is mediatedby the physical association of E7 with cellular regulatory proteins, whose functions are specifically altered by E7, as exemplified by the well-known interaction of E7 with the retinoblastoma protein. In this review, we summarize the available data on the interaction of E7 with cellular regulatory factors and functional consequences of these interactions. We will focus the review on a set of recently identified new target proteins for the E7 oncoprotein, which sheds new light on E7 functions required for cell transformation and immortalization.Similarto the case of the E6 protein of HPV16, whose interaction with p53 was long considered its major activity, it now appears that the interactipn of E7 with the retinoblastoma protein represents just one of many distinct interactions that are relevant for cell transformation. 0 2000 Academic Press.

I. BACKGROUND: THE ROLE OF PAPILLOMAVIRUSES IN HUMAN CANCER

A. Papillomavirus Oncogenes and Their Role in Tumorigenesis Epidemiological and biochemical data support the division of human papillomaviruses (HPVs) into two groups: high-risk (e.g., HPV-16, 18, 5, and 8) and low-risk (e.g., HPV-1,6,11) types (zur Hausen, 1985). Infections with papillomaviruses. of the high-risk type are regularly observed in a subset of human cancers, including cervical cancer and other anogenital cancers (Howley, 1991; zur Hausen and Schneider, 1987). In contrast, infection with papillomaviruses of the low-risk group is associated with beni.gn hyperproliferation (for a recent review, see Alani and Munger, 1998). Three different early genes, referred to as E5, E6, and E7, which code for proteins with known transforming potential in vitro (see below), are regularly present in the genome of both high-risk and low-risk papillomaviruses (for review, see Chan et al., 1995). During infection, expression of these viral genes is controlled by a complex regulatory network of cellular and viral transcription factors (for review, see Hoppe-Seyler and Butz, 1994). In cervical lesions, the HPV sequences are usually integrated into the cellular genome (Diirst et al., 1986), often resulting in the disruption of the E2 gene, which leads to increased transcription of the E6 and E7 genes (reviewed by Demeret et al., 1995). Consequently, E6 and E7 are regularly expressed in cervical cancer cells (Schwarz et al., 1985). While it is generally assumed that tumors result from multiple consecutive genetic alterations, cellular immortalization is generally considered one of the first steps in cancer pathogenesis (Yeager etal., 1998). It was shown that transfection by the DNA of high-risk papillomaviruses is sufficient to immortalize human keratinocytes (Diirst et al., 1987), and the results from in

Cell Transformation by the HPV-16E7 Oncoprotein

3

vitro studies indicate that expression of E6 and E7 is sufficient for the immortalization of primary human keratinocytes (Hawley-Nelson et al., 1989; Miinger et al., 1989a), the natural host cells for papillomavirus infections. More recently, it was demonstrated that both E6 and E7 can immortalize human cells in isolation (Reznikoff et al., 1994, 1996; Wazer et al., 1995), although the frequency of immortalization is significantly reduced when compared to studies where both viral genes were introduced simultaneously (Hawley-Nelson et al., 1989). These results clearly suggest that immortalization by E6 or E7 requires the acquisition of additional genetic changes by random mutation. Unlike the E6/E7 genes encoded by high-risk papillomaviruses, the early genes encoded by low-risk HPVs fail to immortalize human primary keratinocytes (Barbosa et al., 1991). The role of the E5 genes for immortalization, if any, is not clear at the moment.

B. Oncogenic Proteins Encoded by Human Papillomaviruses 1. E5 PROTEIN

The first data on possible functions of the papillomavirus E5 genes were obtained through studies of the E5 gene encoded by bovine papillomavirus 1 (BPV-1) (Settleman et al., 1989). The E5 protein of BPV-1 is localized to the plasma membrane. BPV-1 E5 can cooperate with both the epidermal growth factor (EGF) receptor and the colony stimulating factor-1 receptor to induce transformation of NIH3T3 cells, possibly by enhancing receptormediated signaling (Martin et al., 1989). It was shown that the p type receptor for platelet-derived growth factor (PDGF) is constitutively activated in fibroblasts stably transformed by the BPV-1 E5 protein (Petti et al., 1991). There is a short region of sequence similarity between E5 proteins and PDGF, and E5 binds to the PDGF receptor (Petti and DiMaio, 1992), suggesting that the E5 protein activates the PDGF receptor (PDGFR) by direct binding (Petti and DiMaio, 1994). Several transformation-defective E5 proteins form a complex with the PDGFR and induce receptor tyrosine phosphorylation, indicating that PDGFR activation is necessary but not sufficient for E5-mediated cell transformation (Nilson et al., 1995). The E5 gene encoded by HPV-16 can induce anchorage-independent growth in the presence of EGF (Pim et al., 1992) and transform rodent cells in cooperation with the EGF receptor (EGFR) (Conrad et al., 1994).This indicates that HPV-16 E5 potentiates the mitogenic signals from the EGFR, similar to the results obtained with the E5 gene of BPV-1 and the PDGF receptor. Furthermore, HPV-16 E5 cooperates with HPV-16 E7 to stimulate the proliferation of primary rodent cells (Bouvard et al., 1994).It was shown that HPV-16 E5 binds to a 16-kDa membrane pore protein (Conrad et al.,

4

Werner Zwerschke and Pidder Jansen-Diirr

1993), and HPV-16 E5 can also block the function of cellular gap junctions (Oelze etal., 1995); however, it is not known if these properties of E5 are involved in its in vitro transforming potential. The E5 proteins of the low-risk virus HPV-6 share with HkV-16 E5 the ability to transform rodent cells in the presence of the EGF receptor (Conrad et al., 1994), and to cooperate with HPV-16 E7 in the transformation of primary rodent cells (Valle and Banks, 1995). 2. E6PROTEIN

The E6 genes of various HPV types were shown to transform rodent cells in vitro (Iftner et al., 1988; Neary and Dihdaio, 1989) and cooperate with ’ E7

to immortalize human keratinocytes (see above). Concerning potential mechanisms underlying cell transformation by E6, several functional interactions with cellular proteins have been described.

a. Interaction with p53

It was shown that the E6 oncoprotein encoded by high-risk human papillomaviruses type 16 and 18 promotes the degradation of the cellular tumor suppressor p53 (Scheffner et al., 1990) via binding to the cellular protein E6AP (Huibregtse et al., 1991), and thereby disrupts the p53-mediated cellular response to DNA damage. This results in the failure to exit the cell cycle (Foster et al., 1994; Kessis et al., 1993; Lechner and Laimins, 1994; Li et al., 1995; White et al., 1994), increased radioresistance (Tsang et al., 1995), and the induction of genomic instability (Havre et al,, 1995; White et al., 1994) (for review, see Elbel et al., 1997). In vivo evidence for the ability of HPV-16 E6 to inactivate pS3 function was obtained by the observation that expression of E6 in the eyes of transgenic mice suppresses apoptosis during lens development (Pan and Griep, 1994).

b. Additional Targets for HPV- 16 E6 Although the findings summarized above suggest that a major function of the E6 protein is to inactivate p53, mutational analysis of HPV-18 E6 indicated that the interaction of E6 with p53 is dispensible for the ability to cooperate with activated ras in the transformation of primary rodent fibroblasts (Pim et al., 1994; Storey et al., 1995), suggesting that p53-independent functions of E6 may also contribute to growth regulation. This is supported by the finding that the growth of E6-depleted cervical carcinoma cells can be restored by a mutant of E6 which is unable to target p53 (Spitkovsky et al., 1996) and that the ability of E6 to prevent cell cycle arrest of keratinocytes requires p53-independent functions of E6 (Sherman et al., 1997). Recently, a group of additional cellular proteins with the potential to interact with the HPV-16 E6 oncoprotein were identified, some of which may

Cell Transformation by the HPV-I6 E7 Oncoprotein

5

be involved in the p53-independent functions described above. These include the putative calcium-binding protein ERC-55 (Chen et al., 1995), paxillin (Tong and Howley, 1997), the putative human tumor suppressor protein hDLG (Kiyono et al., 1997), the proapoptotic protein Bak (Thomas and Banks, 1998), and the human minichromosome maintenance protein mcm7 (Kukimotoetal., 1998). Moreover, in addition to p53, two additional proteins, namely the c-Myc oncoprotein (Gross et d., 1998) and a putative GAP protein, referred to as E6TP1 (Gao et al., 1999), were shown to be targeted for proteolytic degradation by E6. However, the role of these interactions for cell transformation remains to be clarified (for a recent review, see Kubbutat and Vousden, 1998). ' The E6 protein encoded by HPV-16 was shown to activate transcription from the E4 promoter of HPV-11 (Tomita et al., 1998), indicating that E6 may play a role in the control of viral gene expression. It was shown that E6 can also activate transcription of several cellular promoters (Desainteset al., 1992), including the c-fos promoter. At least in the latter case, promoter activation requires E6 sequences distinct from the p53-binding region in the E6 protein (Morosov et al., 1994). More recently, it was shown that HPV16 E6 binds to interferon regulatory factor-3 and inhibits its transcriptional activity (Ronco et al., 1998), which may serve to circumvent the normal antiviral response of the infected cell. E6 proteins encoded by low-risk HPVs show a reduced ability to transform rodent cells (Schmitt etal., 1994; Storey etal., 1988; 1990) and to override biochemical functions of p53 (Crook etal., 1994; Lechner and Laimins, 1994);consequently, E6 of HPV-11 displays only a weak elevation in the mutation rate, when expressed in mammalian cells (Havre et al., 1995). Furthermore, when expressed in diploid human cells, HPV-6 E6 is unable to alter the cell cycle profile of these cells (White et al., 1994). Although these data strongly suggest that the ability of E6 to abrogate cell cycle functions of p53 contributes to the enhanced oncogenic potential observed for highrisk HPVs (for a recent review, see Rapp and Chen, 1998), it remains to be determined which of the p53-independent functions of high-risk virus E6 are conserved in low-risk E6.

3. E7PROTEIN In this section, we will only briefly review the biological activities of E7 proteins encoded by various human papillomaviruses, whereas the available data on cellular proteins interacting with HPV-16 E7 (Section 11) and on functional consequences of these interactions (Section 111) will be discussed in detail in separate sections. The E7 gene of HPV-16 can transform established rodent fibroblasts (Kanda et al., 1988) and immortalize primary rodent cells in conjunction with an activated ras oncogene (Phelps et al., 1988). Continued expression of the

6

Werner Zwerschke and Pidder Jansen-Dun

H w - 1 6 E7 gene is required for maintenance of the transformed phenotype in rodent cells (Crook et al., 1989), and expression of HPV-16 E7 in nonmetastatic mouse cell lines makes the cells metastatic in nude mice (Chen et al., 1993). In transgenic mice, coexpression of the E6 and E7 genes of highrisk HPVs elicit epidermal hyperplasia (Auewarakul et al., 1994), verrucose lesions, and papillomas (Greenhalgh et al., 1994); furthermore, E6 and E7 cooperate to induce various tumors on ectopic expression in transgenic mice (Arbeit et al., 1993; Pan and Griep, 1994). E7 of the low-risk HPV types 1,6, and 11efficiently transforms established rodent cellJines (Schmitt et al., 1994; Tsao et al., 1994), and HPV-1 E7 induces anchorage-independent growth in established human keratinocytes (Tsao et al., 1994). However, the E7 genes of the low-risk HPV types are strongly reduced in their ability to cooperate with ras in transforming primary rodent cells (Ciccolini et al., 1994; Storey et al., 1990), to immortalize primary human keratinocytes (Barbosa et al., 1991; Hu et al., 1995), and to induce metastatic growth properties in nude'mice (Chen et al., 1993).

.

11. CELLULAR E7eBINDING PROTEINS

A. Domain Structure of E7

.

By mutational analysis, three oncogenic domains of E7 have been identified (Fig. 1).The very amino-terminal part (conserved domain 1, c d l ) and a stretch of 21 amino acids (referred to as conserved domain 2, cd2), which is homologous to conserved region 2 (CR2; see Moran and Mathews, 1987) of adenovirus E l A, are required for the transformation of rodent cells (Banks et al., 1990a; Watanabe et al., 1990). The cd2 domain contains a sequence motif (LxCxE) that is conserved in other viral oncoproteins, such as adenovirus E1A or the large T antigen of simian virus 40 (SV-40)(Chellappan et al., 1992). Both the cdl and cd2 domains of HPV-16 E7 are also required for epidermal hyperplasia and tumor formation in E7 transgenic mice (GulFig. l Physical interaction of HPV-16 E7 with cellular target proteins. The primary amino acid sequence of the HPV-16 E7 protein (shown in blue) is given. Residues that are known to participate in the transforming functions of E7 are indicated in large uppercase letters. It is assumed that the E7 polypeptide forms a Zinc finger structure at its C-terminus; a ZnZ+ion, probably coordinating the four cysteine residues at positions 55/58 and 91/94, is included in the model. Proteins that are known to bind E7 are grouped according to their putative interaction site on E7 and their function in the cell. Molecular interactions discussed in this review are indicated by red arrows. The s4 subunit of the proteasome was assigned a function in cell cycle control in this figure, since it is clear that the regulated degradation of certain proteins by the proteasome plays a key role in cell cycle regulation; however, a specific function of the S4 subunit in cell cycle control has not been proved so far (see text). For abbreviations, see Table I.

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Cell Transformation by t h e HPV-16E7 Oncoprotein

7

liver et al., 1997). In addition, two CxxC motifs in the C-terminal part of HPV-16 E7 are required for immortalization of human keratinocytes by HPV-16 (Jewers et al., 1992). Although cdl-interacting proteins have not been described so far, both cd2 and the C-terminus of HPV-16 E7 were implicated in direct physical interactions with cellular proteins (Fig. 1).

B. Subcellular Localization of HPV- 16 E7 The subcellular localization of the HPV-16 E7 protein has been intensely studied in the past. The results of biochemical fractionation experiments in E7-expressing cells as well as the results of indirect immunofluorescence analysis (Kanda et al., 1991; Sato et al., 1989; Smotkin and Wettstein, 1987; W. Zwerschke et al., unpublished results, 1998) suggest that a significant proportion of HPV-16 E7 is found in the cytoplasm. By indirect immunofluorescence analysis, E7 can also be found in the nucleus (Fujikawa et al., 1994; Kanda et al., 1991; Sato et al., 1989), and it was reported that the detection of E7 by immunofluorescence depends on the unmasking of specific epitopes, which are hidden due to the interaction of E7 with nuclear and/or cytoplasmic binding partners (Kanda et al., 1991). More recently, it was reported that part of the E7 protein of HPV-16 is associated with the nucleolus (Zatsepina et al., 1997). These findings raise the possibility that, besides the well-established nuclear targets for E7, for example, the proteins of the retinoblastoma gene family (see below), the interaction with cytoplasmic and/or nucleolar proteins may contribute to the oncogenic functions of E7.

C. cd2hteracting Proteins It was shown that HPV-16 E7 binds to the retinoblastoma protein, pRb (Dyson et al., 1989; Miinger et al., 1989b), and binding depends on cd2 of E7 (Barbosa et al., 1990) and the “pocket” domain of pRb (Stirdivant et al., 1992). Subsequently, it was shown that E7 binds to two pRb-related proteins, referred to as p107 and p130 (Arroyo et al., 1993; Davies et al., 1993; Hu et al., 1995). The crystal structure of the pRb “pocket,” bound to a short synthetic E7 peptide containing the LxCxE motif, has been resolved by X-ray crystallography; the data suggest that certain structural motifs within pRb, referred to as “cyclin-folds,” are involved in the interaction with the LxCxE domain of E7 (Lee et al., 1998a). HPV-16 E7 also associates with cyclin A (Daviesetal., 1993; Tommasino etal., 1993),cyclin E (McIntyreetal., 1996), and cyclin-dependent kinase (cdk2) (Davies et al., 1993; Tommasino et al., 1993) through its cd2 domain; however, it is assumed that these interactions are not direct but are mediated by p107 (Davies et al., 1993),which is known to associate with cyclin/cdk2 complexes (Shirodkar et al., 1992).

8

Werner Zwerschke and Pidder Jansen-Durr

The efficiency of binding to both pRb and pl07lcyclin A appears to correlate with the transforming capacity of the E7 oncoproteins of several human papillomaviruses (Ciccolini et al., 1994; Heck et al., 1992; Hu et al., 1995; Phelps et al., 1992). However, the E7 proteins of the high-risk cutaneous types HPV-5 and HPV-8 bind to pRb with rather low affinity (Schmitt et al., 1994; Yamashita et al., 1993), and E7 of the benign type HPV-1 binds pRb and pl07/cyclin A with similar affinity as the high-risk E7 proteins (Ciccolini et al., 1994; Schmitt et al., 1994). Although low-risk viruses cannot immortalize human keratinocytes, immortalization has been reported for mutants of HPV-16 E7 impaired for pRb binding (Jewers et al., 1992). These observations indicate that additional domaips, besides the pRb/plO7 bind, ing site in cd2, determine the oncogenic potential of a given E7 protein. It is now clear that various additional cellular proteins are targeted by E7, and there are first pieces of evidence that binding of E7 affects the normal physiological function of these proteins (see below). Hence, it is not too surprising that the ability of E7 to interact with the pRb family is not the sole parameter determining its oncogenic potential. Serines 31 and 32 in the amino-terminal half of E7 are required for the interaction of E7 with TATA box binding protein (TBP) (Massimiet al., 1996), and probably also the TBP-associated factor TAF110 (Mazzarelliet al., 1995). More recently, it was reported that the C-terminal region of E7 also contributes to the interaction between E7 and TBP (Phillips and Vousden, 1997).

D. Proteins Interacting with the GTerminus of E7 It was shown that HPV-16 E7 forms dimers in vitro, involving a putative zinc finger structure in the C-terminus of E7 (McIntyre et al., 1993), and that the addition of zinc ions to the E7 protein encoded by HPV-18 induces a detkctable change in the protein conformation (Kang et al., 1997). According to the current model, Zn2+ions are bound by E7 via four cysteine residues at positions 55/58 and 91/94, respectively (Fig. 1).In keeping with this assumption, it was demonstrated that E7 from both HPV-16 (Clemens et al., 1995; Zwerschke et al., 1996) and HPV-11 (Zwerschke et al., 1996) can form homodimers also in vivo, and dimerization requires the two C-X-X-C motifs in the C-terminal part of E7 (Clemens et al., 1995; Zwerschke et al., 1996). Mutational analysis in the C-terminal part of E7 suggested that the structure of the Zn finger is essential for cell transformation and immortalization (Banks et al., 1990a; Barbosa et al., 1990; Edmonds and Vousden, 1989; Jewers et al., 1992; Watanabe et al., 1990), suggesting that the zinc finger may mediate the interaction of E7 with specific cellular proteins involved in its oncogenic function. However, some of the data are biased by the fact that mutations in the aforementioned cysteines in general reduce the stability of

Cell Transformation by the HPV-16E7 Oncoprotein

9

the E7 protein. Recently, a mutational analysis demonstrated that mutations in the "loop" region, which do not affect Zn binding and protein stability, reduce the transforming potential of E7 (Massimi et al., 1997);in that analysis, a short peptide sequence (LEDLL) was identified at positions 79-83 which appears to play a role in cell transformation (Massimi et al., 1997). It was found that the C-terminus of E7 is required for the interaction with the inhibitors of cyclin-dependentkinases p2lWAF-'(Funk et al., 1997; Jones et al., 1997a) and ~ 2 7 ~ (Zerfass-Thome '~' et al., 1996), the transcription factor AP-1 (Antinore et al., 1996), and the S4 ATPase subunit of the 26 S proteasome (Berezutskaya and Bagchi, 1997). Finally, a second, weaker interaction site for the retinoblastoma protein was mapped to the E7 C-termi~ U (Patrick S et al., 1994). While the interactions mentioned above locate to the C-terminal part of E7, it is currently unknown which E7 residues are involved. It was also found that the E7 protein interacts with the glycolytic contro1,enzymeM2 pyruvate kinase (M2-PK) (Zwerschke et al., 1999), and this interaction requires the LEDLL motif at positions 79-83.

111. MODULATION OF CELLULAR FUNCTIONS BY E7 Concerning the consequencesof E7 binding to cellular regulatory proteins, three types of functional outcomes have been described (Table I): (i) direct binding of E7 to multiprotein complexes leads to a remodeling of these complexes, as in the case of p107/E2F complexes, (ii) E7 binding to a given protein leads to a modulation in the function of the target protein, as in the cases of p21WAF-',~ 2 7 ~S4, ' , and W-PK, respectively; and (iii) E7 binding triggers the degradation of the target protein by the proteasome, as in the case of pRb. The available data suggest that at least four different cellular processes are influenced by the E7 interactions mentioned above (TableII): ( 1)E7 overrides both transcriptional and nontranscriptional cell cycle checkpoint controls, mainly at the GUS boundary, (2) E7 modulates the expression of additional cellular genes that are not involved in cell cycle control, (3)E7 deregulates the cellular carbohydrate metabolism, and (4) E7 modulates the frequency of programmed cell death (apoptosis).

A. Cell Cycle Deregulation by E7 1. CELL CYCLE CHECKPOINTS TARGETED BY E7

As a major function of E7 during HPV infection we consider its ability to deregulate control of the cell cycle progression, by favoring the exit of qui-

Table I Known E7 Target Proteinsa ~~

~

E7 Target

Cellular function

Localization

E7 Domain

+ C-terminus

Result of E7 binding

Nuclear

cd2

Nuclear

Cd2

Nuclear

Cd2

cyclid, cyclinE, cdk2 p27-l

Transcriptional repressor GI arrest Transcriptional repressor G1 arrest Transcriptional repressor G1 arrest Subunits of cyclin-dependent kinases cdk inhibitor

Nuclear

cd2, via p107?

?

Nuclear

C-terminus

p21WM-1

cdk inhibitor

Nuclear

C-terminus

M2-PK

Glycolyticcontrol enzyme

Cytoplasmic

C-terminus

S4 subunit

Subunit of 26 S proteasome Transcriptional activator Transcriptional activator

?

Nuclear Nuclear

C-terminus C-terminus Ser 31/32

Inactivation of p27-', activation of cyclWcdk2 kinase Inactivation of p2IWAF-',activation ..of cyclidYcdk2 kinase M2-PK shifted to low affinity dimeric form, expansion of phosphometabolite pools Increase of ATPase activity Increase of AP- 1-dependent transcription Modulation of transciption (?), interaction with TAFllO (?)

p107 p130

AP-1 TJ3P

Proteolytic degradation of pRb, derepression of E2F Disruption of E2F-pl07 complexes derepression of E2F Disruption of E2F-pl30 comblexes ( ? ) derepression of E2F (?)

aFor each of the listed target proteins, kown cellular functions and the subcellular localization are indicated. Where h o w , E7 domains required for the binding of a given target protein are indicated, and functional consequences of E7 interaction are listed. For references, see text. Abbreviations: pRb, retinoblastoma protein; c d w , cyclin-dependent b a s e 2; KIP1, kinase-inactivating protein 1; WAF-1, wild type activated fragment 1; M - P K , type M 2 pyruvate kinase; AP-1,activating protein 1; TBP, TATA box binding protein.

Cell Transformation by the HPV-16E7 Oncoprotein

Table 11 Biological Functions of HPV-16E7' E7 Function

Cellular factors involved

Inactivation of cell cycle checkpoints

pRb, p107, p130 (?), P21, P27

Activation of cellular genes

AP-1

Repression of cellular genes Modulation of glycolysis

TBP (?), p53 W - P K , other glycolytic enzymes ( 2 ) S4 proteasome subunit (?)

Induction of proteolysis

Remarks Activation of E2F-driven genes, Derepression of cyclindependent kinases Activation of AP-1driven genes (?) Delay of cell differentiation (?) Expansion of phosphometabolite pools Destabilization of pRb (?)

uThe table provides a list of known biological effects of HPV-16E7 and indicates the cellular factors that are involved in each activity.

escent cells from GO and entry into the S phase. E7 allows rodent cells in G1 to enter S phase (Banksetal., 1990b; Crook etal., 1989; Schulze etal., 1998) and induces an increased capacity for cell proliferation in primary human cells (Foster and Galloway, 1996; White et al., 1994). It was shown that E7 can overcome several forms of G1 arrest that are induced by loss of cell adhesion (Banks et d., 1990a; Schulze et d . , 1998), growth factor withdrawal (Morozov et al., 1997; Zerfass et al., 1995a), DNA damage (Demers et al., 1994; Hickman et al., 1994; Slebos et al., 1994; Vousden et al., 1993), and differentiation signals (Pei et al., 1998; Ruesch and Laimins, 1998). More recently, it was reported that expression of HPV-16 E7 also abrogates the mitotic spindle checkpoint (Thomas and Laimins, 1998). The results summarized in the previous paragraph indicate that E7 can override various cell cycle checkpoint controls in vitro. An increase in the cell proliferation capacity, along with the inhibition of cell differentiation, was also observed when the HPV-16 E7 gene was expressed in the lens of transgenic mice, whereas expression of a cd2 mutant of E7 had no effect (Pan and Griep, 1994). Similar to the findings with E7-expressing cell lines, the radiation-induced DNA damage response is impaired in E7 transgenic mice (Song et al., 1998), indicating that E7 promotes S phase entry in vivo. These results indicate that a major function of E7 is to override cell cycle control, mainly at the Gl/S boundary. According to the current concept, two groups of regulatory interactions contribute to the ability of HPV-16 E7 to override cell cycle control: (i) E7-driven activation of cellular E2F-dependent genes and (ii) the direct interaction of E7 with cell cycle regulatory proteins, in particular with inhibitors of cyclin-dependent k'mases.

12

Werner Zwerschke and Pidder Jansen-Diirr

2. ACTIVATION OF CELL CYCLE REGULATED GENES BY HPV-16 E7

*

By interacting with members of the pRb family, HPV-16 E7 induces the activity of the E2F family of cellular transcription factors (Dyson et al., 1992; Wu et al., 1993), which are known to control the expression of the major cell cycle regulatory genes at the GUS transition, including cyclin E (Botz et al., 1996; Geng et al., 1996; Ohtani et al., 1995), cyclin A (Schulze et al., 1995), cdc6 (Yan et al., 1998), and cdc25A (Iavarone and Massague, 1999) (for review, sesHelin, 1998). Of those, the genes encoding cyclin E (Martin et al., 1998; Zerfass et al., 1995b) and cyclin A (Schylze et al., 1998; Zerfass et al., 1995b) were shown to be direct targets for E7-dependent transactivation. Interestingly, expression of human cyclin E2, a newly identified member of the G1 cyclin family, is also induced by HPV-16 E7 (Zariwala et al., 1998); however, it is unclear at present if E7-dependent activation of cyclin E2 gene expression involves E2F. The mechanism by which E7 overrides the repressor function of pRb family members appears to be distinct between pRb and p107 (see below); only very few data are available about the functional interaction of E7 with p130, the third member of the pRb family (Cobrinik et al., 1993; Mayol et al., 1993). In the case of pRb, it was found that E7 binding triggers proteolytic degradation of pRb via the ubiquitin-proteasome pathway (Boyer et al., 1996), probably resulting in increased levels of “free,” transcriptionally active E2F (for review, see Beijersbergen and Bernards, 1996). Recruitment of proteasome activity by E7 may involve its interaction with the S4 subunit of the proteasome (Berezutskaya and Bagchi, 1997); however, this hypothesis needs to be tested experimentally. In contrast to the findings with pRb, binding of E7 to p107 does not have any influence on the metabolic stability of the p107 protein. It was shown that, as in the case of pRb/E2F complexes, -E7 also triggers the reappearance of “free” E2F from p107/E2F complexes; however, in this case, formation of “free” E2F results from a direct physical sequestration of the p107 protein from E2F/p107 complexes (Lam et al., !994; Zerfass et al., 1995a), which contributes to the activation of cyclin A gene transcription by E7 (Schulze et al., 1998). On the other hand, E7 is unable to disrupt the S phase-specific E2F-plO7-cyclin A complexes (Pagan0 et al., 1992);rather, E7 associates with such complexes (Arroyo et al., 1993). However, the function of the ternary complexes resulting from this interaction is presently unknown. Much of the early work on E2F-dependent gene expression was carried out with the E2 promoter of adenovirus 5, which contains two high affinity E2F binding sites (Jansen-Diirr et al., 1989; Kovesdi et al., 1986) and is strongly trans-activated by HPV-16 E7 (Phelpset al., 1991).The E7proteins of the lowrisk viruses HPV-6 and HPV-11 can also trans-activate the adenovirus E2 pro-

Cell Transformation by the HPV-16E7 Oncoprotein

13

moter (Miinger et al., 1991), although their ability to bind both to pRb (Miingeretal., 1991)and to p107/E2F complexes (Arroyoetal., 1993)is drastically reduced. In the case of the low-risk virus HPV-1, the evidence is conflicting. It was reported that, in spite of its high pRB binding affinity, HPV-1 E7 cannot trans-activate the E2 promoter (Schmitt et al., 1994) and is unable to relieve the E2F-1 transcription factor from repression by Rbl (Ciccolini et al., 1994). However, Ibaraki et al. (1993) reported that the Ad5 E2 promoter is equatly stimulated by the E7 genes of HPV-1 and HPV-16. Although the reason for this apparent contradiction is unclear, it is conceivable that in addition to the E2F binding sites mentioned above, additional regulatory elements of the E2 promoter contribute to trans-activation by E7 proteins.

3. INACTIVATION OF cdk INHIBITORS BY HPV- I6 E7 The observation that HPV-16 E7 overrides cell cycle control mainly at the Gl/S boundary raises the possibility that E7 can neutralize or bypass the inhibitory effects of the cdk inhibitors (cki) p21WAF-*(El-Deiry et al., 1993) and ~ 2 7 (Polyak ~ ' ~et al., ~ 1994), which are induced by various antiproliferative signals, including activation of p53 (El-Deiry et al., 1993), growth factor withdrawal (Firpo et al., 1994),and loss of cell adhesion (Assoianand Zhu, 1997; Fang et al., 1996; Schulze et al., 1996). Indeed, it was found that both p2lWAF-'and ~ 2 7 ~ are ' ~ targeted ' by HPV-16 E7, and this leads to a et al., 1996) and functional inactivation of both ~ 2 7 (Zerfass-Thome ~ ' ~ ~ p21WAF-l(Funk et al., 1997; Jones et al., 1997a). Consequently, both p21WAF-land ~27'~' fail to block the activity of cyclin E/CDK2 complexes in E7-expressing cells (Funk et al., 1997; Jones et al., 1997a; Schulze et al., 1998). Inhibition of cki function by E7 can also be reproduced in vitro, indicating that it is a direct effect (Funk et al., 1997; Jones et al., 1997a; Zerfass-Thome et al., 1996). However, the ability of E7 to override p21WAF-' function appears to depend on the relative expression levels of either protein. Hence, some authors reported that E7 fails to rescue cdk2 kinase activity in cells expressing high levels of p21WAF-l(Hickman et al., 1997; Morozov et al., 1997). Although the precise molecular mechanisms underlying cki inhibition by HPV-16 E7 remain to be established, the available data suggest that targeting of cki function by E7 could play a major role for its ability to override cell cycle control (for review, see Funk and Galloway, 1998). This was also suggested for other transforming oncogenes, such as adenovirus E1A (Ma1et al., 1996),the Tax protein of HTLV-I (Suzukietal., 1996) or the cellular c-myc protooncogene (Vlach et al., 1996). In the case of p21WAF-',it was shown that the binding of E7 also impairs the ability of p2lWAF-lto act as an inhibitor of DNA synthesis in vitro (Funk et al., 1997; Ruesch and Laimins, 1997). Furthermore, it appears that E7 not only inactivates p2lWAF-'by direct complex formation but also controls the

14

Werner Zwerschke and Pidder Jansen-Diirr

intracellular level of this growth-suppressive protein. Thus, it was shown that HPV-18 E7, expressed from the natural HF'V-18 promoter in cultured by posttranscriptional keratinocytes, induces very high levels of p2 lWAF-l mechanisms, and these cells, although expressing E7, fail to reactivate DNA synthesis (Jian et al., 1998). Hence, the functional interplay between p2lWAF-'and E7 appears quite complex, and more work is required to precisely define the role of this interaction for cell transformation in vivo.

B. Regulation of Other Genes by HPVcl6 E7 Besides modulation of the pRb/E2F pathway, which appears to be essential for the ability of E7 to reactivate S phase-promoting genes in quiescent host cells (see above), additional genes are controlled by E7, as is outlined below. It is possible that the interaction of E7 with E2F-unrelated cellular transcription factors serves to regulate viral gene expression; alternatively, it is possible that, as in the case of the E2F-driven genes, regulation of additional cellular genes by E7 is used to facilitate virus reproduction. These questions remain to be analyzed. As a matter of fact, it was shown that E7, by binding to the Jun component of the AP-1 transcription factor, can activate transcription of AP-1-driven genes (Antinore et al., 1996), whereas more recent data suggest that E7, by binding to both c-Jun and pRb, inhibits the ability of pRb to activate a subset of AP-1-driven genes (Nead et al., 1998). Control of AP-1 activity by E7 may be relevant for viral gene expression, since it was shown that AP-1 plays a key role in controlling papillomavirus early gene expression (Hoppe-Seyler and Butz, 1994; Kyo et al., 1997; Rosl et al., 1997). It was shown that HPV-16 E7 stimulates expression of the c-fos gene via a cyclic AMP response element in the c-fos promoter (Morosov et al., 1994, 1995), and E7 was also shown to activate, in a cd2-dependent manner, transcription of the gene encoding a 100-kDa protein related to heat shock proteins (Morozov et al., 1995). Finally, two transcriptional activation domains were identified in the cdl domain and the C-terminus of HPV-16 E7, which ark both independent of E2F/pRb (Zwerschke et al., 1996). While the data suggest that additional, E2F-unrelated transcription factors are targeted by HPV-16 E7, more work is required to understand transcriptional activation of E2F-independent genes by E7. There is some evidence that, in addition to the well-documented role of E7 as transcriptional activator, certain cellular genes are repressed by E7. Thus, it was shown that E7 can block the ability of p53 to stimulate transcription (Massimi and Banks, 1997). This activity involves serines 31 and 32 of E7, which have been shown to mediate binding of E7 to TBP (Massimi et al., 1996), suggesting that the interaction of E7 with TBP may be responsible for

Cell Transformation by the HPV-16E7 Oncoprotein

15

transcriptional repression. It was also shown that the gene encoding smooth muscle a-actin is downregulated in E7-expressing embryonal fibroblasts, and transcriptional repression of this gene by E7 was also observed in transient transfection experiments (Nishida et al., 1995). A transcriptional repressor domain was recently identified in the C-terminal part of E7 (Zwerschke et al., 1996), suggesting that E7 may interact with transcriptional repressors, for example, members of the histone deacetylase protein family (for review, see Hassig and Schreiber, 1997; Wolffe, 1996), which are known to interact with members of the pRb family (Brehm et al., 1998; Ferreira et al., 1998; Luo et al., 1998). Although the interaction with a histone deacetylase was shown to be responsible for transcriptional repression by the Myc dncoprotein (Sommer et al., 1997), the mechanisms underlying transcriptional repression by HPV-16 E7 remain to be identified.

C. Modulation of Cellular Carbohydrate Metabolism by HPVc I6 E7 The interaction of E7 with type M2 pyruvate kinase (M2-PK) (Zwerschke et al., 1999) represents the first example of a cytoplasmic target for the E7 protein, and this finding suggests that E7 can directly influence the carbohydrate metabolism of the host cell, which is a well-known target for oncogenic alterations in the vast majority of all tumors (for review, see Bannasch et al., 1998; Mazurek et al., 1997; Weinhouse, 1972). Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate, which is one of three rate-limiting steps in the glycolytic pathway (Fig. 2). Located at the exit of glycolysis, M2-PK controls the rate of synthesis of pyruvate, which is an essential intermediate for the complete oxidation of glucose. M2-PK comes in two different conformations, a tetrameric enzyme with high affinity for its substrate PEP and a dimeric enzyme with low substrate affinity (Eigenbrodt et al., 1992). When the PEP affinity is high, most of the input glucose will be channeled into the respiratory pathway. When MZPK is shifted to the low affinity dimeric form, the production of pyruvate is reduced, and this leads to an increase in the pool of phosphometabolites upstream of PEP, which are then available for the biosynthesis of nucleotides and complex carbohydrates (Eigenbrodt et al., 1992) (Fig. 2). The binding of E7 to the glycolytic enzyme M2 pyruvate kinase shifts the equilibrium of M2-PK complexes from the tetrameric to the dimeric form, concomitant with a significant loss in the affinity of the enzyme for its substrate PEP (Zwerschke et al., 1999).This leads to an expansion of the intracellular phosphometabolites and an increase in aerobic glycolysis (Zwerschke et al., 1999), suggesting a direct impact of E7 on glycolytic pathways. It is conceivable that the interaction of E7 with MZPK changes the meta-

16

Werner Zwenchke and Pidder Jansen-Dun

E7 OFF: maximal ATP production

I

I

-

1

Glucose

- -b nucleotide precursors - -b 1,BPhosp

E l ON: maximal biosynthetlcactivity

I

nucleotlde precursors

lycerale - - b serine

8 0

Phosphoenolpywvate(PEP)

serine

Phosphoenolpyruvate (PEP)

M2-PK WMM, high PEP afflnlty

M2-PK dlmer, Pyruvate .COz+H&+ATP

Fig. 2 The role of M2 pyruvate kinase in control of the glycolytic flux. At left, the glycolytic flux is shown in E7-nonexpressing resting cells, where M2-PK is predominantly in the tetrameric form. Since under these conditions the conversion of phosphoenolpyruvate (PEP)to pyruvate is very efficient, most of the input glucose will be channeled into the respiratory pathway, and only little biosynthesis can take place. When M2-PK, through binding of E7, is shifted into the low affinity dimeric form (right),the conversion of PEP to pyruvate is slowed down, leading to an expansion of the phosphometabolite pools upstream of PEP, which are in a dynamic equilibrium with PEP. Expansion of the phosphometabolite pools then allows increased biosynthesis of nucleotides and other essential carbohydrates.

*

bolic status of the host cell, providing the cells with increased pools of glycolytic phosphometabolites, which are then available for biosynthetic processes. Since the level of glycolytic metabolites is usually quite low in resting cells (for review, see Mazurek et al., 1997), this property of HPV-16 E7 may well be important for the ability of the viral protein to induce cell proliferation in quiescent host cells.

,D.Modulation of Apoptosis by HPV-16E7 It has long been known that E7 has the potential to interfere with regulatory pathways that control programmed cell death (apoptosis) (for review, see Tommasino and Crawford, 1995). On the one hand, it was demonstrated that expression of E7 induces apoptosis in vivo in transgenic mice (Pan and Griep, 1994) as well as in vitro in cultured cells (Iglesias et al., 1998; Jones et al., 1997b; Puthenveettil et al., 1996; Stoppler et al., 1998). Apoptosis induction by E7 involves both p53-dependent and p53-independent pathways (Howes et al., 1994; Pan and Griep, 1995). On the other hand, it was demonstrated that, under certain circumstances, E7 protects mammalian cells from apoptosis (Lee et al., 1998b; Magal et al., 1998), suggest-

Cell Transformation by the HPV-I6 E7 Oncoprotein

17

ing that the response of the cells to expression of E7 depends on their genetic background. At present it is not clear, however, which genetic factors determine the actual response of a given cell to E7. It was reported that both destabilization of pRb and stabilization of p53 contribute to apoptosis induced by E7 (Jones et al., 1997b). Similar to HF'V-16 E7, the E1A oncogene of adenovirus 5 induces apoptosis in certain cell types, and this correlates with an E1A-dependent stabilization of pS3 (Debbas and White, 1993). In the case of adenovirus ElA, it was shown that stabilization of p53 is mediated by an E1A-dependent upregulation of plSARF(de Stanchina et al., 1998), a cellular protein (Duro et al., 1995; Quelle et al., 1995) that binds to mdm-2 and pievents it from destabilizing p53 (Pomerantz et al., 1998). Although these findings raise the possibility that stabilization of p53 in E7-expressing cells may result from an E7-dependent upregulation of p l 9ARF,this prediction still needs to be experimentally tested.

IV. THE ROLE OF HPW16 E7 IN CELL PROLIFERATION AND IMMORTALIZATION A. Model for the Induction of Cell Proliferation by E7 The data summarized in the previous paragraphs support the following model of how E7 induces proliferation of resting mammalian cells (Fig. 3). According to our model, the cyclin-dependent kinases constitute the driving forces of cell proliferation. In resting cells, the expression of various cyclin subunits is downregulated (Koff et al., 1991; Pagano and Draetta, 1991; Won et al., 1992); consequently, the activity of cyclin-dependent kinases is low. In addition, there are several distinct molecular brakes, which act to block cell proliferation. On the one hand, the members of the retinoblastoma protein family, pRb, p107, and p130, were shown to arrest susceptible cells in the G1 phase of the cell cycle (Claudio et al., 1994; Hinds et al., 1992; Zhu et al., 1993). On the other hand, expression of the CDK inhibitors p2lWAF-'and p27-l also blocks progression through the cell cycle in the G1 phase (El-Deiry et al., 1993; Polyak et al., 1994). When the E7 gene is artificially expressed in resting cells, a rapid transcriptional activation of the genes encoding cyclin E and cyclin A is observed (Zerfass et al., 1995b). In parallel, the molecular brakes defined above, namely, the CDK inhibitors p2lWAF-'and ~ 2 7 ~or~ the ' , pRb family members pRb and p107, are directly targeted by E7 and thereby inactivated. In our model, these events would reactivate the cyclin/cdk machinery and remove the molecular brakes (p21WAF-',p27K1p1,pRb, and p107) and thereby allow safe transit into the next cell cycle phase.

18

Werner Zwerschke and Pidder Jansen-Diirr

Fig. 3 Effects of HPV-I6 E7 on the cell cycle machinery. The cyclin-dependent kinases are the driving forces in cell cycle progression. In the top panel, the situation for a resting mammalian cell is depicted. In the absence of any growth stimulators or viral oncoproteins, the genes encoding cyclin E and cyclin A are not expressed; hence, the cyclinkdk machinery is switched off. Furthermore, progression through the cell cycle is slowed down by several molecular brakes, such as p21WAF-',~ 2 7 ~p107, ~ ' ,and pRb. On expression of E7 (bottom panel), the cyclin E and*A genes are activated and the molecular brakes mentioned above are neutralized by E7 through a physical interaction. This leads to an onset of cell cycle progression, and the modulation of M2-PK activity by E7 provides the glycolytic metabolites required for sustained cell proliferation.

In certain settings, however, there may be additional obstacles to S phase entry that are not related to the cyclin/cdk network. Depending on the physiological status of the cell, the abundance of the glycolytic metabolites, which are required for the de novo biosynthesis of nucleic acids and other cellular constituents, may be rate-limiting for the synthesis of nucleic acids or even preclude it. It is known that the levels of glycolytic metabolites are low in resting cells (for review, see Mazurek et al., 1997).To allow sustained cell proliferation, a reprogramming of the cellular carbohydrate metabolism is required, to ensure the availability of sufficient glycolytic phosphometabolites. A reprogramming of the glycolytic apparatus was also described in rest-

Cell Transformation by the HPV-I6 E7 Oncoprotein

19

ing thymocytes that are stimulated to enter S phase by growth factors (Netzker et al., 1992, 1994). According to our model, in E7-expressing cells a metabolic switch is triggered by the direct interaction of the E7 protein with M2 pyruvare kinase, a rate-limiting control enzyme in the glycolytic pathway (Eigenbrodtet al., 1992).By decreasing the substrate affinity of M2-PK, the conversion of PEP to pyruvate will be inhibited, resulting in the increase of the phosphometabolite pools (Zwerschke et al., 1999), which are then available for increased biosynthetic activity (Fig. 3).

B. The Role of E7 in Immortalization of Human Cells HPV-16 and several other oncogenic viruses, for example, adenovirus 5 and SV-40, immortalize cells by inducing a period of increased proliferative potential that terminates in culture crisis, after which immortalization occurs in a small percentage of the cells, leading to immortal cell lines that have undergone dramatic genome rearrangements (DeSilva et al., 1994). Usually, immortalization is described as a two-step process in which a limited extension of the life span is followed by crisis and the subsequent appearance of immortal cell lines. Expression of the E6 and/or E7 genes of HPV-16 is sufficient to immortalize human keratinocytes (Hawley-Nelson et al., 1989; Munger et al., 1989a). However, immortalization by E7 only is a rare event, and coexpression of E6 greatly increases the immortalization frequency, most likely involving the ability of E6 to induce genomic instability, by abrogating p53 function (Reznikoff et al., 1994; White et al., 1994). In the following section, potential contributions of E7 to the extension of cellular life span and the eventual progression to immortality are discussed. 1. LIFE SPAN EXTENSION

In the case of E7-immortalized cells, it appears that part of the inbuilt cellular growth-suppressive factors are inactivated by E7 through direct binding, whereas other growth-suppressive pathways cannot be targeted by E7 but are to be inactivated by random mutation. During the extension of the cellular life span, E7 inactivates a series of cell cycle checkpoint control systems, mediated by various cdk inhibitors and by the proteins of the retinoblastoma family. This involves the physical binding and functional inactivation of ~ 2 7 ~ ' p2lWAF-l, ~', pRb, p107, and probably p130, allowing additional rounds of cell proliferation. The p2Up27-unrelated cdk inhibitor p16INK4a is usually strongly overexpressed in cells expressing E7 (Nakao et al., 1997; Reznikoff et al., 1996; Sano et al., 1998; Xiong et al., 1996), but an interaction with E7 has not been described. This finding probably reflects the fact that E7-mediated inactivation of pRb appears to cancel transcriptional repression of the p16INK4a gene by pRb (Aagaard et al., 1995;

20

.

Werner Zwerschkeand Pidder lansen-DUrr

Hara et al., 1996); on the other hand, the high abundance of p16INK4a in E7-expressing cells does not interfere with cell proliferation, since cyclin Dassociated kinase, which Is the major physiological target for p16INK4a, is not required for S phase entry in E7-expressing cells (Lukas et al., 1994, 1995). Apparently, the ability of E7 to stimulate proliferation of diploid cells inevitably triggers an increased tendency of the cells to undergo apoptosis. Although the reason for this effect is unclear at the moment, it is possible that the ability of E7 to drive cells into apoptosis may be mediated by an increased expression of pTSARF(see above, Section III,D).As it is known that plSARF gene expression can be induced, probably yia an E2F binding site(s) in the ARF promoter (Bates et al., 1998), by known oncogenes such as adenovirus ElA (de Stanchina et al., 1998) and c-myc (Zindy et al., 1998), it is likely although not formally documented that E7, which shares with Myc and E1A the ability to activate E2F-driven transcription (Chellappan et al., 1992), induces apoptosis via the ARFIp53 pathway. 2. PROGRESSION TO IMMORTALITY Cells escaping from crisis are immortal and bear the potential for indefinite cell proliferation. This requires the activation of a pathway that ensures the conservation of the chromosome ends (telomeres). This is usually brought about by the reactivation of the enzyme telomerase, although other pathways have also been described (reviewed by Reddel et al., 1997). Telomerase is a ribonucleoprotein enzyme, made up of a catalytic protein subunit referred to as hTERT (Meyerson et al., 1997; Nakamura et al., 1997) and a specific RNA subunit (for review, see Shay, 1997). While the RNA component of telomerase appears to be ubiquitously expressed, the protein subunit is expressed in the germ line and a few stem cell types but not expressed in most somatic cells (Shay, 1997). That telomerase plays a key role in determining cellular life span is shown by the fact that reexpression of hTERT in diploid human fibroblasts significantly extends the life span of these cells (Bodnar et al., 1998; for review, see Sedivy, 1998). It was shown early on that immortalization of human cells by SV-40 (Murnane et al., 1994; Shay et al., 1993) and Epstein-Barr virus (Counter et al., 1994) is correlated with an increase of telomerase activity. Subsequently, telomerase activity was found increased in human keratinocytes after retroviral expression of HPV-16 E6, whereas expression of E7 had no effect (Klingelhutz et al., 1996). Although artificial reexpression of hTERT appears to induce infinite cell proliferation in diploid fibroblasts (Bodnar et al., 1998; Counter et al., 1998),it was shown that telomerase activity is not sufficient for immortalization of human keratinocytes or mammary epithelial cells; instead, the inactivation of the RbIpl6INK4a pathway, be it by E7 expression or transcriptional dowriregulation of p16INK4a expression, is also required

21

Cell Transformation by the HPV- I6 E7 Oncoprotein

for immortalization (Kiyono et al., 1998). In keeping with earlier studies, suggesting that at least four distinct pathways contribute to replicative senescence of human cells (Pereira and Smith, 1988), these findings indicate that, besides the protection of the telomeres, additional pathways must be targeted during immortalization. Our model, derived from molecular interactions of the E7 protein encoded by HPV-16, would predict that at least two of these alterations can be triggered by E7. For an E7-expressing cell to become immortal, upregulation of the phosphometabolite pools, required for sustained cell proliferation, could be induced by the ability of E7 to target M2-PK. Furthermore, the known ability of E7 to suppress apoptosis may contribute to the establishment of immortal clones; however, the pathways involved in apoptosis suppression by E7 remain to be identified. According to our model, the loss of additional tumor suppressor genes and the reactivation of telomerase would be required for immortalization. At present there are no hints for a direct involvement of E7 in these processes. Hence, it is predicted that these pathways are activated through secondary genetic rearrangements taking place in E7-expressingcells (Fig. 4). Although mortal cell

Direct E7 effects (€7 targets)

Effects of mutatiodselection

extended life span

Immortal cell

Proliferation ON (pZ1. p27, pRb family) Apoptosis ON (PI s"?

MetabolitesUP

apoptosis OFF

1

~

_

_

_

telomerase ON tumor suppressors lost

Flg. 4 Two-step model for cellular immortalization by HPV-16 E7. Progression of a diploid human cell to E7-induced immortality is schematicallydepicted. In a first step, cell proliferation is induced directly by E7 through the binding and inactivation of cell cycle inhibitors. Concomitant with increased cell proliferation, the cells display a high frequency of apoptosis, probably also induced by E7. The E7 target involved in this activity has not been identified; it a p pears possible that E7-induced modulation of pl9gene expression is involved (see text). Progression of cells through crisis is accompanied by the reactivation of telomerase and most likely the loss of some tumor suppressor proteins. At present, there is no evidence that E7 may directly be involved here, and it is assumed that these steps result from random mutation of cellular genes. On the other hand, the ability of E7 to modulate M2-PK activity may well contribute to immortalization; furthermore, it is conceivable that the ability of E7 to suppress apoptosis contributes to immortalization, although the relevant cellular targets for E7 are still to be identified.

22

Werner Zwerschke and Pidder jansen-Diirr

some aspects of the model shown here still require experimental confirmation, we would expect that, with the discovery of additional cellular target proteins for E7, new properties of this fascinating multifunctional protein will become apparent.

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Tumor Invasion: Role of Growth Factor4nduced Cell Motility Alan Wells* Departments of Pathology and Cell Biology University of Alabama at Birmingham and Birmingham Veterans Administration Medical Center Birmingham, Alabama 35294

I. Introduction 11. Tumor Invasiveness A. Invasion Is Distinct from Metastatic Spread B. Measuring Tumor Invasiveness C. Invasion of Nonneoplastic Cells into the Tumor In. Cell Motility A. HaptokinesidHaptotaxis versus Chemokinesis/Chemotaxis B. Measuring Motility C. Biophysics D. Biochemistry lV. Motility in Tumor Invasion A. Proteases in Invasion and Motility B. Growth Factor-Induced Motility as Rate-Limiting C. Adhesion Receptors in Growth Factor-Induced Invasion D. Motility Suppressors V. Therapeutic Interventions A. Targeting Invasion as an Adjuvant Therapy B. Targeting the Biophysical or Molecular Aspects of Motility C. Targeting Invasion-Associated Motility VI. Summary and Future Directions References

Cancer progression to the invasive and metastatic stage represents the most formidable barrier to successful treatment. To develop rational therapies, we must determine the molecular bases of these transitions. Cell motility is one of the defining characteristicsof invasive tumors, enabling tumors to migrate into adjacent tissues or transmigrate limiting basement membranes and extracellular matrices. Invasive tumor cells have been demonstrated to present dysregulated cell motility in response to extracellular signals from growth factors and cytokines. Recent findings suggest that this growth factor receptor-mediated motility is one of the most common aberrations in tumor cells leading to invasiveness and represents a cellular behavior distinct from adhesion-related hapto*Current address: Department of Pathology, University of Pittsburgh and Pittsburgh Veterans Administration Medical Center, Pittsburgh, Pennsylvania 15661.

Advances in CANCER RESEARCH 0065-23OWOO$30.00

Copyright 8 2000 by Academic Press. All rights of reproduction in any form reserved.

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kinetic and haptotactic migration. This review focuses on the emerging understanding of the biochemical and biophysical foundations of growth factor-induced cell motility and tumor cell invasiveness, and the implications for development of targeted agents, with particular emphasis on-signaling from the epidermal growth factor (EGF) and hepatocyte growth factor (HGF) receptors, as these have most often been associated with tumor invasion. The nascent models highlight the roles of various intracellular signaling pathways including phospholipase C-y (PLCy), phosphatidylinositol (PI)3’-kinase,mitogen-activated protein (MAP) kinase, and aain cytoskeleton-related events. Development of novel agents against tumor invasion will require not only a detailed appreciation of the biochemical regulatory elements of motility but also a paradigm shift in our approach to and assessment of cancer therapy. 0 2000 Academic press.

1. INTRODUCTION The ability of a tumor to breach tissue and matrix barriers and establish growing masses within normal tissue defines progression to the invasive and metastatic stage. This is the most ominous development in tumorigenesis, as localized tumors are, by and large, curable whereas invasive and metastatic tumors account for most of the mortality and morbidity of cancer. These two aspects of progression, invasion and metastasis, complicate our approach to tumor therapy; metastatic spread disseminates the tumor to ectopic sites and renders targeted approaches (surgery, radiation, directed pharmacology) to the primary tumor mass partial at best, and tumor invasion compromises normal tissue and adnexa with the result that targeted approaches are either not feasible or result in significant comorbidity. With significant advances and real successes in treating primary, localized tumors, the next challenges lie in limiting tumor progression. To this end, investigations have sought to define cell properties critical for metastasis and invasion. Invasion occurs when a tumor cell acquires properties that enable it to pene‘trate the surrounding tissue or basement matrix. This consists of a number of definable steps in an oft-described schematic (Fig. 1). Invasiveness is marked by cellular transition to a mesenchymal phenotype (Birchmeier et al., 1993). These dedifferentiated cells then must loosen attachments to the primary tumor mass to allow for a leading edge free of cell-cell constraints. This subset of cells then must recognize the surrounding stroma or matrix and actively migrate into and/or through that space. Often the invasive tumor reorganizes the surrounding barrier to enable penetration, this usually takes the form of proteolytic degradation, but in many instances the cells may migrate along tissue planes and through pores requiring minimal degradation (Fried1et al., 1998; Nehls and Herrmann, 1996). Thus, the active migration of the cells into the surrounding tissue is a defining characteristic of the invasive tumor (Cianchi et al., 1997; Levine et al., 1995). It is becoming evident that cell motility is one of the rate-limiting steps of

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1 matrix I Fig. 1 Steps of tumor invasion. To accomplish tumor invasion of adjacent tissues and transmigration of matrices, a number of steps are envisioned. (1)The primary tumor (hexagonal cells) must undergo a mesenchymal transition to a fibroblastoid phenotype (oblong cells)which is concomitant with (2) a loosening of attachment to the primary tumor mass and acquisition of a fusiform, motile morphometry. These cells then (3)penetrate the surrounding stroma or matrix, usually as a loosely connected sheet of cells. Invasion is only evident if these tumor cells can then survive and proliferate in the ectopic site (4). It should be noted both that the ectopic tumor growth may not retain the properties of the original invasivecells (denoted by oval cells) and that cells of the invasive subclone likely exist in both the primary tumor and the ectopic growth. The presence of tumor microheterogeneityand phenotypic drift, coupled with contamination of defined tumor masses by invasive subclones, confounds easy genetic and biochemical analyses.

invasion. Cell motility is physiologically tightly controlled, with growth factor- and cytokine-inducedmotility being noted predominantly during organogenesis, inflammation, and wound repair. However, late in the tumorigenic cascade induced motility appears to become dysregulated with invasion the result. How this aberrant motility is initiated or maintained is the key to understanding the transition to invasion. One approach has been to identify genetic alterations in cancer. Most of the genetic defects that mark tumorigenesis are linked to earlier neoplastic transitions (Kinder and Vogelstein, 1996; Lenguaer et al., 1998); the few that correlate with invasiveness, such as amplification of the EGF receptor (Libermann et al., 1985), do not, in themselves, provide ready clues to the mechanisms of motility control. This

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lack of d i d genetic data suggests that invasion is either an epigenetic phenomenon or is sufficiently distantly downstream of a mutation to appear so. Therefore, to better understand invasion and highlight potential therapeutic targets, we need to conside; what is known about the regulation of growth factor-induced cell motility. This review focuses on the underlying biophysical and biochemical regulatory controls of cell motility with special reference to how these relate to tumor invasion when induced by growth factors. This discussion is being limited to the invasiveness of solid tumors derived from physiologically adherent cells; tumors of hematopoietic origin likely represent a special case of invasion befitting the different nature and regulation of adhesion and migration of cells that pass a significanf portion of their life cycles in the nonadherent state. However, it is likely that similar if not identical underlying cellular mechanisms operate in both situations. Furthermore, the discussion will focus mainly on growth factor-induced cell motility, rather than adhesion-mediated motility, as much current research suggests that tumor cell responses to growth factors and other cytokines promotes invasion. This is not a hard and fast distinction as matrix components uncovered during matrix remodeling may engage a different panel of adhesion receptors or a switch in integrins may initiate new signaling pathways in a fashion akin to growth factor-induced cell motility. In the absence of well-defined models deciphering tumor invasion, reflecting a nascent field, one is forced to focus on the regulatory aspects of motility to provide a foundation for rational approaches to the biology and treatment of tumor invasion.

11. TUMOR INVASIVENESS Tumor cells often demonstrate an increased ability to penetrate matrices and other tissues. This is noted in de novo human tumor specimens and animal tumor models by histological analyses in which one finds tumor cells inteicalating into adjacent tissues or breaching a basement membrane to gain access to a conduit for dissemination (vasculature or body cavity). This phenomenon has been termed tumor invasion. Usage varies in whether this term includes the entry of tumor cells into capillaries and small lymph vessels, often called ccmicroinvasiveness. ” This uncertainty is reflected in the biological situation in which tumor movement into these small vessels may occur either by transmigration of a basement membrane or by being “trapped” during neoangiogenesis and lymphangiogenesis. Herein, we focus on the tumor transmigration of established matrices and penetration into formed tissues as invasion.

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A. Invasion Is Distinct from Metastatic Spread The cellular processes that enable invasion can be conceptually deconstructed into separate steps: (1) mesenchymal transition, (2)loosening of attachment to primary mass, (3)penetration of surrounding stroma or matrix, and (4) continued survival and growth in the new site (Fig. 1). The aspect that is unique to invasion is cell migration into the surrounding tissue. The mesenchymal transition or dedifferentiation that appears to be requisite for invasiveness (Birchmeier et al., 1993,1995) also occurs in tumors that have not progressed to this state. This epithelial-mesenchymal transition appears to be part of a physiological program that promotes wound healing which can proceed from epithelial cells in addition to stromal precursors (Clark, 1996; Martin, 1997). The breaking of attachments to the primary tumor mass and survival and growth in an ectopic site are properties shared during metastatic spread. Invasion is separable from metastasis both medically and biologically. It is well known that different human tumors display predilections for one type of tumor progression over the other. As examples, glioblastoma multiformes invade the brain parenchyma leading to death well before evidence of metastatic spread, whereas many breast cancers recur after dissemination from at most microinvasive primaries that were excised years earlier. Furthermore, in metastatic spread, the tumor cells need to acquire properties that enable survival during separation from the primary tumor mass during dissemination and those that permit growth separate from the primary tumor. During invasion, the tumor remains, by definition, contiguous with the primary site, allowing for orthopic stromal and paracrine supporting elements and factors. The invading tumor cells may move as a group, maintaining cell-cell connections and junctions to the primary mass (Farina et al., 1998; Fried1 et al., 1995). Other biological differences have been postulated based on the pathologic findings. For metastatic spread, the tumor cells must gain access to conduits for dissemination, often involving lymphatics and vascular beds. This may require transmigration of a matrix barrier. However, during neoangiogenesis and lymphangiogenesis triggered in a tumor by vascular endothelial growth factor (VEGF)and other factors (Folkman, 1997; Folkman and Klagsburn, 1987; Hanahan, 1997), tumor cells may gain access to nascent vascular beds prior to the formation of a mature basement membrane. In fact, recent advances in visualizing live tumor cells during metastatic dissemination suggest that the major rate-limiting step is neither intravasation nor extravasation from vascular conduits but rather growth in the ectopic site (Koop et al., 1996; Luzzi et al., 1998). Thus, metastasis may require little invasive ability, with the resultant metastases grqwing as localized tumors. For invasion, on the other hand, the tumor must penetrate into the surrounding

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parenchyma and obliterate defining boundaries. Obviously, such a variant behavior bespeaks different operative cell processes.

B. Measuring Tumor Invasiveness Standardized methods must exist to study any experimental process; in many ways the assays available define the process under investigation. The “gold standard” for invasion is pathologically identified invasion of tumors in the natural hoq. However, such a yardstick is unattainable for mechanistic studies in humans and extremely cumbersome and expensive even in animal tumor models. Therefore, a number of alternative predictive assays shave been proposed. The most common of these are discussed below, not as an exhaustive review, but rather to provide a flavor for the various approaches to the complex phenomenon of tumor invasion. These are presented in descending order of complexity.

I . HUMAN STUDIES The search for genes that promote invasiveness in human cancer has followed the strategy common for finding disease-related genes. One major effort has involved analyzing de novo tumors in an effort to discern changes that lead to invasion. This has successfully implicated a large number of genes at many stages of carcinogenesis. However, the challenges of identifying causal changes late in tumor development are formidable. In many cases, the nature of the specimens and information available limit the correlations to prognostic outcomes or presence of identifiable metastases. Despite these obstacles, a number of tumor parameters, such as angiogenic density (Pluda, 1997), and genes, including proteases (Stetler-Stevenson et al., 1993a; Yan et al., 1998), integrins (Rabinovitz and Mercurio, 1996; Varner and Cheresh, 1996),and growth factors (Aaronson, 1991; Finn etal., 1997), have been linked to tumor invasiveness. In one illustrative case, EGF receptor signaling has been linked to bladder cancer invasion, with the demonstrdtion of increased levels of EGF receptors being localized to the site of invasion (Neal et al., 1985; Nguyen et al., 1994; Rao et al., 1993). Most of these putative invasion-related genes have been identified through the candidate gene approach in which the search is targeted. Technical advances have enable a more unbiased approach to identifying molecules that promote invasiveness; these include differential display (Sager, 1997; Sotiropoulou et al., 1997) and microarray processing (Iyer et al., 1999; Welford et al., 4998). These approaches also have the advantage of being able to detect epigenetic changes at the level of transcription (but not at the important levels of protein stability, localization, or modification). Current

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efforts are likely to yield many new candidates for molecules involved in tumor invasion. These clinical correlations cannot distinguish between epiphenomenon and true linkage, let alone causality. This would require direct interventiona1 trials. A number of such trials are underway (Parsons et al., 1997) and, it is hop,ed, will highlight therapeutic targets. Still, these targets may not represent true invasion-promoting molecules. For instance, an apoptotic agent may lead to reduced observed invasiveness as the tumor mass involutes, and thus the decreased tumor invasion, though clinically important, represents an indirect effect. 2: ANIMAL MODELS (de Novo AND XENOGRAFI'S)

Animal models of tumorigenesis have yielded important insights into the stages of tumor development and key molecular events. The use of animal models 'reflects the appreciation that tumor progression, like tumor development, represents the interplay of behaviors of both the tumor cell and the host. Host contributions include acquired and innate immunity, physical constraints, and various cytokines and growth factors. Only whole animal models present the span of these processes. The advantages of animal models over human studies are that interventions are possible, not only to limit tumor properties, but, importantly, to enhance them in an attempt to define both events that are required and those that may be modulated by the tumor itself in a rate-limiting manner. However, few models and studies have focused on tumor invasiveness directly. Reasons for this are obvious; the variable penetrance of both genetic and induced tumors and the long lead time to tumor development are further confounded by the fact that many of these tumors are noninvasive. Still a few models are available for investigation that are reported to invade rapidly after development; two are the chemically (BBN)-inducedrat bladder model (Inui et al., 1996) and the T antigen-driven transgenic mouse prostate cancer model (Greenberg et al., 1995). As we learn more from other investigational approaches, human correlations, or in uitro assessments, newer models are likely to be developed for studying the development of invasive tumors (Kim et al., 1999). Tumor grafts into mice are commonly used to investigate invasiveness. This usually takes the form of implanting tumors and cell lines from either humans or other rodents into susceptible mouse hosts, immunoincompetent in the case of xenografts and related strains in the case of allografts. The literature on this is too vast to cite, but a few principles warrant discussion. First, the site of the graft appears critical to tumor growth and progression; orthografts are likely to yield the most faithful recapitulation of progression, whereas ectopic subcutaneous grafts (excepting melanomas which are or-

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thopic for the skin) are often disappointing in terms of little invasion and few, small metastases (Crowley et al., 1993; Fidler, 1991; Gleave et al., 1992; Stephenson et al., 1992). Second, invasiveness needs to be scored in a quantitative manner by defining a>eproducible target of invasion. The diaphragm invasion model following peritoneal seeding of tumors allows such analyses, including both fequency of invasion and extent of isvasiveness (Knox et al., 1993; Turner et al., 1996, 1997). Third, tumors display a natural predilection to mutate enabling the selection of invasive sublines. Determining differences between the noninvasive parental line and the daughter invasive lines can be fruitful for identifying changes that may be sufficient for invasive activity (Bao et al., 1996).And last comes a cautionary note that interventions, whether aimed at augmenting or inhibiting invasion, may identify a mechanism that does not promote invasion in the de novo pathological state. For instance, inhibition of specific metalloproteinase-mediated proteolysis may limit invasion, but it is not clear that the transition to invasion is accompanied by modulation of matrix metalloproteinase (MMP) levels, these events being necessary but not the altered event (Chambers and Matrisian, 1997). Despite these concerns and limitations, the whole animal studies are critical to understanding tumor invasiveness. The overriding advantages of these murine models are that the invasiveness can be scored by established histopathological analyses and that these situations reproduce the important interactions between tumor and host which define tumor behavior.

3. INVASION INTO TISSUES AND MATRICES The quickest and simplest approach to assessing the invasive potential of a tumor is to determine whether a tumor cell or mass can penetrate tissues in vitro. This includes migration into tissue explants and natural and artificial matrices. The advantages of these methods are speed, low cost, reproducibility, and amenability to interventions. Thus, one can easily introduce pharmacological, biological, and molecular agents that may be impractical in whole animal studies due to toxicity or delivery problems. With answers in days rather than weeks to years, these studies are often used to provide a proof-of-concept rationale for animal studies or as initial confirmation of causality in clinical correlations. To re-create the invasive environment, models have been generated that investigate movement of primary tumor cells or derived cell lines into tissue aggregates. This has been used most extensively in evaluating glioma invasion (Engebraaten et al., 1993; Penar et al., 1998). Howevet, this requires that the target tissue can be maintained easily in culture over the many days required for invasion to occur. Invasion into acellular extracellular matrices has been shown to be a first level correlate for invasion in vivo (Albini et al., 1987).In these models, cells

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are assessed for their ability to penetrate into or through either a complex biological matrix or a collagen gel. Cells can be enumerated either within the matrix or after they have transversed a barrier of known thickness; metabolic labeling of the cells prior to application onto the matrix will account for any confounding effects of differential proliferation during the course of these experiments that typically occur over a 2- to 4-day period (Siegalet al., 1993; Xie et al., 1995). The use of biologically active complex matrices is preferred on theoretical grounds as these matrices contain multiple signaling moieties, including both adhesive and antiadhesive components, and can be recognized by many different adhesion receptors (Bornstein, 1995; Fried1 et al., 1998; Murphy-Ullrich, 1995; Stracke et al., 1994). The most commonly used extracellular matrix for this purpose is the Engelbreth-Holm sarcoma-derived Matrigel (Albini et al., 1987). This transformed cell-line-derived matrix has the confounding problem of presenting high levels of growth factors which have been implicated in the invasive process, including transforming growth factor-a (TGFa), platelet-derived growth factor (PDGF), and TGFP (Siegal et al., 1993); even the newer, “growth factor-reduced” product contains levels of these factors that can signal over extended periods. Thus, if one is examining the effects of growth factors on invasion, care must to be taken to account for the presence of these factors in Matrigel. An alternative matrix has been described that is derived from human amniotic membranes, Amgel; the advantage of this matrix is that it does not contain detectable levels of ligands for the EGF, PDGF, fibroblast growth factor (FGF), or TGFP receptors (Siegal et al., 1993). Amgel, which is not commercially available, does contain most of the main matrix components of Matrigel, and can be used similarly. To avoid these problems, one can assess cell migration into or through simple collagen gels (Terranova et al., 1986; Westermark et al., 1991). These gels have the simultaneous advantages and disadvantages of being monotonous, with few confounding variables (see Section III,C,2,c on matrix architecture, below), but lacking the ability to stimulate many biological functions. This diversity of experimental models shows that there is no one clearly superior approach. The choice of experimental system therefore depends on the compatibility of the cells with the matrix and the precise nature of the investigative question. One main caveat in predicting invasive behavior from these in vitro model systems is their lack of a organismal aspect. Tumor development and invasion are becoming recognized as a “field effect” in which the surrounding nonneoplastic tissues and cells contribute to the outcome of the neoplastic transformation. Furthermore, the response of the organism to the tumor is also critical. Not only are there inflammatory and immunological responses, which may either limit or perversely promote tumor progression, but ingrowth of new vessels and nerves may provideconduits for dissemination or natural cleavage planes for invasive migration, as well as producing factors

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to which the tumor may respond (Djakiewet al., 1993).Despite the fact that these extracellular matrices lack the stromal compartments and organismal aspect that greatly influence tumor invasiveness in vivo, they have proved valuable in providing copiok data for further investigation.

C. Invasion of Nonneoplastic Cells into the Tumor Recent excitement has focused attention on invasion into the tumor of blood and lymph yasculature. This mode of cell invasion likely promotes tumor progression. Many studies have shown angiogenic indices to be useful prognostic markers for tumor progression and invasion. As such, it warrants brief mention in this discussion on cell motility in tumor invasion, though any causality vis-8-vis invasion is still speculative; what is evident is the need for angiogenesis to sustain large tumor nodules. What is clear from numerous investigations is that this form of physiological invasion of the endothelial structures utilizes the same biophysical mechanisms for cell transmigration and is signaled by many of the same factors that promote tumor invasion. Angiogenic growth factors that have also been implicated in tumor invasiveness directly include TGFa, HGF, PDGF, TGFP, and various FGFs. In addition, recent reports demonstrate that host-derived molecules, such as PAI-1 (Bajouet al., 1998)and MMPs (Itoh etal., 1998), are required for both angiogenesis and invasion, suggesting that the two may be linked. This has been supported by-a report that the introduction of an antagonist to the VEGF receptor blocked both angiogenesis and keratinocyte invasion in a xenograft model of tumor progression (Skobe et al., 1997). In addition, it is possible that VEGF can function as a growth factor for epithelial cells in addition to its angiogenic role; VEGF receptors were recently shown to be present and functional on thyroid cells, and their levels were increased after cell exposure to other growth factors (Wang et al., 1998). However, it is still uncertain whether these two aspects are causally linked, namely, that angiogenic ingrowth promotes tumor invasiveness or merely represent two aspects of tumor progression. That similar factors and biochemical signals are utilized would not be surprising since these phenomena represent a physiological and pathological consequence of cell motility-invasion of cells into new sites.

111. CELL MOTILITY Cell motility or locomotion consists of moving the cell body from one locale to another, distant site while attached to a surface. This involves progressive extension at the leading edge and detachment and forward movement at the trailing end. This process is operational in many physiological

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and pathological situations; cell motility is most evident as a major component of organognesis, inflammation, and wound repair. This process shares major features and molecular controls with axonal growth, a specialized situation which will not be considered herein. Furthermore, it is becoming evident that some of the same processes are used for cell spreading and extensions even in the absence of cell translocation. The discussion below attempts to synthesize what is known about the regulation of cell motility in terms of biophysical steps and biochemical control elements. This has been best characterized to date in fibroblastoid cells. Data from epithelial cells generally support the findings in the fibroblastoid cells. Certain discrepancies, such as EGF-induced motility coinciding with a loss of focal adhesions in fibroblasts (Xie et al., 1998) but a gain in mammary carcinoma cells (Bailly et al., 1998a), are reconcilable in the context of an overall model of cell motility incorporating these different biophysical and biochemical rheostats (Fig. 2). It is legitimate to extrapolate findings from fibroblastoid cells to carcinoma invasion as these tumor cells undergo a mesenchymal transition during tumor progression in which they regain many of the fibroblastoid behaviors (Birchmeieret al., 1993). As the best characterized growth factor system for induced cell motility is that of the EGF receptor, a growth factor receptor linked to the invasive progression of numerous tumors, we will highlight specifics from this system.

A. Haptokinesis/Haptotaxis versus

Chemokinesis/Chemotaxis Cell locomotion can be triggered by signals generated by receptors that participate in adhesion and receptors for cytokines and growth factors. The

Fig. 2 Steps of cell motility. The various biophysical aspects of cell motility are underlined and given with their associated biochemical intermediary signaling molecules. The active involvement of the italicized molecule, phospholipase C-y (PLCy), at this point appears unique to growth factor-induced motility; active signaling via the 'other molecules seems to be shared by both haptokinetic and chemokinetic motility. The role of rho in adhesion in the protruded lamellipod is as yet unproved. Adapted from Wells et al. (1998).

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former mode of motility is referred to as haptokinesis and haptotaxis, while the latter is constituted by chemokinesis and chemotaxis. The underlying cytoskeletal mechanisms of cell locomotion are likely identical between the two modes of motility as the same biophysical steps and cytoskeletal actions are required. What seems to distinguish the two are quantitative changes at the biophysical level, chemokinesis displays faster motile speeds within a cell type and, possibly, differential involvement of select regulatory elements, whereas growth factor receptors seem to preferentially recruit PLCy signaling. However, one hesitates to make a hard distinction between the growth factor recegtors and adhesion receptors (integrins, CD44, etc.) in that the latter group is quite diverse, with some integrins likely functioning more in the mode of a growth factor receptor ‘with a primary function of triggering downstream elements [e.g., a6P4 controlling CAMPgeneration during motility (O’Conner et al., 1998)Jthan providing traction in the classic concept of adhesion receptors. Furthermore, although in fibroblast systems in vitro it is relatively easy to parse the contributions of adhesion receptors and growth factor receptors, in vivo and in many other in vitro systems the two are intimately intertwined. The adhesion receptors are necessary to provide traction and basal signaling on which the growth factor receptors act, disruption of which will prevent any motility or invasiveness (see below). In addition, many adherent cells have constitutive signaling from both sets of receptors. For example, breast and prostate epithelial cells and tumor cell lines present autocrine EGF receptor-stimulating loops (Ahmed et al., 1991; Cook et al.; 1991; Tillotson and Rose, 1991; Wiley et al., 1998). The behaviors of such cells might be dictated as much by the autocrine signaling as the adhesion receptors engaged. Therefore, disruption of specific “adhesionsignaled” pathways may also affect growth factor receptor regulatory signals, with the combination of the two resulting in the measured outcome (Keely et al., 1997). Despite these caveats and the danger one faces in dissecting the two classes of receptors, such a reductionist tool is necessary to decipher mechanisms at this early stage in our understanding of cell motility. As much current evidence points to chemokinetickhemotactic cell motility as being important in tumor invasion, this discussion will focus on that mode of induced cell motility. 1. GRADIENTS

Cell motility in response to an extracellular signaling factor, such as a growth factor, may occur either in a gradient of the factor (-taxis) or in a macroscopically homogeneous solution of that factor (-kine&). The implications of these two different modes of signaling motility are immense for both the biophysical steps and the biochemical regulatory controls intrinsic to the cell and the generation and source of the signals extrinsic to the cell.

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Establishing a directionality to cell locomotion involves a cellular asymmetry with a leading lamellipod and a trailing edge (Lauffenburger and Horwitz, 1996). It is seemingly self-evident how a cell establishes polarity during chemotaxis; it orients into the face of the oncoming signal based on an increase in receptor signals. However, numerous mathematical models have argued against this simple view on the basis that gradients across cells are too “shallow” to enable easy localization of source (Alt, 1980; Fisher et al., 1989; Maheshwari and Lauffenburger, 1998), though others suggest that minute differences integrated over time would allow for a fine discrimination (Gurdon et al.? 1998). Despite these theoretical reservations, it is evident from classic time-lapse images of motility of Dictyostelium toward CAMP and neutrophils toward chemoattractants that these cells sense the proper source and move productively and progressively toward that source, up to a point at which the local concentration saturates cell receptors. This ability for a cell to discern concentration differences has recently been reinforced by images of mammary carcinoma cells locomoting toward EGF (Badly et al., 1998b) (seehttp://www.ca.aecom.yu.edu/anatomy/segall/ Movies/MTwel.htm for moving images). Thus, either cells can sense minute gradients, or a mechanism for reinforcement exists. This does not likely involve redirecting chemoattractant receptors toward the gradient of factor, as in Dictyostelium the receptors are not concentrated in the lamellipod and do not align with the gradient (Xiao et al., 1997). Rather, such a mechanism might involve directing the motility machinery toward the cell front. This has been observed with integrins and other adhesion receptors recycled to the front of migrating neutrophils and fibroblasts (Bretscher, 1996; Lawson and Maxfield, 1995; Shaw et al., 1998), talin, which links the adhesion receptors to the cytoskeleton, to the leading edge in Dictyostelium (Lawson and Maxfield, 1995), and the actin modifying protein profilin in the extending lamellae of fibroblasts (Buss et al., 1992). However, these intracelMar redistributions may reflect changes required for persistence rather than localization of a gradient. Further investigations in which these molecular asymmetries are disrupted will determine their causal role in chemotactic locomotion. For chemokinesis the story is likely more complicated. Herein the aggregate cell movement is random reflecting the homogeneous nature of the signal, though each cell at any given time moves in its own vectorial direction. Establishing this cell directionality is likely stochastic. But how a cell then determines a predominant lamellipod and begins to locomote are still uncertain. In the case of EGF, a strong chemokinetic agent, directional motility does not become predominant until some 4 hr after stimulation (Ware et al., 1998), though whether this reflects required changes in the cell proteome or an adaptive refractile time (to allow alteratims in substratum adhesions, for instance) is unknown. It has been proposed that establishing a polarity

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may require specific intracellular switches such as cdc42 (Nobes and Hall, 1999), but this may function to suppress secondary, nondirectional lamellipodia from forming. Interestingly, it also has been suggested that downstream elements activated during chemokinetic motility niay be different than during chemotactic motility (Anand-Apte and Zetter, 1997). During chemotaxis, pancellular activation or distribution of a messenger would disrupt the asymmetry and end locomotion, whereas in a chemokinetic situation the intracellular milieu is adjusted to distinguish between the directional and nondirectional signals. Experimental evidence for this attractive hypothesis is still lacking. The major motility parameter that is differentially affected by signaling gradients or lack thereof is persistence. Persistence is defined as the continuation of a locomoting cell proceeding in a constant direction, and it is often expressed in terms of time or distance; at a persistence of 0 the cells would not move from a stationary position (Maheshwari and Lauffenburger, 1998; Ware et al., 1998). In the face of a signal gradient,’chemotaxis, persistence is maintained by the gradient, with directional changes concurrent with alterations in the source of the signal (Bailly et al., 1998b) or when the cell reaches saturation of receptors. Thus, the external gradient imposes the persistence. During chemokinesis, in which there is no extrinsically defined “directionality,” intrinsic cell mechanisms define the persistence. How persistence is maintained is likely due to reinforcement of motility signals and recruitment of the motility machinery as discussed above and below. However, by persistence being intrinsically defined and changeable during chemokinesis, a cell can proceed in a random walk manner to sample a large area rather than proceed in a preordained direction (Maheshwari and Lauffenburger, 1998). This, in fact, is one of the major properties of EGF-induced motility, that while EGF increases motile speed, it decreases persistence time dramatically, with the result that a small cell population becomes highly dispersed to engage a large surface area (Ware et al., 1998). For tumor invasiveness, the consequences are obvious. As cells must find a permissive environment to survive and grow, the ability to sample large areas would confer an advantage. Therefore, one may predict that the growth factors that promote tumor invasiveness are those that provide primarily chemokinetic signals, such as EGF and HGF, rather than chemotactic signals. A large part of the distinction between chemotaxis and chemokinesis resides in the source of the external motility signal. For instance, EGF, which functions as a strong chemokinetic signal (Ware et al., 1998), can direct chemotactic events (Baillyet al., 1998b; Blay and Brown, 1985),possibly via a biased random walk up a chemokinetic gradient (Alt, 1980; Maheshwari and Lauffenburger, 1998). Growth factor signals can be presented in a number of ways. There is little support for classic endocrine signaling in tumor invasion or cell motility. Rather the growth factor, and adhesion receptor,

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signals appear to be locally derived. Paracrine release of factors could provide for both chemotaxis, if coming from a point source such as in ingrowing vessel or nerve, or chemokinesis, if produced by intercalated stromal elements or neighboring tumor cells. There is growing evidence that much of the induced cell motility in tumor cells is from autocrine signals, both of classic growth factors, such as TGFa and HGF (Kim et al., 1999; Matsumoto et al., 1995; VandeWoude et al., 1997), and of relatively tumor-specific factors, as autotaxin (Klominek et al., 1998; Stracke et al., 1992).This autocrine situation may arise from the mesenchymal transition that epithelial tumors undergo (Birchmeieret al., 1993),with the consequent loss of polarity and tight cell-cell junctions disrupting the physiological separation of receptors and ligands (Fig. 3). In secretory epithelial cells of the urogenital and mammary systems, the separation of apically produced EGF and TGFa from the basolateral EGF receptors is lost during tumorigenic dedifferentiation and enables an active autocrine stimulatory loop (Kim et d., 1999). Thus, molecules (e.g., cadherins, catenins) that prevent this loss of cell polarization and segregation of ligands and cognate receptors may function as tumor suppressors. The autocrine growth factors would be predicted to function as chemokinetic signals, and thus one is not surprised that the chemokinetic growth factors TGFa and HGF are often identified in these situations. Two other cell extrinsic forces would promote tumor invasion, or at least movement away from the tumor mass. First is the predilection for cells to

Fig. 3 Dedifferentiation enables autocrine signaling. Shown is a model wherein loss of cellcell junctions leads to autocrine signaling in secretory epithelium, as exemplified by prostate cancer progression. In the normal physiological situation (left), tight junctions established by homotypic cadherin binding spatially segregates apically secreted EGF from the basolaterally presented EGF receptors (which respond to stromally derived TGFa). During early tumorigenesis (middle),genetic and epigenetic signals destabilize the cell tight junctions, enabling the lumenal EGF access to the EGF receptors. This autocrine signaling leads to further breakdown of tight junctions and cell proliferation secondary to EGF receptor signaling (Hazan and Norton, 1998). Consequently, the full dysplastic phenotype can be observed (right). In addition, EGF receptor signaling enhances TGFa expression through a positive feedback loop (Bjorgeand Kudlow, 1987). At the edges of the dysplasticcell mass, the ligalrd would serve as an autocrine sonar promoting cell dispersal. Adapted from Kim et al. (1999).

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move into an acellular area. This is noted acutely on creation of wounds in a monolayer of cells, even in the absence of matrix components in the space. The cells at the wound front actively migrate into the denuded area with restricted activation of various small G protein molecular switches indicative of cell motility (Nobes and Hall, 1999).In isolated colonies of epithelial cells, EGF receptor activation results in the outermost two rows of cells migrating outward, while the inner cells proliferate to increase the colony numbers (Barrandon and Green, 1987). It is likely that this cell movement from the existing mass is dictated by cell-cell contacts and physical considerations. Autocrine signaling may even play a role in directing the cells away from others. In this proposed scheme, autocrine signaling acts as sonar; when other cells are present in one direction that side df the cell senses a deficit of factor as the neighboring cells bind the factor, with the result that the cell locomotes toward the opposite side with the more active autocrine signaling loop. Still, invasion often occurs into an established cellular organ such as soft tissues or through a smooth muscle capsule ‘(especiallyfor urogenital carcinomas). In these situations, a putative mechanism of motility signaling may occur, one which has been termed “sequestrine” (Wells et al., 1998). In this proposed system, as the cell moves into and remodels a matrix or foreign tissue, signaling components are uncovered. These could either be predeposited growth factors that are bound to matrix, such as heparin-binding EGF (HB-EGF) and other heparin-binding factors, or matrix components themselves-new integrins may be engaged to signal as a cell enters a novel tissue and its constituent matrix components (O’Conner et al., 1998; Shaw et al., 1997; Weber et al., 1996). Last, it is possible that matrix components harbor cryptic signaling domains that are liberated during matrix degradation (see below). Any of these possibilities would provide for a chemoattractant gradient, whether chemotactic or directed chemokinetic, that would promote further tumor cell incursion.

B. Measuring Motility As with invasion, cell motility is defined by the methods of assessment. Here, the different assays are designed to quantitate various aspects of motility or different end points. In addition, specialized assays measure distinct events and stages of motility, but those will not be discussed herein. 1. TRANSWELL CHAMBERS

One of the simplest and commonly used methods determines the extent of cells moving across a porous filter. This is similar to the transmigration assay for invasiveness. In this assay, one places a known number of cells on top

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of a filter with pores slightly smaller than spread cell area and after a predefined time, usually a few hours, enumerates the percentage of cells that have translocated to the lower side of the filter. For chemotaxidchemokinesis, various attractants or factors are added to either the upper or lower chamber; a checkerboard analysis will distinguish chemotaxis from chemokinesis (i.e., for chemotaxis, factor in the lower well only has greater translocation than factor in both than factor only in top well). For haptotaxis/haptokinesis, the filter is coated on either surface or both, and translocation assessed. Caveats limit the widespread applicability of this method. The first concerns the physical nature of the barrier pores. For a cell to translocate to the bottom side of the filter, it must change its shape and effective diameter. Thus, co‘mparing between two cells or two treatments runs the risk of anomalous results based on differences in cell deformability as much as motility. One example of this would be fibroblasts derived from gelsolin-knockout mice: these cells display close haptokinetic motility, but the gelsolin-devoid cells cover many times the surface area of the heterozygous fibroblasts (Witke et al., 1995; Xie et al., 1998);this size difference would exaggerate any differences in motility. Second, the distance a cell must travel is uneven, uncontrolled, and unquantified. Obviously cells that are seeded adjacent to or even on the pores need travel only short distances. In the case of haptotactic analyses, it is possible that cells adjacent to the pores can “sample” the bottom side without cell body translocation and thus move to the bottom based not on motility per se but on differential adhesiveness (DiMilla et al., 1993). A third concern is that the motility event can reasonably only be studied from the initiation of signals and not after a time delay. In the case of fibroblasts induced by EGF, there is a refractory window of about 1-2 hr, during which locomotion is actually reduced to below the haptokinetic baseline (Maheshwari etal., 1999);in the transwell chamber assay, this situation would be interpreted incorrectly over the initial few hours. Last, this is strictly an end point assay in which the various steps of motility cannot be discerned. Despite these inherent problems, this assay has been used successfully to yield many important insights into mechanisms of motility. 2. In Vitro SURFACE ASSAYS

The so-called “wound healing” assay represents a second simple in vitro method to determine motility in a quantitative manner. In this assay, cells are grown to a monolayer and a defined edge is generated by physically removing the cells. Cells are then assessed, usually at discrete time points but possibly by time-lapse videomicroscopy, for the extent of movement into the denuded area. Often the denuded area is in the form of a gap, and the time to closure of the “wound” is measured. This method avoids the need for cellular deformation required during transwell translocation, and allows

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for assessment of intermediary time points and quantification of the extent of translocation. In addition, a delay in any response can be controlled for by introducing the acellular_area after stimulating the cells. However, this method is designed for chemokinesis and haptokinesis, and is not easily adapted for chemotactic or haptotactic analyses. One can measure movement into an acellular area from a cell colony. Here the cells are grown as compact colonies before introduction of signals and subsequent assessment of cell movement from the original colony center. For epithelial cells, this often entails both disruption of cell-cell adhesions and locomotion. For this reason, this assay is referred to as “colony scattering.” The advantage of this over the wound healing assay is that there is no need for mechanical denuding of cells with its concomitant mechanosensory signals and disturbance of a defined substratum or matrix. A variant of these assays aims to enable hapto- and chemotactic assessments. This can be accomplished by overlaying the cells with a semisolid inert medium (soft agar or methylcellulose).Introduction of small molecules into wells or pellets in the medium establishes a gradient. Recent technical advances aiming to generate continuous gradients on surfaces should soon allow haptotactic measurements.

3. DIRECT VISUALIZATION The most obvious but technically challenging and time-consuming method to measure motility is direct visualization. With time-lapse microscopy not only can overall motility be assessed, but also the individual parameters of speed and persistence can be parsed (Maheshwari and Lauffenburger, 1998). In addition, the individual biophysical steps of lamellipod protrusion and rear detachment are evident (Bailly et al., 1998b). And these can be determined at different times after stimulating. Locomotion can be tracked for both single cells and from acellular fronts of cell masses. Technical advances in vital labeling of cells and computer autofocusing of microscopes have extended this approach to movement in three-dimensional structures (Fried1et al., 1998). Chemotaxis is approached by watching cells move toward a souice of factors, usually either a pellet or a pipet tip. The chief limitations are the need for expensive instrumentation, the reliance on single cell assessment (with the need for many such assays for statistical purposes), and the time-consuming nature of these assays which may last up to 48 hr for a single experimental point. However, the wealth of data make this a gold standard for motility assessment ilz vitro, and the method is often used to confirm or expand on findings from the simpler above methods. Recent reports have demonstrated the feasibility of visualizing cell motility in intact organisms (Farina et al., 1998; Koop et al., 1996). These advances are sure to change paradigms of cell motility.

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C. Biophysics I . INTRINSIC BIOPHYSICS OF MOTILITY For simplicity of discussion, locomotion of adherent cells can be considered to be governed by distinct but orchestrated processes. These are lamellipod extension, cell-substratum adhesion at the leading edge, contractility to move the cell body forward, and release at the trailing uropodia (Fig. 2). Cytoskeletal reorganization is necessary to convert the cell from a sessile phenotype to a motile, asymmetric morphology, and is required throughout motility to maintain lamellipod growth and forward movement. These processes are interdependent, and alterations in one affect other aspects; for instance, decreasing integrin levels reduces cell adhesion and limits motility (Keely et al., 1995) likely by decreasing the contractile force that can be generated (Huttenlocher et al., 1995). Thus, it is not surprising that multiple processes may be affected by a single initial biochemical event; current data point to the possibility of cytoskeleton contraction directly leading to uropod detachment and forward movement of the cell body simultaneously, with this initial event being actuated by the erk MAP kinases (Klemke et al., 1997; Xie et al., 1998). Further upstream, activation of a single growth factor receptor can modulate many of these processes; the EGF receptor leads to direct regulation of cytoskeleton organization, cell contractility, and deadhesion (Wells et al., 1998), and the ligand for the c-met receptor, HGF/scatter factor (SF), is known by its dual phenotypic effects of promoting both cell dispersion and motility (Birchmeier et al., 1997; VandeWoude et al., 1997). This working model of cell motility does not define the rate-limiting steps; rather, each process is presented as required for induced cell motility. Since many of the biophysical processes occur in a basal state, signaled by the same receptors that provide adhesion to substratum, the integrins, syndecans, and CD44, it is possible that induced motility may be accomplished by upregulating just a subset of these processes, such that each individual one may appear to be sufficient for induced motility. In the following discussion, cell motility is deconstructed to individual cyclical steps to provide for testable hypotheses, realizing that this simplistic situation likely does not fully reflect the physiological nuances of cell motility.

a. Lamellipod Extension Lamellipod extension is the most obvious process in cell motility, as this protrusion extends the cell body to create new, distal adhesion sites. This extension of the cell membrane often occurs with membrane ruffling, a phenomenon in which the structure of membrane regions lacking focal contact sites produces extensions perpendicular to the surface under the actions of several small G proteins (Downward, 1992; Ridley et al., 1995).The link be-

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tween these two phenomena is uncertain at present. Ruffling in itself is insufficient for lamellipodia extension (Ridley et al., 1995; Welsh et al., 1991). Recently, it has been proposgd that ruffling and lamellipod extension are antithetical, with protrusion occuring on the cessation of ruffling (Rottner et al., 1999). This is supported by data which demonstrate that while EGF induces ruffling acutely (Ridley et al., 1992) lamellipod extension continues during progressive cell locomotion when ruffling is practically absent (Maheshwari et ul., 1999). This latter situation is qualitatively similar to the fish keratocyte in which motility occurs in the absence of observable ruffles (Small et al., 1995). Extension appears to require localized actin polymerization. This motility-associated actin reorganization, with possible concomitant polymerization (Chan et al., 1998), is controlled by numerous actin inodifying proteins (AMP).The activity and functioning of these proteins are modulated by interaction with polyphosphoinositides (Janmey and Stossel, 1987; Schafer et al., 1996; Sun et al., 1995), allowing for regulatory control by downstream effectors of growth factor receptors (Chen et al., 1996a; Kauffmann-Zeh et al., 1995; Kundra et al., 1994). Regardless of the underlying events, lamellipodia are required to extend the cell body. Lamellipod protrusion activity is increased by many growth factors and matrix components (Baillyet al., 199813; Chen et al., 1996a; O’Conner et al., 1998; Shaw et al., 1997; Ware et al., 1998). The kinetics of this effect depends on the cell type; in fibroblasts, lamellipodia extension is most marked after an initial retraction of the cell body (Maheshwari et al., 1999; Welsh et al., 1991), whereas’in epithelial tumor cells, lamellipodia are extended within minutes after EGF or HGF exposure (Bailly et al., 1998a). The difference in the timing of extension reflects the immediate post-EGF motility behavior of these cell types; epithelial tumor cells chemotax toward EGF within minutes, while fibroblasts require an induction period of hours before significant locomotion. This may represent either a fundamental difference between fibroblasts and epithelial cells or simply differences in cytoskeletal organization of the unstimulated cells, that is, rigid stress fibers that require reorganization prior to membrane extension may not be present in unstimulated epithelial cells; although this is of fundamental interest, it would have few implications for interventional strategies. A second possibility is the need for de novo transcription for fibroblasts to be capable of induced motility, whereas in epithelial cells the entire regulation of motility is epigenetic; this scenario has significant implications for targeted therapies against tumor invasion. Further investigation is needed to shed light on this important step. Cellular asymmetry and polarity are defined by the lamellipod. Acutely on growth factor stimulation, multiple lamellipodia and/or filopodia may be formed. Shortly thereafteq a dominant lamellipod emerges as other lamellipodia and filopodia are retracted and formation of new protusions suppressed (Bailly et al., 1998a). This lamellipod persists for at least as long as

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the cell continues in that original direction (persistence time). The mechanisms by which the dominant lamellipod is established and maintained, and competing lamellae suppressed, are presently not known. The relocalization of elements of the motility machinery may be involved; these would include adhesion receptors (Bretscher, 1996; Shaw et al., 1998) and intracellular molecules that link adhesions to the actin cytoskeleton (Buss et al., 1992; Lawson and Maxfield, 1995).The localization of subsets of translated mRNA to the base of the dominant lamellipod may further the persistence of this extension (Kislauskiset al., 1997). However, these redistributions may be an effect rather than a cause; in the case of the mRNA localization, the specific mRNA will shift to a new localization within minutes of changes in the dominidnt lamellipod or even new tension on the cytoskeleton (Chicurel et al., 1998). A change in migratory direction may be accomplished by either the dominant lamellipod altering the direction of extension, as seen in epithelial cells durbg haptotaxis (Bailly et al., 1998a), or by a second lamellipod forming and becoming dominant, as often noted in fibroblasts (Ware et al., 1998). Of great intrigue is that EGF treatment of fibroblasts, while increasing cell speed 3- to 7-fold, decreases persistence time and thus promotes the change in dominant lamellipod. Thus, the increase in cell motility may promote not the ability to traverse a specific distance, as would be important for axonal growth toward targeted innervation, but rather an increase in “sampling” of an area (Fig. 4). In this latter situation, speed would be intrinsic but final localization would result from the cell “finding” an appropriate environment. Thus, during tumor invasion, induced motility would enhance the ability of the tumor cells to locate a permissive environment or “soil” (Fidler, 1991).

b. Cell-Substratum Adhesion Cell adhesion is accomplished mainly by integrin and nonintegrin receptors binding to specific extracellular matrix protein domains (Faassen et al.,

no EGF

EGF

Fig. 4 Cell scattering is increased by EGF receptor signaling. Cell tracts of NR6 fibroblasts expressing EGF receptors were determined by time-lapse videomicroscopy. Each plot presents representative cell tracks for five cells, with the origins of migration superimposed at 0,O for clarity of presentation.Tracks were acquired during constant motility, from 8 to 20 hr after exposure to EGF (25 nM). Distance between hatch marks on’both axes is 50 pm. Adapted from Ware et al. (1998) with permission from the Company of Biologists Ltd.

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1992; Huttenlocher et al., 1995; Hynes, 1992; Thiery and Boyer, 1992). Attachments between cell and substratum are formed at or near the leading edge of migrating cells a-nd are reported to persist until they are detached at the cell rear (Lauffenburger and Horwitz, 1996). These interactions occur in a variety of physical forms which include the close (10-15 nm) and very structured focal adhesions, the more labile focal contacts, and the more diffuse close contacts (Burridgeet al., 1988). All of these attachments comprise transmembrane aggregations of integrins and syndecan 4 that serve to organize a defined set of cytoskeletal and signal transduction molecules (Burridge and Chrzanowska-Wodnicka, 1996; Miyamoto et al., 1995; Stossel, 1993). The strength of these attachments is proportional to density of both the extracellular matrix ligands (DiMilla et al., 1993) and the transmembrane receptors (Chen et al., 1993; Keely et al., 1995) as well as the composition of the cytoplasmic side of the adhesion plaque (Miyamoto et al., 1995). Thus, changes in cell adhesiveness would mirror alterations in the number and/or nature and composition of the cell-substratum contacts (Maheshwari and Lauffenburger, 1998; Xie et al., 1998). It is uncertain what roles these different forms of cell-substratum contacts play during cell motility. Part of this is due to the duality of the changes in adhesions-new attachments at the front, loss of adhesion at the rear. Fibroblasts lose their focal adhesions within minutes of exposure to EGF (Dunlevy and Couchman, 1993; Xie et al., 1998); this coincides with an acute decrease in adhesiveness. A large part of that adhesiveness is regained within a few hours even in the continued presence of EGF, though the focal adhesions are still absent (Maheshwari et al., 1999; Xie et al., 1998). Mammary carcinoma cells display an opposite response; chemotactic signaling by EGF results in new focal contacts forming at the base of the lamellipod (Bailly et al., 1998a). It is possible to reconcile these difference by positing that the epithelial cell focal contacts are more organized than the IRM-negative fibroblast close contacts but .still less structured or looser than IRM-positive focal adhesions indicative of a sessile fibroblast. If the extended lamellipodia do not attach to substratum, they are retracted and the cell does not move forward (Bailly et al., 1998a). In any case, the cell must present a minimal level of adhesiveness or locomotion will not occur. It has been documented for both haptokinetic and chemokinetic motility that there is a biphasic effect of adhesion on cell motility-at too low an adhesive level the cells retract and stay in place, and at too high an adhesiveness level the cells remain spread and incapable of detaching to move forward (DiMilla et al., 1993; Palecek et al., 1997; Ware et al., 1998). This has been measured for EGF-induced motility on various densities of fibronectin; on very low adhesive surfaces, EGF receptor-signaled deadhesion results in near complete detachment sustained for hours, and on very adhesive surfaces, EGF fails to initiate detectable detachment (Maheshwari et al., 1999). Interestingly at both ex-

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tremes, EGF actually reduces locomotion to well below basal haptokinetic motility. In sum, modulation of adhesiveness is required for cell motility. This is critical at opposite ends of the cells in opposite directions, involving the formation of new adhesions at or near the front and the release of adhesions at peripheral and trailing parts of the cell. Adhesiveness is modulated not only by soluble growth factors, but also by “anti-adhesive” molecules in the matrix (includingSPARC and tenascin);whether these function by competing for adhesive “sites” or initiating counteradhesive signals is currently being determined (Greenwood and Murphy-Ullrich, 1998;Murphy-Ullrich, 1995).How these various classes of molecules, growth factors, matrix components, and adhesion complex constituents interact to modulate focal adhesion aggregation and the avidity for the substratum is the subject of intense investigation.

c. Contraction Molecular motors are responsible for moving the cell body forward over the substratum, probably initiated by contractile forces being generated at or near the leading edge. Myosin-based motors likely generate the forces necessary (Goeckler and Wysolmerski, 1995; Lauffenburger and Horwitz, 1996). Contractile force is required in at least two processes; to pull the cell body toward the leading edge and concurrently to reduce the rear-end resistive adhesion to the substratum. A third situation may provide protrusive force for the extending lamellipod; however, this may be independent of molecular motors and generated by actin polymerization or even chemiosmotic coupling (Savareseet al., 1992; Sjaastad et al., 1996). Different myosin isoforms have been implicated in these situations to accomplish the asymmetry of motility (Conrad et al., 1995; Mermall et al., 1998);myosin I1 has been shown to be important for retraction of the uropod, while myosin 11 and myosin V can be found concentrated in the lamellipodia (Verkhovsky et al., 1995;Wang et al., 1996);myosin IIB but not IIA is found in the axon growth cone (Cheng et al., 1992). Others have postulated that it is not subcellular localization of myosin isoforms per se, but differences in quaternary complexes of myosin I1 with its regulatory proteins, such as specific kinases (Kimura et al., 1996; Matsumura et al., 1998) and phosphatases (Murata et al., 1997).The machinery of these intracellular motors in motility is an area of fervent investigation (Mitchison and Cramer, 1996; Stossel, 1993) that may provide therapeutic targets for limiting tumor invasion. However, these details will not be discussed herein, due to the extensive nature of these studies and the current focus on regulatory control of motility.

d. Detachment at the Rear Release of the trailing cell edges is required for locomotion over any distance. This detachment can be effected by numerous mechanisms. During fi-

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broblast and epithelial cell haptokinesis many focal contacts are severed from the actin cytoskeleton and are left behind attached to the substratum (Cooray et al., 1996; Crowley and Horwitz, 1995; Du et al., 1995; Huttenlocher et al., 1997; Inomafa et al., 1996; Regen and Horwitz, 1992; Yamaguchi et al., 1994). Another mechanism involves the dissolution of the focal contacts with recycling (endocytic or directed within the membrane) of the elements to the leading edge; this appears to be the predominant mode of detachment in hematopoietic cells (Hughes et al., 1997; Lauffenburger and Horwitz, 1996;Lawson and Maxfield, 1995). It is likely that these two processes are neither cell-specific nor mutually exclusive, but rather reflect the biophysics and metabolic requirements of motility. We have proposed that fast moving cells, such as neutrophils, pieferentially recycle their adheasion elements and that in slower moving cells, such as fibroblasts and epithelial cells, the majority of adhesion components are shed (Wells et al., 1998). Experimental studies will determine whether recycling or shedding is dictated by the faster motility exacting too high a metabolic cost to allow for de novo synthesis of these molecules or by the larger size of fibroblasts and epithelial cells precluding efficient trafficking required for recycling. 2. EXTERNAL DETERMINANTS OF CELL MOTILITY

a. Detachment from Tumor Cell Mass The cell-cell connections that characterize epithelial organs and the tumors which develop therein present constraints to cell migration from the original site. Thus, cells must overcome these limitations, which occur either through complete loss of attachments or, as more commonly seen in invasive tumors, decreasing the number and circumcellular nature of these connections. These intercellular connections are primarily composed of adherens junctions (cadherins) and desmosomes (Barth et aL, 1997; Cowin and Burke, 1996). Loss or loosening of these structures correlates with, and appears to contribute to, tumor invasion (Birchmeier et al., 1995). However, the mechanism whereby disruption of these junctions confers advantages for cell invasiveness are not fully determined. One hypothesis suggests that disruption of these intercellular junctions alters established and creates new intracellular signaling pathways, such as p-catenin “switching” from a structural element to a transcription (co)factor (Behrens et al., 1996; Huber et al., 1996; Morin et al., 1997). As it is unclear if and how these altered signaling pathways contribute to invasion or motility, they are mentioned only briefly. Another possibility is that the loss of cell polarity allows for autocrine signaling that is spatially restricted in intact epithelia (Fig. 3) (Kaech et al., 1998; Kim et al., 1999). For instance, development and maintenance of urogenital epithelia are dependent on a tightly controlled stromal-epithelial interaction which results in EGF receptor activation on basolateral aspects,

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while at the same time secreting copious amounts of EGF from the apical surface of the epithelial cells. With the loss of a tight segregation of the ligand and receptor, one can envision dysregulated signaling promoting tumorigenic phenotypes including motility. Such a situation has been reported for mammary epithelial cells presenting a dysregulated intracrine EGF-EGF receptor signaling loop (Wiley et af., 1998). A third proposed mechanism directly relates to the physical nature of cell motiliv-tight cell-cell connections prevent motility by anchoring the cells to the original site. Most growth factors that promote epithelial cell motility also disrupt intercellularconnections via defined signalingpathways (Birchmeier et al., 1995; Potempa and Ridley, 1998; VandeWoude et al., 1997).This is host dramatic in the case of scatter factor (SF),also known as HGF, which signals via the c-met receptor protein tyrosine kinase. Activation of the c-met receptor promotes both motility and tumor invasiveness in vitro (Giordano et al., 1993), and by pathways separable from cell proliferation (Hartmann et al., 1992; Royal et al., 1997). These pathways are shared with those that promote dissociation (Hartmann et al., 1992; Potempa and Ridley, 1998). Thus, the signaling program that leads to epithelial cell motility is connected with pathways that cause disruption of intercellular junctions. Still, as a tumor may invade as a coordinated mass (Fried1et al., 1995), this dissociation need not be complete but rather just sufficient to enable a cell to assume a motility morphology and allow for a relaxation of plasma membrane constraints to enable forward protrusion.

b. Cell-Substratum Attachments There is a burgeoning literature on the roles of matrix attachments in modulating cell motility and tumor invasion. Much of this focuses on the specific haptokinetic and haptotactic signals elicited by specific integrins and other adhesion receptors. A biophysical aspect of these interactions involves the relationship between adhesion and actual motility (Lauffenburger and Horwitz, 1996). It has been shown that on simple surfaces there is a biphasic relationship between adhesiveness and speed with optimal speed at an intermediary adhesiveness. On fibronectin- or collagen-coated surfaces at low adhesiveness the cells are nonmotile, likely due to lack of adequate traction for new attachments, and at high adhesiveness the inability to break rear attachments limits progressive extensions (DiMilla et al., 1993; Maheshwari et al., 1999; Palecek et al., 1996, 1998). It is possible that the adhesion dependency of motility is dictated not by the physical adhesiveness but by the level of signaling via the extent of adhesion receptor occupancy; however, this is unlikely as a similar adhesion dependency was found by altering the avidity of integrins rather than matrix density (Palecek et al., 1997). These limitations point to the numerous rate-limiting events that may predominate under different conditions.

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Complex matrices present a somewhat less clear situation. When we determined the rate of motility of fibroblasts on the human extracellular matrix Amgel (Siegal et ul., 1993), we did not note a strong biphasic effect of matrix concentration on haptokinetic motility (Ware et al., 1998).This complex, biologically active matrix contains both adhesive and anti-adhesive domains (Murphy-Ullrich, 1995), the respective contributions of which likely vary with concentration. For instance, at lower densities the adhesive domains (e.g., integrin binding sites) may predominate, whereas at greater densities the antiadhesive domains of tenascin and entactin may reach critical signaling levels and offset high levels or excess numbers of adhesive sites. However, the concept of adhesiveness dictating functional motility holds validity even on these complex surfaces. EGF-induced chemokinesis demonstrated a strong biphasic response dependent on Amgel concentration despite no pronounced adhesion-related haptokinesis effect (Ware et al., 1998). At low Amgel concentrations EGF induced a deadhesion that resulted in cell speeds less than the haptokinetic level, and at high Amgel concentrations EGF-induced motility was absent. It was across only a one-to-two log order of Amgel at which EGF exposure resulted in significantly increased motility. This effect also may reflect signaling from adhesion receptors rather than a physical consequence; this is unlikely as haptokinesis is relatively unaffected and the initial EGF-induced retraction is similar across the range of permissive and nonpermissive surfaces. In short, these few reports highlight the physical nature of motility, and the importance of quantitative approaches to each process.

c. Matrix Architecture Most invasion occurs into a three-dimensional structure, though aspects of invasiveness and subsequent spreading are accomplished on essentially two-dimensional surfaces. The biophysical resistance that must be overcome to penetrate a three-dimensional interconnected matrix adds another level of complexity. As most tumor cells are on the same scale as or larger than the spaces encountered in biological matrices, cells must overcome this resistance (Friedl et al., 1998). This is accomplished by shape change as well as mat'rix remodeling with proteolysis. In this situation migration may be ratelimited by matrix remodeling dependent on proteases. That cells adapt to surmount these physical constraints is supported by the fact that in fibroblasts and osteoblasts interactions with a three-dimensional but not twodimensional matrix increases production of matrix remodeling MMPs (Langholz et al., 1995). This is likely due to a switch and/or upregulation in adhesion receptors in response to the altered extracellular topography (Klein et al., 1991), resulting in outside-in signaling that alters the transcription profile of the cell (Rijkonen et al., 1995). In addition, during movement through tight pores and remodeled space, adhesion receptors are shed (Friedl

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et al., 1997). Thus, to move through many compacted matrices (>1 mg/ml collagen density), the proteolytic processing to “open upyythe lattice and adhesion to the matrix appear to be rate-limiting (Friedl et al., 1998; Hiraoka et al., 1998). Motility, however, would play a predominant role in moving through a less compacted space. Tumor cells often move in a coordinated fashion with the mass following a path of low biophysical resistance generated by the initial cells (Friedl et aL, 1995). In addition, any structural alteration to the matrix that enlarges the pores or creates planes within the lattice would present a situation in which motility is rate-limiting (Friedl et al., 1998). Thus, gels with large pores and thick fibers promote endothelial migration while dense1y.cross-linked pores prevent this ingrowth (Nehls and Herrmann, 1996). It has been proposed that hyaluronan, the levels of which correlate positively with tumor progression, promotes tumor invasion, at least in part, by creating pores and channels in matrices (Docherty et al., 1989); addition of hyaluronan during wound repair results in a more open architecture of the granulation tissue (Iocono et al., 1998). In sum, the physical construction of the matrix dictates the rate-limiting biological processes in cell ingrowth, and in situations of an open packing or one with channels, motility rather than remodeling becomes a predominant event.

D. Biochemistry The goal of our research, and others investigating the link between induced cell motility and tumor progression, is to define the alterations or aberrations in the control of cell motility that occur during tumorigenic transformation. One main avenue of investigation aims to link the biochemical regulatory machinery with the resultant biophysical processes. In some instances, these pathways have been deciphered with specific reference to cell motility, either haptokinetic or chemokinetic. However, more often the evidence for a role in cell motility is indirect; the signaling pathways have been investigated in terms of short-term changes in adhesion to substratum or alterations in cell morphology and actin cytoskeletal organization, and effects on cell motility extrapolated from these limited responses. The following discussion of specific molecular signaling pathways focuses on the regulatory pathways downstream from cell surface signals, with the EGF receptor and other growth factor receptors being used as the main points of reference. Unfortunately, the state of the art does not yet enable us to discuss biochemical pathways in terms of biophysical events, but rather forces us to present intermediary effectors and discuss possible roles in the various biophysical processes. The importance of defining these signaling pathways is that they represent rational targets for therapeutic interventions aimed at this critical stage of tumor

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progression. Furthermore, it is proposed that the intracellular pathways will represent convergent elements in common to many stimulators of motility and thus can be targeted for a broad spectrum of tumors. Two distinctions need clarification. First, for the most part this discussion will focus on induced motility, whether chemokinetic or chemotactic, as opposed to adhesion-initiated haptokinetic or haptotactic motility. Current findings suggest that it is this growth factor- and cytokine-induced motility that contributes to invasion. However, one should not draw too fine a distinction; for instance, a switch in integrins during tumor progression may represent a form of induced motility that functions via haptokinetic mechanisms. Second, the regulatory pathways are not necessarily the operative pathways. The actual machinery that underlies motility involves the actin cytoskeleton. However, the structural elements and basic biology of this structure appear to be largely unaffected by tumor progression; instead, there seems to be a dysregulation of the normal cytoskeleton functioning and activity cycles. In addition, any single stimulus might'activate only a subset of signaling pathways with the other, required signals being provided at low but sufficient levels. Thus, one may find that disruption or inhibition of a specific biochemical process blocks motility and invasion, but that this process may not be upregulated per se during tumor invasion. In many ways, the discrepancy of much current literature on many proteases seems to fit this conundrum. But even this clarification highlights the duality of the situation in which cell invasion is a complex constellation of cellular phenomena which only becomes evident on the coordinate activation of a myriad of functions, many of which are similarly or even more present in noninvasive cells. 1 . ACTIN CYTOSKELETAL MACHINERY

Cell motility can be considered the consequence of dynamic alterations in the actin cytoskeleton. This involves multiple steps (Mitchison and Cramer, 1996; Small etal., 1996; Stossel, 1993):(1)global reorganization of the actin cytoskeleton from a polarized morphology for epithelial cells or sessile morphology in the case of fibroblasts and dedifferentiated epithelial cells, (2) actin polymerization and scaffolding in the protruding lamellipod, (3) depolymerization in the trailing edges, and (4) contraction to bring the cell body and trailing edge forward (Fig. 2). Numerous investigators have determined the functioning of the actin cytoskeleton and the molecular motors that drive motility, which will not be discussed herein. While obviously critical to induced cell motility during invasion and metastasis, the structural proteins of the actin cytoskeleton have not, for the most part, been implicated directly in this stage of tumor progression; rather, regulatory and accessory molecules have been observed as mutated, dysregulated, or modulated. These are discussed below.

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One possible exception to this involves the targeted transcription and translation of cytoskeletal elements. In fibroblasts, full motility occurs only after an induction period of many hours (6 to 8 hr in 3T3-derived NR6 cells) (Maheshwari et ul., 1999). This is likely related to the need for de novo transcription, as low doses of the transcription inhibitors actinomycin D or puromycin prevent induced motility (Bauer et al., 1992; Chen et al., 1994a; Gordon and Staley, 1990; Kislauskis et al., 1997). The specific messages induced remain unknown. However, there are numerous obvious candidates. First would be elements of the adhesion sites, since these are shed and must be replaced in locomoting fibroblasts (Regen and Honvitz, 1992) and epithelial tumor cells (Fried1et al., 1998). A second group would consist of the signaling effectors and intermediaries discussed below, as many of these are degraded as an attenuation mechanism and must be replaced for extended signaling as required for persistent motility. Also proposed are the structural components of the actin cytoskeleton. P- Actin transcription increases acutely on exposure to the same growth factors that stimulate motility. More intriguing is the spatial control of translation of these molecules. P-Actin mRNA is directed to the base of the extending lamellipod (Kislauskiset al., 1997) presumably in response to formation of new adhesions and the generation of tension (Chicurel et al., 1998). Thus, new synthesis of the cytoskeleton is localized to regions of high polymerization (Bailly et al., 1998b). Although this has not been reported as being aberrant during tumor progression, such investigations are still in their infancy. 2. GROWTH FACTORS AND COGNATE RECEPTORS Many different growth factors and their cognate receptors are found to be upregulated during tumorigenesis (Aaronson, 1991). The underlying mechanisms span the complete genetic and epigenetic range including gene amplification, increased transcription and translation, dysregulated cellular localization, autocrine stimulation, and decreased signal attenuation. However, as these receptors signal pleiotropic responses, the nature of the operational cell response is uncertain. Disruption of growth factor receptor signaling has prevented tumor growth and progression in a large number of experimental situations and models. However, because of the multitude of cell responses, including mitogenesis, motility, dedifferentiation, and protease production, the mechanistic links are difficult to assign. Initially, as many of these growth factors and receptors constitute protooncogenes, mitogenesis was proposed as being the critical signal. In many in vitro and animal studies, dysregulation and/or mutation of these factors, including TGFa, EGF receptor, erbB2, c-sis, and various FGFs, results in single-step tumorigenic changes. However, this has not been noted consistently in human tumors. More recently, investigators have focused on the strong

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correlation between growth factodgrowth factor receptor signaling and the invasive and metastatic step, with many investigators proposing that dysregulated cell motility, not proliferation, is critical for this transition (Barrack, 1997; ChicoLe and Silbergeld, 1997; Finn et al., 1997; Kim et d., 1999; Levine et al., 1995; Woodhouse et al., 1997). Many of the growth factors identified as upregulated in human tumors have been shown to stimulate cell motility (see Levine et al., 1995, for review). Although experimental data demonstrating tumor invasiveness is still sparse, we have provided evidence for EGF receptor-mediated motility being rate-limitbg for prostate tumor cell invasion both in vitro and in vivo. Briefly, the transmigration of a human extracellular matrix, Amgel (Siegalet al., 1993), by DU-145 human prostite carcinoma cells was dependent on EGF receptor, which signaled motility; a signaling-restricted EGF receptor construct that was fully mitogenic failed to increase transmigration (Xie et al., 1995). When these constructs were implanted as mouse xenografts, the pattern was similar. Inhibition of the motility-associated PLCy pathway by pharmacological (Turner et al., 1996) or genetic (Turner et al., 1997) means abrogated tumor invasion but not growth (Fig. 5). We have recently extended this proof of concept to other prostate, bladder, and breast carcinoma lines in vitro (Kassis et al., 1999). Furthermore, disruption of this convergent signaling intermediary prevents the EGF-, insulin-like growth factor I (IGF-I)-, and PDGF-induced motility of human glioblastoma primary explants, and nearly completely prevents invasion into normal brain tissue (Khoshyomn et al., 1999). Thus, at least in a few neoplasias, invasion into normal tissue has been shown to be prevented by abrogating growth factorinduced motility. One motility factor that warrants special mention is autotaxin (Levine et al., 1995; Stracke et al., 1992). This secreted molecule was found to be produced by many tumor cells and stimulate both chemotactic and chemokinetic motility of a wide variety of cells, seemingly by initiating protrusion. What makes this motility factor interesting is that it appears to stimulate motility by acting as an extracellular exophosphodiesterase rather than binding to and activating a specific receptor (Stracke et al., 1997). Whether this implicates small nucleosides or alterations of matrix components and/or cell surfaces as motility signals will shed much insight into the manifold mechanisms of motility. Fig. 5 Inhibition of PLCy signaling prevents invasiveness but not tumor growth. Human prostate carcinoma cell (DU-145) derivatives expressing either the dominant-neganve PLCy fragment PLCz (left) or a transfection/selection control plasmid (right)were inoculated into the peritonea! cavity of athymic mice. After 45 days, tumor growth was extensive for both subvariant populations. However, the cells expressing PLCz formed noninvasive tumors as demonstrated by failure to penetrate the diaphragm (top) and the pancreas (bottom). Adapted from Turner et a[. (1997).

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3. PHOSPHOLIPASE C-y The signaling pathway involving phospholipase C-y (PLCy) has been delineated from a growth factor receptor to a biophysical process in motility (Fig. 6). We have shown that EGF-induced cell motility is inhibited by decreasing PLCy activity (Chen et al., 1994b). A recent study with fibroblasts devoid of PLCy-1 demonstrates the requirement of PLCy signaling to observe the full motility response from EGF receptor (Ji et al., 1998). PLCy activation also plays a required role in PDGF-, IGF-1-, and HGF-induced motility (Bornfeldt et al., 1994; Derman et al., 1996; Kundra et al., 1994). PLCy is also required for growth factor-induced motility in epithelial cells (Dcrman et al., 1996; Polk, 1998; Xie et al., 1995). On the other hand, activation of PLCy alone is not sufficient for cell motility, as one would posit for a cellular process that entails multiple biophysical processes. Inhibition of MEK prevents EGF-induced motility (Xie et al., 1998) and PDGF- and HGF-indbced motility requires PU-kinase and/or ras activation as well (Derman et al., 1996; Kundra et al., 1994,1995). In addition, basic FGP (bFGF) activation of the FGF receptor-1 isoform does not induce fibroblast motility, despite activating PLCy and these other intermediaries (Wennstrom et al.,

EGFR

Flg. 6 Motility signaling through phospholipase C-y. On ligand binding the EGF receptor (EGFR)activates PLCy via its kinase domain (gray box)and autophosphorylated tyrosines (PY and YP). This effector molecule then hydrolyzes phosphoinositide 4,s-bisphosphate (PIP,) to generate inositol trisphosphate (IP,) and diacylglycerol (DAG).The hydrolysis of PIP, leads to mobilization of actin modifying proteins (AMP) that are then free to act on both monomeric (G-actin)and polymerized (F-actin)actin as discussed in the text. Activation of protein kinase C (PKC)leads to a negative feedback attenuation of EGF receptor signaling (Chen etal., 1996b). The motility-related actions downstream of DAG and IP, are by inference and remain speculative. Adapted from Wells et al. (1998).

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1994). Furthermore, in endothelial cells that respond to basic fibroblast growth factor (bFGF)with chemotaxis, PLCy activation seems not to be required but phospholipase A2 activity is (Clyman et al., 1994; Sa and Fox, 1994), suggestingthat some modes of induced motility may bypass the function subsumed by PLCy activation. The mechanisms by which PLCy induces cell motility are being deciphered, and will be discussed below. Abrogation of PLCy signaling does not seem to affect haptokinetic motility. This could due to either PLCy not being required for haptokinetic motility or constitutive low level PLCy signaling providing the essential functions during haptokincsis. This would be settled by determining whether inhibition of PLCy reduces haptokinesis. Unfortunately these data are not clear, in part due to the only partial inhibition achievable by pharmacological in’ hibitors of PLC. Reduction of PLCy activity by pharmacological (U73122) or molecular agents (antisense and dominant-negative constructs) diminished, in parallel, EGF-induced motility in mouse fibroblasts while not affecting basal, haptokinetic motility (Chen et al., 1994b). On the other hand, U73 122 reduced basal, haptokinetic motility of human primary dermal fibroblasts, Hs68, but to a lesser extent than the reduction in EGF receptormediated motility (K. Gupta and A. Wells, unpublished data, 1998); these data must be considered in light of the high endogenous activity of PLCy which may hint at an autocrine stimulatory loop in these cells. Others have reported that PDGF-stimulated but not adhesion-related motility is correlated with PLCy activation (Carloni et al., 1997). PLCy-1 devoid fibroblasts presented seemingly normal unstimulated motility, though possible expression of PLCy-2 may have subsumed this function (Ji et al., 1998). In sum, at present, we propose that PLCy is a required, active element of growth factor signaling, but not for haptokinetic movement, and thus represents a target that distinguishes between these two classes of motility stimuli. Recently, attention has focused on the roles of phosphatidylinositol transfer protein (PITP) and phosphatidylinositol4’-kinase(PI4-kinase)in the regulation of PLCy signaling. It has been proposed that these two molecules are coordinately modulated with PLCy by growth factor receptor signaling to provide for regeneration of the PIP2 substrate (Hsuan and Tan, 1997).PITP hai been reported to be required for EGF receptor activation of PLCy and of P14-kinase (Kauffmann-Zeh et al., 1995).Although it is uncertain what roles these two molecules play in motility, it is likely that modulation of PLCy directly or indirectly through modulation of substrate availability will impact growth factor-induced motility. A report of PI4-kinase being recruited and activated by integrin-containing complexes during haptokinesis (Yauchet al., 1998) suggests a molecular mechanism by which adhesion-related signals may “prime” the cell to respond motogenically to growth factors. EGF-induced fibroblast cell motility has been deconstructed in an attempt to link specific growth factor signaling events to their resultant biophysical

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responses (Maheshwari and Lauffenburger, 1998; Ware et al., 1998). PLCy activation and subsequent mobilization of actin modifying proteins is related to both the actin cytoskeletal reorganization required to establish cellular asymmetry and lamellipod formation and/or extension. However, until the other required elements and signaling pathways are defined for these biophysical processes, the exact role of PLCy signaling will remain imprecise. 4. ACTIN MODIFYING PROTEINS It is the downstream cellular events triggered by PLCy activation that affect the cell motility machinery. These effects can be quite diverse, due to the generation of multiple second messengers. PLC hydrolyzes phosphoinositide bisphosphate (PIP,) generating diacylglycerol (DAG) [which activates protein kinase C (PKC)] and inositol trisphosphate (IP,) which releases intracellular calcium stores. PLCy also induces signals that do not require subsequent activation of PKC or mobilization of calcium. By hydrolyzing PIP,, the submembrane milieu is altered and docking sites for many proteins are lost. Many cytoplasmic actin modifying proteins (AMP),such as gelsolin, cofilin, and profilin, bind PIP,. These molecules inhibit PIP, hydrolysis by PLCy which is overcome by EGF receptor tyrosyl-phosphorylation of PLCy (Banno et al., 1992; Goldschmidt-Clermont et al., 1991; Janmey and Stossel, 1987; Sun et al., 1997). In the PIP2-bound state, the actin modifying actions of gelsolin appear to be inactive (Goldschmidt-Clermont et al., 1990; Janmey et al., 1992; Lamb et al., 1993; Onoda et al., 1993). It is logical to postulate that PIP, sequestration of profilin and cofilin also limits their functioning either by direct interference or compartmentalization. Unbound profilin, cofilin, and gelsolin could affect actin polymerization by locally sequestering actin monomers, severing actin filaments, or nucleating new filament formation (Aderem, 1992; Laham et al., 1993; Weeds and Maciver, 1993). Mobilized profilin could promote new actin filament formation by increasing nucleotide exchange (Theriot and Mitchison, 1993); this activity may account for the increase in actin filaments noted after EGF treatment of A431 cells (Dadabay et al., 1991; Rijken et al., 1995). Cofilin could contribute to the formation and growth of the submembrane orthogonal array of actin seen at the extending lamellipod by generating uncapped actin filaments (Bailly et al., 1998b; Chan et al., 1998). Capping proteins may also contribute to growth factor regulation of cell motility, since their functioning is controlled by PIP, (Heiss and Cooper, 1991; Schafer et al., 1996) and their levels correlate with speed of cell movement (Eddy et al., 1997; Sun et al., 1995). In support of the contention that seemingly subtle alterations in actin assembly and availability directly impacts motility, and its effect on tumor invasion, a small actin sequestering molecule, thymosin pl5, was found to be upregulated in invasive prostate carcinomas, and it was further demon-

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strated that alterations in levels of thymosin p l 5 causally correlated with cell motility (Bao et al., 1996). Thus, hydrolysis of PIP, can directly alter actin cytoskeleton organization by mobilizing and thereby “activating” this class of effector molecules. Hydrolysis of PIP, also could contribute to cytoskeletal reorganization by dirsupting normal bundling of stress fibers at focal adhesions. PIP, is required for a-actinin, vinculin, and ezrin functioning as links between adhesion sites and the cytoskeleton (Fukami et al., 1992; Gilmore and Burridge, 1996; Niggli et al., 1995).Recently, a novel actin bundling actinin has been reported to be concentrated at the leading edge of motile, invasive cells; this molecule might cause the formation of adhesion sites which promote motility rather than the more stable focal adhesions that seem to limit cell motility (Honda ‘et al., 1998). Thus, we can define a direct pathway from a growth factor receptor to the actin cytoskeleton machinery which drives motility (Fig. 6). What remains to be determined is which biophysical processes are promoted by the mobilization and activation of this class of molecules; current data point to morphological changes and lamellipod extension. Severing of actin filaments has been proposed as causing localized swelling and extension of pseudopodia at the leading edge of movement (Leeet al., 1993; Maxfield, 1993). In support of this hypothetical link between actin severing and motility, excess gelsolin directly increases cell motility (Cunningham et al., 1991), and mobilization of these molecules by expression of a competitive binder of PIP, results in enhanced motility and a motility-competent actin cytoskeletal morphology (Chen et al., 1996a). Overexpression of another PIP,-binding, actin modifying protein, CapG, augments the motility response of fibroblasts to fetal bovine sera or PDGF; these cells demonstrate increased phosphoinositide turnover in response to PDGF (Sun et al., 1995). Release of actin modifying proteins from the plasma membrane would not be expected to produce sustained movement unless there were an asymmetry in either the actin modifying proteins or the signal. Both of these conditions exist in chemotaxis; therein, the receptor ligand is at greatest concentration at the leading edge of the cell, and PIP, would be preferentially hydrolyzed in this location. Second, while gelsolin is disperse throughout the cell, profilin, which serves to organize new actin filaments (Carlier and Pantaloni, 1994), is concentrated in extending lamellipodia (Buss et al., 1992). Thus, we can provide a mechanistic framework for EGF receptor activation leading to localized actin remodeling and enhanced cell motility. Despite the opportunity for actin severing to be linked to detachment from the substratum secondary to disconnection of the cytoskeleton to the adhesion plaques, this is not supported by the available evidence. EGF receptor mutants that neither activate PLCy nor mobilize AMP still signal focal adhesion disassembly and detachment from substratum (Welsh et al., 1991; Xie et al., 1998). Furthermore, it is possible that detachment of trailing regions

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of the cells requires an intact cytoskeleton to exert pull on the attachment sites (Klemke et al., 1997; Palecek et al., 1997). Still, it is possible that that the actin cytoskeletal reorganization induced by AMP activation reduces or rearranges cell-substratum connections at the leading lamellipod, a process important for extension.

5. CALCIUM-REGULATED EVENTS

'

The PLCy hydrolysis of PIP, liberates two second messengers, IP, and DAG, which may actuate events in cell motility. Intracellular calcium stores are released on IP, binding to intracellular receptors. Elevated cytosolic calcium levels in tumor cells are associated with increased cell motility (Savarese et al., 1992) and transmigration of an extracellular matrix (Fong et al., 1992), though the causality and mechanisms of this correlation remain illdefined. Furthermore, calcium regulates actin polymerization and stability and form of the actin cytoskeleton (Janmey, 1994; Mafield, 1993);high levels of intracellular calcium lead to increases in filamentous actin content (Rijken et al., 1995). A second biophysical process that calcium transients may modulate is contractility. Myosin I1 activity is enhanced in elevated calcium (Kamm and Stull, 1989). However, it is not known if growth factorinduced transient releases from intracellular stores are an active signal during induced motility. In fact, calcium mobilization is not required for EGFinduced actin polymerization (Rijken et al., 1995), suggesting that the EGF receptor-mediated motility pathway requires other second messengers, and that calcium plays a permissive rather than an active role in EGF-induced cytoskeletal reorganization. The protease calpain has been proposed as a downstream effector triggered by calcium fluxes. Calpain cleavage of focal adhesion constituents (Cooray et al., 1996; Du et al., 1995; Huttenlocher et al., 1997; Inomata et al., 1996; Yamaguchi et al., 1994) may contribute to cell motility by decreasing the cell-substratum adhesion. Our recent evidence points to EGF causing an increase in calpain activity and leading to calpeptin- and calpain inhibitor I-inhibitable proteolysis of focal adhesion components. This would favor a role for calcium being involved in rear detachment during EGFinduced motility. On the other hand, two recent reports present evidence for calpain being required for formation of new adhesions and cell spreading (Potter et al., 1998; Stewart et al., 1998). Whether calpain functions positively in spreading or whether it is required to alter (dissolve)temporary contacts during cell spreading remains to be determined. Interestingly, recent findings have hinted at a calcium-independent mechanism by which calpain may be activated (Johnson and Guttman, 1997; Sorimachi et al., 1997). Our preliminary investigations suggest that calpain,is activated downstream of the erk MAP kinases and does not require PLCy-mediated calcium mobi-

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lization (A. Glading and A. Wells, unpublished data, 1999) (Fig. 7). So while calpain-mediated proteolysis is gaining a solid footing in regulation of cellsubstratum attachments, the inductive role of calcium transients is being questioned. 6 . PROTEIN KlNASE C The role of PKC in cell motility is still controversial. PKC activity clearly affects the cell attachments either directly (Vuori and Ruoslahti, 1993) or over a longer time by upregulating CD44 levels (Ladeda et al., 1998), and thereby likely alters motility. Strong activation of PKC by phorbol esters inhibits growth factor-induced cell motility (Ando et al., 19.93;Koyama et al., '1992), presumably through stabilization of focal adhesions (Chun and Jacobson, 1993). On the other hand, inhibitors of PKC kinase activity limit GFR

Integrins

ssI PY

YP

a:;..

Fig. 7 Roles for the erk MAP kinases in cell motility. The erk MAP kinases (erk/h4APk)are activated via a well-described cascade in which growth factor receptors (GFR) trigger ras signallfng via grb2 coupling to autophosphorylated tyrosines (PY and YP) or tyrosyl-phosphorylated shc. Adhesion receptors, particularly the integrins, also stimulate erk MAP kinases, though to a quantitatively lesser degree than growth factor receptors. Although inhibition of MEK blocks motilitv. ,, the downstream events are still beine defined as discussed in the text. Activation of calpain appears to be required for motility on adhesive surfaces, though the necessity of this protease for motility over low adhesive surfaces or during the rapid locomotion of hematopoietic cells is unknown. Two putative mechanisms to effect haptokinetic motility, through altered integrin avidity or cytoskeletal contraction on activation of myosin light chain (MLC) kinase (MLCK), are shown, as these mechanisms may also be operative in growth factor-induced motility. Motility also requires de novo transcription, though whether this is achieved through the erk MAP kinases is speculative at present. Adapted from Wells et al. (1998). Y

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cell motility and invasiveness (Schwartz et al., 1993; Zimmerman and Keller, 1992). Combined, this suggests that physiological activation of PKC may promote cell motility. An intriguing possibility that may reconcile these data is that PKC serves to communicate between adhesion receptors and growth factor receptors, especially the EGF receptor. The classic isoforms of PKC, which are the major forms found in focal adhesions (Woods and Couchman, 1992) and are activated by syndecan 4 binding to matrix (Oh et al., 1997), are important in negative transmodulation of EGF receptor signaling (Davis and Czech, 1987; Welsh et al., 1991). We have proposed that adhesionregulated PKC activqtion modulates EGF receptor-mediated motility signals. Th,us, in addition to regulating adhesion-mediated basal motility via effects on’cell-substratum interaction, PKC may serve to communicate between adhesion receptors and growth factor receptors to modulate motility signaling. 7. PHOSPHATlDYLINOSITOL 3’-KINASE

Phosphatidylinositol3’-kinase(PI3-kinase) functioning has been shown to be required for cell motility in various model systems. Site-directed mutants of the PDGFP receptor have demonstrated a requirement in PDGF-induced motility for PI3-kinase (Kundra et al., 1994; Wennstrom et al., 1994). Use of specific inhibitors of PI3-kinase has shown this molecule to be important for PDGF-directed chemotaxis of mesangial cells (Choudhury et al., 1997) and HGF-induced cell scattering in MDCK cells (Royal et al., 1997). Met (the receptor for HGF) activation of PU-kinase, in the absence of concomitant ras activation, has been reported to be sufficient for induced motility (Bardelli and Comoglio, 1997; Royal et al., 1997). Thus, PI3-kinase can be a key regulator of motility. This connection between PI3-kinase and induced cell motility is not universal. Its role in EGF-induced cell motility is less certain. While inhibitors of PI3-kinase block EGF-directed chemotaxis in breast carcinoma cells (Bailly et al., 1998b), we have not seen the same inhibition of chemokinesis in our 3T3-derived NR6 cells (A. Wells, unpublished data, 1994). This difference between cell lines may reflect the presence of erbB3 in the carcinoma cells [it is absent from NR6 cells (David etal., 1996)]which may serve as the intermediate between EGF receptor activation and PI3-kinase since EGF receptor directly activates PI3-kinase only weakly if at all. Alternatively, it may point to two distinct classes of growth factor-induced motility, with one group, exemplified by PDGF, actively using PI3-kinase to signal motility, and the other group, exemplified by EGF and VEGF (Abedi and Zachary, 1997), signaling motility independent of PI3-kinase activation. This would not be an unprecedented situation of parallel pathways to the same outcome; haptokinesis and chemokinesis also likely share some pathways but also have unique signaling events.

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The role of P13-kinase in growth factor-induced motility would be clarified if the mechanism by which this contributes to motility were defined. However, the current literature supports a number of different models. Most evidence points to rac as the key effector for PU-kinase (Hooshmand-Rad et al., 1997; Keely et al., 1997). The most likely biophysical outcome would be the disruption of cell-substratum adhesions and. increased membrane activity; this scenario is supported by thrombospondin-induced focal adhesion disassembly being mediated by P13-kinase (Greenwood et al., 1998), and in Dictyostelium disruption of the two PU-kinase genes prevents membrane ruffling (Zhou et al., 1998). However, the role of membrane ruffling in cell motility is brought into question as the genetic deletion of the P13-k’inase genes does not prevent Dictyostelium mbtility; maximal EGF receptormediated motility in fibroblasts occurs after 6-8 hr of stimulation, at which time membrane ruffling is not evident (Maheshwari and Lauffenburger, 1998), and that forward protrusive activity of fibroblasts is antithetical to ruffling (Rottner et al., 1999). Another proposed mechanism is that P13-kinase activity alters integrin binding and signaling (Shimizu, 1996) and thus signals chemokinetic motility essentially via modulation of haptokinetic motility. That basal and haptokinetic motility is reduced by inhibition of P13-kinase supports this model but does not distinguish whether P13-kinase is upstream or downstream of integrin signaling. The presence of growth factors which do or do not require PU-kinase for motility would allow for this to be tested. In this situation, PDGF- and HFG-induced motility would occur as augmentation of haptokinetic motility. This would be detectable as a simple increase in the amplitude of the biphasic response curves in terms of substratum adhesiveness (DiMilla et al., 1993). That EGF receptor-mediated fibroblast chemokinesis is biophysically and biochemically separable from haptokinesis is seen by resulting in a different dependency of motility on substratum adhesiveness (Maheshwari et al., 1999; Ware et al., 1998). Therefore, this may explain why PDGF receptor- and met-mediated motility are distinguishable from EGF receptor-mediated motility based on PU-kinase dependency. A most intriguing speculation centers on PU-kinase signaling by altering phospholipid composition. In one version, the D3 phosphorylated phospholipids serve to activate PKC, rac, or even PLCy and induce cell motility via those elements (Bae et al., 1998; Derman et al., 1997; Han et al., 1998). An alternate possibility, which we have put forward, involves reduction of PIP, binding sites for AMP on the activation of PU-kinase (Wells et al., 1998). In this particular aspect PU-kinase would be acting somewhat similarly to PLCy in altering the balance between membrane-associated and cytoskeleton-associated AMP. However, the differential rates of regenerating the PIP, binding sites for the AMP would have significant implications and may provide a rationale for the distinction between chemokinetic and chemo-

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tactic motility, with the latter being more compatible with slow turnover. Thus, one might postulate a role for P13-kinase in chemotaxis but not chemokinesis, a hypothesis supported by the reports of PU-kinase being required for mammary carcinoma chemotaxis toward EGF (Bailly et al., 199813) but not for fibroblast chemokinesis (Wells et al., 1998). One,further possibility is that P13-kinase isoforms a and y can act as protein kinases in addition to lipid kinases (Dhand et al., 1994). This activity has been demonstrated to lead to MAP kinase activation (Bondeva et al., 1998). As such, this modulation of MAP kinase activity may impact cell motility (see below). 8.’ Rho AND rac GTPases

The small GTPases are molecular switches involved in cell motility via their effects on the actin cytoskeleton and focal adhesions (Tapon and Hall, 1997). These molecules have been extensively reviewed elsewhere (Hall, 1998; Zigmond, 1996); here we discuss how they might actively participate in growth factor-induced cell motility and tumor invasion. Rac has been implicated in membrane ruffling as induced by EGF and PDGF (Ridley et al., 1992); this may be secondary to alterations in cell-substratum adhesion. How this phenomenon relates to motility is unknown, but if one posits that the ruffling noted is an experimentally induced exaggeration of deadhesion, the physiological role of rac in cell motility may be to destabilize the focal adhesions. Alternately, the ruffling may represent the nondirected counterpart of lamellipodial membrane extensions prior to the establishment of the protrusive machinery of motility. Rho has been linked most closely to formation of new adhesions and stress fibers in response to growth factors (Ridley and Hall, 1992),through two divergent downstream signals (Hall, 1998), though complete formation of the focal adhesion complexes requires both rho and rac (Hotchin and Hall, 1995; Machesky and Hall, 1997). Th’is action, which seemingly would lead to a highly adherent “stuck” cell, may be an experimental exaggeration of punctated in vivo signaling leading to formation of new adhesions as the cell moves forward. That the in vivo response may require both inactivation and then activation of these small G proteins is supported by finding sequential changes in activity of these proteins during MDCK epithelial cell morphological changes (Imamura et al., 1998). Thus, in one model, these two switches would act in concert to regulate adhesiveness of the cell at the protruding lamellipod. How these molecules function during growth factor-induced cell motility is still being deciphered. Growth factors may activate these small GTPases secondary to ras, guanine exchange factors (GEFs),or P13-kinase (Han et al., 1998; Keely et al., 1997; Kozasa et al., 1998; Nimnual et al., 1998). The exact mechanism by which these molecules alter the actin cytoskeleton or fo-

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cal adhesions is not known, but it may involve regulation of myosin-based contraction (Fukata et al., 1998; Kimura et al., 1996).A new intriguing possibility is that rac modulates actin modifying proteins, in one instance by activating gelsolin (Azuma ethl., 1998), or translocating cortactin, an ancillary protein that induces motility and invasion (Pate1et ai., 1998),to the cell membrane via PAK (Weed et al., 1998), or, via UM-kinase, phosphorylating cofilin, with both these actions altering the stability and formation of Factin (Arber et al., 1998; Yang et al., 1998). One of the downstream targets of rho, rho kinase, has recently been shown to contribute to transmigration of hepatoma cells across an endothelial layer; introduction of an active form was sufficient for this increased invasiveness, and a dominant-negative kinase blocked rho-induced invasion (Itoh et d:, 1999). A fourth possibility is that a new member of the rho family, rnd, directly disassembles focal adhesions (Nobes et al., 1998), though this latter possibility of inducing motility via altering adhesion is more likely to be important for haptokinetic than chemokinetic motility. Obviously, while much has been learned about these molecular switches, they will continue to be fertile grounds for investigation. Despite these uncertainties in mechanisms, reports have strongly implicated these small GTPases in tumor invasion (Keely et al., 1998). The are a number of possible molecular underpinnings of this connection. In line with the effects that these molecules have on motility, phosphorylation of myosin light chain and was shown to be necessary for rhoA-mediated invasion of hepatoma cells (Yoshioka etal., 1998). In a number of in vim models a second aspect may come into play. Evidence is mounting that rho is involved in maintaining tight cell-cell contacts (Vouret-Craviari et al., 1998), and thus the small GTPases may take part in loosening the attachments of the invading tumor front from the tumor mass. Still, the finding that the rac inactivator Tiaml limits tumor invasiveness (Hordijk et al., 1997) strongly suggests that these molecules are potential targets for intervention.

9. MAP KINASES Growth factor stimulation leads to disassembly of focal adhesions and arutk deadhesion from substratum, though the precise mechanism by which this is accomplished is only now being deciphered. We have shown that blocking of focal adhesion disassembly also prevents both unstimulated and EGF-induced cell motility (Xie et al., 1998). One possible signaling pathway has been proposed as leading to focal adhesion disassembly (Hughes et al., 1997; Klemke et al., 1997; Xie et al., 1998). The erk species of MAP (mitogen-activated or microtubule-associated protein) kinases is activated on integrin occupancy and clustering as well as by growth factors. Klemke and colleagues (1997) propose that these molecules, in turn, phosphorylate myosin light chain kinase which then phosphorylates myosin light chain, re-

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sulting in contraction. Inhibitors of this pathway prevent adhesion-signaled haptotaxis. Alternately, the activated erks have been postulated as phosphorylating integrins directly and thereby reduce the avidity to the extracelMar matrix (Hughes et al., 1997). This would then, presumably, result in cell motility due to shifting of the adhesiveness to a permissive level for haptokinesis (DiMilla et al., 1993; Palecek et al., 1997). However, this alteration in the biphasic motility response would not necessarily apply to chemokinesis. The adhesion turnover would be required both during the acute cytoskeletal re-organization and subsequently during rear detachment (Lauffenburger and Horwitz, 1996; Wells et al., 1998). We have recently shown that EGF-induced detachment also involves calpain activation (Shiraha et al., 1999). Interestingly, our preliminary data suggest that this occurs not through a PLCy-mediated calcium flux but downstream of the erk MAP kinases. In addition, the activation of MAP kinases directly suggests the possibility of nuclear events in induced motility. This would be consistent with the finding that EGF-induced motility requires an induction period of many hours for maximal motility (Maheshwari et al., 1999) and is blocked by actinomycin D (Chen et al., 1994a). It has been reported that AP-1 is required for EGF-induced motility of A431 cells (Malliri et al., 1998). It is tempting to speculate that this is initiated via erk activation, though longterm transcriptional changes may also be downstream of STAT signaling (David et al., 1996). Despite uncertainty about the actual mechanism of induced motility, the erk MAP kinase pathway seems to be required for both adhesion-signaled and growth factor-signaled cell movement (Choudhury et al., 1997; Klemke et al., 1997; Xie et al., 1998) (Fig. 7). 10. MATRIX-DERIVED SIGNALS IN GROWTH FACTOR-MEDIATED CELL MOTILITY

Physiological fibroblast cell motility requires interaction with the substratum not only to provide traction but also to initiate motility-inductive or -permissive signals. Intense research has provided many insights into haptotaxis signaled by specific extracellular matrix components; these important advances will not be considered here due to space limitations and conceptual focus on growth factor motility and invasion. Other recent studies suggest models in which both adhesion and growth factor receptors provide signals that combine to dictate a cell response, such as motility or proliferation (Alford et al., 1998; Assoian, 1997; Bornfeldt et al., 1995; Cybulsky etal., 1994). One testable model envisions adhesion receptors providing a permissive or restrictive intracellular state for a particular cell response to growth factor signaling. This is not at odds the numerous reports of integrin signaling in tumor cell invasion and motility, as it must be appreciated that in many cell

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systems, especially involving transformed epithelial cells, growth factor autocrine signaling loops exist. Thus alterations in adhesion receptor occupancy and signaling is in the background of concomitant growth factor signaling. That the manipulation of one arm of the external signals to a cell drastically affects overall behavior only points to the tight intracellular integration of these signals. For instance, specific integrin occupancy and activation may “prime” a particular intracellular pathway for subsequent growth factor signaling (Miyamot0 et al., 1996). EGF receptor-mediated motility has been reported to reJ ~ ~ occupancy (Klemke et al., 1994). This may work through quire O L integrin integrin activation of the erk MAP kinase pathway (Klemke et al., 1997), which is necessary for EGF receptor-mediated cell motility (Xie et al., 1998), with full EGF receptor activation of erk MAP kinases requiring integrin clustering and occupancy (Miyamoto et al., 1996). The nature of the adhesion also dictates signaling; integrin occupancy by a stretched matrix is required for PDGF receptor autophosphorylation (Lin and Grinnell, 1993), and the presence of a stretched matrix modulates cellular alteratidns of the matrix (Halliday and Tomasek, 1995). Specific matrix components also determine which genes are transcribed (Xu and Clark, 1996; Xu et al., 1998), and thus alter the proteome keyboard on which growth factor signals play. All these provide a mechanism by which the adhesion receptor may “prime” the cell for responses to growth factors. An additional intriguing mechanism by which matrix alters growth factor signaling is the sequestration of predeposited, or the presence of, cryptic growth factors. We have proposed the term “sequestrine” for this type of signaling (Wells et al., 1998). Extracellular matrices contain many growth factors; however, it is not known if the presence of these factors are coincidental or are purposely predeposited. The heparin-binding factors, including the FGFs and HB-EGF and amphiregulin, may be sequestered via heparin interactions, and they might be liberated for signaling on matrix degradation during invasion. Matrix components have been hypothesized to contain cryptic growth factors. Recently, collagen has been shown to directly activate receptors with intrinsic tyrosine kinase activity similar to growth facto; signaling (Shrivastava et al., 1997; Vogel et al., 1997). Furthermore, EGF-like repeats in laminin and tenascin modulate cell adhesion and motility (Lin and Bertics, 1995; Nelson et al., 1995: Prieto, 1992; Spring et al., 1989). A fragment containing the EGF-like repeats of laminin elicits mitogenesis in Swiss 3T3 cells but not in their EGF receptor-negative NR6 derivatives (Engel, 1989; Panayotou et al., 1989), indicating that these matrix proteins may signal via the EGF receptor. However, these EGF-like repeats have not been shown to bind to the EGF receptor or block binding of EGF to its receptor. This would suggest that the EGF-like repeats function as very low affinity, if at all, ligands for EGF receptor. As these cryptic ligands would

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only be liberated during matrix degradation and be physically restrained on a subcellular scale, high local concentrations could be achieved to allow transient signaling by even very low affinity ligands. 1 1 . SIGNALING BETWEEN ADHESION AND GROWTH FACTOR RECEPTORS Growth factor-induced motility must be accomplished through sites of adhesion provided by integrins and other adhesion receptors, such as syndecans and CD44. Thus, it is intuitive to propose that growth factor signals would affect these adhesions. It has been shown that growth factor signaling alters the profile of expressed integrins (Chen et al., 1993; Ye et al., 1996) and levels of CD44 (Zhang et al., 1996, 1997a). This change in integrin expression is likely the result of transcriptional alterations that also lead to growth factor-inducedmesenchymal transitions in epithelialcells (Fujii, 1996; Kustikova et al., 1998; Malliri et al., 1998). Interestingly the changes in integrin expression patterns may actually require co-incident matrix signals that depend on composition (Xu and Clark, 1996), three-dimensional structure (Wang et al., 1998), and “stiffness” (Lin and Grinnell, 1993) of the matrix, and are likely signaled through the integrins themselves; however, whether this is facilitated by the new integrins or promoted by the initial integrins awaits further investigation. Over a shorter time scale, there is mounting evidence that growth factor receptors can directly affect integrin functioning to promote motility (Hughesetal., 1997; Klemke etal., 1994,1997). This may be accomplished by tyrosyl phosphorylation of the cytoplasmicdomains of the integrins (Mainiero et al., 1996) or by changing the localization and promoting clustering of integrins at the leading edge (Trusolino et al., 1998), or a combination of both. Interestingly, this cross-communication seems to be bidirectional; integrins not only ‘prime’ the cells to respond to growth factors as discussed above, but may directly activate signaling from at least the PDGF and EGF receptors even in the absence of their respective ligands (Lin and Bertics, 1995; Sundberg and Rubin, 1996). These data provide for models of interlacing and overlapping signaling networks to modulate motility (Fig. 8). The cross talk between adhesion and growth factor receptors may occur within the focal adhesion and contacts (Miyamoto et al., 1996; Plopper et al., 1995). Both classes of receptors are concentrated in these structures and activate an overlapping set of downstream effectors. The close proximity of adhesion and growth factor receptors in adhesion aggregates provides for a free flow of both positive and negative regulatory signals between the two. We propose that integrins may promote a motile response to growth factor signaling via “priming” of the erk MAP kinases and calpain (Huttenlocher et al., 1997; Miyamoto et al., 1996; Palecek et al., 1998). Syndecan 4 en-

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GFR

AdhR

I'

locomotidh

.

Fig. 8 Cross talk between growth factor receptors and adhesion receptors in cell motility. There is extensive cross talk between adhesion receptors (AdhR), including integrins (a@)and syndecans (S), and growth factor receptors (GFR). Direct, or nearly so, interactions have been reported as growth factors directly phosphorylating integrin cytoplasmicdomains and integrinassociated kinases activating growth factor receptors through phosphorylation of their phosphotyrosine motifs. Adhesion receptors, primarily syndecan 4, may negatively transmodulate growth factor signaling via PKC (Oh et al., 1997).Integrins and growth factors also combine to induce erk MAP kinases. Growth factor signaling also alters the expression profile of integrins and other adhesion receptors; part of this may be via erk MAP kinase-regulated transcription. On the other hand, current evidence points to PLCy signaling of cell motility to be relatively specific for growth factor-induced motility.

gaged by proteoglycans and collagens would suppress cell responses to EGF receptor activation by intracellular transmodulation via PKC-mediated attenuation of EGF receptor (Welsh et al., 1991). As the basis of activation for many signaling elements is highly localized, for instance, calcium fluxes and translocation to the membrane, close juxtapositioning of the two classes of receptors would be critical. Arguing against this requirement for close juxtapositioning of growth factor receptors and adhesions is the fact that EGF induces a rapid loss of focal adhesions throughout the cell, seemingly by pan-cellular signaling (Xie et al., 1998). This important question can be settled only by engineered surfaces that present either coclustered or physically separated adhesion and growth factor receptor ligands. 12. De Novo MESSAGE TRANSCRIPTION

Another area that warrants mention as it is likely to draw increasing attention in future investigations is that of altered transcription profiles in motility and invasion. That invasive tumor cells present a qualitatively and quantitatively different proteome from their noninvasive parental cells has been attributed to differential transcriptional controls. However, whether these are due to genetic alterations or epigenetic responses to extracellular signals requires further investigations of specific elements. The generation of spontaneous cell sublines that demonstrate enhanced motility and invasive-

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ness in a stable pattern would argue for, at the least, a distant upstream genetic event (Bao et al., 1996). However, a small number of controlled experiments suggest that increased motility and invasiveness can be signaled epigenetically by altering growth factor signaling levels. This is in addition to the reported upregulated production of specific proteins during motility. The previously described induction period for EGF-induced motility may reflect the need for altered transcript (Maheshwari et al., 1999), though this required de novo transcription has been more convincingly if crudely shown by the sensitivity of motility to transcription inhibitors (Bauer et al., 1992; Chen et al., 1994a; Gordon and Staley, 1990). Increasing EGF receptor levels in DU-145 prostate carcinoma cells increases their invasiveness both in viho and in viwo (Turner et al., 1996; Xie et al., 1995); although we do not know if this requires new transcription, in collaboration with David Jones (University of Utah, 1999) we have found a number of specific mRNA species to be either upregulated or downregulated in these cells. Interestingly, urokinase-type plasminogen activator binding to its receptor and signaling via the erk MAP kinases for only 30 min can stimulate mammary carcinoma cell motility as measured over 24 hr, suggesting a persistent change in the transcription program of the cell (Nguyen et al., 1998). A few reports have pointed to the fos family of transcription factors as the mechanism by which growth factor signals alter transcription to promote motility and invasion (Kustikova et al., 1998; Malliri et al., 1998). It is tempting to speculate that this element is modulated by growth factor activation of MAP kinase species (both erk and ink), but this remains to be demonstrated. 13. COUNTERREGULATORYSIGNALS

Factors and signals that directly counter growth factor-induced motility are being identified. These are in addition to matrix compositions or states that do not support or actively prevent motility, such as high and low adhesion surfaces and anti-adhesive signals. Such signals would have to be overcome or ignored for invasion to occur. Select chemokines, produced during the coagulation and inflammation phases of wound healing (Furie and Randolph, 1995),have been shown to inhibit fibroblast and endothelial cell function. While most chemokines act on hematopoietic cells, responses to chemokines have been reported for mesenchymal and carcinoma cells (Neote et al., 1994; Peiper et al., 1995; Youngs et al., 1997; Yue et al., 1994). The ELR-negative members of the C-X-C family of chemokines, PF-4 and IP-10, inhibit endothelial cell proliferation and migration (Gupta and Singh, 1994; Luster et al., 1995; Strieter et al., 1995). They act dominantly in this aspect over promitogenic and promotility chemokines. Thus, these factors are candidates for limiting cell motility. We have found (Shiraha et al., 1999) that these chemokines inhibit EGF receptor-mediated motility of human fibro-

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blasts, without impinging on ligand-induced mitogenesis. IP-10 does not diminish motility in the unstimulated fibroblasts (if anything there is a slight increase) and therefore seems to specifically counter growth factor signaling of motility. IP-10 exerts it; negative modulation via a protein kinase Amediated pathway disrupting EGF-induced calpain activation and the resultant cell deadhesion. Although there are no reports af this class of chemokines altering cancer cell invasion, breast cancer cell lines respond chemotactically to some chemokines (Youngs et al., 1997). Thus, these molecules, present during inflammatory responses, including those to tumors, may alter the invasiveness of t u m q cells.

&. MOTILITY IN TUMOR INVASION Tumor invasion is proposed as a disease of disregulated cell motility. This concept has been gaining recognition over the past few years. Coupled with advances in our understanding of the molecular signals and events of cell motility, investigations into the role of cell motility in tumor progression, especially invasion and metastasis, has burgeoned. The concept that invasion respresents a distinct tumor property first required experimental demonstration. This has been buttressed by the ability to block invasion but not affect tumor growth. Numerous in vitro studies have utilized compounds or molecular agents to block invasiveness. For example, the tumor suppressor KAI blocks motility and invasion but not growth of colon cancer cells (Takaoka et al., 1998), and the calcium channel inhibitor CAI prevents transmigration of Matrigel even in tumor cell lines in which proliferation is unaffected (Jacobs et al., 1997). Interestingly, estrogen can increase tumor growth even while suppressing invasiveness (Rochefort et al., 1998). On the other hand, TGFP, which usually is antiproliferative, can enhance tumor progression, possibly via its motility promoting effects (Barrack, 1997). Despite these illustrative examples, these two cell behaviors critical for tumor progression, invasion and growth, usually have' not been separated experimentally. CAI limits, in a cytostatic manner, the growth of many tumor lines, often concomitant with its anti-invasive effects. Additionally, inhibitors of metalloproteinases and integrin binding have pleiotropic effects. Still, the distinction between invasion and growth has been clearly demonstrated by an in uivo xenograft model of human prostate carcinoma, in which disruption of PLCy signaling prevents tumor invasiveness but not tumor growth (Turner et al., 1996, 1997). On the other hand the reciprocal independence has yet to be rigorously demonstrated in animal models, that clinically significant invasion can occur in the absence of tumor growth. Still the independence of invasion from growth has been

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shown in vitro by transmigration and invasion assays in the presence of inhibitors of proliferation (Xie et al., 1995). That invasiveness is a separable and independent cell property from tumor growth enabled investigators to focus on the key regulatory events that promote tumor invasiveness. Investigations into invasiveness have focused on the three key events required, to transmigrate a matrix-recognition of the matrix, proteolytic degradation and remodeling of the matrix, and active motility through the matrix. Obviously, each might be rate-limiting if prevented, and thus each represents a therapeutic target. However, to better understand the process and determine prognostic markers, one major goal is to decipher which event is modulated during tumor cell progression to promote invasiveness. One caveat must be mentioned: it is possible that more than one of these events is altered in concert, or that various tumors accomplish invasion by modulating the various aspects differently.

A. Proteases in Invasion and Motility Initially, much work focused on the role in invasion of proteases, especially the matrix-degrading metalloproteinases. A key theoretical underpinning for proposing proteases as the key modulatory element was the perception that matrix formed a physical barrier to tumor cell penetration. This was buttressed by in vitro assays in which inhibition of invasiveness was accomplished by blocking the proteolytic activity of select proteases (Aznavoorian et al., 1993; Cresson et al., 1986; Stetler-Stevenson et al., 1993b). Clinical correlations between tumor progression and protease activity further supported this model (Liotta et al., 1980; Ponton et al., 1991; Rozhin et al., 1990; Shima et al., 1992). Recently, the elucidation of membrane receptors for metalloproteinases has suggested that directed proteolysis at the invading edge is critical to tumor invasion (Hiraoka et al., 1998; Sat0 et al., 1994). These studies have proved useful in suggesting a number of prognostic markers and therapeutic agents for trials, though none have yet gained acceptance in clinical use. These concepts of protease activity in invasion have evolved concurrently with the demonstration that transmigration of an extracellular matrix may require little to no proteolytic remodeling (Docherty et al., 1989; Fried1 et al., 1995,1998; Iocono et al., 1998; Nehls and Herrmann, 1996). Thus, the observed correlation of protease activity and tumor invasion may belie actions in addition to simple proteolytic “opening” of a matrix. This has been exemplified by the report that keratinocyte migration over a two-dimensional substratum requires collagenase-1activity, a situation wherein no physical barrier is present to prevent forward motility (Pilcher et al., 1997).Two avenues have been pursued: one concerns the role of proteases as signaling

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molecules in and of themselves (Chambers and Matrisian, 1997), the other as processors and activators of extracellular signals (Blobel, 1997; Werb, 1997). In the former scenariq, the cell surface receptors for proteases are implicated in transmitting signals that promote tumor progression (Lochter et al., 1997). The urokinase-type plasminogen activator, on binding to its receptor in MCF-7 mammary carcinoma cells, initiates an iotracellular signaling cascade involving the erk MAP kinases and leading to increased cell motility (Nguyen et al., 1998). What is most remarkable about this finding, and which strongly suggests that proteolytic activity per se is not operative, is that this effect on motility can be seen up to 24 hr after only a brief 30min exposure to protease. In addition, it has,been proposed that the proteases activate or librate latent growth factors such as TGFP and IGF-I, as Kas been shown for plasmin (Martel-Pelletier et al., 1998; Munger et al., 1997). Cathepsin D has been reported to both increase growth factor availability by degrading the matrix (Briozzo et al., 1991) and inactivate extracellular inhibitors of such signaling (Liaudet et al., 1995; Belien et al., 1999), and metalloproteases have been proposed as liberating cell surface-bound growth factors and cytokines, such as TGFa and tumor necrosis factor (TNF) (Blobel, 1997; Werb, 1997). In either situation, that of direct signaling or indirect signaling through activation of growth factors, proteases are now proposed as inducers of cell motility. Complicating our understanding of the role of proteases is the fact that many of the same factors that induce cell motility also upregulate protease production. For instance, EGF stimulates the production of stromelysin (McDonne11 et al., 1990), a metalloproteinase that has been associated with increased invasiveness (Masson et al., 1998; Matrisian et al., 1986; Sreenath et al., 1992), and interferon decreases invasiveness and motility concurrent with downregulation of collagenase (Qin et al., 1998). In sum, a model is constructed in which proteases promote invasion through the dual roles of limited proteolytic degradation and increased cell locomotion (Fig. 9).

B. Growth Factor-Induced Motility as Rate-Limiting It is almost tautological to argue that blocking of motility would limit cell invasion; obvioulsy without cell translocation, movement into a new, ectopic space could not occur. What is proposed is that growth factor- and cytokineinduced motility promotes tumor invasion and that this process is upregulated during tumor progression to the invasive state. Indirect evidence mainly leads to this model, but experimental verification is increasingly being reported. Many growth factors, EGF/TGFa and HGF in particular (VandeWoudeet al., 1997), have been correlated with tumor invasiveness. For instance, EGF

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Fig. 9 Actions of extracellular proteases in enabling tumor invasion. The major postulated roles for extracellular proteases (designated as MMP) are four: (1) degrading the matrix as a soluble protease; (2)degradingthe matrix at the "invadopodia"effected by soluble M M P binding to surface receptors or membrane associated MT-MMP; (3) liberating or activating mauix(or cell surface-) associated growth factors (GF) or inactivating inhibitors (I); or (4)signaling invasion on binding to specific receptors (lightning bolt).

receptor signaling is upregulated in over half of the invasive glioblastoma multiformes compared to the localized gliomas (Libermann et al., 1984), in the vast majority of invasive but not superficial bladder carcinomas (Neal et al., 1985), and in advanced invasive gastric carcinomas (Yasui et al., 1988). These three cases focus on the invasive behavior of these tumors. In numerous other human tumors, EGF receptor signaling correlates with tumor progression and poorer prognosis; the two clinical parameters may be impacted by increased invasiveness, but the studies were not specific to that particular point. Experimental verification of EGF-induced invasion has highlighted the role of motility. EGF exposure increases the invasiveness of many normal and tumorigenic cell lines (Brunton et al., 1997; Chakrabarty et al., 1995; Engebraaten et al., 1993; Hamada et al., 1995; Hoking et al., 1995; Jarrard et al., 1994; Li et al., 1993). This is similar to invasion induced by other growth factors. Unfortunately, as these receptors initiate pleiotropic signals, the precise cell behavior responsible for invasiveness is open. In a few cases the investigators have assessed the various responses, and have found that invasiveness tracks with growth factor-induced motility and not protease production or altered adhesiveness (Hamada et al., 1995). We also found a correlation between EGF receptor-mediated invasiveness of prostate carci-

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noma cells and cell motility but not protease production or altered adhesiveness (Xie et al., 1995). Still, as the changes in protease profiles and adhesion to matrix may be subtle or involve molecules not tested for by the specific assays, such conclusions are indirect. Rather, one would like to isolate and limit one downstream behavior and determine the consequences. This approach has recently been utilized for dissecting invasion in human ovarian cancer cells in which PKC inhibitors prevented invasion along with curtailing motility, but without altering protease production or adhesion to substratum (Szaniawska et al., 1998). These correlations suggest, but do not demonstrate,.that motility is a critically regulated behaviqr responsible for increased invasiveness. We have taken a slightly different approach, that of failing to activate or disrupting the activation of downstream pathways specific for EGF receptormediated motility. We have demonstrated that EGF receptor-mediated motility requires PLCy signaling (Chen etal., 1994b).EGF does not induce motility if it fails to activate this molecule, this being accomplished either by a signaling-restrictedEGF receptor construct or by pharmacological and molecular agents that block lipase activity of PLCy. In the absence of PLCy activity, haptokinetic motility is unaffected (Chen et al., 1994b; Ware et al., 1998), and mitogenesis is actually increased by the lack of a competing cell response (Chen et al., 1996b). Thus, we have a molecular target for EGFinduced cell motility as opposed to mitogenesis. Overexpressing EGF receptors in DU-145 human prostate carcinoma cells promoted EGF receptordependent invasiveness both in vitro (Xie et al., 1995) and in vivo (Turner et al., 1996); invasion was absent in cells expressing a fully mitogenic EGF receptor construct that failed to activate PLCy.Targeting PLCy signaling in the mouse xenografts, utilizing either pharmacological (U73122) or molecular (dominant-negative PLCz fragment of PLCy-1) agents, abrogated invasiveness (Turner et al., 1996, 1997). Still, these carcinoma cells, and those expressing the signaling-restrictedtruncated EGF receptor construct, formed progressively growing tumors, ones that failed to invade locally (Fig. 5 ) . In all situations, we did not provide exogenous ligand, either in vivo or in vitYO; the activating ligands for the EGF receptor were derived either from autocrine TGFa production or from the host environment. Thus, it seems reasonable to conclude that the motility signaled via the endogenous EGF receptor is the dysregulated event promoting invasiveness of DU-145 cells. Although these experiments provide a proof of concept, this must be extrapolated to other tumors and tumor types for pathophysiological validity. In an initial series of experiments, we have found that the de novo TRAMP murine prostate tumors (Greenberg et al., 1995) present autocrine stimulation of the EGF receptor, as is usual for human prostate carcinomas (Ching et al., 1993; Glynne-Jones et al., 1996). Blocking EGF receptor signaling or PLCy signaling in these cells prevents transmigration of extracellular matrix

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similar to the DU-145 cells (Kassis et al., 1999). To extend this to other cell types in which upregulation of EGF receptor signaling has been correlated with tumor invasion or progression, we have found that in vitro transmigration of a Matrigel barrier by human breast and bladder carcinoma cells can be abrogated by targeting either the EGF receptor or PLCy activity (Kassis et al., 1999). A collaborative investigation with Paul Penar examined the role of PLC signaling in invasiveness of the sentinel tumor, glioblastoma multiformes (Khoshyomn et al., 1999). Inhibition of PLC prevented primary human glioblastoma explants from invading into normal brain spheroids. Interestingly, even in the presence of U73 122, these tumor cells locomoted over the surface of the spheroids to engulf them, but failed to penetrate the pirenchyma. Measuring the rate of locomotion of these cells demonstrated that U73122 had no effect on haptokinetic (“basal”) motility or proliferation. However, this inhibition abrogated motility in response to multiple different growth factors, EGF, PDGF, or IGF-1, suggesting that PLCy is a point of convergence in growth factor signaling of motility. Thus, in sum, these findings not only suggest that growth factor receptor-mediated motility is a rate-limiting aspect of tumor cell invasion, but point strongly to dysregulated signaling being the operative cellular alteration promoting invasion in situ.

C. Adhesion Receptors in Growth FactoFInduced Invasion Much has been reported about the roles of adhesion receptors in tumor progression, both from the point of the tumor cell events and from their role in neoangiogenesis (Clark and Brugge, 1995; Keely et al., 1998; Varner and Cheresh, 1996). In addition to providing for traction, these receptors actively signal cell motility that contributes to invasiveness. However, the interrelatedness and similarities of these receptors with the growth factor receptors are only now being fully recognized. It is likely that some integrins, or others in specific circumstances, primarily function akin to growth factor receptors. This probably underlies the CAMP gating signal induced by the a6P4integrin (O’Conner et al., 1998). It also has been well appreciated that growth factors must affect adhesion receptor functioning to accomplish cell motility and invasion (Hanningan and Dedhar, 1997; Matsumoto et al., 1995; Schwartz, 1997). However, only recently has the reverse been approached (Schwartz, 1997). Integrins are found colocalized with growth factor receptors (Plopper et al., 1995). Recently, it has been shown that integrin activation leads to physical interaction with and activation of the orphan growth factor receptor erbB2 in carcinoma cells (Falcioni et al., 1997). Furthermore, it has been reported that B82L cells require EGF receptors to re-

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spond to laminin in a chemotactic and invasive manner (Lin and Bertics, 1995). These data suggest that integrin responses may require growth factors as intermediaries in specific signaling pathways. A second mechanism by which integrins require grovltth factor receptor signaling in promoting invasiveness may be more subtle. As many, if not most, human cancers present dysregulated growth factor and cytokine receptor signaling due to autostimulatory loops or receptor upregulation, any adhesion receptor signaling is superimposed on this background signaling. Thus, it needs to be determined whether specific integrin-associated events require this concomitant signaling. As an example, mammary carcinoma lines possess TGFa/EGF receptor autocrine signaling, so that the observed integrin-induced invasiveness (Keely et al., 1997) may require this EGF receptor-mediated signaling in addition to adhesion-related signals. Such interrelationships will likely attract increasing attention in the near future. Although this distinction may matter little for direct interventional strategies, the implications for the biology of cancer invasion and for future rational therapies are profound.

D. Motility Suppressors If motility is a key regulator of invasion one might expect to find clinical correlations between invasiveness and loss of so-called motility suppressor proteins or gain of motility inducers. Few have been noted, but this may represent more of a bias in experimental approach than in the biology of tumor invasiveness. For instance, one would have to match the invasive part of the tumor with pieces from the primary mass not involved in invasion, a difficult and somewhat arbitrary distinction to make; more usually the distinction is made between metastatic mass and primary mass, with the confounding problem that the primary tumor may still harbor metastatic and invasive potential whereas the metastatic growth may no longer require this cell behavior. Still, two candidates have been identified, MRP/CD9 and maspin. MRP/CD9 is a member of the tetraspan family of transmembrane rnol&ules and has been linked to cell motility, though the mechanism by which it promotes motility is still undeciphered. Furthermore, the motility that is effected by CD9 is haptokinetic; data on growth factor induced motility are lacking. Interestingly antibodies to CD9 block motility of keratinocytes (Jones et al., 1996)and colon carinoma cells (Cajot et al., 1997)but increase motility of Schwann cells (Anton et al., 1995); in all cases, cell adhesion was noted to be unaffected. Loss of CD9 correlates with poor prognosis and tumor progression-in breast and colon cancers (Cajot et al., 1997; Huang et al., 1998), and with venous invasion and liver metastases in human colon cancers (Mori et al., 1998). It may be counterintuitive that loss of a motili-

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ty-associated protein is associated with tumor progression, but until the mechanism of action is determined, such a finding can be reconciled with a switch to growth factor-induced motility and increased invasion. Motility suppressors and inducers may have been found but are not labeled as such, being misclassified. We contend that this is the mechanistic basis for the correlation of invasion with upregulated growth factor receptor signaling, secondary to increased receptor levels or production of an autocrine stimulatory loop (seeabove). This misclassification may apply to other molecules, as illustrated by the putative serpin, maspin (Sageret al., 1997). The downregulation or loss of expression of this molecule has been correlated with tumor progression of breast and prostate carcinomas, among others. Addition of maspin to cells blocks motility on a two-dimensional substratum and invasiveness through Matrigel in vitro (Jiang et al., 1997; Zhang et al., 1997b). However, exogenous expression in the highly metastatic Dunning prostate tumor line AT3 did not prevent metastases on allografting (Umekita et al., 1997), though this might bespeak the difference between invasion and metastasis with only the former necessitating induced cell motility. We have reported that the incidence of metastatic spread to lung from abdominal or prostatic DU-145 tumors appeared to be independent of their invasive potential and activity (Turner et al., 1996). Interestingly, and consistent with effects of motility inhibition even on plastic tissue culture dishes, maspin is unlikely to act as a classic serpin (Pemberton et al., 1995) but rather acts via binding to specific cell surface receptors (Sager et af., 1997). Maspin might represent the first described invasion suppressor gene that acts via inhibiting tumor cell motility.

V. THERAPEUTIC INTERVENTIONS The overarching goal of these investigations is to identify targets for rational therapies aimed at preventing tumor invasion and its resulting morbidity and mortality. The majority of current therapies either remove tumor cells (surgicallyor by killing them in sit# using radiation) or kill them by exploiting a weak therapeutic index based on most tumors proliferating at a slightly faster rate than normal host cells (in a few cases, unique metabolic requirements allow for cytotoxic and cytostatic therapies with large therapeutic indices). These approaches have been very successful for localized tumors and select fast growing tumors having high proliferative fractions. However, these interventions are not curative in a large number of cancer cases. This is either due to tumor spread or invasion into surrounding parenchyma, making extirpation as detrimental as the cancer, or to tumors that have low mitotic indices or large nonproliferating fractions. As an example,

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prostate cancer presents myriad obstacles to successful therapy. Not only are most clinically evident tumors already invasive, making radical prostatectomy or wide area irradiation necessary with little improvement in survival but with significant iatrogenic complications, but because prostate cancer cells have a mitotic index of only -5% and a substantial noncycling fraction, antimitotic agents are ineffective (Bihrle, 1996; Gittes, 1991; Schultz, 1996; Surya and Provet, 1989). Even early detection is fraught with problems as the natural progression of these lesions suggest that the majority remain clinically silent, and the current treatment entails significant physical and psychological comorbidity (Moul, 1996; Wilt, 1996). Breast cancer, with a significant percentage of cases of early cryptic dissemination, presents similar dilemmas for treatment and detection (Dickson et al., 1996). For these and other tumors, new paradigms of treatment must be developed to limit further extension of the disease even in noncurative situations. For this to be accomplished, we first need to understand the basic biology of tumor invasion.

A. Targeting Invasion as an Adjuvant Therapy Preventing tumor invasiveness would be beneficial in limiting much of the comorbidity of even noncurable cancers. Invasiveness per se has been determined to be an independent indicator of poor outcome (Cianchi et al., 1997). By stopping the penetration and destruction of ectopic sites, the compromise in functioning of adjacent organs and destruction of transiting structures such as nerves and vasculature would be minimized. However, tumors cause significant morbidity and mortality secondary to enlargment by cell growth; these space-occupying lesions can destroy adjacent and internal structures even without actual cellular penetration. Thus, abrogation of invasiveness alone would likely be of limited effect. Therefore, it may be beneficial to consider the invasive tumor as a chronic disease for which the goal is to limit further spread while periodically reducing the primary tumor burden. Invasion must be targeted as part of a broader approach to cancer that also deals 'with the tumor mass. Thus, anti-invasive interventions are to be considered and designed as adjuvant therapies (Moustafa and Nicolson, 1997). This simple fact complicates the development of such agents, as the parameters to determine efficacy are ill-defined. Current therapeutic trials often measure tumor mass or growth in addition to clinical parameters. As antiinvasives are not likely to prevent or reverse this tumor growth, they would be determined as ineffectual. Clinical outcomes, such as survival and measures of tumor-associated morbidity, are long, costly, and subject to the combined effects of tumor load and progression; the anti-invasive agents would only affect one part of the latter event. The recent move to determine inter-

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mediary biomarkers for early evaluation of new anticancer therapies also fails to accurately assess invasiveness. The reasons for this inability lie with our lack of understanding of the basic biology of invasion preventing the identification of appropriate markers and reliance on histological determination of extent of invasion (presumably the selected tumors in any initial trials would already be invasive, rendering ex vivo imaging of invasion into organs less valuable due to the difficulty of distinguishing enlargement of mass from greater invasion). Potential anti-invasive therapies, because they do not target tumor growth, would be evaluated in conjunction with other therapies. This would further confound the analyses. Despite these limitations, the contribution of tumor invasion to mortality and morbidity warrants development of anti-invasive agents. A few tumors may serve well for anti-invasive trials. Prostate cancer, due to its slow growth and rapid development of hormone independence,escapes current antimitotic therapies (Bihrle, 1996; Wilt, 1996). Its pattern of invasiveness into adnexa and adjacent body cavities not only limits the use of surgical extirpation but also enables invasiveness to be readily determined. However, the slow growth with long survival times and variable clinical course and penetrance of early lesions require large, cumbersome trials to define positive outcome. A second rumor that presents clinically relevant invasiveness is the glioblastoma multiformes. As this tumor is rapidly fatal, clinical results would be readily evident. However, as tumor mass is a major concern in intracranial lesions, concurrent antimitotic or debulking therapies, with significant comorbidity, would be necessary. Because of the difficulties and expenses in designing such trials, it is even more important that the putative agents be based on solid mechanistic studies.

B. Targeting the Biophysical or Molecular Aspects of Motility Invasion can be prevented by limiting cell motility. In vitro motility and invasion have been abrogated by a number of approaches. These are rationally aimed at either biophysical events or cellular signals, or a combination of both. The three biophysical aspects that have drawn the greatest attention are matrix architecture, cell adhesion, and lamellipod protrusion. Many studies have proposed to prevent invasion by limiting proteolytic degradation of extracellular matrices (see above). However, these have suffered from theoretical and practical concerns. Conceptually, emerging evidence suggests that tumor cells can transmigrate extracellular matrices with little if any proteolytic remodeling of the matrix, by exploiting fiormal planes and pores (Fried1et al., 1998).And invasiveness of some tumors, such as giioblastoma multiformes, does not require the transmigration of obvious, barrier extra-

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cellular matrices. Operationally, the plethora of different proteases that may accomplish similar matrix “opening” argues against a simple and effective agent; this is further compounded by the lack of evidence that upregulated production of a specific proiease is the rate-limiting step,in tumor invasion. In addition the ubiquity of these same proteases and their use in normal cell and organismal functioning suggests significant limiting toxicity. However, it is likely that specific antiprotease agents will be useful in limiting progression of specific tumors. Ironically, this clinical utility may not be secondary to any effects on matrix remodeling but rather on the newly recognized role of certain proteases as integral parts of signaling cascades, either directly or by liberating latent ligands, leading to cell motility or enhanced cell survival (Pilcher et al., 1997). The advanced understanding of the molecular bases of cell adhesion, critical for motility and invasion, has engendered proposals for preventing tumor progression. The various integrins, as adhesion receptors, have drawn most interest, with select agents currently in preclinical trials (Varner and Cheresh, 1996). The attractiveness of this approach is that adhesion modulation will prevent motility (DiMilla et al., 1993; Xie etal., 1998) and thus stop invasion. However, technical challenges are limiting. In a study to evaluate the feasibility of preventing glioma invasion by blocking integrins, the inter- and intraindividual variation in integrins prevented successful application of this approach (Tonn et al., 1998). The conclusion is that integrin inhibition therapy would need be tailored to each patient (not just to tumor type) and altered as the tumor evolved, a painstaking and challenging process. An ancillary approach has been to ablate not the tumor but the host responses that inadvertently advance tumor progression. Thus, cell motility required for endothelial ingrowth during neoangiogenesis is a target of interest (Arap etal., 1998; Folkman, 1997; Pluda, 1997; Skobe etal., 1997). As endothelial cells are less plastic than tumor cells, the display of integrins to target will not change and thus allows for a more controlled and universal agent (Boehm et al., 1997). Still, as the role of neoangiogenesis in tumor progression is uncertain (it is important for bulk growth but the necessity for adjacent invasiveness or distant metastasis is unproved), this’approach may be of greater utility in reducing tumor bulk than limiting invasion. CAI,an agent that limits calcium influx (Jacobs et al., 1997; Sjaastad et al., 1996), is proceeding into Phase II clinical trials (Kohn et al., 1997). Two of the underlying rationales for its efficacy are that by preventing gated calcium influx one abolishes either integrin-mediated adhesion (Sjaastad et al., 1996) or osmotic flux-initiated lamellipod protrusion (Dong et al., 1994). Success during Phase I trials in stabilizing disease (Kohn et al., 1997) may be due to these actions that limit cell motility, or to other effects on tumor cell growth (Jacobs et al., 1997).

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The application of agents against biochemical targets of cell motility to limit invasion is less advanced, reflecting the state of the knowledge of the underlying regulatory and active elements that directly impact tumor invasiveness. Many of the specific proteins that have been implicated in the motility machinery have been proposed as targets for anti-invasive, anti-metastasis, and anti-angiogenesisagents. These include the small GTPases that control actin cytoskeleton organization and connections to the substratum (Keely et al., 1998), signaling intermediaries (Shimizu, 1996; Turner et al., 1996, 1997), and ligands and their cognate receptors (Drebin et al., 1985; Harris, 1994; Engebraaten et al., 1993; Matsumoto et al., 1995; Sunada et al., 1986). The AP-1 transcription response element, required for sustained mbtility and invasion induced by EGF (Malliri et al., 1998), has been proposed as a target (McCarty, 1998). However, in almost all these cases, disruption of these molecules has multiple effects including growth inhibition or direct killing, with this often being the desired outcome (Drebin et al., 1986; Harris et al., 1994; Wels et al., 1995). However, as these molecules are neither tumor specific, nor mutated, nor even usually upregulated in tumor tissues, the toxicities of targeting these molecules would be expected to be quite severe. In addition, gene deletion experiments are demonstrating that cells possess functional redundancies for many of these targets, and thus epigenetic alteration within tumor microheterogeneity would likely quickly lead to resistant clones. To overcome the propensity of tumors to adapt to targeted therapies, one would target a required and convergent element in cell motility. Targeting growth factors or cognate receptors, though often the invasion-related cellular dysregulation, would likely be circumvented by expression of a different ligand or receptor to subsume the functioning of that receptor. For instance, metastatic and invasive breast cancers demonstrated upregulation of erbB2, EGF receptor, IGF-I receptor, or other growth factor receptors, and invasiveness of glioblastoma multiformes is thought to be driven through the EGF, PDGF, or IGF-I receptors (Khoshyomn et al., 1999); all of these receptors are presumed to function in the same role of promoting tumor cell motility and thus invasion. These different receptors share the property of activating PLCy, among other intermediaries, to promote motility and tumor invasion (Bornfeldt et al., 1994; Kassis et al., 1999; Khoshyomn et al., 1999; Kundra et al., 1994). Thus, PLCy would serve as a convergent bottleneck by which to inhibit growth factor induced cell motility. The requirement for PLCy in cell motility has been further supported by finding deficient locomotion in cells derived from embryos lacking PLCy (Ji et al., 1998).We have determined that PLCy blockade, by either pharmacological or molecular agents, can prevent invasiveness both in vitro and in vivo (Turner et al., 1996, 1997; Xie et al., 1995) (Fig. 5 ) . Originally, we investigated whether this prevents EGF receptor-mediated invasion in prostate cancer cells, but recently

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we have extended these studies to include invasiveness of glioblastoma multiformes, and breast and bladder carcinomas; the invasiveness of these cells is signaled through dysregulated signaling of erbB2 and PDGF and IGF-I receptors in addition to EGF receptor (Kassis et al., 1999; Khoshyomn et al., 1999).These promising preclinical findings suggest that agents which inhibit PLCy can be used against a variety of tumors regardless of the specific dysregulated growth factor and/or receptor.

C. Targeting Invasion4ssociated Motility Potential toxicity remains the major concern about targeting motility to inhibit tumor invasion. Inhibition of all cell locomotion would have devastating effects on many tissues, especially normal cellular turnover throughout the gastrointestinal tract and skin. Fortunately, much of this cell locomotion and organ function seems to be accomplished by haptokinetic and haptotactic motility signaled through adhesion receptors. Tumor invasion, on the other hand, likely requires growth factor receptor-mediated motility in addition to the haptokinetic signals. The evidence for this comes from two directions. First, growth factors induce invasion into matrices in vitro to a significantly greater quantitative extent than in the increase in cell speed; there seems to be a qualitative difference vis-A-vis invasion for receptormediated motility compared to adhesion-mediated motility. This is dramatically seen for the glioblastoma situation in which inhibition of PLCy prevents invasion into a normal brain spheroid but not cell migration on the surface to surround the spheroid (Fig. 10) (Khoshyomn et al., 1999).In vivo we note equivalent spreading of prostate cancers on the serosal surfaces when inoculated into the abdominal cavity, but penetration into tissues requires a functional EGF receptorlPLCy signaling pathway (Turner et al., 1996, 1997). Disruption of PLCy signaling and its downstream events does not affect haptokinetic motility (Chen et al., 1994b; Ji et al., 1998; Ware et al., 1998; Witke et al., 1995; Xie et al., 1998).Thus, growth factor-induced motility seems to be preferential for tumor invasiveness. The physiological roles for growth factor-induced and cytokine-induced cell motility are postulated as wound repair and inflammation in the adult Fig. 10 Phospholipase signaling is required for tumor cell invasion but not superficial migration. Rat glioblastoma cells were genetically engineered to express a dominant-negative PLCy fragment (PLCz; bottom) or not (control; top). A spheroid of these vitally stained cells (bright)were juxtapositioned to a spheroid of normal rat brain (dark).Invasion of the glioblastoma cells into the normal brain was followed over a week (5.5 days shown). Expression of PLCz prevented invasion into the brain parenchyma but not migration over the surface of the brain tissue. Invasion by human glioblastomaexplants of normal brain tissue was similarly prevented by a pharmacological inhibitor of PLC (U73122). She Khoshyomn et al. (1999).

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animal. Growth factor-induced motility also has been implicated during embryogenesis, in promoting the branching morphogenesis of epithelial organs and vasculature (Folkman, 1997; Hogan, 1999; Matsumoto et al., 1996; Wang et al., 1999; Yancopoulos et al., 1998) as well as playing a critical role in axonal outgrowth and guidance (Hall et al., 1996; Jay, 1996; TessierLavigne and Goodman, 1996).Thus, targeting growth factor-induced motility wauld likely impair these functions. Interestingly, this may actually be beneficial in controlling tumor progression. Obviously, limiting wound repair would be detrimental in postsurgical extirpation, but it may prevent excess scarring with morbidity after radiation or chemotherapy to a tumor bed. Also the production of growth factors and cytokines during wound repair may promote Cancer growth and progression. And limiting vascularization (angiogenesis and lymphangiogenesis) is, in itself, a strategy aimed at preventing cancer progression. Even blunting the inflammatory response may be of benefit, in that many of the cytokines, growth factors, and metabolites produced during inflammation may stimulate either tumor cell growth and motility or mutational plasticity. Furthermore, tumor infiltration by macrophages and lymphocytes may, peversely, promote further growth and dissemination. While our initial, preclinical investigations demonstrated no macroscopic toxicity by intraperitoneal administration of a PLCy inhibitory agent (Turner et al., 1996), the side effects of such therapies remain to be determined. Thus, on the basis of theoretical considerations, growth factor-induced cell motility holds promise as a therapeutic target with manageable toxicities.

VI. SUMMARY AND FUTURE DIRECTIONS Tumor invasion into surrounding tissues constitutes a major barrier to successful therapy of cancer. Advances in treating this ominous aspect of tumor progression will require an understanding of the underlying cell and molecular biology. Current evidence points to cell motility as being the major ratelimiting step many tumors utilize to promote invasiveness. Dysregulated growth factor receptor signaling directly results in uncontrolled motility with resultant tumor invasion. Thus, one of the critical keys to deciphering the mechanisms of tumor invasion and identifying targets for novel therapeutic agents is delineating the regulation of cell motility. Recent advances have defined the biophysical processes involved and have begun to parse the biochemical regulatory and signaling elements. These findings, in turn, have suggested numerous therapeutic targets; some of these agents have advanced to early phase clinical trials. However promising the preclinical and early clinical investigations are, these first generation agents will be limited in their

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utility and breadth of application. Future investigations should focus on providing a detailed understanding not only of the exact molecular controls of growth factor-induced motility, but also of the balance in signaling pathways that promote this cellular re5ponse over others. Furthermore, these studies should highlight points of convergence from the plethora of signals that induce motility, so that any targeted agent could be applied to a spectrum of tumors. In parallel, work on the physiological roles of induced cell motility will not only illuminate this fascinating biology, but will enable practitioners to anticipate toxicities and design therapeutic regimens to account for and minimize theg. The next decade of burgeoning investigations into cell motility holds excitement and promise as to uncovering cellular events that will be key to clinically controlling tumor invasion.

ACKNOWLEDGMENTS I thank the members of my laboratory and my collaborators and their laboratories, in particular Tim Turner, Paul Penar, Robert Radinsky, Doug Lauffenburger, Linda Griffith, and Alan Hall, for their helpful discussions and insights that have shaped this review. The studies reported on herein were supported by grants from the Veterans Administration, National Institutes of Health (NIGMS and NCI), and the Department of Defense. This review was written, in part, during a sabbatical supported by a Wellcome Research Travel grant.

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Witke, W., Sharpe, A. H., Hartwig, J. H., Azuma, T., Stossel, T. P., and Kwiatkowski, D. J. (1995). Cell 81,41-51. Woodhouse, E. C., Chuaqui, R. F., and Liotta, L. A. (1997). Cancer 80,1529-1537. Woods, A., and Couchman, J. R. (1992).J. Cell Sci. 101,277-290. Xiao, Z., Zhang, N., Murphy, D. B., and Devreotes, P. N. (1997).J. Cell Biol. 139,365-374. Xie, H., Turner, T., Wang, M.-H., Singh, R. K., Siegal, G. P., and Wells, A. (1995). Clin. Exp. Metastasis 13,407-419. Xie, H., Pallero, M. A., Gupta, D., Chang, P., Ware, M. F., Witke, W., Kwiatkowski, D. J., Lauffenburger, D. A., Murphy-Ullrich, J. E., and Wells, A. (1998).]. Cell Sci. 111,615-624. Xu, J., and Clark, R. A. F. (1996).J. Cell Biol. 132,239-249. Xu, J., Zutter, M. M., Santoro, S. A., and Clark, R. A. E (1998).J. Cell Biol. 140, 709-719. Yamaguchi, R., Maki, M., Hatanaka, M., and Sabe, H. (1994).FEBS Lett. 356,114-116. Yan, S., Sameni, M., and Sloane, B. F. (1998).Biol. Chem. 379,113-123. Yahcopoulos, G. D., Klagsbrun, M., and Folkman, J. (1998).Cell 93,661-664. Yang, N., Higuchi, O., Ohashi, K., Nagata, K., Wada, A., Kangawa, K., Nishida, E., and Mizuno, K. (1998).Nature (London) 393,809-812. Yasui, W., Sumiyoshi, H., Hata, J., Kameda, T., Ochiai, A., Ito, H., and Tahara, E. (1988).Cancer Res..48,137-141. Yauch, R. L., Berdichevski,F., Harler, M. B., Reichner, J., and Hemler, M. E. (1998).Mol. Biol. CeN 9,275 1-2765. Ye, J., Xu,R. H., Taylor-Papadimitriou,J., and Pitha, P. M. (1996).Mol. Cell. Biol. 16,61786189. Yoshioka, K., Matsumura, F., Akedo, H., and Itoh, K. (1998).J.Biol. Chem. 273,5146-5154. Youngs, S. J., Ali, S. A., Taub, D. D., and Rees, R. C. (1997). Int. J. Cancer 71,257-266. Yue, T. L., Wang, X., Sung, C . P., Olson, B., McKenna, P. J., Gu, J. L., and Feuerstein, G. Z. (1994).Circ. Res. 75, 1-7. Zhang, M., Singh, R. K., Wang, M.-H., Wells, A., and Siegal, G. P. (1996). Clin. Exp. Metastasis 14,268-276. Zhang, M., Wang, M.-H., Singh, R. K., Wells, A., and Siegal, G. P. (1997a).J.Biol. Chem. 272, 14139-14146. Zhang, M., Sheng, S., Maass, N., and Sager, R. (1997b). Mol. Med. 3,49-59. Zhou, K., Pandol, S., Bokoch, G., and Traynor-Kaplan, A. E. (1998).J.Cell Sci. 111,283-294. Zigmond, S . H. (1996). Curr. Opin. Cell Biol. 8,66-73. Zimmerman, A., and Keller, H. (1992). Br. J. Cancer 66,1077-1082.

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Nonenzymatic Interactions between Proteinases and the Cell Surface: Novel Roles in Normal and Malignant Cell Physiology Paolo Mignatti'.Zand Daniel B. RLfldnZ~3 'Department of Surgery S. A. Localio General Surgery Research Laboratory and Cardiovascular Surgery Research Laboratory 'Department of Cell Biology and 3The Kaplan Cancer Center New York University School of Medicine New York, New York 1001 6

I. Introduction 11. Extracellular Matrix-Degrading Proteinases: Classification and Structural Features A. Serine Proteinases: The Plasminogen Activator-Plasmin System B. The Matrix Metalloproteinases: Classification C. Common Features of Extracellular Matrix-Degrading Proteinases 111. Proteolysis-Independent Roles of Extracellular Matrix-Degrading Proteinases A. Proteinase-Extracellular Matrix Interactions: Modulation of Cell Adhesion and Migration B. Proteinase Interactions with Transmembrane Proteins C. Extracellular Proteolysis-Independent Generation of Intracellular Signaling D. Proteinase-MediatedControl of Gene Expression IV. Conclusions and Perspectives References

I. INTRODUCTION

Since the 1970s a consistent body of experimental evidence has demonstrated the involvement of proteolytic activities in a variety of biological processes that involve extracellular matrix (ECM) degradation (for reviews, see Andreasen et al., 1997; Basbaum and Werb, 1996; Dan0 etal., 1985; DeClerck and Laug, 1996; DeClerck et al., 1997; Kleiner and Stetler-Stevenson, 1993; Mignatti and Rifkin, 1993; Werb, 1997). These processes are important to several stages of tumor progression, such as local invasion, angio-

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Paolo Mignatti and Daniel B. Rifkin

genesis, and metastasis, as well as to a variety of physiological or pathological conditions, for example, ovulation, spermatogenesis, trophoblast implantation, mammary invo!ution following lactation, uterine involution, nerve regeneration, and rheumatoid arthritis. Common features of these processes are not only the degradation of histological structures-basement membranes, basal laminae, and interstitial stroma-but also cell migration, proliferation, and, in some instances, differentiation or dedifferentiation. The ECM is organized into complex structures consisting of various collagen types, glycoproteins, and proteoglycans. Because these ECM components have selectivs requirements for their degradation, an array of proteinases have been implicated as effectors of ECM degradation. Recent data, however, now indicate more complex interactions of proteinases both with cells and with the ECM. Binding sites for several proteinases have been identified on the cell membrane and in the ECM. These “receptors” include both high-affinitytransmembrane or glycosylphosphatidylinositol(GP1)-anchored proteins and several ECM glycoproteins and glycosaminoglycans (GAGs). Proteinase binding to these molecules modulates a variety of cell functions including adhesion, migration, differentiation, proliferation, and apoptosis. A notable example of a proteinase that induces intracellular signaling is thrombin. This component of the blood coagulation cascade acts on many cells through the cleavage of specific membrane receptors, the protease-activated receptors, or PAR (for reviews, see Goldsack et al., 1998; Grand et al., 1996). In other cases the modulation of cell function is mediated by noncatalytic interactions of a proteinase with other molecules, suggesting that some extracellular proteinases may have roles independent of their degradative activity. In this review we discuss experimental evidence supporting this hypothesis with respect to the structure of ECM-degrading proteinases and their interactions with cell membrane and ECM components.

11. EXTRACELLULAR MATRIX-DEGRADING

PROTEINASES: CLASSIFICATION

AND STRUCTURAL FEATURES The ECM-degrading proteinases produced by most cells can be subdivided into three families: (1) serine proteinases (plasminogen activators); (2) matrix metalloproteinases (MMPs), and (3)cysteine proteinases (cathepsins) (Mignatti and Rifkin, 1993). In addition, a number of endo- and exoglycosidases selectively degrade GAGs and the aminosugar moieties of proteoglycans. However, the plasminogen activators and MMPs play the major roles in most physiological and pathological states involving tissue remodeling. Therefore, this review will focus on these two families of enzymes. Com-

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prehensive discussion of cysteine proteinases or glycosidases can be found elsewhere (Chapman et al., 1997; Ernst et al., 1995).

A. Serine Proteinases: The Plasminogen Activator-Plasmin System The principal serine proteinases involved in tissue remodeling include the plasminogen activators (PAS),leukocyte elastase, and cathepsin G. Of these, the former are the best characterized and have been implicated in a variety of tissue remodeling processes (for reviews, see Andreasen et al., 1997; Basset et al., 1997; Dan0 et al., 1985; Mignatti and Rifkin, 1993, 1996; Moscatelli and Rifkin, 1988; Saksela and Rifkin, 1988; Testa and Quigley, 1990). PAS convert the zymogen plasminogen, a plasma protein ubiquitous in the body, to plasmin. In addition to the PAS and plasmin(ogen), this proteolytic system-referred to as the PA-plasmin system-comprises several components including cell surface binding sites for urokinase-type plasminogen activator ( uPA), tissue-type plasminogen activator (tPA), and plasmin(ogen), and specific PA inhibitors. The physiological role of plasmin is the degradation of the fibrin clot, as supported by the characterization of the phenotype of mice genetically deficient in plasminogen (knockout mice) (Bugge et al., 1995a). However, plasmin has a broad, trypsinlike substrate specificity, and it can degrade several ECM components including fibronectins, laminin, and the protein core of proteoglycans (Werb et al., 1980). It does not degrade elastin and native collagens but can degrade gelatins, the partially degraded or denatured forms of the collagens. In addition, plasmin also activates certain prometalloproteinases (see below) (He et al., 1989; Mazzieri et al., 1997; Murphy and Docherty, 1992; Werb et al., 1977) as well as latent elastase (Chapman and Stone, 1984). 1. THE PLASMINOGEN ACTIVATORS: STRUCTURAL FEATURES

Although plasma kallikrein, the blood coagulation factors XI and XII, and the bacterial protein streptokinase can activate plasminogen (Bouma and Griffin, 1978; Colman, 1969; Mandle and Kaplan, 1977; Summaria et al., 1982), the term PA currently refers to two enzymes, the urokinase-type PA ( uPA) and the tissue-type PA (tPA), which are kinetically very efficient activators of plasminogen. Urokinase (55 kDa) and tPA (70 kDa) are the products of two distinct genes that have evolved from a common ancestor by duplication and subsequent mutations (Edlund et al., 1983; Pennica et al., 1983; Verde et al.,

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1984). The amino acid and nucleotide sequences of uPA and tPA show interesting features. Both PAS are typical “mosaic” proteins consisting of distinct domains with structu_ral and/or functional homology to functional units of ECM structural proteins or growth factors. These modular structures result from “exon shuffling” or “exon insertion,” a process by which domains are exchanged between proteins during evolution. tPA was the first mosaic protein to be described (Novokhatny et al., 1991; Li and Graur, 1991). Three functional “modules” are present both in tPA and uPA: (1)a C-terminal catalytic domain homologous to the proteinase domain of trypsin and other trypsidike proteinases, and containing the His, Asp, and Ser residues typical of the charge relay in the active site of all serine proteinases; (2) a “kringle” module, a cysteine-rich sequence characterized by three internal disulfide bridges to form a structure resembling the Danish cake that bears his name; and (3)an N-terminal, cysteine-rich “growth factor” domain with high homology to epidermal growth factor (EGF) (Li and Graur, 1991). uPA has one kringle and tPA has two. In addition, the 43 N-terminal residues of tPA have no counterpart in uPA. This sequence forms a fingerlike structure homologous to the “finger” domains that confer fibrin affinity on the ECM protein fibronectin. tPA, unlike uPA, has a strong affinity for fibrin. Because plasminogen also binds to fibrin, the juxtaposition of plasminogen and tPA on the common ligand results in a 60-fold decrease of the Km (increase in the affinity) for plasminogen activation (Dans et al., 1985).Whereas tPA is poorly active in the absence of fibrin, its activity is strongly enhanced by fibrin (Dans et al., 1985). In the light of these features, it has been proposed that the two PAS have different physiological roles: tPA controlling clot lysis, and uPA primarily mediating tissue-remodeling processes. However, this separation is not absolute. In tPA knockout mice, uPA supplies sufficient fibrinolytic potential to clear fibrin deposits from most tissues (Carmeliet et al., 1994; Bugge et al., 1996). Kringle domains are also present in other proteins, and they define a family of polypeptides evolved from an ancestral gene that contained a single kringle domain, a serine proteinase domain, and an activation peptide connecting the two domains (Gherardi et al., 1997). These proteins include coagulation factors and growth factors. Plasminogen has five kringles and prothrombin has two (Li and Graur, 1991; Novokhatny et al., 1984). Interestingly, other kringle proteins devoid of catalytic activity also have structural and sequence homology to plasminogen. Apolipoprotein( a) [Lp(a)] possesses a number of kringles but is devoid of an active proteinase domain (Ichinose, 1992); hepatocyte growth factor/scatter factor (HGF/SF)has four kringles and a proteinase domain but lacks catalytic activity because of a mutation in the active site (Birchmeier and Gherardi, 1998; Gherardi et al., 1997; Nakamura, 1991; Ponting et al., 1992; Tashiro et al., 1990). Macrophage-stimulating protein (HGFUMSP), a 78-kDa protein produced

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by the liver and initially identified as a HGF/SF-like protein, has 45% sequence homology to HGF/SF and also possesses four kringles (Skeel et al., 1991; Yoshimura et al., 1993).It is noteworthy that, like plasminogen, HGF/ SF is also activated by uPA or other serine proteinases (Naldini et al., 1995). In addition, HGF/SF stimulates uPA expression in epithelial and endothelial cells (Grant et al., 1993; Pepper et al., 1992). Similarly, MSP is also activated by a pericellular proteinase(s) (Wang et al., 1994b). “Growth factor” domains are also present in the blood coagulation factors IX and X, and in protein C (Wang et al., 1994a,b). Catalytically active uPA and tPA consist of two polypeptide chains (A and B). The B chains of both enzymes, which include the C-terminal portion of thk proteins, are similar and contain the active site. The A chains also show a high degree of homology but differ considerably in size, the difference being accounted for by the second kringle and the finger domains of tPA. In uPA, the amino acid sequence encompassing residues 13-30 of the Nterminal,’EGF-like domain of the noncatalytic A chain mediates binding to the cell membrane receptor for uPA (uPAR, see below) (Appella et al., 1987).

2. CONTROL OF PLASMINOGEN ACTIVATOR ACTIVITY

’

Plasminogen activator activity is regulated both transcriptionally and posttranscriptionally. Similar mechanisms also control MMP activities (see below). PA gene transcription is modulated by a number of agents, including tumor promoters, oncogenes, growth factors, cyclic AMP,retinoids, prostaglandins, and W light in a variety of cell types. Posttranscriptional control of enzyme activity occurs at different levels: (1) proenzyme activation, (2)interaction with cell membrane or ECM binding sites, and (3) inhibition by specific tissue inhibitors (Dan0 et al., 1985; Mignatti and Rifkin, 1993). As is the case for all extracellular serine proteinases and MMPs (see below), PAS are secreted in the form of inactive, single-chain zymogens (prouPA or sc-uPA, pro-tPA or sc-tPA)that are converted to the active, two-chain form by limited proteolysis (Dan0 et al., 1985; Petersen et al., 1988). One of the more important features of the PA-plasmin system is the amplification loop resulting from plasminogen and pro-uPA activation. Trace amounts of plasmin activate pro-uPA (Petersen et al., 1988), generating a feedback mechanism of pro-uPA and plasminogen activation. As the plasminogen concentration in plasma is relatively high (-2 pM, or -200 pg/ml) and approximately 40% of plasminogen is located in extravascular sites (Robbins and Summaria, 1976), small amounts of PA can generate high local concentrations of plasmin. Plasmin probably does not represent the only mechanism of pro-uPA activation in vivo, as other enzymes including cathepsin B can activate pro-uPA in vitro (Berkenpas and Quigley, 1991; Goretzki et al., 1992; Kobayashi et al., 1991). However, the relatively high concentrations

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of plasminogen present in virtually all vertebrate tissues implicate plasmin as the most important activator of pro-uPA.

3. THE UROKINASE RECEPTOR The amplification loop achieved by plasmin activation of pro-uPA is further modulated by the high-affinity interaction (Kd = 50-150 pM) of (pro)uPA with the uPA receptor (UPAR).uPAR is a highly glycosylated, 55to 60-kDA protein linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (Behrendt et al., 1990, 1991, 1993; Estreicher et al., 1989; Nielsen et al., 1988; Ploug et al., 1991; Roldan et al., 1990; for a review, see Behrendt and Stephens, 1998). The level of glycosylation modulates uPAR affinity for its ligand (Meller et al., 1993). uPAR is synthesized as a 313-residue (35-kDA) polypeptide containing 28 cysteine residues, the pattern of which defines three homologous repeats (domains 1to 3), each consisting of approximately 90 amino acid residues. This domain structure has been proposed to represent a novel superfamily of glycolipid-anchored membrane proteins (Ploug et al., 1993). The N-terminal 87 amino acid residues (domain 1) constitute the ligand-binding domain (Ploug et al., 1993). Residues located at equivalent positions in uPAR domains 1and 3 participate in the assembly of the ligand-binding site (Ploug, 1998; Ploug et al., 1998). Domains 2 and 3 increase the affinity of uPA binding to domain 1 (Ploug et al., 1994) by providing a second binding site for the ligand (Higazi et al., 1997). These domains may also mediate the affinity of uPAR binding to the ECM protein vitronectin (see below). On the cell surface uPAR is present in two forms: a three-domain form with ligand-binding properties and a two-domain form devoid of domain 1 [uPAR(2+3)] and uPA-binding activity. The two-domain form results from uPA or plasmin cleavage of the three-domain molecule. The cleavage of uPAR by uPA or plasmin may represent a control mechanism by which these proteinases modulate their own activity on the cell surface (H0yer-Hansen et al., 1992,1997a). In addition, soluble forms of uPAR lacking the GPI anchor are found both in vitro and in vivo (Pedersen et al., 1993; Ploug et al., 1992; Pyke et al., 1993). As is the case for uPA, uPAR expression is also modulated by several agents, including cytokines, hormones, and tumor promoters (Estreicher et al., 1989; Kirchheimer et al., 1988a; Lu et al., 1988; Lund et al., 1991a,b, 1996; Mignatti et al., 1991; Picone et al., 1989; Stoppelli et al., 1985). For example, in human U937 monocytelike cells, the tumor promoter phorbol 12-myristate 13-acetate (PMA), which induces macrophage differentiation, strongly increases uPAR gene transcription and the cells’ uPA-binding capacity (Lund et al., 1991a,b; Picone et al., 1989). Fibroblast growth factor-2 (FGF-2 or bFGF) and vascular endothelial growth factor (VEGF), two

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potent angiogenesis inducers, upregulate uPAR expression in vascular endothelial cells (Mandriota et al., 1995; Mignatti et al., 1991). Following secretion, uPA binds to uPAR in its inactive pro-uPA form through a specific N-terminal sequence of its noncatalytic A chain (Appella et al., 1987). The bound zymogen is then activated by proteolytic cleavage (Petersenet al., 1988).Unlike most plasma membrane receptors, uPAR is not phosphorylated after binding the ligand, and formation of uPA-uPAR complexes does not result in internalization or receptor downregulation. The interaction of uPA with uPAR on the plasma membrane has (at least) four important consequences: (1)the localization of enzyme activity at focal contact sites (Hebert and Baker, 1988; Pollanen et al., 1988); (2)a dramatic lowerink (40-fold) of the K , for plasminogen activation (Lee et al., 1994; Ellis et al., 1991); (3)the internalization and rapid degradation of uPAR-bound uPA after complex formation with the plasminogen activator inhibitors (PAIs; see below) (Cubellis et al., 1990; Estreicher et al., 1990); and (4)the generation of intracellular signaling and upregulation of several cell functions (see below). 4. BINDING SITES FOR TISSUE-TYPE PLASMINOGEN ACTIVATOR AND PLASMINOGEN

Binding sites for tPA and/or tPA-inhibitor (PAI-1)complexes (see below) have been described on the membranes of fibroblasts, vascular smooth muscle cells, endothelial cells, monocytes/macrophages, and melanoma cells (Bizik et al,. 1993; Carroll et al., 1993; Ellis and Whawell, 1997; Felez et al., 1991, 1993; Hajjar, 1991; Hajjar and Hamel, 1990; Morton et al., 1990; for a review, see Felez, 1998). On human vascular endothelial cells, high-affinity (Kd = 25 nM) binding for tPA is associated with annexin II (Ann 11),a calciumand phospholipid-binding protein that also binds plasminogen and plasmin with high affinity (Kd = 161 and 75 nM, respectively) (Hajjar et al., 1994, 1998). The Ann 11-mediated assembly of plasminogen and tPA confers on tPA-dependent activation of plasminogen an approximately 60-fold increase in catalytic efficiency, and localizes constitutive plasmin generation on the vessel wall (Cesarman et al., 1994). PAI-1 associated with the surface of endothelial cells also appears to be a major binding site for tPA (Ramakrishnan et al., 1990; Russell et al., 1990; Wittwer and Sanzo, 1990).Unlike uPAuPAR interactions, binding of tPA*PAI-1complex requires elements of the PAI-1 moiety and/or regions of the proteinase domain of tPA (Morton et al., 1990). Binding of plasmin and tPA on the surface of endothelial cells protects these enzymes from their physiological inhibitors, a,-antiplasmin and PAI-1 (Shih and Hajjar, 1993). Although high-affinity plasma membrane binding sites for plasminogen have not yet been characterized, an a-enolase-related molecule and the

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Hyman nephritis autoantigen (gp330)have been implicated as candidate receptors (Kanalas and Makker, 1991; Miles et al., 1991). In general, cell surface proteins with C-terminal lysyl residues appear to function as plasminogen-binding sites; a-enolase-is a prominent representative bf this class of receptors (Miles et al., 1991). This molecule also interacts with tPA (Miles et al., 1991; Felez et al., 1993), suggesting that several high-affinity binding sites for tPA may be shared with plasminogen. In addition, low-affinity, highcapacity binding sites for plasminogen appear to be present in the chondroitin sulfate proteoglycans of the ECM and cell surface (Hajjar et al., 1986; Miles and Plow, 1285; Plow and Miles, 1990; Plow et al., 1986). Interestingly, uPA has a significant affinity for heparin and heparan sul'fate proteoglycans (Andrade-Gordon and Strickland, 1986). Binding sites for plasminogen and tPA are also present on fibronectin and laminin (Moser et al., 1993; Stack and Pizzo, 1993). Fibronectin binds both plasminogen and tPA via a 55-kDa N-terminal fragment (Moser et al., 1993). Unlike fibrin, intact fibronectin does not enhance the rate of tPA-catalyzed plasminogen activation; however, a mixture of proteolytically degraded fibronectin fragments stimulates the activation reaction, resulting in an 11-fold increase in the kc,,/ Km (Stack and Pizzo, 1993).Therefore, both plasminogen and PAS are colocalized either on the cell surface and/or in the ECM (Plow et al., 1986).

5. THE PJASMINOGEN ACTIVATOR INHIBITORS An additional mechanism for the extracellular control of PA activity is mediated by specific protein inhibitors present in most tissues and often expressed by PA-producing cells. The expression of these inhibitors can be modulated by a number of biological agents, including tumor promoters and growth factors. Although PAS can form complexes with several members of the serine proteinase inhibitor (serpin) superfamily (Carrel and Travis, 1985), only three inhibitors have a sufficiently high affinity to be effective in vivo. The first of these, the type 1 PA inhibitor (PAI-1) is a 45-kDa protein produced by a variety of cell types and present in platelets and plasma (Erickson et al., 1985; Hekman and Loskutoff, 1985; Loskutoff and Edgington, 1977). The second inhibitor, the type 2 PA inhibitor (PAI-2), is a 46.6-kDa protein expressed most notably by cells of the monocyte-macrophage lineage (Astedt et al., 1985; Kawano et al., 1970; Kruithoff et al., 1986). The third inhibitor, protease nexin 1 (PN-l),is a 45-kDa protein originally purified from cultured fibroblasts but also produced by several other cell types (Baker et al., 1980; Eaton et al., 1984). A fourth, less characterized inhibitor, PAI-3, has been isolated from human urine and is identical to the protein C inactivator; it is eonsiderably less efficient than the other inhibitors (Heeb et al., 1987). Both PAI-1 and PAI-2 bind the active two-chain forms of uPA and tPA, rapidly forming 1:l molar complexes, but have a poor affinity for the

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one-chain zymogen forms of both PAS. The association rate constant of PAI1for two-chain uPA and two-chain tPA (K, = 107-108 M-' sec-l) is higher than that of PAI-2 ( K , = 105-106 M-l sec-l). PN-1 is less specific for PA than PAI-1 and PAI-2. It inhibits two-chain uPA efficiently (K, = lo5 M-l sec-l) but has virtually no effect on single-chain uPA, single-chain tPA, and two-chain tPA. In contrast, PN-1 is an extremely rapid inhibitor of thrombin, and also inactivates trypsin and plasmin (Saksela and Rifkin, 1988). Plasminogen activator inhibitors participate in the turnover of PAS. When uPAR-bound uPA reacts with PAI-1, PAI-2, or PN-1, uPAR*uPA*inhibitor complexes are rapidly internalized and degraded (Conese et al., 1994,1995; Cubellis et al., 1990; Estreicher et al., 1990), and uPAR is recycled to the cell surface (Nykjaer et al., 1997). The process requires interaction with other cell membrane components PA*PAI-1 and PAI-l*PN-1 complexes as well as uncomplexed tPA bind to a,-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP or a,MR), a multifunctional receptor shared by a variety of ligands including a,-macroglobulin, apoprotein Eenriched P-very low density lipoprotein, tPA, and Pseudomonas exotoxin A. This receptor mediates endocytosis and degradation of the uPAR*uPA*PAI1 or uPAR*uPA*PN-Icomplexes on the cell surface and participates, in cooperation with other receptors, in the hepatic clearance of PA*PAI-1 complexes and uncomplexed tPA from blood plasma (Andreasen et al., 1994; Bu et al., 1993; Conese et al., 1995; Grobmyer et al., 1993; Herz et al., 1992; Iadonato et af., 1993; Kounnas et al., 1993). This mechanism represents a novel type of molecular recognition of serine proteinases and serpins by their cellular binding sites.

B. The Matrix Metalloproteinases: Classification The MMPs, or matrixins, are a family of at least 21 zinc- and calcium-dependent proteinases that can collectively degrade virtually all protein components of the ECM (for reviews, see Kleiner and Stetler-Stevenson, 1993; Massova et al., 1998; Matrisian, 1990, 1992; Mignatti and Rifkin, 1993; Parsons et al., 1997; Werb and Chin, 1998). These proteinases share the following features: (a) they show consistent structural and sequence homology; (b) they have a zinc- and calcium-binding domain of about 165 amino acid residues, and are inhibited by chelating agents (hence the name metalloproteinases); (c)their active site mediates both substrate hydrolysis and autolytic cleavage; (d)with few exceptions, they are secreted as inactive zymogens that become activated by partial proteolytic cleavage and subsequent autolysis; and (e) they are inhibited by specific tissue inhibitors of metalloproteinases (TIMPs, see below). All members of the MMP family have broad substrate specificity. In the

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past, this feature has led to the use of a multiplicity of terms to name individual MMPs after their substrates. The current, widely accepted nomenclature uses the acronym MMP followed by a number that reflects the order in which the MMPs were identified. Although this system has obvious advantages, it does not afford easy identification of the distinctive features of a given MMP, and may generate some confusion. Some MMPs that were assigned a number were later shown to be identical to previously discovered members of the same family. However, the number of subsequently identified MMPs was not changed accordingly. Thus, although the name proposed for the latest addition to the MMP family is MMP-24, only 21 distinct MMPs have so far been identified (Table I). On the basis of their structure and substrate specificity, most MMPs can be grouped into four subclasses (Table 11): (1)collagenases, which include MMP-1 (collagenase, collagenase-1, or interstitial collagenase), produced

Table I Matrix Metalloproteinases M M P number

5 6 7

8 9 10 11 12 13 14 15 16 17 18

19 20 21 22 23 24

.

Enzyme Collagenase-1, or collagenase Gelatinase A, or 72-kDa type IV collagenase Stromelysin-1 Neutral metalloproteinase from articular cartilage, identical to MMP-2 Neutral “telopeptidase” from osteoblasts and periodontal fibroblasts, probably corresponding to MMP-3 or MMP-13 Acid metalloroteinase from articular cartilage, identical to MMP-3 Matrilysin Collagenase-2, or polymorphonuclear neutrophil collagenase Gelatinase B, or 92-kDa type IV collagenase Stromelysin-2 Stromelysin-3 Metalloelastase Collagenase-3 Membrane-type 1 matrix metalloproteinase (MT1-MMP) Membrane-type 2 matrix metalloproteinase (MT2-MMP) Membrane-type 3 matrix metalloproteinase (MT3-MMP) Membrane-type 4 matrix metalloproteinase (MT4-MMP) MMP-18, novel cDNA amplified by PCR from human mammary gland cDNA MMP-19, identical to rasi-1 (rheumatoid arthritis-associated autoantigen) Enamelysin X-MMP, novel cDNA cloned from Xenopus laevis C-MMP, novel cDNA cloned from chicken embryo fibroblasts Novel MMP cloned from a human ovary cDNA library Membrane-type 5 matrix metalloproteinase (MT5-MMP)

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Table 11 Classification of the Matrix Metalloproteinases Based on Their Structure and Substrate Specificity Collagenases MMP-I, collagenase, collagenase-1, interstitial collagenase, fibroblast collagenase MMP-I, collagenase-2, neutrophil collagenase, PMN collagenase MMP-13, collagenase-3 Gelatinases MMP-2, gelatinase A, or 72-kDa type IV collagenase MMP-9, gelatinase B, or 92-kDa type IV collagenase Stromelysins MMPJ, stromelysin-1, transin-1, proteoglycanase, procallagen-activatingfactor W P - 7 , matrilysin, PUMP-1 (putative or punctuated metalloproteinase 1) MMP-10, stromelysin-2, transin-2 MMP-11, stromelysin-3 Membrane-type MMPs MMP-14 or MT1-Mh4P MMP-19 or MT2-MMP MMP-16 or MT3-MMP MMP-17 or MT4-MMP MMP-24 or MTS-MMP Unclassified MMP-12, metalloelastase, murine macrophage elastase MMP-18

MMP-19 MMP-20, enamelysin MMP-21, or XMMF' MMP-22, or CMMP MMP-23

by a variety of cells including fibroblasts, endothelial cells, and smooth muscle cells, MMP-8 (collagenase-2, neutrophil collagenase, or PMN collagenase) contained only within neutrophil granules, in distinction to all other MMPs which are rapidly secreted (Hasty et al., 1990), and MMP-13 (collagenase-3), an enzyme originally described in breast carcinomas and which may represent the predominant collagenase in some rodents (Freije et al., 1994; Quinn et al,. 1990); (2) gelatinases, originally referred to as type IV collagenases, which include MMP-2 (gelatinaseA, or 72-kDa type IV collagenase) and MMP-9 (gelatinaseB, or 92-kDa type IV collagenase) (Okada etal., 1990,1992; Rantala-Ryhanen etal., 1983; Salo etal., 1983); (3)stromelysins, which include four members: MMP-7 (matrilysin),MMP3 (stromelysin-1),MMP-10 (stromelysin-2),and MMP-11 (stromelysin-3) (Basset et al., 1990; Chin et al., 1985; Goldberg et al., 1986; Nicholson et al., 1989; Quantin et al., 1989; Sanchez-Lopez etal., 1988; Wilhelm et al., 1987, 1989; Woessner and Taplin, 1988); and (4) membrane-type MMPs (MT-MMP), which comprise five members (MT1-, MT2-, MT3-, MT4MMP, and MT5-MMP) (Pei, 1999; Puente et al., 1996; Sato et al., 1994;

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Takino et al., 1995; Will and Hinzmann, 1995). Unlike the other MMPs, which are secretory proteins, MT-MMPs have the unique property of possessing a hydrophobic sequpnce close to the C-terminus, which allows insertion of the protein into the cell membrane. MT-MMPs are therefore transmembrane enzymes, with the catalytic domain exposed on the cell surface and a short cytoplasmic domain (20-26 amino acids). However, soluble MT-MMP forms have also been described in the culture medium of some tumor cell lines (Imai et al., 1996; Matsumoto et al., 1997). MT1-, MT2-, MT3-, and MT5-MMP have been implicated as physiological activators of MMP-L(Pei, 1999; Sat0 et al., 1994, 1997; Ueno et al., 1997); MT4-MMP is structurally different from the other MT-MMPs and its physiological role is unclear (Puente et al., 1996). ’ Some MMPs cannot be grouped into any of these classes. MMP-12 (metalloelastase, or murine macrophage elastase) degrades type IV collagen and insoluble elastin but, unlike the gelatinases, does not digest gelatins (Shapiro et al., 1993).MMP-18 has closest structural identity to both collagenase and stromelysins (Cossins et al., 1996). MMP-19 has unique structural features absent in other mammalian MMPs, suggesting that it may represent a novel class of these enzymes (Sedlacek et al., 1998). MMP-20 (enamelysin), recently cloned from odontoblastic cells, lacks a series of structural features distinctive of the four MMP classes (Llano et al., 1997). MMP-23 has a unique domain structure that has no equivalent in other MMPs (see below) (Velasco et al., 1999).

I . STRUCTURAL FEATURES OF THE MATRIX METALLOPROTEINASES The analysis of the nucleotide sequences of the cloned MMPs has shown the presence of distinct domains that are conserved among the members of this family (Fig. 1).The smallest (28 kDa), and perhaps ancestral MMF’, matrilysin, possesses only three domaim. The first N-terminal domain is a transient signal peptide. The second is an 8- to 10-kDa propeptide (“pro” domain), which is responsible for maintaining the enzyme in an inactive, zymogen state. The “pro” domain contains the highly conserved sequence PRCGVPDV, in which the C residue represents a ligand for the active site Zn2+. This interaction confers latency on the MMP. The third domain is a highly conserved, zinc- and calcium-binding catalytic domain of about 165 amino acid residues (21 kDa); this domain contains the sequence HEXGHXXGLXH, in which the conserved H residues represent three Zn2+-interactive ligands. Single amino acid mutations introduced into this sequence render the enzyme catalytically inactive (Goldberg et al., 1986; Matrisian, 1990, 1992; Sanchez-Lopez et ul., 1988; Woessner, 1991). The catalytic domain is also the site of interaction with the specific MMP inhibitors

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(TIMPs, see below) and contributes to the determination of the substrate specificity (Murphy and Docherty, 1992). In addition to these domains, all MMPs except matrilysin possess a fourth C-terminal domain, referred to as the “hemopexin” domain. This domain has strong sequence similarity to members of the hemopexin family of proteins, which includes the ECM component vitronectin. In the case of collagenase-1, -2, and -3 and stromelysin-1, this domain mediates binding to native collagen and is important for the cleavage of its triple helix. In several MMPs, except matrilysin, the hemopexin domain also mediates binding to the noncatalytic C-terminal domain of the TIMPs (see below). This binding to the hemopexin domain accelerates interaction of the catalytic site of the MMPs with the N-terminal, active site of the inhibitors. The interaction of the hemopexin domain with the TIMPs is particularly relevant for the gelatinases, less important for collagenase-1, and negligible for stromelysin-1 and MT1-MMP (Murphy et al., 1992a,b; Murphy and Knauper, 1997). In all MMPs, except matrilysin, a hyperflexible linker, or “hinge” region, of variable length bridges the catalytic domain to the C-terminal, hemopexin domain. In addition, some MMPs have distinctive structural features. The gelatinases (MMP-2 and MMP-9) possess a unique “collagen-binding” domain consisting of three tandem repeats of a 58-amino acid fibronectin type 11-like module. In the primary structure this sequence is N-terminal to the Zn2+binding region of the catalytic domain; however, secondary structure predictions suggest that the “fibronectin” domain is a separate folding unit. This module confers on the gelatinases the ability to bind to denatured type I, IV, and V collagens (gelatins), as well as to native type I collagen (MMP-2) and elastin (Murphy and Knauper, 1997). Binding occurs to multiple sites of the collagen molecule, primarily within the telopeptide region. Gelatinase B (MMP-9) possesses an additional “collagen domain” with sequence homology to the a2 chain of type IV collagen. MT-MMPs possess a 10- to 15-amino acid hydrophobic sequence close to the C-terminal end of the hemopexin domain. This sequence directs insertion of the MT-MMPs into the cell membrane, leaving the C-terminal20- to 26-amino acid peptide of the hemopexin domain as a cytoplasmic tail. The “pro” domain of the four MT-MMPs and of stromelysin-3 contains consensus cleavage sites for the prohormone convertase furin; this enzyme may, indeed, be involved in MT1-MMP and stromelysin-3 activation (Murphy and Knauper, 1997; Pei and Weiss, 1995,1996). The “pro” domain of MT1MMP is also required for TIMP-2 binding and subsequent proMMP-2 activation (Cao et al., 1998). MMP- 19 has a unique catalytic domain Cys residue, which is also present in Xenopus MMP (XMMP, or MMP-21) and in a recently characterized MMP produced by cultured primary chicken embryo fibroblasts (CMMP, or

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I

MMP STRUCTURE

COLLAGENASE STROMELYSIN

NH+

OELATINASEA

NH+

GELATINASEB

MI+

.

coo-

z. coo.

an

z.

Fig. 1 Modular structures of MMPs. H and C represent His and Cys residues, respectively. The figure does not represent the actual relative dimensions of the different domains.

MMP-22). Phylogenetic analysis suggests that XMMP and human MMP-19 represent founding members of the MMP family, whereas CMMP is related to the collagenase MMPs. MMP-19 may thus represent an ancestral member of the mammalian MMP family (Pendas et al., 1997; Yang and Kurkinen, 1998; Yang et al., 1997). Interestingly, MMP-19 shows sequence identity with rasi-1, a gene expressed in the inflamed synovium of rheumatoid arthritis*(RA)patients. MMP-19 is recognized by autoantibodies from the serum of patients with RA or systemic lupus erythematosus (SLE), suggesting that it may play a role in RA- and SLE-associated tissue destruction (Sedlacek et al., 1998). The recently cloned MMP-23 (Velasco et al., 1999) displays a unique domain structure: it lacks a recognizable signal sequence, it has a short prodomain, and its C-terminal domain is very short and has no sequence similarity to hemopexin. MMP-23 is predominantly expressed in ovary, testis, and prostate, suggesting that it may play a specialized role in reproduction. 2. SUBSTRATE SPECIFICITY OF THE MATRIX METALLOPROTEINASES The members of the four subclasses of MMPs have different but partly overlapping substrate specificities. Interstitial collagenases degrade intersti-

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1 I7

DOMAINS

00

Signalpeptide

Pro peptide Catalytic domain za

Fibronectin type 11-like repeats Collagen domain

Hemopexin domain Transmembrane domain Fig. 1 (continued).

tial collagens (types I, 11, and 111), as well as collagen types VII and X, but cannot degrade gelatins, type IV collagen, or other protein components of the ECM (Miller et al., 1976a,b; Schmid et al., 1986). Both gelatinase A (MMP-2) and B (MMP-9) degrade basement membrane (type IV)collagen, as well as collagen types I (gelatinaseA), V, VII, and X. In addition, they efficiently degrade gelatins (hence the name gelatinases) and fibronectins (Aimesand Quigley, 1995; Collier et al., 1988; Liotta et a!., 1979; Seltzer et al., 1989a,b; Welgus et al., 1990; Wilhelm et al., 1989). The stromelysinsare so designated because of their broad substrate specificity: stromelysin-1 (MMP-3)and -2 (MMP-10)degrade fibronectins, laminin, elastin, and the protein core of proteoglycans. Unlike collagenase (MMP1)or the gelatinases, these stromelysins cleave collagen types IV, V, VIII, and IX only in the nonhelical region and type I collagen in the N-terminal region (Chin et al., 1985; G . Murphy et al., 1991, 1993; Okada et al., 1986; Wilhelm et al., 1987). In contrast, stromelysin-3 (MMP-11) appears to exhibit only weak proteolytic activity (G. Murphy et al., 1993). Stromelysin-1 (MMP-3) cleaves fibrinogen and cross-linked fibrin (Bini et al., 1996). Matrilysin (MMP-7), the smallest MMP (28 kDa), possesses broad and potent catalytic activity: it is a stronger proteoglycanase than stromelysin-1 or -2, and also degrades type IV collagen, insoluble elastin, laminin, fibronectin, gelatin, and entactin (Murphy etal., 1991; Sires et al., 1993).Matrilysin ap-

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pears to be produced by a select population of cell types, the most prominent of which are glandular epithelia (Rodgers et al., 1993). Both gelatinases degrade type IV collagen-more efficiently at higher temperatures than the stromelysins and matrilysin (Murphy et al., 1991). The gelatinases and matrilysin are markedly more active than the stromelysins in the degradation of elastin. Matrilysin and the stromelysins are more potent proteoglycan-degrading enzymes than the gelatinases. MT1-MMP cleaves within the “pro” domain of MMP-2 (gelatinase A) and MMP-13 (collagenase-3)and activates these MMPs (Knauper et al., 1996; Sat0 et a1.,.1994; Will et al., 1996). In addition, the analysis of recombinant MT1-MMP forms lacking the transmembrane ‘domain has shown that MT1-MMP degrades gelatin (Imd et al., 1996) as well as native types I, 11, and 111collagens into characteristic J and fragments (Ohuchi et al., 1997). Purified MT1-MMP also degrades fibronectin, tenascin, nidogen, aggrecan, and perlecan. Only MT2-MMP shows activity against laminin (d’Ortho et al., 1997). MT1-MMP and membrane-bound MMP-2 degrade polymerized, insoluble fibrin, although with low efficiency (Hiraoka et al., 1998). MT1-MMP hydrolyzes type I collagen 6.5- or 4-fold more efficiently than type I1 or 111collagen, whereas MMP-1 (collagenase)digests type I11 collagen more efficiently than the other two collagens (Ohuchi et al., 1997). MT1-MMP also digests cartilage proteoglycan, fibronectin, vitronectin, and laminin-1 as well as a,-proteinase inhibitor and a,-macroglobulin. Its type I collagenolytic activity is synergistically increased by coincubation with MMP-2, indicating that MT1-MMP shares substrate specificity with both MMP-1 and MMP-2, and that its proteolytic activity is both direct and mediated by proMMP-2 activation (Ohuchi et al., 1997). However, it should be noted that most of these data have been obtained with recombinant MTMMP lacking the transmembrane domain: it is possible that the specificity of cell-bound MT1-MMP is different from that of the soluble enzyme. 3. CONTROL OF MATRIX METALLOPROTEINASE ACTIVITY

The expression of MMP activity is controlled at three levels: (1)gene transcription, (2) proenzyme activation, and (3) inhibition by specific tissue inhibitors. In most cell types the MMP genes are not constitutively expressed but transcription can be induced by a number of agents, including tumor promoters, growth factors, and oncogene products (Collier et d., 1988; Kerr et al., 1988a,b, 1990; Masure et al., 1990; McDonnell et al., 1990; Salo et al., 1985; Schonthal et al., 1988; Wilhelm et al., 1989).The induction of MMPs in several cell types by phorbol esters, interleukin-1(IL-1),and tumor necrosis factor a ( m a )is dependent on the binding of transcription factors to activator protein-1 ( AP-1) and polyomavirus enhancer A-binding protein (PEA-3)elements located in the MMP promoters (Buttice and Kurkinen,

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1993; Buttice et al., 1991; Curran and Franza, 1988; Gaire et al., 1994; Vincenti et al., 1994). Both protein kinase C and tyrosine kinase signal transduction pathways have been implicated in MMP induction (Sudbeck et al., 1994). Protooncogene induction by growth factors or tumor promoters appears to be involved in both the positive and negative regulation of MMP gene expression. In some cases, the expression of more than one MMP is coordinately regulated, as is the case for the phorbol ester induction of both stromelysin and interstitial collagenase in macrophages and endothelial cells (Frisch et al., 1987). In other situations, the expression of these two genes can be dissociated, indicating the existence of separate regulatory mechanisms. As is the case for PAS, glucocorticoids and transforming growth factot-Pl (TGF-P1) block the induction of MMP expression by other cytokines in a number of cell types, including vascular endothelial cells (DiBattista et al., 1991; Lund et al,. 1996; Matrisian et al., 1992). With the exceptions of neutrophil collagenase (MMP-8), which is stored in cytoplasmic granules, and stromelysin-3 and MT1-MMP, which are activated intracellularly by furin (Pei and Weiss, 1995, 1996), all MMPs are secreted in a proenzyme form. In the proenzyme form the ‘ ‘ p r ~domain ~’ may be folded so that the Cys residue of the PRCGVPDV sequence is coordinated with the zinc ion in the active site and blocks the enzyme’s activity. In the test tube, latent MMPs can be activated by a number of agents, including organomercurials, chaotropic agents, and proteinases. The conformational changes induced by these MMP activators disrupt the Cys-zinc interaction and render the zinc ion available for catalysis. MMP activation in vitro is effected by initial cleavage of amino acid residues between the pro and the catalytic domain, with the resulting destabilization of the interaction between the Cys residue in the pro domain and the active site Zn2+ (the “cysteine switch’’ mechanism) (Springman et al., 1990). The activated enzyme removes the N-terminal “pro’’ domain by autoproteolysis, thus becoming permanently activated (Kleiner and Stetler-Stevenson, 1993; Matrisian, 1992; Mignatti and Rifkin, 1993). In cell cultures the activation of latent MMPs appears to require a series of proteolytic reactions that involves several enzymes, including plasmin. In the first reaction, plasmin cleaves within the 84 amino acid residues in the N-terminal region that includes the “pro” domain. The activated enzyme can subsequently activate other proenzyme molecules, initiating an amplification loop similar to the feedback mechanism of pro-uPA and plasmin activation. In addition, plasmin-activatedstromelysinremoves approximately 15residues from the C-terminal end of the collagenase molecules. This “superactivation” results in a 5- to 8-fold increase in collagenase activity (He et al., 1989). Plasmin activation is not the only mechanism by which active M M P s can be generated in vivo; additional mechanisms invplve the action of other proteinases, including MT-MMPs, cathepsin G, arid tissue kallikrein (Cao et al.,

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1995; Desrivieres et al., 1993; Okada et al., 1992; Sat0 et al., 1994; Watanabe et al,. 1993). In several cases, one MMP can activate another MMP. MT1-, MT2-, MT3-, and MT5-MMP can activate proMMP-2 (Pei, 1999; Sat0 et al., 1994,1997; Ueno et al., 1997). MT1-MMP and MMP-2 activate proMMP-13 (Knauperet al., 1996).The gelatinasescan also be activated by MMP-1, MMP-3, and MMP-7 (Cao et al., 1995; Imai et al., 1995; Kleiner and Stetler-Stevenson, 1993; Matrisian, 1990, 1992; Mignatti and Rifkin, 1993; Ogata et al., 1992; Okada et al., 1992; Sat0 et al., 1994). MMP-2, in turn, can activate proMMP-9 (Fridman et al., 1995). However, it should be noted,that most, if not all, of these reactions have been characterized in the test tube or in cell cultures: the physiological mechanism(s)of MMP activation in vivo remains undetermined. 4. THE TISSUE INHIBITORS OF METALLOPROTEINASES

In the extracellular milieu MMPs can be inhibited by interaction with the TIMPs. Like the serpins (see Section II,A,5), the TIMPs are members of a multigene family. Four TIMPs have been characterized, TIMP-1, -2, -3, and -4 (for a review, see Gomez et al., 1997). The prototypical member, TIMP1,is a 26- to 28-kDa protein produced by many cell types (Albinet al., 1987; Campbell et al., 1987; Hanemaaijer et al., 1993; Herron et al., 1986a,b; Stricklin and Welgus, 1983; Welgus et al., 1979). TIMP-1 is identical to human erythroid-potentiatingactivity (EPA),a previously identified glycoprotein that stimulates the growth of erythroid progenitors in vitro and enhances colony formation by the K562 human erythroleukemia cell line (Avalos et al., 1988). The second member of the TIMP family, TIMP-2, is a 21-kDa protein originally isolated from human melanoma cells (Stetler-Stevensonet al., 1989). TIMP-2 shows a significant (66%) sequence homology to TIMP-1, including conservation of the positions of 12 Cys residues and 3 of 4 Trp residues. The homology appears closer at the protein than at the nucleotide level, suggesting that the two inhibitors diverged early in the evolution of this gene family (Stetler-Stevenson et al., 1990). With few exceptions, TIMP-1 and TIMP-2 inhibit all MMPs. Interestingly, TIMP-2 also possesses erythroidpotentiating activity, probably because it shares with TIMP-1 a common structuraldomain responsible for such activity (Stetler-Stevensonet al., 1992). Thus, TIMP-1 and TIM€’-2 are bifunctional molecules with both growth factor and antiproteolytic activities. TIMP-3 is the only member of the TIMP family found exclusively in the ECM. Its expression is regulated in a cell cycle-dependent fashion in certain cell-typesand may serve as a marker for terminal differentiation (Leco et al., 1994).The most recently discovered TIMP, TIMP-4, is essentially expressed only in adult heart; very low levels of TIM€’-4 mRNA are found in kidney,

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placenta, colon, and testis. This unique expression pattern suggests a tissuespecific role for TIMP-4 in extracellular matrix homeostasis (Gomez et al., 1997; Greene et al., 1996). Both TIMP-1 and TIMP-2 bind noncovalently to active MMPs in a 1:l molar ratio and specifically inhibit their activity. TIMP-MMP interactions are reversible and kinetically less restrictive than serine proteinase inhibition by the serpins. TIMP inhibition of active MMPs requires interactions primarily with the MMP catalytic domain but secondarily with the hemopexinlike domain, which contributes to the binding affinity (Baragi et al., 1994; Murphy and Knauper, 1997; Murphy et al., 1992a). No complex formation occurs between the TLMPs and the proenzyme form of interstitial coilagenase (Srricklin and Welgus, 1983; Stricklin et al., 1978). However, TIMP-1 forms a complex with the pro form of MMP-9, whereas TIMP-2 appears to form a complex specificallywith proMMP-2 (Goldberget al., 1989; Stetler-Stevenson et al., 1989). TIMP-2 binds through its noninhibitory Cterminal' domain to the hemopexinlike domain of proMMP-2, and the enzyme is secreted from cells as a 72-kDa progelatinase-TIMP-2 complex (Murphy et al., 1992b). The binding between the C-terminal domains of the enzyme and inhibitor molecules is required for effective inhibition of the gelatinases, which involves interaction of the N-terminal domain of TIMP2 with the catalytic domain of MMP-2 (Murphy and Knauper, 1997; Taylor et al., 1996). TIMP-2 also binds through its N-terminal domain to the catalytic domain of MTI-MMT (Zucker et al,. 1998) providing a bridging function between MT1-MMP and proMMP-2 (Fig. 2). The formation of a MT1-MMP*TIMP-2*proMMP-2complex on the cell surface is required for proMMP-2 activation (Strongin et al., 1995). As is the case for the PA inhibitors, TIMP expression is modulated by a

W P k

a a 4

Fig. 2 The cell surface-bound trimolecular complex consisting of MT1-MMP, TIMP-2, and MMP-2. TIMP-2 makes up a bridge between MT1-Mh4P and W - 2 . The figure does not represent the actual relative dimensions of the different molecules and their domains.

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number of biological agents. TIMP-1 and TIMP-2 appear to be regulated independently: in a number of cell lines TIMP-1 expression is upregulated by phorbol esters, whereas TIMP-2 expression is unaffected (Stetler-Stevenson et al., 1989). Interestingly, in some cases opposing patterns of regulation of MMPs and TIMP have been described. The cellular response to TGF-PI is a notable example. In a number of cell types, including fibroblasts and endothelial cells, TGF-P1 upregulates TIMP expression and at the same time represses stromelysin and collagenase expression (Edwards et al., 1987). It is noteworthy that TGF-P1 can have an identical effect on the PAplasmin system: ul capillary endothelial cells it stimulates production of PAI-1 while repressing uPA expression (Pepper et al., 1990; Saksela et al., 1987).

C. Common Features of Extracellular Matrix-Degrading Proteinases The analysis of ECM-degrading serine proteinases and metalloproteinases raises several interesting points. First, PAS and MMPs share a number of common features: (1)the expression of both families of proteinases is modulated at the transcriptional level by oncogenes, tumor promoters, and growth factors; (2) both PAS and MMPs are secreted in the form of inactive proenzymes; (3) the conversion of a variety of proenzymes to active enzymes requires the action of plasmin or other proteinases; (4) the active enzymes are inhibited by specific inhibitors produced locally in tissues; ( 5 )the expression of both PA inhibitors and TIMPs is modulated by growth factors, hormones, and cytokines; and (6) several growth factors and cytokines coordinately modulate the parallel expression of both PAS and MMPs and their respective tissue inhibitors in a variety of cell types (Bikfalvi et al,. 1997; Edwards et al., 1987; Moscatelli et al,. 1986; Pepper et al., 1990; Rifkin and Moscatelli, 1989; Saksela et al., 1987). These similarities illustrate that the division of the ECM-degrading proteinases into classes and subclasses is useful only for descriptive purposes. Physiologically, these enzymes act in concert via a cascade of proteolytic events whose end result is the generation of a broad spectrum of degradative activities. The PA-plasmin system appears to play a pivotal role in this cascade. The production of small amounts of PA results in the generation of high local concentrations of broad-spectrum enzymes, plasmin and stromelysins, and of enzymes with more restricted substrate specificity, such as collagenase, the gelatinases, and elastase (Chapman and Stone, 1984; He et al., 1989; Mazzieri et al., 1997; Werb et al., 1977). This cascade can be blocked at different levels by tissue inhibitors. The blockade of plasminogen activation by PAIs will inhibit subsequent activation reactions, and result in

Proteolysis-IndependentRoles of Proteinases

I23

the repression of MMP and elastase activation. In contrast, inhibition of MMPs by TIMPs will block these enzymes but leave plasmin unaffected. Second, the proteolytic systems of mammalian cells appear to be highly redundant. Two different PASshare the common role of activating one proenzyme, plasminogen. There exist at least 21 different MMPs, many of which have averlapping substrate specificity. Many protein components of the ECM can be degraded both by plasmin and by certain MMPs, although with different efficiency. The substrate specificity of plasmin overlaps with that of several MMPs, for example, the stromelysins. Several collagen types can be degraded by a variety of MMPs. Third, both ECM-degrading serine proteinases (PAS and plasmin) and MMPs have modular structures, and they possess noncatalytic domains evolutionarily derived from growth factors, ECM components, or other proteins devoid of proteolytic activity. Fourth, some of the noncatalytic domains of both PAS and MMPs mediate interactions with multiple binding sites on the cell membrane and ECM. These interactions do not result in degradation of the binding site but localize the enzyme activity to specific regions of the cell surface. Finally, some PA inhibitors, most notably PAI-1, and some TIMPs also have binding sites on the cell membrane and the ECM. The presence of binding sites for the respective proteinases results in the formation of multimolecular complexes. This feature is well illustrated by uPAR and membrane-type MMPs (at least by MT1-MMP), which show interestingsimilarities and differences. Both proteins form trimolecular complexes on the cell membrane: uPAR with uPA and PAI-1, PAI-2, or PN-1; MT1-MMP with MMP-2 and TIMP-2. Both uPAuPAR and MT1-MMP-MMP-2 interactions result in dramatic upregulation of the proteinase activity. Howevel; whereas TIMP-2 binding to MT1-MMP is required for activation of the gelatinase and the trimolecular complex is not internalized, PAI-1 interaction with uPAR-bound uPA results in internalization and degradation of the proteinase-inhibitor complex. Several hypotheses can explain the redundancy of ECM-degrading proteinases. The structural and biochemical complexity of the ECM demands that proteinases act in concert to achieve a degree of degradation quantitatively and qualitatively sufficient for cell migration and tissue remodeling. This process may require both a temporal and spatial coordination of the action of different proteinases. For example, one enzyme may initiate the partial degradation of an ECM component, which would leave an anchoring site(s) for cell migration unaffected and/or generate protein degradation products with chemotactic activity. A second proteinase may subsequently contribute to the complete digestion of the substrate. Degradation of one ECM component may be required for a second ECM protein to become accessible to another proteinase. Many of the substrate specificities of ECMdegrading proteinases have been characterized in the test tube or under in

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vitro conditions. Differences in the activity of these enzymes may be more relevant in vivo and dependent on the tissue microenvironment. This hypothesis is supported by the_observation that some proteinases have specific patterns of expression in different tissues or within different areas of the same tissue. The tissue specificity of some proteinases may be related to the turnover of specific components. For example, matrilysin is expressed almost exclusivelyby glandular epithelia; in a variety of tumors, uPA and stromelysin3 expression is confined almost uniquely to stromal fibroblasts or infiltrating macrophages. Interestingly, whereas uPA is catalytically very efficient, stromelysin-3 is virtually devoid of proteolytic activity, suggesting that the two proteinases may have different roles in the same tissue compartment. The presence of noncatalytic domains in both PASand MMPs, along with their interaction with a variety of cell membrane and ECM components, suggests an additional hypothesis to explain the redundancy of ECM-degrading proteinases. These proteinases may have distinct, as yet unidentified proteolysis-independent roles, although they possess partially overlapping catalytic activities. Evidence has emerged from numerous studies in the past few years to support this hypothesis.

111. PROTEOLYSIS-INDEPENDENT ROLES OF EXTRACELLULAR MATRIX-DEGRADING PROTEINASES. When the structure of uPA was first characterized, the finding of an EGFlike domain in its noncatalytic chain generated the conjecture that this enzyme might have growth factor-like activity. Early studies indicated mitogenic or chemotactic effects of uPA on a variety of cell types (Fibbi et al., 1988; He et al., 1991; Kirchheimer et al., 1987a,b, 1988b, 1989a,b; Rabbani et al., 1990). However, these results were received with skepticism by the scientific community, not only because of the modest effects elicited by relatively high concentrations of uPA, but mainly because a cell membrane reciptor that could transduce the mitogenic signal of uPA to the inside of the cell was not known. In spite of earlier observations of uPA associated with cell membrane fractions (Quigley, 1976), extravascular proteolytic reactions were believed to occur in the soluble phase, and the hypothesis that tissue proteinases could localize on the cell surface was largely disregarded. The effect of uPA on cell proliferation and/or migration was explained as an indirect result of cytoskeletal modifications generated by plasmin-mediated degradation of the ECM. The subsequent identification and characterization of uPAR as a GPI-anchored protein (Behrendt et al., 1990; Nielsen et al., 1988; Roldan et al., 1990; Vassalli et al., 1985) did not add much credibili-

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ty to a growth factor-like activity of uPA. Because of the lack of a cytoplasmic domain, GPI-anchored receptors were considered incapable of transducing extracellular signals across the cell membrane. However, the recent findings of a dose association between signal transduction proteins and GPIlinked receptors (Cervello et al., 1996; Shenoy-Scaria et al., 1993; Solomon et al., 1996; Thomas and Samelson, 1992; for a review, see Maxfield and Mayor, 1997) has given impetus to numerous investigations aimed at identifying roles for uPAR other than the control of uPA activity. These studies have now identified interactions of uPAR with ECM components, and with transmembrane and cytoplasmic proteins. These associations unequivocally activate several signal transduction pathways and modulate cell functions including adhesion and migration, as well as the expression of a variety of genes. In addition, recent findings have identified MMP-2 interactions both with MT1-MMP and a,P3 integrin on the cell membrane (Brooksetal., 1996, 1998; Strongin et al., 1993, indicating potential proteolysis-independent roles for MMPs.

A. Proteinase-Extracellular Matrix Interactions:

Modulation of Cell Adhesion and Migration The interactions between cell membrane receptors and the ECM regulate a number of cell functions, including migration and proliferation, tumor cell growth and metastasis, and cellular responses to mechanical stress. Intracellular actin filaments (stress fibers) bind to the plasma membrane and establish indirect interactions with the ECM, thus facilitating cell adhesion and migration. The links between the cytoskeleton and the ECM are mediated by transmembrane linker proteins, and they are morphologically defined by regions of close proximity of the cell to the substratum (focal contacts). In cultured cells, uPAR and uPA colocalize with the intracellular actinbinding protein vinculin at focal contacts, and with vitronectin, a plasma and ECM component with multiple binding properties (for reviews, see Preissner, 1991; Preissner and Seiffert, 1998). In addition to binding uPA on the cell surface, uPAR is also a high affinity (Kd< 30 nM) receptor for vitronectin (Wei et al., 1994). Vitronectin binding probably occurs through regions of domains 2 and 3 of uPAR that do not interact directly with uPA, and the Nterminal somatomedin B domain of vitronectin, which contains the PAI-1 binding site (Fig. 3A) (Deng et al., 1996a,b). However, intact uPAR is required for efficient vitronectin binding (Hsyer-Hansen et al., 1997b). Cells that express membrane-anchored uPAR, but not cells expressing soluble uPAR (suPAR),become strongly adhesive with altered morphology in the absence of uPA (Wei et al., 1994). However, vitronectin binding and cell adhesion are enhanced 8- to 10-fold by uPA or uPA peptides containing the uPAR-

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A

D

I

nPAR-VN bindlog: cell adbaian

I

B. T

Elfec PAI-I: det.chmen1 and inbibltlon of migration

E A

R uPA-VN blndine: Lnaepscd cell sdhaiion

I

Ex-

u P A ineread attachment and migration

F

Fig. 3 Effects of uPAR, uPA, PAI-1, and avp3integrin on cell attachment, spreading, and migration. VN stands for vitroneain. The two strands with globular ends represent the aVand p3 integrin chains. Soluble uPA andlor uPA-PAI-1 complexes are shown in D and E to represent conditions of excess PAI-1 or excess uPA (three moleculesof PAI-1 versus two of uPA in D; three molecules of uPA versus two of PAI-1 in E). The figures does not represent the actual relative dimensions of the different molecules and their domains.

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binding domain (Fig. 3B). Vascular smooth muscle cell (SMC) adhesion to vitronectin is stimulated dose-dependently by single-chain by uPA, by the amino-terminal fragment of uPA, or by two-chain uPA but not by low-molecular-weight uPA lacking the receptor-binding domain. This effect is not modified by aprotinin, a plasmin inhibitor, indicating a plasmin-independent process. Preincubation of single-chain uPA with soluble uPAR inhibits single-chain uPA stimulation of adhesion, as does pretreatment of SMCs with phosphatidylinositol-specific phospholipase C, which removes uPAR from the cell membrane. Vitronectin binding by its specific integrin receptors, avp3 or avps, is required for cell spreading and migration (Fig. 3C). However, antibodies to avp3or avpsintegrins, which inhibit vitronectin binding, do not abiolish single-chain uPA-mediated cell adhesion (Chang et al., 1998). Thus, uPAR possesses a dual function: matrix adhesion and regulation of proteinase activity. The observed increase in uPAR expression associated with a variety of malignancies may reflect not only the ability of uPAR to localize uPA on the tumor cell membrane, but also its role in modulating cell functions by promoting adhesion to the ECM. The dynamic balance between cell adhesion and detachment required for migration is modulated in part by PAI-1, which binds both to uPA and to the central region of the N-terminal, somatomedin B of vitronectin (Deng et al., 1996a,b). Migrating SMCs increase expression both of integrin receptors for vitronectin and of uPA. Vitronectin enhances cell migration, and the specific vitronectin receptor, avp3integrin, is required for cell motility (Fig. 3C). Active PAI-1 blocks cell migration through a mechanism independent of its ability to inhibit PAS, because the avp3binding site on vitronectin overlaps with the binding site for PAI-1 (Fig. 3D) (Deng et al., 1996a). Formation of the PAI-lwPA complex results in loss of PAI-1 affinity for vitronectin and restores cell adhesion and migration, showing that PAI-1 can control cellmatrix interactions by regulating the accessibility of specific cell-attachment sites (Fig. 3E). Therefore, cell spreading and migration on a vitronectin-rich ECM depend on the relative amounts of PAI-1 and uPA: excess uPA promotes migration by favoring uPAR-mediated binding and removing PAI-1 from the integrin binding site of vitronectin; excess PAI-1 inhibits integrin binding and blocks cell spreading on vitronectin (Fig. 3D,E). Thus, the localization of uPA at focal contact sites not only initiates a proteolytic cascade leading to ECM degradation, but is also required to expose cryptic cellattachment sites necessary for SMC migration (Stefansson and Lawrence, 1996).PAI-1 inhibits human amnion WISH cells and human epidermoid carcinoma HEp-2 cell migration on vitronectin- but not on fibronectin-coated surfaces. A PAI-1 mutant without ability to inhibit plasminogen activation has been shown to be as active as wild-type PAI-1 as a migration inhibitor, showing that this effect is independent of the inhibition of plasminogen activation. PAI- 1 specifically interferes with integrin- and vitronectin-mediat-

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ed migration: active PAI-1, but not latent or reactive center-cleaved PAI-1, inhibits vitronectin binding to integrins without affecting binding of fibronectin. Thus, PAI-1, independently of its role as a proteinase inhibitor, inhibits cell migration by -competing for vitronectin binding to integrins (Kjoller et al., 1997). Contrasting results have been reported on the inhibitory effect of PAI-1 on migration of melanoma cells. In these cells, adhesion to vitronectin, but not to type I collagen, is significantly enhanced by uPA or its amino-terminal fragment (ATF). Soluble uPAR (suPAR) inhibits this effect, indicating that uPA*uPAR also functions as a vitronectin receptor in melanoma cells. However, melanoma cell chemotaxis on a vitronectin matrix is inhibited by uPA and ATF, implying that cell motility decreases when uPA*uPAR acts as a vitaronectin receptor. In contrast, PAI-1 stimulates melanoma cell migration on vitronectin, presumably by inhibiting uPA*uPAR-mediatedcell adhesion and reversing the inhibitory effect of uPA on cell migration (Stahl and Mueller, 1997). Recent studies also indicate a role in cell adhesion and migration for the soluble form of uPAR. Cultured human vascular smooth muscle cells (HVSMC), human umbilical vein endothelial cells (HUVEC), and monocytic cells release suPAR into the culture medium, suPAR does not bind to HVSMC or HWEC, and it inhibits uPA binding to the cells. However, with cells depleted of membrane-bound uPAR by treatment with phosphatidylinositol-specificphospholipase C, or with a uPAR-deficientcell line (LM-TK-),suPAR increases uPA binding to the cell surface in a dose-dependent manner. This effect is specifically inhibited by vitronectin or by a monoclonal antibody to vitronectin, showing that vitronectin-mediated binding of uPA to cells is modulated by suPAR. The soluble uPAR*uPA complex may bind to cell surface-associated vitronectin and compete with cell-bound uPAR for uPA binding. On the basis of these findings it has been proposed that vitronectin can concentrate uPA*suPAR complexes at the cell surface and thus modulate the level of local PA activity required for tissue remodeling (Chavakis et al., 1998). An additional mechanism of regulation of the complex interactions between uPAR and vitronectin is provided by the internalization of the uPARwPA*PA inhibitor (PAI-1, PAI-2, PN-1) complex. On binding of PAI to uPAR-bound uPA, the trimolecular complex is internalized; the enzyme-inhibitor complex is degraded intracellularly, while uPAR is recycled to the cell membrane (Fig. 3F). Thus, the dynamic process of cell migration can be envisaged as a series of alternating attachment and detachment events regulated by uPAR*uPA and PAI-1 (Fig. 3). The binding of uPAR*uPA to vitronectin and the resulting increase in cell-substratum adhesion are inhibited by active PAI-1. Conversely, the formation of uPAR*uPA*PAI-1 complex restores cell adhesion and

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motility, a process facilitated by internalization of the uPAR*uPA complex. The subsequent recycling of ligand-free uPAR provides the cell surface with newly available binding sites for uPA and/or vitronectin and increases adhesion. Although the contribution of uPA to uPAR-vitronectin interaction is independent of its proteolytic activity, the ability of uPA to generate plasminwhich degrades vitronectin-also appears to have important implications for cell migration. Limited cleavage of vitronectin by plasmin abolishes cell adhesion and promotes detachment, an effect comparable to that of PAI1 (Waltz et al., 1997). Thus, although active uPA promotes cell adhesion through uPAR-vitronectin binding, it also mediates subsequent cell detachment through both plasmin and PAI-1 complex formation. This observation illustrates a significant example of the dual nature of uPA, a proteolytic enzyme that can regulate cell functions through both proteolysis-dependent and -independent mechanisms. The internalization of uPAR*uPA*PAIcomplex requires the presence on the cell membrane of the a,-macroglobulin receptor/low density lipoprotein receptor-related protein (a,MR/LRP), a transmembrane protein that mediates the endocytosis of diverse ligands (Lestaveland Fruchart, 1994). a,MR/LRP associates with and internalizes uPA-bound PAI-1 and can also internalize PAI-1 or uPAR (Conese et al., 1995; Herz et al., 1992; Nykjaer et al., 1992). uPAR-bound pro-uPA is not internalized and degraded unless it is activated to uPA and complexed with PAI-1. It has been proposed that uPAR-associated pro-uPA is protected against degradation via a,MR/LRP because its a,MR/LRP-binding site on the A chain is shielded by binding to uPAR; in contrast, the affinity of uPAR-uPA*PAI-l complex for a,MR/LRP is sufficiently high to allow rapid internalization and degradation (Nykjaer et al., 1994). a,MR/LRP-deficient murine embryonic fibroblasts produce higher uPA levels and migrate more rapidly than wild-type cells. This effect is much more relevant when the cells are grown in culture wells coated with serum or vitronectin or fibronectin, suggesting that the higher levels of uPA produced by the mutant cells may account for their enhanced migration. Therefore, uPA and uPAR may form an autocrine loop for promoting fibroblast migration, and LRP may counteract the activity of this system (Weaveret al., 1997). Because a,MR/LRP is a transmembrane protein, these results suggest that binding of the uPAR*uPA*PAI-1complex may also generate intracellular signaling. However, this hypothesis has not been proved. Small biochemical modifications of uPA can also affect uPAR-mediated cell adhesion and migration. Serine phosphorylation of human pro-uPA reduces its sensitivity to inhibition by PAI-1 without affecting its catalytic activity or affinity for uPAR. However, phosphorylation of serines 138 and 303 abolishes the proadhesive and chemotactic activity of uPA. These residues are not involved in modulating pro-uPA binding to vitronectin but are im-

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portant in the regulation of uPAR polarization in migrating human U937 monocytoid cells, indicating that pro-uPA phosphorylation on Ser138/303 can modulate the signal transducing ability of uPAR (Franc0 et al., 1997). Serine phosphorylation may, therefore, represent a mechanism by which cells can regulate extracellular signals for adhesion and migration. These findings show that uPA can provide a bridging function between the cell membrane and the ECM by potentiating the physical association of uPAR with vitronectin. The dynamic modulation of this trimolecular complex by PAI-1, which binds both uPA and vitronectin, has profound implications for cell migration. In spite of the variety of in vitro results that clearly demonstrate a role for components of the uPA-plasmin system in cell migration, it should be not* ed here that mice genetically deficient in either uPAR, uPA, plasminogen, or PAI-1, or combinations thereof, do not display major phenotypes related to impaired cell migration during normal development (Bugge et al., 1995a,b; Carmeliet etal., 1994). Although discrepancies between in vitro data and results obtained with knockout mice for a variety of genes are not uncommon, this observation prompts consideration of the redundancy of the extracellular proteolytic systems of mammalian cells and of the complex array of molecules that control such fundamental processes for development as cell migration. In this light, it does not appear inappropriate to hypothesize that the lack of one or several components of a proteolytic system can be complemented by other proteinases or other molecules. It should also be noted that in adult animals the lack of components of the uPA-plasmin system has a variety of effects on physiological and pathological processes involving cell migration and proliferation. Wound healing and ovulation efficiency are severely impaired in plasminogen knockout mice (Kao et al., 1998; Leonardsson et al., 1995; b m e r et al., 1996). Plasminogen- or uPA-deficient mice show reduced development of intimal hyperplasia following arterial injury (Carmeliet et al., 1997a,b). Conversely, plasminogen deficiency greatly accelerates the formation of atherosclerotic lesions in apolipoprotein E-deficient animals (Xiao et al., 1997). Plasminogen deficiency in host mice also impairs the growth and local invasion of Lewis lung carcinomas, although sufficient proteolytic activity is generated for tumor development and metastasis (Bugge et al., 1997). Similarly, deficient PAI-1 expression in host mice prevents tumor invasion and vascularization of transplanted malignant keratinocytes (Bajou et al., 1998). A recent, intriguing finding that uPAR-deficient cells enter a state of dormancy reminiscent of that observed in human cancer metastasis implicates uPAR as a potential modulator of tumor cell proliferation in vivo. Metastatic tumor cells transfected with antisense uPAR mRNA, which have uPAR levels 2- to 5-fold lower than and a proliferation rate in vitro indistinguish-

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able from parental cells, remain dormant for up to 5 months when inoculated in vivo. The mechanism responsible for their dormancy is decreased proliferation. Surprisingly, this phenotype is not permanent: although they maintain low uPAR levels, the antisense transfectants reemerge from dormancy to initiate progressive growth and form metastases. It has been proposed that other factors whose expression is dependent on prolonged in vivo growth can compensate for the lack of uPAR and confer on the cells the malignant properties induced by the receptor (Yu et ul., 1997). Thus far, effects of MMPs mediated by nonproteolytic interactions with the ECM have not been described. The hemopexin domain present in all MMPs except matrilysin mediates binding to the collagen triple helix (see Section II,B,l). However, this interaction appears to be transient in nature and finalized to substrate degradation (Murphy and Knauper, 1997). The fibronectin type II-like modules, or “collagen-binding” domain, of the gelatinases confer high affinity for type IV collagen and gelatins on these MMPs (Banyai et al., 1994). In addition, the collagen-binding domain of MMP-2 mediates specific binding to native collagen types I, V, and X, to elastin, and to heparin (Steffensen et al., 1998a, 1995). However, it has not been determined whether gelatinase interactions with collagenous components of the ECM mediate effects other than their degradation. In cultured cells both gelatinases are found associated with the cell surface but not with the ECM (Mazzieri et al., 1997). MMP-2 association with the cell surface occurs primarily through binding to MT-MMPs and aVp3 (see Section III,B). Two forms of MMP-9 with M, 92,000 and 82,000/85,000 can be labeled on the surface of human HT1080 fibrosarcoma cells and of phorbol ester-treated MCFlOA breast epithelial cells (Mazzieri et al., 1997; Toth et al., 1997). Although the Mr of the 82,000/85,000 form of MMP-9 is consistent with that of active gelatinase B, both 92-kDa and 82/85-kDA forms are recognized by antibody to the N-terminal “pro” domain, indicating that they are in a latent or only partially activated form (Toth et d., 1997). The MMP-9 binding site on the surface of MCFlOA cells has recently been identified as the 012 chain of collagen IV, a protein hydrolyzed by MMP-9 (Olson etal., 1998). Pro-MMP-90TIMP-1complex or MMP-9 alone bind a2(IV) with high a&ity (Kd = -22 nM)but do not associate with triple-helical collagen IV. Binding appears to occur through a site(s) other than the “collagen-binding” domain, as gelatinase A (MMP-2), which also possesses this domain, does not bind 012(IV)(Olson et al., 1998). The cell surface localization of MMP-9 is certainly important for targeting the proteolytic degradation of collagen IV and/or other substrates for MMP-9. However, the finding that the cell-associated forms of MMP-9 represent latent enzymes may indicate-by analogy to cell membrane-associated uPA-that their cell surface localization may mediate functions independent of their proteolytic activity.

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B. Protelnase Interactions with Transmembrane Proteins Evidence indicates that components of the uPA-plasmin system, as well as some MMPs, physically interact with a variety of transmembrane proteins. These interactions have important implications for the transduction of extracellular signals across the cell membrane. uPAR forms stable complexes with a variety of integrins. This both inhibits the adhesive function of the integrins and promotes uPAR-mediated cell adhesion to vitronectin (Wei et al., 1996). Studies show that uPA, uPAR, and PAI-1 regulate the adhesive properties of integrins, at least in part, through the generation of intracellular signals. These findings illustrate the intricate mnnections between proteolysis and adhesion that operate at the cell surface to regulate migration (Chapman, 1997). In human HT1080 fibrosarcoma cells uPAR associateswith members of the and p3 integrin families whose expression is induced by certain ECM molecules. Double-immunofluorescencelabeling showed that the staining patterns of uPAR and p1 integrins are strikingly similar when the cells adhere to fibronectin, laminin, or vitronectin but not when polylysine is used as an adhesion substrate. Resonance energy transfer (RET) between uPAR and p1 integrins was observed, especially at focal adhesion plaques, indicating that these molecules are within about 7 nm of each other on the membrane of HT1080 cells. uPAR and p3 integrin coclustering and RET were also observed on tumor cells adherent to vitronection- but not to fibronectin-, laminin-, or polylysine-coated surfaces. In addition, as colocalizes with uPAR on cells attached to fibronectin-coated surfaces; as and aV colocalize with uPAR on cells adherent to vitronectin; and a3and a6 associate with uPAR on cells attached to laminin. Thus, uPAR may laterally associate with integrins of tumor cells when attached to specific extracellular matrix elements to enable directional proteolysis for tumor cell migration and invasion (Xue et al., 1997). In leukocytes uPAR is found in close association with p2 integrins. The p2 integrin-dependent recruitment of leukocytes to the inflamed peritoneum of uPAR knockout mice is significantlylower than in wild-type animals. In vitro, p2 integrin-mediated adhesion of leukocytes to endothelium is lost on removal of uPAR from the leukocyte surface by phosphatidylinositol-specific phospholipase C. Leukocyte adhesion can be reconstituted by addition of soluble intact uPAR, but not uPAR lacking the uPA-binding domain. A monoclonal antibody to uPAR enhances adhesion of monocytic cells and neutrophils to endothelium; in contrast, addition of inactivated uPA reduces cellto-cell adhesion irrespective of the p2integrin-stimulating pathway. Thus, pZ. integrin-mediated leukocyte-endothelial cell interactions and recruitment to inflamed areas require the presence of uPAR. Ligand binding to uPAR modulates leukocyte adhesion to endothelium (May et al., 1998).

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Antibodies to avp3integrin completely inhibit cell adhesion on PAI-1, suggesting that this integrin may be the transmembrane molecule that physically connects uPAR*uPA*PAI-1complex to the cytoskeleton. Confocal microscopy analysis of filamentous actin in cells grown in PAI-l-coated dishes has shown a dose-dependent increase of filopodia and cytoskeletal reorganization, suggesting a migratory profile. Thus, PAI-1 plays a direct role in dynamic cell adhesion, particularly at the cell’s leading edge, where increased uPA and uPAR levels are localized in migrating cells. Immobilized PAI-1 could bridge the cell membrane with the ECM through the formation of a multimolecular complex that includes avp3integrin (Planus et al,. 1997). Carcinoma cell migration on vitronectin is mediated by avpsintegrin and rehuires upregulation of uPA and uPAR by growth factors or phorbol ester. Cell migration on vitronectin is blocked by either SUPARor an antibody that disrupts uPA binding to uPAR, or by a monoclonal antibody to avp5.PAI-2 blocks cell migration but does not affect adhesion, suggesting that in these cells only migration is mediated by uPAR-bound uPA. However, carcinoma cells that express avp3integrin can also migrate on vitronectin by a mechanism independent of growth factors or uPA and uPAR expression (Yebra et al., 1996). Integrin avp3is a marker of progression in malignant melanoma. The metastases-derived melanoma cell lines MeWo LNI 61 and MIM/8 LNI have markedly increased expression of a, mRNA transcripts and higher avp3relative to their parent cell lines. These cells also express elevated levels of uPAR mRNA and higher levels of surface-bound uPA. Blockade of avp3synthesis by transfection with a, antisense phosphorothioate oligonucleotides results in a marked decrease in cell adhesion to vitronectin and approximately 50% downregulation of uPAR mRNA levels. Vice versa, ligation of avp3integrin by immobilized antibody results in a 2-fold increase in uPAR mRNA, suggesting that in these metastatic melanoma cells uPAR expression is linked to the expression of the vitronectin receptor, avp3integrin (Nip et al., 1995). uPAR-integrin interactions also play important roles in modulating lymphocyte and neutrophil functions. Lymphocyte migration from the bloodstream into tissues involves integrin-mediated adhesion to vascular endothelium and migration across the endothelial and subendothelial barriers. Resting T lymphocytes do not express uPAR. However, coclustering of antigen receptor complex with PI or pz integrins results in rapid upregulation of uPAR mRNA and protein expression through signaling pathways involving both protein kinase C activation and increased intracellular cyclic AMP. Plasminogen activation and in vitro ECM invasion by uPAR-expressing T cells is blocked by anti-uPAR antibodies as efficiently as by plasmin or urokinase inhibitors. It is noteworthy that uPAR-expressing T cells can be detected in human tumor specimens by cytofluorimetric or immunohistochemical analysis, indicating that integrin-mediated expression of uPAR may play a

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role in T cell migration in vivo (Bianchi et al., 1996). In resting neutrophils, the leukocyte integrin aMP2(complement receptor type 3, CR3, or C D l l b / CD18) is physically associated with uPAR. uPAR-CR3 interactions are reversible: the two receptors dissociate during cell polarization and migration, and they reassociate when the cells return to a resting state (Kindzelskii et al., 1996). The interaction of uPAR with vitronectin and uPA also modulates fibrin degradation by macrophages through an integrin-mediated mechanism independent of plasmin. uPAR and the leukocyte integrin Mac-1 (CDllb/ CD 18) mediate complementary functions in myelomonocytic cells, as both receptors promote fibrin(ogen) degradation and bind vitronectin and fibrin, respectively. Fibrin and vitronectin colocalize at exudative sites in which macrophages bearing the two receptors accumulate. With monocytes, vitronectin attachment promotes subsequent Mac-1-mediated fibrin(ogen) degradation. In contrast, uPAR occupancy by exogenous uPA or by uPA peptides containing the uPAR-binding sequence inhibits Mac-1-mediated fibrinogen binding and degradation. These effects may be relevant during the tissue remodeling processes that occur in wound healing. Vitronectinenhanced fibrin(ogen)turnover by Mac-1 may promote clearance of the provisional matrix and subsequent healing (Simon et d., 1996). A physical association between uPAR and intracellular components may also be mediated by uPAR interactions that occur at specific sites of the cell membrane referred to as caveolae (Latin for “small cavities”). These are flask-shaped microinvaginations of the plasma membrane found on a variety of cell types, and whose biological role is unknown. Caveolin, a 22-kDa transmembrane phosphoprotein, initially identified in v-src-transformed cells, is a characteristic component of caveolae. Molecular cloning has identified three different caveolin genes (for reviews, see Anderson, 1998; Okamoto et al., 1998). On the surface of a number of cell types both uPAR and uPA colocalize in caveolae, as well as at focal contact sites and at cell-cell contacts. The colocalization of uPAR and uPA in caveolae enhances plasminogen activation, indicating that caveolae may promote efficient plasmin generation by clustering uPAR, uPA, and possibly other protease receptors in one’membranecompartment (Stahl and Mueller, 1995). Caveolin can be coprecipitated from cell extracts with uPAR-specific antibodies, suggesting a close spatial association between these two proteins. It has been proposed that caveolin oligomerizes with integrins within caveolae and forms clusters with GPI-anchored proteins (Stahl and Mueller, 1994). Caveolin is phosphorylated on tyrosine residues and forms complexes with a variety of intracellular signal transduction effectors, including G proteins, nonreceptor tyrosine kinases (c-yes), and ras. Thus, caveolin may represent a transmembrane “adaptor” through which uPAR and uPA can transduce extracellular signals across the plasma membrane.

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Recent data show an interesting interaction of uPAR with the cationindependent mannose 6-phosphate receptor/insulin-like growth factor-II (IGF-11) receptor (CIMPR). The two purified receptors interact with high affinity (in the low micromolar range) through domains 2 and 3 of uPAR and a site on CIMPR different from the mannose 6-phosphate- or the IGF11-bindingsites. The uPAR-CIMPR interaction is not perturbed by uPA. Immunofluorescence and electron microscopy studies show that CIMPR modulates the subcellular distribution of uPAR, and can direct this protein to lysosomes. These effects require the sorting signal of CIMPR (Nykjaer et al., 1998).It is noteworthy that both CIMPR and components of the uPA-plasmin system including uPAR have been implicated as physiological activators of latent transforming growth factor-pl (Dennis and Rifkin, 1991; Lyons et al., 1988; Odekon et al., 1994; Sat0 and Rifkin, 1989). The functional implication(s) of the physical association of CIMPR with uPAR in this process, as well as in the control of other cell functions, remains to be investigated. Interactions of uPAR with ECM and membrane proteins are summarized in Table 111. In spite of the multitude of data showing proteolysis-independent roles for components of the uPA-plasmin system, only a few studies have so far focused on similar functions for the MMPs or their inhibitors. However, recent data show very interesting interactions of gelatinase A (MMP-2) and TIMP-2 with several cell surface components. Proteolytically active MMP2 is found associated with aVp3integrin on the surface of invasive cells. MMP-2 and avp3colocalize on angiogenic blood vessels and melanoma cells Table 111 Cell Surface and Intracellular Proteins That Interact with Urokinase-Type Plasminogen Activator Receptor

Protein

Type Extracellular matrix proteins Integrins

Vitronectin pl, f32,83 integrins aVp3(coregulatedwith uPAR)

Transmembrane receptors

a,-Macroglobulin receptor (a,MMow density lipoprotein receptor-related protein (LRP) Complement receptor type 3 Cation-independentmannose 6-phosphate receptor/ insulin-like growth factor-I1 receptor Caveolin

OVPS

Transmembrane “adaptor proteins” Intracellular proteins (indirectinteractions)

Non-receptor tyrosine kinases p53/56’yn, ~ S 8 / 6 4 ~ & p59‘V , G protein subunits (through caveolin)

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in vivo and in vitro. In cultured melanoma cells expression of avp3mediates binding of MMP-2 in a proteolytically active form and accelerates cellmediated collagen degradation. The formation of an SDS-stable aVP3*MMP2 complex is mediated by the noncatalytic C-terminus of MMP-2, as a truncation mutant devoid of the hemopexin domain lacks the ability to bind avp3 (Brooks et al., 1996). The interaction of avp3 with MMP-2 has important implications for angiogenesis, a process that requires a fine modulation of both cell adhesion and extracellular proteolysis. MMP-2 and avp3integrin are functionally associated on the endothelial surface of forming blood vessels. A fragment of MMP-2 that comprises the C-terminal hemopexin domain, termed PEX, prevents binding of the gelatinase to avp3and bloiks cell surface-associated collagenolytic activity. PEX also blocks angiogenesis and tumor growth on the chick chorioallantoic membrane. Interestingly, a naturally occurring form of PEX can be detected in vivo in avp3-expressingtumors and during developmental vascularization of the retina. On the basis of these findings it has been proposed that in vascularized tissues PEX interaction with endothelial cell avp3modulates MMP-2 activity and the invasive behavior of new blood vessels (Brooks et al., 1998). These findings implicate a p integrin as an important mediator of proY 3 teinase-cytoskeleton interactions. It is noteworthy that the avP3binding domain of MMP-2 has sequence similarity to vitronectin, a specific ligand for this integrin. It is not known whether MMP-2 binding to avP3is required for activation or whether the gelatinase is bound by the integrin after it becomes activated. More important, it is unknown if the interaction of its Cterminal domain with the integrin prevents binding of TIMP-2, which would indicate that the integrin-bound MMP-2 is at least partly protected from inhibition by its specific tissue inhibitor. These molecular mechanisms have not been explored. It should also be noted that vitronectin is a ligand for both avp3and uPAR, and that MMP-2 binds both avp3and MT-MMP. This integrin may thus provide a bridging function between components of the PAplasmin system and MMPs. In addition, the sequence similarity between the C-terminal, hemopexin domain of MMPs and vitronectin suggests that uPAk may bind members of the MMP family as well as vitronectin. To our knowledge, this hypothesis has not been investigated. Although avintegrins have been implicated in many developmental processes including angiogenesis, a recent study of the phenotype of avintegrin-null (knockout) mice considerably reduces the importance of avintegrins in these processes. The deficiency of all five avintegrins, although causing lethality, allows considerable development and organogenesis including vasculogenesis and angiogenesis (Bader et al., 1998). Very recent data also show proteolysis-independent associations of MMP2 mediated by its fibronectin type 11-like module, or “collagen-binding” do-

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main. The collagen-binding domain of human MMP-2 mediates binding of this proteinase to collagen al(1)and a2(I)chains, and facilitates adhesion of fibroblasts to collagen. This effect is inhibited by a recombinant peptide corresponding to the collagen-binding domain of MMP-2 and by neutralizing antibodies to the p1 integrin chain but not to the p2 integrin subunit. This observation reveals the presence on the cell surface of an adhesion complex consisting of MMP-2, type I collagen, and integrins as a result of interactions involving the collagen-binding domain of MMP-2. MMP-2 binds pericelltllar collagen, which in turn is anchored to the cell membrane through alpl, a2Pl,and a& integrins. The integrin receptors and the collagen-binding domain of MMP-2 appear to recognize different binding sites on the collagen molecule, It is not known if the collagen-binding domain of MMP-2 can directly bind integrins, as this MMP possesses no RGD sequence. The collagen-binding domain also accelerates proMMP-2 activation, probably through an MT-MMP-independent mechanism whose details remain to be elucidated (Steffensen et al., 1998b). It is not known whether MMP-2 association with collagen and integrins on the cell surface can affect cell functions other than ECM degradation, as is the case for uPAR-uPA-vitronectin interaction. It is worth recalling that MMP-2 also binds MT-MMPs through TIMP-2, and that MT1-MMP can cleave interstitial collagens (Ohuchi et al., 1997). The collagen-binding domain of MMP-2 promotes cell adhesion and spreading (Steffensen et al,. 1998b). These observations are reminiscent of the dynamic balance between adhesion and migration regulated by uPA and PAI-1 interactions with uPAR and vitronectin, and by plasmin degradation of this ECM protein. MMP-2 has multiple interactions with the cell surface: with MT1-MMP*TIMP-2 complex and with avp3through the hemopexin domain, and with interstitial collagen through the fibronectin type 11-like module. These multiple associations illustrate a significant example of the variety of properties conferred on proteinases by their modular structure, and they strongly suggest additional roles for these enzymes. Thus, although no evidence has been provided for a proteolysis-independent role of MMP-2 or other MMPs in cell adhesion and migration, this possibility certainly deserves consideration. Gelatinase interactions with cell surface and ECM components are summarized in Table IV.

C. Extracellular Proteolysis-Independent Generation of Intracellular Signaling The finding of multiple, proteolysis-independent interactions of components of the extracellular proteolytic systems with a variety of transmembrane proteins provides support for the hypothesis that ECM-degrading

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Table Ill Gelatinase Interactions with Cell Surface and Extracellular Matrix Components Enzyme MMP-2

MMP-9

Interaction with MT1-h4MP.TIMP-2 complex aY& integrin Elastin and heparin Collagen al(1)and a2(I)chains; native collagen types I, V, and X (through the fibronectin type 11-like module, or “collagen-binding”domain; facilitates fibroblast adhesion to collagen) Indirect interactions with alp1,azpl,and a,P1 integrins through pericellular collagen a 2 chain of collageq IV (through sites other than the “collagen-binding” domain?)

proteinases can also generate intracellular signals through proteolysis-independent mechanisms. Evidence has shown a variety of intracellular effects generated by components of the uPA-plasmin system, and several signal transduction pathways are activated by the cell surface binding of uPA to uPAR. With human WISH epithelial cells that express high levels of uPAR, addition of exogenous, inactive pro-uPA results in increased cell migration and phosphorylation of serines in cytokeratins 8 and 18 (CK8, CK18). Binding of pro-uPA to uPAR elicits a time-dependent increase in CK8 phosphorylation, a change in cell morphology, and redistribution of cytokeratin filaments. These effects are mediated by the association of uPAR with an atypical isoform of protein kinase C (PKCJ. A similar pattern of phosphorylation effects is elicited by uPA in several normal epithelial cells or tumors but not in other cell types, suggesting that this uPAR-mediated mechanism of signal transduction may be unique to epithelia (Busso et al., 1994). The PKCEisoform has been implicated in activation of raf, which in turn activates the mitogen-activated protein (MAP) kinase cascade. In human MCF-7 mammary carcinoma cells, which are also of epithelial origin, treatment with pro-uPA or with the noncatalytic N-terminal fragment (ATF)of uPA that contains the uPAR-binding site induces phosphorylation of the MAP kinases ERKl and ERK2. ERK activation is maximal after 1 min of exposure of the cells to pro-uPA or ATF, and returns to baseline levels by 5 min. Pro-uPA- or ATF-treated MCF-7 cells also show increased motility, which is maintained for many hours, even after removal of the ligands from the cell surface. This effect is completely abolished by PD098059, a specific inhibitor of ERK, showing that uPA upregulates MCF-7 cell migration through MAP kinase activation (Nguyen et al., 1998).

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Recent evidence also implicates the localization of uPAR*uPA complexes at focal contacts with the activation of focal adhesion-associated proteins. Treatment of cultured endothelial cells with uPA results in tyrosine phosphorylation of focal adhesion kinase (FAK),of the focal adhesion-proteins paxillin and p130cas,and of p44/42 MAP kinase. uPA-induced activation of MAP kinase is blocked by removal of uPA from the cell surface or by treatment of the cells with the tyrosine kinase inhibitor genistein. Cytochalasin D abolishes FAK and MAP kinase activation, indicating a role for cytoskeletal organization in the cell response to uPA. However, MAP kinase activation in response to uPA treatment is not blocked by specific inhibitors of src tyrosine kinases, indicating that in endothelial cells MAP kinase can be activated by a pathway involving FAK and the cytoskeleton but not src kinase activation (Tang et al., 1998). A variety of nonreceptor tyrosine kinases are also found associated with uPAR. In human TLC-598 kidney epithelial cells, components of the JAK1/ STATl signal transduction pathway, including the transmembrane adapter gp130, are colocalized with uPAR in caveolae, and can be coimmunoprecipitated with uPAR. On formation of uPA*uPARcomplex, JAKl associates with uPAR. This effect results in subsequent STATl phosphorylation, dimerization, and binding to DNA, and in the eventual activation of gene expression. Downregulation of uPAR expression abolishes STATl activation by uPA but leaves STATl activation by interferon-? unaffected, showing that uPAR can activate the JAKl/STATl pathway through a specific mechanism independent of other membrane receptors (Koshelnick et al,. 1997). A similar signal transduction pathway appears to be utilized by vascular smooth muscle cells (VSMC). Binding of uPA to VSMC rapidly induces increased tyrosine phosphorylation of JAK1, Tyk2, and p59fyn, p53/56'Yn, p, of the src family of nonreceptor proteinp53/5Shck, and ~ 5 5 ~members tyrosine kinases involved in signaling from antigen or cytokine receptors. In response to uPA treatment of VSMC, STATl is rapidly phosphorylated, translocates to the nucleus, and binds to specific DNA regulatory elements. Migrating VSMC show colocalization of uPAR, JAK1, and Tyk2 to the leading edge, whereas other nonreceptor tyrosine kinases remain randomly distributed. Thus, in VSMC uPA can activate at least two signal transduction pathways: the JAKl /STAT1 cascade and src-like protein kinases. Although the former pathway is likely involved in regulating cell migration, the functional consequences of the activation of the latter pathway remain to be determined (Dumler et al., 1998). Tumor cells of nonepithelial origin can also activate src-type protein kinases in response to uPA. In human HT1080 fibrosarcoma cells uPAR is associated with fyn, hck, and lck. Treatment of the cells with catalytically inactive uPA generates activation of hck but has no effect on fyn or Ick phosphorylation. Both p38 and ERK2 appear to be downstream effectors of the

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HTlO8O cell response to uPA, which ultimately results in increased c-fos gene transcription. Activation of c-fos likely generates formation of the transcription factor activator protein-1 (AP-l), which induces expression of a variety of genes including PAL2 (Konakova et al., 1998) (see below). Thus, although a specific transmembrane adapter protein that transduces uPAR-generated signals across the plasma membrane has not been identified, several intracellular signal transduction pathways are activated through the proteolysis-independent interactions of uPAR with uPA and multiple cell surface proteins. As a variety of transmembrane molecules, including integrins, a,MR/LRP, CIMPR, and caveolin, can physically interact with uPAR, it is not surprising that a multitude of intracellular events can be triggered by uPA binding to uPAR. The enzyme-receptor interaction, which was inizially envisaged as a simple two-molecule process, actually involves a variety of cell surface components depending on the tissue microenvironment and on specific regions of the cell surface. The colocalization of uPAR and integrins within caveolae and the recent findings of physical association between caveolin and G protein subunits imply that the G protein signaling pathway may also be utilized to transduce signals generated by uPAR interaction with uPA and/or vitronectin. This hypothesis is also supported by the observation of complexes consisting of GPI-anchored proteins with heterodimeric G protein subunits in lymphocytes, and by the recent finding of increased levels of G protein subunits in uPA-treated human colon cancer cells (Wang et al., 1996). Other nonmitogenic signals in response to uPA interaction with uPAR have also been described in cultured endothelial cells and keratinocytes (Del Rosso et al., 1990, 1992; Fibbi et al., 1988, 1990). These include production of diacylglycerol, mobilization of intracellular calcium, and activation of glucose transporters (Anichini et al., 1994,1997; Del Rosso et al., 1993). Conversely, the involvement of CAMPand protein kinase A in transducing signals generated by uPA-uPAR interaction has not been described. The main intracellular effects mediated by uPAR are summarized in Table V.

Table V Intracellular Effects Mediated by uPAR Activation of signal transduction effectors (PKC, nonreceptor tyrosine kinases, ERKl and ERK2, JNSTAT, FAK) Phosphorylationof cytokeratins 8 and 18 Generation of diacylglycerol Induction of gene expression (c-fos, c-jun, c-myc, PAI-2, cathepsin B, gelatinase) Generation of mitogenic and nonmitogenic signals Maintenance of malignant phenotype (dormancy of metastasis in vivo)

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Cell functions may also be modulated by uPA through mechanisms independent of uPAR binding. In IF6 and M14 human melanoma cells, purified native uPA (35-60 nM),but not defucosylated ATF or recombinant uPA from Escherichia coli lacking posttranslational modifications, elicits a mitogenic response ranging from 25 to 40% of that induced by fetal calf serum. Blocking the proteolytic activity of uPA partially decreases its mitogenic activity, whereas plasmin inhibition is ineffective. Low-Mr uPA lacking the growth factor and the kringle domains is not mitogenic; in contrast, the amino-terminal domain (ATF)of uPA has a mitogenic activity similar to full-length uPA. Interestingly, a monoclonal antibody that blocks uPA interaction with uPAR does not inhibit the mitogenic effect of uPA. Thus, proteolytically inactive uPA can induce a mitogenic response in melanoma cells in vitro by a mechanism that involves its ATF domain but is independent of high-affinity binding to uPAR (Koopman et al., 1998). The finding that uPA lacking posttranslational modifications is devoid of mitogenic activity indicates that low-affinity interaction(s) of uPA with cell surface or ECM components-for example, heparin or heparan sulfate proteoglycans (Andrade-Gordon and Strickland, 1986)-might mediate the mitogenic effect and represent a novel mechanism by which uPA can generate intracellular signaling. Among the MMPs, the most suitable candidates for potential transducers of extracellular signals across the cell membrane appear to be the MTMMPs. The presence of a cytoplasmic tail in MT-MMPs strongly suggests that this domain may exert an intracellular function(s). Surprisingly, this hypothesis has not yet been tested, as most studies on this class of MMP have focused on the function(s) and mechanism of action of the extracellular domain. A recent report, however, clearly indicates a role for the transmembrane and cytoplasmic domains of MT1-MMP in determining the localization of this proteinase at specific cell regions involved in ECM invasion. Human RPMIl7951 cells that overexpress MT1-MMP activate soluble and cell-associated MMP-2, and invade an in vitro reconstituted ECM. In these cells MT1-MMP is localized at invadopodia (or podosomes), specialized cell surface structures that focalize ECM degradation by invasive cells (Chen, 1990,1996). When the cells are transfected with a mutant MT1-MMP lacking the transmembrane and cytoplasmic domain or with a chimeric protein consisting of the extracellular domain of MT1-MMP spliced to the interleukin-2 receptor transmembrane and cytoplasmic domains, these modified MT1-MMP molecules do not localize at invadopodia. Concomitantly, ECM degradation and invasion are greatly reduced, although the cells retain their ability to activate MMP-2. In contrast, a chimeric protein containing the transmembrane and cytoplasmic domains of MT1-MMP spliced to TIMP-1 is concentrated at invadopodia. These results show that the transmembrane and cytoplasmic domains of MT1-MMP regulate the cell membrane localization of this MMP (Nakahara et al., 1997).

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In addition to this study, several observations suggest the hypothesis that MT-MMPs may regulate cell functions through proteolysis-independent mechanisms. Their similarity to the uPAR-uPA system (outlined in Section II,C), the presence of a cytoplasmic domain, and their capacity to serve as a cell membrane binding site for MMP-2-TIMP-2 complex all support this hypothesis. It is noteworthy that TIMP-2, a ligand for MT1-MMP, has growth factor activity. Both TIMPs have erythroid-potentiating activity (EPA; see Section II,B,4). In addition, TIMP-2 but not TIMP-1 stimulates the proliferation of human HT1080 fibrosarcoma cells or of normal dermal fibroblasts (Hs68). This effect is completely blocked by inhibitors of adenylate cyclase. Treatment of the cells with TIMP-2 upregulates CAMPprpduction through activation of adenylate cyclase (Corcoran and Stetler-Stevenson, 1995). Inferestingly, TIMP-2 inhibits bFGF-induced endothelial cell proliferation (A. N. Murphy et al., 1993). The cell membrane receptor(s) that mediates the growth factor activity of TIMP-2 has not been identified. The finding that TIMP-2, but not TIMP-1, binds MT1-MMP through its N-terminal, inhibitory domain makes MT-MMP(s) the most likely candidate(s) for this role. However, the capacity of MT-MMPs to transduce extracellular signals across the cell membrane has not been investigated. It should also be remembered that MMP-2 has the capacity to bind both TIMP-2 and a&. This integrin binds vitronectin, a ligand for uPAR and PAI-1. Thus, vitronectin might also indirectly interact with MT1-MMP, and provide a physical link between components of the uPA-plasmin system and other members of the MMP family of proteinases and their inhibitors. In view of the involvement of vitronectin in cell adhesion and migration, it is likely that MT-MMPs may also participate in the control of these and possibly other cell functions.

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D. Proteinase-Mediated Control of Gene Expression The finding that uPAR interaction with uPA and vitronectin can activate mulFiple signal transduction pathways has prompted investigation of the potential effects of these interactions on gene expression. The initial observation that uPA treatment of tumor cells results in increased c-fos gene expression (Dumler et al., 1994; Konakova and Schleuning, 1998) generated the hypothesis that binding to uPAR may induce the activity of the transcription factor AP-1, which consists of a c-fos/c-jun heterodimer. Results have shown that uPA can activate signal transduction pathways that induce AP-1 binding to DNA (Dear et al., 1997). AP-1 binding sites are present in the promoter regions of a number of genes, including those of other proteinases and proteinase inhibitors. uPA binding to uPAR results in activation of the PAI-2 gene promoter-most likely mediated by AP-1 (Dear et al.,

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1997)-indicating a potential regulatory mechanism for plasminogen activation. In macrophages uPA interaction with uPAR results in increased expression of c-myc and c-jun, and subsequent modulation of cathepsin B and gelatinase expression (Rabbani et al., 1997). Thus, the signaling pathways activated by uPA binding to uPAR can modulate expression of a variety of genes, including those for other ECM-degrading proteinases.

IV. CONCLUSIONS AND PERSPECTIVES Since the initial observations of high levels of proteolytic activities associated with transformed cells, our understanding of the roles of extracellular proteinases in a variety of physiological and pathological processes, including tumor invasion, angiogenesis, and metastasis, has evolved considerably. Initially, few proteinases-mainly the plasminogen activators-were envisaged as effectors of the ECM degradation required for cell migration and invasion. By virtue of its broad substrate specificity and the relative abundance of its zymogen form in virtually all tissues, plasmin was considered as “the” proteinase that played the major role in this process. The identification of a variety of other proteinases with different substrate specificities-the MMPs, which interact with plasmin-introduced the concept of a cascade of proteolytic reactions required for cell invasion. The subsequent discovery of cell surface binding sites for some proteinases, the identification of families of tissue inhibitors of PAS and MMPs, and the recent, considerable expansion of the MMP family of proteinases substantially increased the complexity of the proteolytic reactions involved in ECM degradation. In the light of these findings, it has become apparent that the proteolytic mechanisms of cell invasion not only involve degradative enzymes but also include complex interactions with inhibitors and multiple binding sites on the cell surface. Two additional important features of the extracellular proteolytic systems prompt consideration: the redundancy of ECM-degrading enzymes and their respective inhibitors, and the modular structures of serine proteinases and MMPs. These observations suggest that, although some of these enzymes have similar proteolytic activities, their noncatalytic domains may mediate interactions with other molecules and functions other than ECM degradation. The findings discussed in this review have established several new concepts on the roles of the extracellular proteolytic systems. First, components of both the uPA-plasmin system and the MMP family associate with a variety of cell surface proteins. These interactions modulate cell functions including adhesion, migration, and proliferation through ,the activation of a number of intracellular signal transduction pathways. uPAR appears to play a major

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role in these reactions, due to its multiple associations with several cell surface proteins. The finding that uPAR is a high-affinity receptor for vitronectin, along with the profound implications of uPAR-uPA-vitronectin binding for cell adhesion and migration, has shed a totally new light on the role of cell surface-associated uPA. The recent identification of MT-MMPs has also provided evidence for a specific cell membrane binding site for some MMPs. The biological significance of the MT1-MMP*TIMP-2*MMP-2complex on the cell membrane is not fully understood. Although the formation of this complex appears to be required for pro-MMP-2 activation, by analogy to the uPAR*uPA*PAI-1complex, MT-MMPs may mediate proteolysisindependent roles similar to those of uPAR. This hypothesis is strengthened by the presence of a cytoplasmic tail in MT~MMPs,the function of which has thus far remained unexplored. A second, important concept arises from the numerous findings that have implicated integrins and other transmembrane proteins as potential mediators of intracellular signaling generated by the extracellular interaction of proteinases with multiple binding sites. Among these molecules, avp3integrin appears to be of particular importance as it mediates multiple interactions both with components of the uPA-plasmin system and with some MMPs. Vitronectin, the extracellular ligand of avp3integrin and uPAR, may represent the molecular scaffold that affords the assembly of a large comMTplex consisting of transmembrane or membrane-linked proteins (avp3, MMP, and uPAR), proteinases (uPA and MMP-2), and proteinase inhibitors (PAI-1 and TIMP-2). Multiple direct and indirect interactions occur among the components of this complex (Fig. 4). This has important implications for a variety of cell functions including regulation of extracellular proteolysis, cell adhesion, migration, and proliferation. A third, important aspect is illustrated by the findings of novel roles for proteinase inhibitors. Initially, these molecules were considered as negative regulators of cell invasion because of their inhibitory effect on ECM degradation. This view was in contrast with the observation that in a variety of tumors elevated levels of some inhibitors-PAL1 in particular-were predictors not of low invasiveness and metastatic potential but of high tumor aggressiveness. The recent characterization of the noncatalytic role of PAI-1 on cell migration can provide an explanation for this discrepancy, in the light of the dynamic balance between cell spreading and detachment required for cell locomotion. Other observations point to similar dual roles also for the TIMPs. Although numerous reports have shown the inhibitory effect of TIMP-1 and -2 on cell invasion, both inhibitors have growth factor activity. Interestingly, TIMP-2 stimulates the growth of tumor cells and inhibits growth factor-induced endothelial cell proliferation in vitro. These findings may have important implications in the use of this MMP inhibitor for cancer treatment. It is also important to note that both proteinases and in-

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Fig. 4 Vitronectin (VN), ayp3integrin, and uPAR constitute the scaffold of a multimolecular complex consisting of transmembrane or membrane-linked proteins ( a J 3 MT-MiW, and uPAR), proteinases (uPA and MMP-2), and proteinase inhibitors (PAI-1 and TIMP-2). Multiple direct and indirect interactions occur among the components of this complex. Vitronectin and avP3may afford a physical interaction between the MMPs and the PA-plasmin systems. The sizes of the different molecules and their domains are not depicted in their actual relative proportions in the illustration.

hibitors can generate intracellular signals. Although these molecules have opposing effects on ECM degradation, they may have similar growth factor effects on a variety of cells. Finally, it is noteworthy that proteinases can generate intracellular signals and regulate the expression of a variety of genes by interacting with multiple cell surface components and activating a number of intracellular signaling pathways. Unlike growth factors, which interact with specific cell membrane receptors, proteinases and inhibitors appear to transduce extracellular signals across the cell membrane through multiple mechanisms. These observations have important implications for the involvement of proteinases in tumor biology and clinical oncology. The presence of high levels of proteinases in tumors can no longer be simply associated with a high capacity of the tumor cells to degrade the ECM and thus to invade locally and/or to metastasize. Similarly, numerous studies have shown that the expression of high levels of some proteinase inhibitors by a variety of tumors correlates with high aggressiveness. Both proteinases and inhibitors can be involved in the regulation of tumor cell functions including proliferation, in addition to migration and invasion. Treatment of certain tumors with lowmolecular-weight inhibitors of serine- or metalloproteinases has shown a reduction in tumor size. This effect is currently believed to result from inhibi-

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tion of tumor angiogenesis, but it might reflect a direct inhibition of tumor cell functions. More importantly, the new concepts derived from the findings of proteolysis-independent roles of ECM-degrading proteinases should provide substantial indications for the development of antiproteolytic therapies aimed at blocking tumor invasion and metastasis. Antiproteolytic therapies have thus far been aimed at inhibiting the catalytic activity of proteinases, mainly by using molecules designed to specifically block their active site. In the light of our knowledge of the variety of nonenzymatic roles of proteinases, this approach can no longer be held sufficient, as it does not affect proteinase interactions with cell surface components. This may actually explain why antiproteolytic therapies have thus far been onlybartially successful. New therapeutic tools need to be designed to inhibit the interactions not only of proteinases but also of proteinase inhibitors with a variety of molecules in the ECM and on the cell surface. The multitude of molecular interactions occurring among the numerous components of the extracellular proteolytic systems and a variety of different cell surface components makes this task much more challenging than the design of molecules aimed at blocking the catalytic site of proteinases. The characterization of the nonproteolytic roles of proteinases is only in its initial stage, and certainly far from complete. A more detailed understanding of the interactions of proteinases and their inhibitors with cell surface components is required before we can exploit our knowledge of the biology and biochemistry of proteinases for clinical purposes. Further investigations may provide additional insights into the roles of proteinases in tumor metastasis and present novel therapeutic approaches.

ACKNOWLEDGMENTS Work was supported by grants from -.e National Institutes of Health to D. B. R. and from the U.S. Department of Defense and from the S. A. Localio Laboratory for General Surgery Research to P. M.

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Molecular Pathogenesis of AIDS4ssociated Kaposi’s Sarcoma: Growth and Apoptosis Kaoru Murakami-Mod,’ Shunsuke Mod, and Benjamin Bonavida Department of Microbiology and Immunology UCLA School of Medicine University of California, Los Angeles Los Angeles, California 9009.5

I. Introduction A. History of Kaposi’s Sarcoma B. Enigma of Kaposi’s Sarcoma 11. Histopathogenesis

A. Histological Stages B. Cell Surface Markers C. Cellular Ploidy III. Clinical Features A. Symptoms B. Immunological Status C. Spontaneous Regression and Rapid Progression D. Opportunistic Infections E. Use of Glucocorticoids F. Multiple Lesions: Metastasis or Proliferative Lesions? IV. In Vitro and in Vivo Models A. Kaposi’s Sarcoma Cell Cultures B. Development of Kaposi’s Sarcoma Lesions in Nude Mice C. Model for AIDS-Kaposi’s Sarcoma Pathogenesis V. Molecular Mechanisms of Kaposi’s Sarcoma Cell Growth A. Oncostatin M B. Soluble Interleukin-6 Receptor/Interleukin-6 Complex C. Glucocorticoids D. Tumor Necrosis Factor Q and Interleukin-lp E. Basic Fibroblast Growth Factor VI. Roles of Virus Infections in Kaposi’s Sarcoma Development VII. Apoptosis in Kaposi’s Sarcoma Cells A. Resistance of Kaposi’s Sarcoma Cells to Anticancer Drugs B. Expression of Antiapoptotic Molecules in Kaposi’s Sarcoma Cells C. Death Receptor-MediatedApoptosis D. Human Herpesvirus-8-EncodedAntiapoptotic Molecules

* Address correspondence to Dr. Kaoru Murakami-Mori, Department of Pathology I, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860,Japan. Advances in CANCER RESEARCH 0065-23OWOO$30.00

Copyright 0 2000 by Academic Press. All rights of reproducrion in any form reserved.

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VIII. Concluding Remarks and Therapeutic Implications References

1. INTRODUCTION

A. History of Kaposi’s Sarcoma Kaposi’s sarcoma (KS) is a multicentral, multiorgan tumor, with individual lesions characterized by proliferating spindle-shaped cells, ongoing angiogenesis, infiltration of inflammatory monoriuclear cells, extravasated erythrocytes, and edema. Historically, KS was first described by Moriz Kaposi as an idiopathic multiple pigmented sarcoma of skin (Kaposi, 1982). Before onset of the AIDS epidemic, the incidence of KS was rare and the disease occurred mainly in elderly white men of Mediterranean descent (classic KS) (Digiovanna and Safai, 1981). Typically, lesions appear on the lower extremities, and visceral involvement is uncommon. This form of KS generally runs an indolent benign course and seldom causes death. A more aggressive form of KS was noted to occur predominantly in young black men in sub-Saharan Africa (endemicKS) (Taylor et al., 1971). Unlike classic KS, this form of KS is variable from a benign, localized cutaneous lesion to a lethal, disseminated, rapidly progressive disease with visceral and lymph node involvement. In particular, lymphadenopathic KS is an aggressive form found in African children and young adults, and the prognosis is poor (Slavinet al., 1970). In addition, KS has occurred in transplant recipients and in patients on immunosuppressive therapy (iatrogenic KS), particularly in individuals treated with corticosteroids. These patients are 400 to 500 times more likely to develop KS than the control population (Penn, 1979; Hanvood et al., 1979). This form of KS has limited lesions in the skin and oral mucosa, but these lesions occasionally disseminate widely in the skin, lymph nodes, and internal organs. Interestingly, these lesions often regress with discontinuation of the immunosuppressive therapy. Since 1981, KS has become common in patients seropositive for human particularly in homosexual men (epidemicor immunodeficiency virus (HIV), AIDS-KS). A 20,000 times greater risk of developing KS has been estimated in case of HIV-infected patients, in comparison with the uninfected population (Friedman-Kien et al., 1982; Havercos et al., 1985; Beral et al., 1990). This form is a highly aggressive and lethal disease. Lesions are widespread in the skin and oral mucosa, and often involve lymph nodes and various internal organs, induding lung, liver, gastrointestinal tract, and spleen. Most cases have a rapid fulminant clinical course, and approximately 20% of patients succumb to complications of KS (Miles, 1994). Thus, during the

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1990s, KS has shifted from a rare, asymptomatic disease to a relatively common problem causing significant morbidity and mortality for AIDS patients.

B. Enigma of Kaposi’s Sarcoma A great deal of effort has been made to understand the pathogenesis of KS; however, it remains controversial whether KS is a reactive proliferative lesion or a true malignancy. The origin of tumor cells is also unclear. The higher prevalence of KS gmong AIDS patients suggests that H N infection may be an important contributor to development of this disease; however, there is at present no definitive answer regarding the exact role of HIV in the pathogenesis of KS. Epidemiological observations have implicated a possible infectious etiology in developing KS (Beral et al., 1990). Several DNA viruses, ipcluding cytomegalovirus (CMV), Epstein-Barr-like virus (EBV), human herpesvirus-6 (HHV-6), and human papillomavirus (HPV), have been suspected of causing KS; however, no clear evidence for the involvement of these viruses in the pathogenesis of KS has been established. DNA sequences of a new herpesvirus, termed human herpesvirus-8 (HHV-8), have been identified in all epidemiological forms of KS, at a high frequency (Chang et al., 1994). Current serological assays confirm a strong association of HHV-8 infection with the risk of KS development (Weiss et al., 1998),thereby suggesting that this virus may play an important, if not essential, role in KS development; however, the mechanism by which HHV-8 may drive KS is unknown. In this review, we address these issues and outline recent advances in the molecular pathogenesis of KS. Using cultured KS tumor cells, we have studied intracellular molecular events underlying the growth and apoptosis of these cells. These data are expected to provide new insights into the nature of KS tumor cells and ultimately lead to effective strategies for treating subjects with KS and for preventing the onset of this unusual tumor.

11. HISTOPATHOGENESIS A. Histological Stages Kaposi’s sarcoma lesions have complex histological features, ranging from early-stage lesions that are reminiscent of reactive granulomatous inflammatory foci to late-stage lesions that closely resemble angiosarcomas or fibrosarcomas. Lesions vary from patches to plaques to nodules that can occur at any site of the body (Tapper0 et al., 1993). In the early stage, KS le-

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sions consiHt of a collection of irregular, small-sized capillarylike structures in the dermis; these lesions are accompanied by an inflammatory infiltrate of mononuclear cells (patch stage). This stage is followed by an expansion of spindle-shaped cells which surround characteristic slitlike vascular spaces containing erythrocytes (plaque stage). These cells have a low mitotic index and are normal diploid. In late-stage KS lesions, aggregates of spindle-shaped cells become more predominant, and vascular spaces filled with erythrocytes are present between individual bundles and fascicles of spindle-shaped cells (nodule stage). In the late stages, spindle-shaped cells with a high mitotic rate and nuclear polymorphism may be prominent. Extravasated erythrocytes and hemosiderin-laden macrophages are common. Taken together, KS lesions have three histological features, namely; angiogenesis, inflammation, and proliferation. Despite a variety of clinical behavior and epidemiology, the histological features are indistinguishable in all epidemiological forms of KS, which suggests that a common mechanism may be involved in the pathogenesis of these different forms of KS.

B. Cell Surface Markers The spindle-shaped cell population is a predominant component of KS lesions; therefore, this cell is generally considered to be a tumor cell (KS cells). Examinations of cell surface antigens showed that most KS cells express endothelial cell markers, such as factor VIII and CD34, but some cells express characteristic antigens of smooth muscle cells, macrophages, or dermal dendritic cells (Rutgers et al., 1986; Nickoloff and Griffiths, 1989; Sturzl et al., 1992; Regezi et al., 1993; Kaaya et al., 1995). These findings suggest that the KS cell population of this lesion represents a heterogeneous mixture of endothelial cells, smooth muscle cells, macrophages, and dermal dendritic cells Such a mixed cellularity may support the idea that KS cells are not neoplastic, since neoplastic cells usually arise from one lineage. Some KS cells simultaneously express characteristic antigens of these different cell types, a finding which may suggest that KS cells are derived from pluripotential mesenchymal progenitor cells (Uccini et al., 1994).

C. Cellular Ploidy Flow cytometry analysis of cellular DNA content has shown that most KS lesions are normal diploid (Fukunaga and Silverberg, 1990; Kaaya et al., 1992; Eto et a1.,-1992). Similar data were obtained using cultured KS cells isolated from various lesions. Other evidence in favor of the hypothesis that

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KS represents a chronic reactive lesion is the absence of nuclear atypia. These findings support the idea that KS may be not a true neoplasm, but rather it may represent a proliferative and hyperplastic lesion.

111. CLINICAL FEATURES A. Symptoms Kaposi’s sarcoma has a variable clinical course, ranging from limited, asymptomatic cutaneous disease to a widespread, explosively progressive disease with visceral involvement and edema. For the majority of AIDS patients, however, this disease is ultimately progressive and sometimes lethal (Dezube, 1996). Skin lesions appear anywhere on the body, but they are often concentrated on the lower extremities, face, trunk, and genitalia. These lesions may often cause substantial pain and dysfunction, especially in edematous areas. Extracutaneous spread is common, involving most frequently the oral cavity, gastrointestinal tract, lung, and lymph nodes. Although oral and gastrointestinal involvement is often asymptomatic, it can cause pain, bleeding, ulceration, diarrhea, nausea, vomiting, and functional abnormalities. Pulmonary involvement is also quite common and may present as nonspecific respiratory symptoms, such as cough, chest pain, shortness of breath, and fever. Pleural effusion is associated with KS lesions on the visceral pleura, which sometimes cause bleeding into the pleural space.

B. Immunological Status Differences in clinical manifestation and prognosis among the four epidemiological forms of KS may be due to the immunological status of the patient. In HIV-infected patients with more profound immune dysfunction, KS tends to become a more aggressive, widespread, and rapidly progressive form that often affects internal organs (Dezube, 1996). Impaired immune responses have been noted in the poor-prognostic variants of endemic KS (Matondo and Zumla, 1996). KS occurs in immunosuppressed organ transplant recipients, and these lesions regress on discontinuation of the immunosuppressive therapy (Penn, 1979; Harwood et al., 1979). Therefore, it has been hypothesized that KS may arise as a consequence of immune dysregulation, and impairment of immune surveillance may promote the survival of tumor cells.

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C. Spontbneous Regression and Rapid Progression Kaposi’s sarcoma can somstimes regress spontaneously, even in severely immunocompromised patients (Real and Krown, 1985; Jhnier et al., 1985). Conversely, this lesion may be rapidly exacerbated with involvement of visceral organs and lymph nodes, even in patients with a relatively preserved immunological status (Miles, 1994). Thus, KS may represent a reactive proliferative lesion, which progresses or regresses depending on environmental factors such as the availability of persistent stimulation by various mitogenic factors (Costa and Rabson, 1983; Brooks, 1986). HIV infection could generate not only dysregulation of the immune surveillance system but also abnormal production of inflammatory cytokines with mitogenic ability for KS cell growth and maintenance (Levy and Ziegler, 1983).

D. Opportunistic Infections Opportunistic infections have been associated with a new development of KS and with exacerbation of the preexisting KS (Miles, 1994). Various cytokines, such as tumor necrosis factor a (TNFa) and interleukin-lp (ILlB), are generally recognized as potent mitogens of KS cells (Ensoli et al., 1992). Serum levels of these cytokines are initially increased following HIV infection and are further augmented by complications of infection, a finding that may explain the aggravated development of KS lesions in the context of opportunistic infections (Lepe-Zuniga et al., 1987; Lahdevirta et al., 1988).

E. Use of Glucocorticoids Numerous clinical observations revealed that glucocorticoid use is a possible risk factor in KS. Development of new KS as well as progression of preexisting KS have been noted during treatment of HIV-infected patients with glucocorticoids (Real et al., 1986; Schulhafer et al., 1987; Gill et al., 1989), and even with the use of immunosuppressive therapy with glucocorticoids for organ transplant recipients (Penn, 1979; Harwood et al., 1979). The induction of KS lesions is also frequent in patients on glucocorticoid therapy for the treatment of autoimmune diseases, lymphoproliferative disorders, and diseases unrelated to the immune system (Trattner et al., 1993a,b). It is of interest that K5 lesions often regress on reduction or discontinuation of this therapy.

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F. Multiple Lesions: Metastasis or Proliferative Lesions? Kaposi’s sarcoma often presents as multifocal, widely disseminating mucocutaneous lesions. The nature of these lesions has been debatable; do individual lesions spread from a primary single lesion (metastasis)or are they nonneoplastic benign proliferative lesions? Clonality is a fundamental characteristic of neoplastic lesions: Malignant tumors arise from clonal replication of a single cell, whereas reactive processes are derived from polyclonal proliferation. The inactivation pattern of X chromosomes is a useful marker for determiqation of clonality (Vogelstein et al., 1985). Using this marker,’Rabkin etal. (1995,1997) showed that KS is a disseminated monoclonal cancer. Conversely, Delabesse et al. (1997) reported a polyclonal inactivation pattern of X chromosomes from multiple skin lesions, suggesting that these individual lesions arose from different cellular origins. Gill et al. (1998) showed that more than half the number of patients develop multiple lesions with only a polyclonal inactivation pattern, while monoclonal lesions were observed in other cases. Interestingly, some patients develop both polyclona1 and monoclonal lesions. In addition, the individual monoclonal lesions in a given patient sometimes have different inactivation patterns. These data suggest that multiple KS lesions begin as polyclonal proliferative processes with angiogenic and inflammatory reactions, whereas some lesions may evolve to clonal outgrowth from independent cellular origins.

W. InVltro AND InVlvo MODELS A. Kaposi’s Sarcoma Cell Cultures The unusual features of KS suggest that this lesion is not a true neoplasm, at least early in the disease course, but rather it is a benign and reversible hyperplastic focus (Costa and Rabson, 1983; Brooks, 1986). Studies of the nature and pathogenesis of KS had been hampered mainly by the inability to maintain long-term cultures of KS cells in vitro. However, Nakamura et al. (1988)established long term culture of AIDS-KS-derivedcells, using conditioned medium (CM) from human T lymphotropic virus type-11 (HTLV11)-infectedCD4’ T cells. They found that the long-term culture of KS cells requires a growth-promoting factor(s) released from HTLV-11-infected cells. In the presence of this CM-derived factor, KS cells acquire a peculiar spindle-shaped morphology. Such growth factor dependency of KS cells may relate to clinical observations that KS lesions rapidly progress and sometimes

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regress spontaneously. These data also support the idea that retrovirustransformed or activated T cells are possible sources of KS cell growth factors in lesions. The vast majority of cultured KS cells were normal diploid, and these cells were able to giow only transiently after inoculation into nude mice (Salahuddin et al., 1988). Thus, the isolated KS cells were not immortal nor malignant. In contrast, only a few neoplastic clones have been isolated from both epidemic and iatrogenic forms of KS (Herndier et al., 1994; Lunardi-Iskandar et al., 1995; Popescu et al., 1996). These cells grew independently of endogenous growth factors, and produced tumors in nude mice, at the injection sites, as well as metastases in a variety of internal organs. In addition, these immortalized cells had chromosomal abnormalities. These data indicate that although the majoritjr (if not all) of KS cells in lesions are hyperplastic normal diploid cells, neoplastic cells are present in some patients. It is conceivable that some preexisting benign KS cells acquire malignant properties through genetic alterations, and in these cells neoplastic transformation does occur, the result being development of a true clonal malignancy.

B. Development of Kaposi’s Sarcoma

Lesions in Nude Mice When the cultured KS cells are subcutaneously inoculated into nude mice, a lesion of mouse origin resembling an early KS lesion develops at the site of injection within 5 days. In contrast, no reaction is observed in case of formalin-fixed cells. The KS-like lesions regress when the growth of inoculated KS cells terminates (Salahuddin et al., 1988). CM derived from KS cell cultures can also induce such a KS-like lesion, a finding which provides evidence that KS cells produce and release biological activities which contribute to the induction of angiogenesis and infiltration of inflammatory mononuclear cells. These data also favor the idea that the KS cell is a key element of the lesion that is inducible and reversible. Our group and others have found that KS cell-derived basic fibroblast growth factor (bFGF) and vascular endothelid ckll growth factor (VEGF)are major factors responsible for the proliferation of vascular endothelial cells in vitro and angiogenesis in vivo (Cornali et al., 1996; Nakamura et al., 1997; Samaniego et al., 1998). In addition to bFGF and VEGF, KS cells have been reported to produce various cytokines and growth factors, such as interleukins (IL-6, IL-lp, IL-8), leukemia inhibitory factor (LIF),granulocyte-monocyte colony stimulating factor (GMCSF), platelet-derived growth factor (PDGF), and transforming growth factor p (TGF-9) (Ensoli et al., 1989,1992; Corbeil et al., 1991).These factors may also play important roles in the development of KS lesions with the characteristic histological features.

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C. Model for AIDSeKaposi’s Sarcoma Pathogenesis A possible model for the pathogenesis of KS development has been proposed (Fig. 1).Under excessive stimulation or disturbance of the immune system, activated mononuclear cells (monocyte-macrophages and lymphocytes) begin to produce and release cytokines and growth factors that exhibit mitogenic activities for KS progenitor cells of mesenchymal origin, an event which probably triggers the activation and proliferation of the KS progenitors. In addition, these cytokines and factors further enhance the activation of mononuclear cells. The activated KS cells, in turn, acquire the characteristic spindle-shaped morphology and begin to produce and release a variety of rytokines and growth factors, which presumably promote angiogenesis, inflammatory cell infiltration, and proliferation of normal endothelial cells, smooth muscle cells, and fibroblasts. The KS cell-derived factors may also support the proliferation of KS cells per se. Such cellular responses could contribute to the development and maintenance of the typical histological features of KS. The infiltrated inflammatory cells further amplify the activation and proliferation of KS progenitors through cytokine production. Thus, the cytokine-mediated proliferative cascade is established. Persistent activation of KS cells may lead to malignant transformation in some cells.

Ks progenitors

Fig. I Possible model of AIDS-KS pathogenesis. The cytokine-mediated vicious circle may contribute to development and aggravation of KS. MNC, Mononuclear cell; EC, endothelial cell; SMC, smooth muscle cell; FB, fibroblast.

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V. MOLECULAR MECHANISMS OF KAPOSI’S SARCOMA CELL GROWTH A. Oncostatin M The CM from HTLV-11-infected T cells is required for the long-term growth of KS cells (Nakamura et al., 1988). Nair et al. (1992)identified this mitogenic activity as oncostatin M (OM). OM, a 30-kDa glycoprotein, was originally identified from activated human T cells, based on the potential to inhibit the growth of melanoma cells (Brown et al., 1987; Malik etal., 1989). Subsequently, this cytokine has been characterized as a regulator for growth and differentiation of normal and tumor cells. OM shares functional similarity and structural homology with LIF and IL-6, both members of the IL6 family (Rose and Bruce, 1991; Nishimoto etal., 1994; Zhang etal., 1994). However, we have found that KS cell growth is exclusively induced by OM and not by LIF or IL-6 (Murakami-Mori et al., 1995). It is generally accepted that the biological effects of IL-6 family members are mediated through multimeric receptor complexes involving a common signal transducing subunit (gp130)and respective cytokine-specific subunits (Fig. 2) (Kishimoto et af., 1992; Taga et al., 1992). Gearing et af. (1992) showed that OM and LIF can bind with high affinity to a LIF/OM receptor complex, which consists of a heterodimer of gp130 and a LIF receptor binding subunit (LIFR). Mosley et al. (1996) reported the existence of an OM-specific receptor complex that is a heterodimer of gp130 and an OM receptor-specific subunit (OMR). LIF cannot bind this receptor. Although gp130 functions as a high-affinity converting and signal-transducing subunit in a number of receptor complexes for the IL-6 family members, OM is apparently the only one that can bind specifically and directly to gp130, with low affinity (Gearing et al., 1992; Liu et al., 1992). The interaction of OM with gp130 alone is incapable of inducing intracellular signal-transducing events, and subsequent association of LIFR or OMR is required for the generation of a high-affinity signal-transducing complex (Mosley et al., 1996). Thus, OM utilizes two types of heterodimer complexes for binding and signal transduction (OMR/gpl30 and LIFR/gpl30). Such a dual receptor system may provide an explanation for the redundancy and specificity in biological responses mediated by OM and LIF, that is, some of the OM activities are shared with LIF while others are OM-specific (Thoma et al., 1994). Using Scatchard plot analysis, we obtained evidence for the existence of two affinity classes of OM binding sites on KS cells, with Kd values of 6-12 pM (high affinity) and 521-815 pM (low affinity) (Murakami-Mori et al., 1995). Conversely, these cells lacked a binding property for LIE Using com-

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Molecular Pathogenesis of Kaposi's Sarcoma LIF

OM

11-6

0

0

@Q

LIF/OM moptor (LIFWgpl30)

Expressionon KS cells

o M (OMWgpl30)

(+I

(-1

11-6 ~ receptor ~ (lL-8Wgp130)

~

(-1

Fig. 2 Binding specificity of the gp 130-related cytokine receptors. OM can bind to the OMspecific receptor (OMR/gp 130) and the LIF/OM receptor (LIFR/gpl30). Neither LIF nor IL6 is capable of binding the OM-specific receptor. AIDS-KS cells express the OM-specific receptor but lack the LIFlOM receptor and the IL-6 receptor.

petition binding assays, we showed that the OM-specific receptor (OMR/ gp130), but not the LIF/OM receptor (LIFR/gpl30), contributes to OM binding to KS cells. Anti-gpl30 antibodies abolished the OM binding as well as OM-induced growth stimulation of KS cells, thereby indicating that gp130 plays a key role in OM binding and signaling of the OM-specific receptor. PCR amplification clearly reveals the expression of mRNAs for gp130 and OMR in KS cells, while the transcript of LIFR is not observed (Fig. 3). Therefore, we conclude that the lack of a biological response of KS cells to LIF is due to absence of the LIF/OM receptor, and that the OM-specific receptor is responsible for the OM-induced mitogenic signal for KS cells (Fig. 4). Control

KS3

KSlOB

KS22

Flg. 3 Detection of transcripts for the gpl30-related receptor subunits in AIDS-KS cells. Two micrograms of total RNA of KS3, KSlOB, and KS22 cells were subjected to cDNA synthesis and PCR amplification. The amplificationproducts were electrophoresedon 1.5% agarose gels. The primers for gp130, OMR, LIFR, ILdR, and @-actindirected the amplification products corresponding to 960,743,791, 826, and 838 bp, respectively. Approximately 100 attomoles of the double-strandedcDNA fragments encoding the respectiveproducts were included as templates in PCR amplifications (positivecontrols).

~

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LIF OMWgpl3O gp130/gp130 (OM-specMc receptor)

1 Proliferation of AIDSKS

cells

IL-6

I

Fig. 4 Roles of the gpl30-related cytokines in AIDS-KS cell proliferation. OM and soluble IL-6 receptorlIL-6 complex transduce the growth-promoting signals in KS cells through the OM-specific receptor (OMR/gpl30) and the gp130 homodirner, respectively.

B. Soluble Interleukin-6 Receptor/

Interleukin-6 Complex Miles et al. (1990) reported that IL-6 is a growth factor for KS cells, as determined by the use of antisense oligonucleotides for IL-6. However, we have found that the mitogenic response of KS cells is not induced by IL-6 treatment. IL-6 exerts biological activity through a high-affinity IL-6 receptor complex consisting of an IL-6 receptor binding subunit (IL-6R) and a homodimer of gp130 (Fig. 2) (Taga et al., 1989; Hibi et al., 1990). IL-6 first binds to IL-6R with low affinity, an event which induces high-affinity binding to gp130 and its homodimerization. As shown in Fig. 3, KS cells express a large amount of gp130 mRNA, but IL-6R is not expressed in these cells. Accordingly, this phenot);pe can account for the lack of IL-6 responsiveness of KS cells. It has been reported that a soluble form of IL-6R (sIL-~R),when complexed with IL-6, induces high-affinity binding to gp130 and its homodimerization (Taga et al., 1989; Hibi et al., 1990; Murakami et al., 1993). We have found that sIL-~R,lacking transmembrane and cytoplasmic regions, functions as a potent growth factor for KS cells by making these cells responsive to IL-6 (Murakami-Mori et al., 1996). After exposure to sIL-6R together with IE-6 in culture, KS cells assumed a spindle-shaped morphology and showed a remarkable augmentation of growth. Even without the addition of IL-6, sIL-6R induced significant levels of growth. Since KS cells ex-

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press high levels of IL-6, it is likely that, in the presence of sIL-~R,these cells acquire an IL-6 growth loop (Fig. 4). In recent years, naturally produced sIL6R has been detected in sera from healthy individuals (Novick et al., 1989), and increased levels of sIL-6R as well as IL-6 have been noted in the sera of HIV-infected patients (Breen et al., 1990; Honda et al., 1990, 1992). The perturbed productions of sIL-6R and IL-6 may play crucial roles in the development of KS lesions by directly acting on the growth of KS cells. Antigp130 antibodies blocked the mitogenic effects of sIL-6R/IL-6 and OM on KS cells; therefore, we refer to sIL-6R/IL-6 and OM as gpl30-related KS cell growth factors (Murakami-Mori et al., 1995, 1996).

C. Glucocorticoids We have found that dexamethasone (Dex), in a synergistic manner, enhances OM- or sIL-6R/IL-6-inducedgrowth of KS cells, whereas Dex alone slightly augments the basal growth of these cells (Fig. 5 ) (Murakami-Mori et

4 MC Growth enhancement Qpl3O-Stat3 activation

OM

s’L-6w IL-6

Synergistic

(+I

11-18

TNFa

Additive

(-1

Fig. 5 Dexamethasone enhancement of the basal and factor-induced proliferation of AIDSKS cells. KS cells (3 X lo3 cells) in triplicate wells were incubated on 24-well plates for 6 days in medium (RPMI1640,10%FBS) alone (MC)or supplemented with OM (10 ng/ml), sIL-6R (50 ng/ml)/IL-6 (20 ng/ml), TNFa (10 ng/ml), or IL-lP (10 ng/ml), in the presence or absence of lO-’M Dex. Cell growth was determined using a Coulter particle counter. Values are means 2 SD of triplicate determinations of two independent experiments. Open and closed bars denote experimental results obtained in the absence and presence, respectively, of Dex.

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MC

DeX

OM

QM/Dex

Fig. 6 Morphological effects of OM and Dex on AIDS-KS cells. KS cells (3 X lo3 cells) were grown for 6 days in medium (RPMI1640,10% FBS) alone (MC) or with lo-’ M Dex, 10 ngl ml OM, or a combination of both in 24-well plates. Cells were fixed, stained with Giemsa, and 6bserved under a light microscope ( X 125).

al., 1997). KS cells have a polygonal shape in medium alone, and their exposure to sIL-6R/IL-6 or OM induces a characteristic spindle shape (Fig. 6). The induction of such a spindle-shaped morphology is observed with Dex alone, but is minimal. When the cells are stimulated by Dex given together with OM or sIL-6R/IL-6, spindle-shaped cells are more prevalent compared with cultures that were stimulated with Dex alone or with OM or sIL-6R/ IL-6 alone. In addition, anti-gpl30 antibodies or a glucocorticoid antagonist RU-486 abolished this synergistic effect. Dex has additive but not synergistic effects on the stimulation of KS cell growth with IL-1p or TNFa,the mitogenic signals of which are not mediated through gp130 (Fig. 5 ) . Thus, Dex has specific interactions with the gpl30-related growth factors. Guo et al. (1996) showed that the expression of glucocorticoid receptors is increased in KS cells in the presence of OM, TNFa,or IL-1p. This finding may partly explain the combined effects of Dex and these growth factors on KS cell growth. However, since we observed a selective synergy between Dex and sIL6R/IL-6 or OM in KS cell proliferation, it seems reasonable to assume that there is a more specific cross talk between the signaling pathways of the glucocorticoids and the gpl30-related growth factors. The gpl30-related growth factors activate a cytoplasmic transcription factor STAT3 (signal transduction and activation of transcription) through tyrosine phosphorylation. Once activated, STAT3 dimerizes, translocates into the nucleus, and binds to specific DNA elements, resulting in transcriptional activation of target genes (Zhong et al., 1994; Schindler and Darnell, 1995; Ihle, 1996). Immunoblot analysis revealed that sIL-6R/IL-6 or OM can induce rapid tyrosine phosphorylation of STAT3 in KS cells, and Dex significantly enhanced the accumulation of tyrosine-phosphorylated STAT3. Electrophoreticmobility shift assays showed sIL-6R/IL-6- or OM-induced DNA binding activity of STAT3 in KS cells, and Dex further increased this activity (Murakami-Mori et al., 1997). Thus, Dex appears to participate in

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the gpl30-STAT3 signaling and transcriptional events by enhancing STAT3 activation, thereby leading to the selective synergistic stimulation of KS cell growth with Dex and the gpl30-related growth factors (Fig. 7). It has been reported that Dex upregulates the expression of gp130 and IL-6R in a number of cells, a finding that may explain the frequently observed synergistic effects of IL-6 and Dex (Rose-John etal., 1990; Snyers etal., 1990; Schooltink et al., 1992; Mwi et d., 1998). However, regarding KS cells, this possibility can be ruled out, since Dex has little or no effects on these molecules in KS cells. Dex appears to participate in molecular events for STAT3 activation at postreceptor levels of the gpl30-STAT3 signaling pathway. Studies showed that the activated glucocorticoid receptor functions as a coactivator or a negative regulator for several transcription factors, through direct protein-protein interactions (Yang-Yen etal., 1990; Nishio et al., 1993). A similar mechanism may be involved in the enhancement of STAT3 activity with Dex and the gpl30-related factors. Evidence has accumulated that glucocorticoid secretion from the hypothalamic-pituitary-adrenal axis is increased during immune responses and inflammatory reactions (Navarra et al., 1990; Lyson and McCann, 1991). Indeed, elevated levels of serum glucocorticoids, in addition to increased amounts of serum sIL-6R and IL-6, have been noted in HN-infected patients (Christeff et al., 1988; Villette et al., 1990). These events could lead to a cyOM

slL-BwlL-6

4

4

OM

Fig. 7 Enhancement of the gpl30-mediated tyrosine phosphorylation of STAT3 and its DNA binding activity in Dex-treated AIDS-KS cells. Dex appears to participate in the gpl30-STAT3 signaling and transcriptional events by enhancing STAT3 activation, thereby leading to selective synergy in KS cell growth stimulation between Dex and OM or sIL-6R/IL-6.

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tokine-rich and glucocorticoid-rich environment that is favorable for the development of KS lesions. The synergy between the gpl30-related factors and glucocorticoids may explain, at least in part, the increased risk and high frequency of KS in HIV-infected patients. Furthermore, KS development has been noted with the use of glucocorticoids, both for transplant recipients and for treatment of subjects with autoimmune diseases (Penn, 1979; Harwood et al., 1979; Trattner et al., 1993a,b). As sIL-6R has been identified even in the sera of healthy individuals and KS cells produce large amounts of IL-6 (Novick et al., 1989), KS in association with glucocorticoid therapy may be due to direct effects of Dex. Dex augments the susceptibility of KS cells to sIL-6R/IL-6 complexes by enhancing STAT3 activity throygh tyrosine phosphorylation. Our findings, therefore, provide suggestive evidence that the gp130-STAT3 growth signaling pathway is involved in the pathogenesis of Dex-induced and Dex-aggravated KS.

D. Tumor Necrosis Factor a and Interleukinel f3 The cytokines TNFa and IL-1p are now recognized as key pathogenic mediators of infectious and inflammatory diseases (Aggarwal and Natarajan, 1996; Dinarello, 1996). They are mainly produced by activated macrophages and monocytes. HIV infection stimulates and dysregulates the immune system, leading to abnormal production of these cytokines in patients (Molina et al., 1989; Merrill et al., 1989). Opportunistic infections further augment the serum levels of these inflammatory cytokines (Lahdevirta et al., 1988; Lepe-Zuniga et al., 1987). It has been noted that TNFa functions as a growth stimulator for KS cells (Ensoli et al., 1992). The TNF ligand-receptor system is unique in that one ligand is able to interact with two cell surface receptors containing very different intracellular domains: one with a molecular mass of 55 kDa (termed TNFR-I) and another of 75 kDa (termed TNFR-11) (Tartaglia and Goeddel, 1992; Vandenabeele et al., 1995). The significance of the existence of two separate types of TNF receptors is unclear. Using neutralizing anti-TNFR-I and'anti-TNFR-I1 antibodies, we have found that the mitogenic activity of TNFa for KS cells is mediated exclusively through TNFR-I, not through TNFR-11, although PCR data revealed that both types of TNF receptors are expressed in KS cells (Murakami-Mori et al., 1999). Mitogen-activated protein kinases, p44MAPK and p42MAPK (also termed extracellular signal-regulated kinases, ERKl and ERK2, respectively), have been reported to play pivotal roles in transmitting and integrating extracellular signals required for the regulation of cell proliferation, differentiation, and apoptosis (Moriguchi et al., 1996). Several lines of evidence show that ERK1/2 activator kinases are MEK1/2 (MAPK/ERK activator ki-

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nases 1 and 2) that are located upstream in the ERK1/2 signaling pathway (Zheng and Guan, 1993).We found that ERK1/2 in KS cells are significantly activated by TNFa, through tyrosinekhreonine phosphorylation by MEK1/ 2. This activation was not observed in anti-TNFR-I antibody-treated KS cells, thereby indicating that TNFa activates ERK1/2 through KS cell surface TNFR-I (Murakami-Mori et d.,1999). A selective MEK1/2 inhibitor, PD98059, profoundly inhibited not only the activation of ERK1/2 but also the TNFol-induced KS cell proliferation. On the basis of these findings, we propose that TNFol elicits the mitogenic response of KS cells through the TNFR-I-MEK1/2-ERK1/2 signaling pathway (Fig. 8). Since TNFR-I lacks intrinsic kinase activities, TNFR-I-associated proteins may be important for ERK1/2 activation. Schievella et al. (1997) isolated a

TNFR-I

MADD

I

ifi

I

ActD I

f i i 4-J

x

holhntlon d AIDS-KS cells

Fig. 8 The TNFa signaling pathway in AIDS-KS cells. TNFa induces KS cell proliferation through direct activation of the TNFR-I-MEK1/2-ERK1/2 pathway. Involvement of TNFR-II in KS cell growth seems unlikely, although these cells express TNFR-I and TNFR-I1 at similar levels. Expression of the TNFR-I-associated protein MADD is selectively abolished in actinomycin D-treated KS cells, in which TNFa failed to induce the activation of ERKll2 activity. Thus, MADD may provide a link between TNFR-I and ERK112 in KS cells. Although TNFRI is depicted as a monomer, it is presumed to function in a multimeric form.

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novel deaih domain protein called MADD (MAPK-activatingdeath domain protein) that associates with TNFR-I through the death domain. They also showed that overexpression of MADD induced ERK2 activation in COS cells. We noted that actinomycin D (Act D) treatment of KS cells selectively abolished the expression of MADD. In contrast, Act D treatment did not affect the expression of other TNFR-I-associated proteins such as TRADD, TRAF2, RIP, and FADD. Further, we showed that TNFa failed to induce ERK1/2 activation in Act D-treated cells, suggesting that MADD provides a link between TNFR-I and ERK1/2 in KS cells (Murakami-Mori etal., 1999). Brown and Howe (1998) reported that MADD is highly homologous to a member of the GDP/GTP exchange protein family that plays an important role in the activation of G proteins such as RAS, CDC42, and RAC. Therefore, MADD may participate in the TNFR-I-ERK1/2 signaling pathway through the activation of G proteins (Fig. 8). The cytokine IL-lp is capable of stimulating KS cell growth in culture (Ensoli et al., 1992). Like TNFa, IL-1p has been reported to phosphorylate and activate ERK1/2 in various types of cells. Thus, ERK1/2 may also be involved in transmitting the IL-1p-induced mitogenic signal for KS cells.

E. Basic Fibroblast Growth Factor Kaposi’s sarcoma cells can prosper in response to a variety of external cytokines including OM, IL-6R/IL-6, TNFa, and IL-1p. Such a functional redundancy of the diverse cytokines may suggest that signaling pathways specific for the respective growth factors converge on the activation of a common intracellular molecule in KS cells. In addition to these external growth factors, the involvement of KS cell-derived growth factors has been implicated (Ensoli et al., 1989). KS cells produce and release several growth factoss, including bFGF, VEGF, TGF-P, and PDGF (Ensoli et al., 1992). Using antisense oligonucleotides, Ensoli et al. (1994a)reported that the KS cell-derived bFGF functions as an autocrine growth factor. We have found that blocking of the endogenous bFGF activity abolished the growth of KS cells by preventing entry into the S phase of the cell cycle, even in the presence of external growth factors (Murakami-Mori et al., 1998). For example, treatment of KS cells with anti-bFGF antibody completely inhibits both the basal growth and the OM-induced proliferation (Fig. 9). The synergistic effects achieved with Dex and OM were also abrogated by this antibody. Thus, the existence of a signaling loop of endogenous bFGF is essential for KS cell growth, regardless of the presence or absence of external growth factors (Fig. 10). Conversely, anti-VEGF, anti-PDGF, or anti-TGF-P antibody had no effect on the basal and cytokine-induced growth of KS cells (K. MurakamiMori and S. Mori, unpublished data, 1999).

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There is general agreement that as positive regulatory subunits of cyclindependent kinases (CDKs), G1 cyclins (cyclins D and E) are rate-limiting controllers of G1 progression or entry into S phase of the cell cycle of mammalian cells (Pines, 1993; Sherr, 1994; Draetta, 1994). Cyclin D and cyclin E bind to and activate CDK4 and CDK2, respectively. These cyclins control different events, as evidenced in experiments using microinjection of anti-cyclin D or anti-cyclin E antibody and overexpression of cyclin D or cyclin E molecules (Baldin et al., 1993; Ohtsubo et al., 1995; Resnitzky and Reed, 1995). We have found that block of the endogenous bFGF action profoundly inhibited cyclin E expression and CDK2 activity in KS cells, but not cyclin D expression and CDK4 activity (Murakami-Mori et al., 1998). The inhibition of cyclin E expression and CDK2 activity resulted in G1 growth arrest of KS cells, while cyclin D-CDK4 activity remained at a steady-state level. Therefore, the cyclin E-CDK2 activity apparently contributes to the Gl-to-

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Fig. 10 Dual control of AIDS-KScell growth and cyclin E-CDK2 activity by endogenous bFGF and external signals. After release from KS cells, bFGF supports the constitutive activation of FGFR-1 (flg) and FGFR-2 (bek), an event which leads to transduction of FGF-specific signals to intracellular machinery. In addition, the external growth factors markedly induce KS cell growth through the respective cell surface receptors. Both bFGF-specific action and external factor-dependentmitogenic signals are definitely required for cyclin E expression, CDK2 activity, and S phase entry in KS cells. These molecular events govern the cell cycle progression and the continuous proliferation of KS cells.

S phase transition of KS cell cycle, and this role is indispensable even in KS cells containing active forms of cyclin D-CDK4. It,has been reported that bFGF uses two cell surface receptors FGFRl (flg) and FGFR2 (bek)for signal transduction (Dionne et al., 1990). We have obtained evidence that FGFRl and FGFR2 were constitutively tyrosine-phosphorylated in KS cells, even in the absence of external mitogens, and that the addition of bFGF further enhanced this phosphorylation (Murakami-Mori et al., 1998). Exogenously added acidic FGF (aFGF),which generated a rapid tyrosine phosphorylation of FGFRl and FGFR2 in KS cells, reversed the inhibitory effects of anti-bFGF antibody on cyclin E expression and CDK2 activity. These findings support the idea that the FGF-specific action is exerted through the extracellular interactions of FGF with cell surface FGF receptors. Presumably, after release from KS cells, bFGF binds to and activates

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these receptors through tyrosine phosphorylation, leading to transduction of FGF-specific signals to intracellular machinery that could be crucial for cyclin E expression in KS cells. Thus, the endogenous bFGF signaling loop appears to induce the constitutive activation of FGF receptors, an event which contributes to cyclin E-CDK2 activity and S phase entry in KS cells. In addition, we have observed that the presence of external growth factors markedly induced cyclin E-CDK2 activity, S phase entrance, and KS cell proliferation. The external growth factors had little effect on expression and activation of FGFRl and FGFR2 in KS cells, therefore, it is unlikely that the proliferative effects of external growth factors are due to the simple augmentation of KS cell responsiveness to extracellular FGF. Further, we found that the addition of aFGF or bFGF alone was insufficient to induce the cyclin E expression and S phase entrance in KS cells. Thus, the FGF activity cannot compensate for the lack of external growth factors. All this evidence shows that integration of the activities of external growth factors and endogenous bFGF is required for full activation of cyclin E-CDK2 activity (Fig. 10). This dual control system may play a central role in the multiple factorinduced proliferation of KS cells.

VI. ROLES OF VIRUS INFECTIONS IN KAPOSI’S SARCOMA DEVELOPMENT Immunosuppression induced by HIV may relate to the risk of KS. For example, the impaired immunity in patients may support tumor cell survival and may promote replication of oncogenic viruses. However, KS is still 300 times more common in people with HIV infection than in other immunosuppressed groups (Beral et al., 1990), and AIDS-KS has a more aggressive nature than the other epidemiological forms of KS (Dezube, 1996). These findings suggest that HIV may play a more specific role(s) in the pathogenesis of KS. Ensoli et al. (1990) reported that Tat, an HIV-encoded protein, is actively released from HIV-infected T cells into the extracellular fluid, and that the addition of recombinant Tat promotes KS cell growth in culture. They also showed that bFGF and Tat synergistically induce KS-like lesions in nude mice (Ensoli et al., 1994b). These data may explain the high frequency and aggressiveness of AIDS-KS. A direct transforming involvement of HIV in KS development seems unlikely because DNA sequences of the virus have not been detected in these lesions. The etiologic role of additional sexually transmissible agents has been suggested by the following studies: the risk of KS is generally greater among those who acquired HIV by sexual contact than through mother-to-child transmission; KS occurs predominantly among homosexual and bisexual

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men who are sexually active; KS is rare in those acquiring HIV through transfusion or intravenous drug use; and women are more likely to have KS if their partners are bisexual men rather than intravenous drug users or transfusion recipients (Beral et ai., 1990). A new gamma herpesvirus recently identified from AIDS-KS biopsies, HHV-8, shares sequence similarity with oncogenic herpesviruses, such as EBV and herpesvirus saimiri (HVS) (Chang et al., 1994). HHV-8 has been suggested to function as a transforming virus in KS development, since this virus encodes several proteins that may have an oncogenic capability (Moore and Chang, 1998). For example, K1, K9 (viral interferon regulatory factor or vIRF), K12 (also known as kaposin), and ORF 74 (viral G-protein-coupled receptor or vGPCR) were reported to induce cellular transformation in rodent fibroblast cells and NIH3T3 cells as well as tumorigenesis in nude mice after inoculation (Gao et al., 1997; Bais et al., 1998; Lee et al., 1998; Muralidhar et al., 1998). Flore et al. (1998) have reported that HHV-8 infection caused long-term proliferation and survival of endothelial cell cultures in comparison with uninfected control cultures, although HHV-8 was present in only a minority of the cells (1-5% of the cells in each infected culture). The prolonged growth was dependent on the presence of high concentrations of VEGF, whereas its lower concentrations induced apoptosis in these cells. These authors suggest that the survival of uninfected cells (the majority of the cells) in the same culture is supported by upregulation of a VEGF receptor through a paracrine mechanism. HHV-8-positive cells acquired anchorage-independent growth, but they still required endothelial cell growth supplement. They did not show tumorigenesis in an animal model. Thus, it needs to be determined if the effects of HHV-8 infection on endothelial cells are due to neoplastic transformation or other growth-promoting mechanisms. Recent studies show that HHV-8 encodes a cyclin D homologue that is capable of stimulating CDK6 to phosphorylate pRB and histone H1 (GoddenKent et al., 1997; Li et al., 1997a). The finding that transcripts of viral cyclin are expressed in KS tissues may suggest an active role of viral cyclin for KS cell growth (Cesarman et al., 1996). This type of viral proteins may interfkre with cell cycle progression, ultimately leading to cellular transformation. We found that the anti-bFGF antibody-induced inhibition of cyclin E expression and cyclin E-CDK2 activity resulted in G1 growth arrest of KS cells, although cyclin D-CDK4 activity remained at a steady-state level (Murakami-Mori et d., 1998). Whether or not ectopic expression of viral cyclin can overcome the bFGF antibody-induced growth arrest of KS cells will be addressed in ongoing investigations. One of the most intriguing aspects of KS is the involvement of numerous cytokines in its pathogenesis. There is accumulating evidence that HIV in-

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fection dysregulates the immune system, leading to the perturbed production of various inflammatory cytokines in patients, at systemic and local levels (Lepe-Zuniga et al., 1987; Wright et al., 1988; Breen et al., 1990; Honda et al., 1990, 1992). Recent reports show that both circulating and tumor-infiltratingmononuclear cells are productively infected with HHV-8, suggesting that this virus may trigger inflammatory responses and abnormal production of several inflammatory cytokines (Decker et al., 1996; Blasig et al., 1997; Sirianni et al., 1998). It seems likely that these cytokines contribute to the stimulation of KS cell growth, the induction of angiogenesis, and the accumulation of inflammatory cells in lesions. Besides, the HHV-8 genome encodes an It-6-like protein and three chemokine homologues that share functional properties with cellular IL-6and chemokines, respectively (Moore et al., 1996; Nicholas et al., 1997; Molden et al., 1997). Such virus infections might lead to a cellular and viral cytokine-rich environment favorable for the development of KS lesions in patients, even though HHV-8 could not directly transform KS precursor cells. It has been reported that cultured KS cells do not contain HHV-8 sequences but do maintain KS cell features, responsiveness to KS cell growth factors, and the capability of inducing KS-like lesions in nude mice (Dictor et al., 1996). Further, all established transformed clones of KS are HHV-8 negative (Flamand et al., 1996). These findings suggest that HHV-8 is not likely to cause KS, as a transforming virus (Gallo, 1998). Thus, the discovery of HHV-8 has renewed interest in the nature of KS; is KS a cytokine-mediated proliferative disease or a virus-transformed malignancy in the clinical setting of loss of immune surveillance?

VII. APOPTOSIS IN KAPOSI’SSARCOMA CELLS A. Resistance of Kaposi’sSarcoma Cells

to Anticancer Drugs Cells are continuously exposed to a variety of stress signals such as virus infection and genotoxic agents. To eliminate virus-infected or transformed cells, the multicellular organism has developed a mechanism of programmed cell death or apoptosis. For example, cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, which serve as important effectors for immune surveillance, induce death in target cells through apoptosis (Arase et al., 1995; Kagi et al., 1994a,b; Mori et al., 1997; Rouvier et al., 1993). Anticancer drugs and radiotherapy also kill malignant cells by induction of apoptosis (Fisher, 1994; Kaufmann et al., 1993). Tumor cells, in turn, take a counter-

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measure(s) by developing antiapoptotic features. Indeed, many types of tumor cells are highly resistant to anticancer treatments by chemotherapeutic drugs and radiation. We haye examined the sensitivity of AIDS-KS cells to various anticancer drugs including cycloheximide (CHX), Act D, cisplatinum, paclitaxel, etoposide, and adriamycin (Fig. 11).Among them, Act D was the most effective drug to induce apoptosis in. KS cells: the concentration required to achieve 50% cell death was 30-100 ng/ml. In contrast, KS cells were relatively resistant to other drugs (CHX and adriamycin, 0.3-1 pg/ml; cisplatinum, 3-10 pg/ml; paclitaxel, >10 pg/ml). Etoposide had no cytotoxic effect on KS cells. To acquire such a drug resistance, KS cells may express antiapoptotic molecules. AIDS-KS cells are also highly resistant to the cytotoxic effects of NK cells (Reiter et al., "1992).Studies examining the niolecular basis of antiapoptotic mechanisms in KS cells are needed in order to develop effective strategies for treating KS patients.

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B. Expression of Antiapoptotic Molecules in Kaposi’s Sarcoma Cells Bcl-2 is an antiapoptotic molecule that confers survival advantage on various tumor cells (Adams and Cory, 1998; Green and Reed, 1998; Kroemer, 1997). Overexpression of Bcl-2 was noted in spindle-shaped cells in lesions of AIDS-KS and classic KS. The level of Bcl-2 expression was increased in advanced stages of KS, suggesting that Bcl-2 may play a critical role for the prolonged survival of KS tumor cells (Bohan Morris et al., 1996). Bcl-x, a Bcl-2-related gene, generates two different sizes of mRNA by alternative splicing. BcI-X, (a long form of Bcl-x) prevents apoptosis, whereas a short form of Bcl-x (Bcl-xs)promotes apoptosis (Adams and Cory, 1998; Boise et al., 1993,1995). Foreman et al. (1996)reported that the spindle-shaped cells isolated from AIDS-KS lesions express significantly higher levels of Bcl-x,, compared with Bcl-2. We observed a large amount of Bcl-x, expression in cultured AIDS-KS cells, while only a small amount of Bcl-2 expression was detected in these cells (Mori et al., 1996, 1999a). These findings favor the idea that overexpression of such antiapoptotic gene products may contribute to the prolonged survival of KS cells.

C. Death ReceptorcMediated Apoptosis 1 . DEATH RECEPTORS Death receptors, identified as apoptosis-inducing molecules, belong to the TNF receptor superfamily, which is defined by 2-6 repeats of a cysteine-rich extracellular domain (Ashkenazi and Dixit, 1998; Schulze-Osthoff et al., 1998). In addition, death receptors share a homologous cytoplasmic sequence, termed the “death domain,” which is essential for transduction of apoptotic signals. To date, five death receptors have been isolated, namely, TNFR-I, CD95 (Fas/Apo-1), DR3, DR4, and DR5 (Schall et al., 1990; Itoh etal., 1991; Chinnaiyan et al., 1996; Schneider et al., 1997; Pan et al., 1997a, 1997b). The death receptors are activated through cross-ligation by their cognate death ligands, an initial step that induces clustering of cytoplasmic death domains and recruitment of adaptor molecules and zymogen forms of initiator-caspases (pro-caspases). This assembly is called a death-inducing signaling complex (DISC).The DISC formation is required for activation of initiator-caspases, which leads to activation of downstream cascades of effector-caspases, and eventually causes cell death. AIDS-KS cells express significant amounts of TNFR-I (Murakami-Mori et al., 1999), Fas (Mori et al., 1996), DR4, and DR5 (Mori et al., 1999a); therefore, the respective ligands may induce apoptosis in these cells. TNFa, a specific ligand of TNFR-I, in-

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duced the growth stimulation of KS cells through ERK1/2 activation. The TNFR-I-associated death domain protein, MADD, seems to provide a biological connection between TNFR-I and ERK1/2 (Murakami-Mori et al., 1999). Conversely, we havebbtained evidence that in Act D-treated KS cells, the cross-ligation of Fas, DR4, or DR5 induced apoptosis rapidly and rigorously (Mori et al., 1996,1999a). Here, we will focus on the molecular mechanisms underlying Act D-induced sensitization of KS ceils to Fas-, DR4-, or DRS-mediated apoptosis in KS cells. 2. Fas-MEDIATED APOPTOSIS Accumulating evidence shows that most tumor cells have acquired a relative or absolute resistance to Fas-mediated apoptosis. Since the Fas system is important for tumor cell killing by CTL and NK cells, the resistance of tumor cells to Fas-mediated apoptosis may lead to their escape from immune surveillance in the host (Krammer et al., 1998). In addition, the Fas system may participate in the anticancer drug-induced apoptosis. Anticancer drugs upregulate the expressions of Fas and its natural ligand (Fas-L) on certain types of tumor cells, thereby leading to enhancement of interactions between Fas and Fas-L (Fluda et al., 1997; Friesen et al., 1996; Miiller et al., 1997). Min et al. (1996) showed a positive correlation between the Fas expression on acute myeloid leukemia cells and their cytotoxic responses to anticancer drugs. Thus, it seems reasonable to assume that Fas-mediated apoptosis is involved in the development and progression of certain tumors in patients. A number of studies have been done to examine the intracellular molecular events occurring in the Fas-mediated signaling pathway (Ashkenazi and Dixit, 1998; Scaffidi et al., 1998; Schulze-Osthoff et al., 1998) (Fig. 12). Binding of Fas-L to Fas rapidly induces oligomerization of Fas and clustering of the death domains, and then recruits the adaptor protein FADD and the pro-caspase-8 (DISC formation) (Kischkel et al., 1995). Subsequently, pro-caspase-8 is autoproteolytically activated, leading to activation of the effector-caspase cascades (Medema et al., 1997; Muzio et al., 1996). We showed that AIDS-KS cells are resistant to Fas-mediated apoptosis; however, dn agonistic anti-Fas monoclonal antibody (CH-11) induces cell death in more than 80% of Act D-treated KS cells (Mori etal., 1996).The single treatment of CH-11 or Act D alone had little effect on the viability of KS cells. We consider that caspase-8 activation is a critical step to induce the Fas-mediated apoptotic signals in KS, since (a) Fas-mediated caspase-8 activation was not observed in the Act D-untreated KS cell, and (b) the selective caspase-8 inhibitor completely inhibited the Fas-mediated apoptosis in Act Dtreated KS (Mori et al., 1999b). Actinomycin D may reduce the expression of an antiapoptotic molecule(s) that could interfere with the activation of caspase-8 in the Fas signalingpath-

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Fig. 12 Apoptotic signaling pathway induced by Fas. Fas-L induces oligomerization of Fas and triggers clustering of death domains, an initial event that recruits FADD and pro-caspase8 (DISC formation). Pro-caspase-8 is autoproteolytically activated in the DISC and then initiates downstream effector caspases. FAP-1 can block Fas-mediated apoptosis through the interaction with the suppressive domain of Fas. cFLIP interacts with FADD through the deatheffector domain and inhibits DISC formation and caspase-8 activation. Bcl-2 and Bcl-x, also inhibit the Fas-mediated apoptosis through an unknown mechanism(s). DD, Death domain; DED, death-effector domain; SD, suppressive domain.

way. Fas-associated phosphatase-1 (FAP-1) is reported to inhibit the Fasinduced apoptosis through its interaction with the carboxyl-terminal 15amino acid polypeptide of Fas, termed the “suppressive domain” (Itoh and Nagata, 1993; Sat0 et al., 1995). We found that Act D treatment downregulated the FAP-1 expression (Mori et al., 1996), thereby suggesting that the Act D-induced sensitization may be due to downregulation of FAP-1 expression in KS cells. A similar observation was noted in a colon cancer cell line (Yanagisawaetal., 1997).In addition, Bcl-x, expression was reduced in Act D-treated KS cells, whereas Act D had no effect on Bcl-2 expression (Mori et al., 1996, 1999a). As Bcl-2 and Bcl-x, have been suggested to interrupt the activation of caspase-8 through inhibition of DISC formation (Kawahara et al., 1998), Bcl-x, may also contribute to the resistance of KS cells to Fas-mediated apoptosis. Irmler et al. (1997)reported that the FLICE

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(caspase-8J-inhibitoryproteins (FLIPs) interrupt the interaction of FADD and pro-caspase-8 through their death-effector domains, and thereby inhibit the caspase-8 activation. The existence of FLIPs may provide a possible explanation for the fact that the majority of tumor cells are resistant to the Fasmediated apoptosis. However, it seems unlikely that FLIP contributes to Fas-mediated apoptosis, since we have found a high level of FLIP expression even in Act D-treated KS cells (Mori et al., 1999a).

3. APO-~L/TRAIL-MEDIATED APOPTOSIS Possible therapeutic applications of TNFa and Fas have been considered for the treatment of several malignant diseases; however, many studies have been limited because of the high incidence of severe side effects (Aggarwal and Natarajan, 1996). Unlike Fas-L and TNFa, Apo-2L/TRAIL, a specific ligand of DR4 and DRS, is widely expressed in normal tissues. In addition, the expression of DR4 and DRS is observed in most normal tissues (Chaudhary et al., 1997; Pan et al., 1997a,b; Schneider et al., 1997). These findings support the idea that Apo-2L/TRAIL is less toxic for normal cells, in comparison with Fas-L and TNFa. Indeed, Apo-2L/TRAIL had no cytotoxic effect on peripheral blood mononuclear cells and human endothelial cells (Sheridan et al., 1997). There may exist a mechanism(s) that protects normal cells from apoptosis induction by Apo-2L/TRAIL. It has been hypothesized that resistance of normal cells to Apo-2L/TRAIL may be due to the expression of decoy receptors, which bind Apo-2L/TRAIL and modulate the signal transduction required for induction of apoptosis (Degli-Espostiet al., 1997a,b; Pan et al., 1998; Sheridan et al., 1997). Although AIDS-KS cells showed relative or absolute resistance to Apo-2L/TRAIL, the combination of Act D and Apo-2L/TRAIL synergistically induced apoptosis in these cells (Mori et al., 1999a). Similar effects were observed with melanoma cells (G~ffithet al., 1998). Thus, the combined use of anticancer drugs and Apo2L/TRAIL appears to reduce the doses of anticancer drugs required for the induction of cytotoxic effects. Because AIDS-KS patients generally have a progressive immunodeficiency,the use of anticancer drugs is restricted to the minimum. In this regard, the combination therapy of Apo-2L/TRAIL and anticancer drugs may prove to be a safe and effective strategy to prevent untoward side effects. AIDS-KS cells are relatively resistant to chemotherapeutic drugs (Fig. 11).The combined effects of these anticancer drugs with Apo2LlTRAIL are currently under investigation. Apo-2LITRAIL binds to two death receptors, DR4 and DRS, and then triggers rapid apoptosis in sensitive cells. Although little is known of players that are involved in the intracellular signaling pathways mediated by DR4 and DRS, it has been accepted that apoptosis induction by Apo-2L/TRAIL requires caspase activities (Griffith et al., 1998; Mariani et al., 1997). Using

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specific caspase inhibitors, we have found that caspase-3-like activities and caspase-8-like (caspase-8 or -10) activities are both essential for the Apo-2L/ TRAIL-induced apoptosis in Act D-treated AIDS-KS cells (Mori et al., 1999a). Apo-2L/TRAIL-induced activation of caspase-8-likeactivities is not observed in Act D-untreated KS cells. Thus, the sensitizing effect of Act D may be directed toward molecular events required for activation of caspase8-like activities. Pan et al. (1997b) showed that Apo-2L/TRAIL preferentially activates caspase-10, whereas caspase-8 is strongly activated in Fasmediated apoptosis. Which type of caspase-8-like activities predominantly functions in the Apo-2L/TRAIL-mediated apoptosis in KS cells has yet to be determined. Further, we noted that a caspase-9 inhibitor, zLHED-fmk, attehuated the Apo-2L/TRAIL-induced apoptosis in KS cells, a finding that indicates that caspase-9 participates in the Apo-2L/TRAIL signaling pathway (S. Mori, K. Murakami-Mori, and B. Bonavida, unpublished data, 1999). Since it has been proposed that caspase-9 activation plays a pivotal role in the mitochondria-dependent apoptotic pathway (Li et al., 1997b), Apo-2L/TRAIL-gedicated signaling may be relayed to effector-caspasgs through the mitochondria-dependent pathway in KS cells. Bcl-x, is capable of blocking the mitochondria activation induced by apoptotic stimuli (Adams and Cory, 1998; Pan et al., 1998), and Act D treatment of KS cells profoundly reduced the expression of Bcl-x, (Mori et al., 1999a). Thus, the constitutive expression of this survival protein may be associated with the resistance of KS cells to Apo-2L/TRAIL-mediated apoptosis.

D. Human Herpesvirus-8-Encoded

Antlapoptotic Molecules Apoptosis induction in virus-infected cells is one of the most important antivirus responses. As counteracting mechanisms, many viruses have evolved genes encoding antiapoptotic molecules, which prevent the death of virus-infected cells until the viral replication cycle is complete. Prolonged survival of the infected cells is not only favorable for high levels of propagation but also may contribute to the persistent infection and oncogenesis of viruses. HHV8 encodes two peptides with sequence similarity to cellular antiapoptotic molecules Bcl-2 (Cheng et d., 1997; Sarid et d., 1997) and FLIP (Thome et al., 1997). Cellular Bcl-2 is known to inhibit Bax-mediated apoptosis by its heterodimerization with Bax, through a highly conserved domain termed the “BH3 domain” (Adams and Cory, 1998). It has been shown that viral Bcl-2 (vBcl-2)can overcome Bax-induced apoptosis in human fibroblasts, although vBcl-2 lacks the BH3 domain (Cheng et a/., 1997). vBcl-2 may inhibit apoptosis using a BH3-independent mechanism. We suggested that cellular Bcl-x, may play an antiapoptotic role against the death receptor-mediated apopto-

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sis in KS cells, although the Bcl-2 role is unclear in these cells (Mori et al., 1996,1999a). Whether ectopic expression of vBcl-2 may overcome the death receptor-mediated apoptosis in KS cells needs to be determined. A newly identified family of viral inhibitors, vFLIPs, was found to inhibit death receptor-mediated apoptosis (Thome et al., 1997). vFLIPs contain two death-effector domains that interact with FADD, resulting in the inhibition of recruitment and activation of caspase-8. We showed that AIDS-KS cells are resistant to Fas-mediated apoptosis, and Act D treatment confers Fas sensitivity on these cells (Mori et d.,1996). Since the KS cell cultures used in this study contain no HHV-8 DNA sequences, it is unlikely that vFLIF's function as antiapoptotic molecules in these cells.

VIII. CONCLUDING R E W K S AND THERAPEUTIC IMPLICATIONS Recent progress in tumor biology has provided answers to several central questions regarding the regulatory mechanism of growth and apoptosis of tumor cells. However, it remains to be established how these research efforts would result in improved survival of patients. Indeed, there is no decreased mortality among KS patients. We therefore emphasize the need of translational research, which applies advances in basic science to specific clinical problems. It is commonly accepted that the process of KS development involves multiple steps and complex interactions of numerous cytokines, growth factors, and hormones (Fig. 1).We identified several intracellular signaling events that are responsible for the control of KS cell proliferation, and definitions of their exact roles have provided an appropriate explanation for the unusual biological.behavior of this tumor. In addition, these components may prove to be potential targets for the molecular-based approach to KS treatment, since the KS cell is a regulatory element in the development, aggravation, and resolutionpf KS lesions (Fig. 13).For example, we showed that block of gpl30-mediated signaling by anti-gpl30 antibodies abolished the synergistic growthstimulating effects with Dex and gpl30-related factors. This synergism was also abrogated by the glucocorticoid receptor antagonist RU-486. Because of the high probability of synergistic action between Dex and gpl30-related factors in KS lesions, the block of either Dex or these cytokines may yield a greater anti-KS effect in patients than would be expected from the activity of either alone. Since we have obtained supportive evidence for the existence of specificcross tatk between Rex and the gpl30-STAT3 signaling pathway, further understanding of the molecular basis of their interactions is expected to lead to development of compounds that specifically inhibit this pathway.

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Fig. 13 Prospective anti-KS approaches. The process of KS development involves multiple steps and complex interactions of numerous cytokines, growth factors, and hormones. Better understanding of intracellular molecular events allows identification of new targets for therapeutic intervention in KS.

We showed that TNFa induces KS cell proliferation through direct activation of the TNFR-I-ERKlI2 pathway, and that treatment of KS cells with anti-TNFR-I antibody or the MEK1/2 inhibitor PD98059 profoundly inhibited proliferation of these cells. Such knowledge should also lead to a therapeutic approach for anti-KS treatment. One particular problem is the functional redundancy of the diverse external cytokines as KS cell growth factors, which may work in parallel at the lesions. This may increase a potential challenge to effective therapy. In addition to the external growth factors, the specific action of KS cell-derived bFGF is essential for KS cell proliferation. We obtained evidence of a correlation between KS cell growth and cyclin E-CDK2 activity, events tightly regulated through integration of the external mitogenic signal and the endogenous FGF signal. Block of either of these two signals can decrease cyclin E-CDK2 activity, and growth arrest follows. Thus, different mitogenic signaling pathways converge regarding activation of the common intracellular molecule. New treatments that interfere with such a common molecular event underlying proliferation of KS cells will provide the best opportunity to control this tumor. Existing chemotherapeutic treatments and radiation therapy for KS are only partially effective and often cause significant adverse effects; therefore, cure or long-term remission is unlikely with currently available treatments.

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We indicated that AIDS-KS cells express high numbers of antiapoptotic molecules including BcI-xL, FAP-l, and FLIPS, molecules which may contribute to the resistance of KS cells to various apoptotic signals such as chemotherapeutic drugs, radiation theiapy, and immune surveillance mechanisms. In addition, KS cells are highly resistant to the apoptosis induced by Apo-2L1 TRAIL or CH-11; however, these molecules induced rapid and violent cell death in Act D-treated KS cells (Fig. 13).Since Act D treatment significantly downregulated the expression of Bcl-x, and FAP-1 in KS cells, these antiapoptotic molecules may determine the therapeutic responsiveness of KS cells through modulation of intracellular death signaling. These findings should lead to identification of new targets for therapeutic intervention for KS. We noted that the combined use of Apo-2L/TRAIL and Act D can reduce the amount of Act D required to induce effective cytotoxic effects on KS cells. Although selection of chemotherapeutic drugs depends on their effectiveness, the clinical state of the patient, and the related toxicity, singleagent therapy is not effective. The combined therapy of Apo-2L/TRAJL and ' c anticancer drugs is worthy of consideration. Epidemiological studies have suggested that all forms of KS are associated with HHV-8 infection. HIV infection also contributes to the development and maintenance of KS lesions. Persistent production of KS cell growth factors by virus-infected cells may lead to the cytokine-rich environment in favor of KS development and disease aggravation, and impaired immunity with increasing HIV replication may support the survival of KS tumor cells. Control of viral replication and maintenance of the low viral load are critical for long-term management of KS patients.

ACKNOWLEDGMENTS We acknowledge the contributions of our collaborators, Dr. Avi Ashkenazi, Department of Molecular Oncology, Genentech, Inc., Dr. Tadamitsu Kishimoto, Osaka University, Dr. Tetsuya Taga, Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, and Dr. Shuji Nakamura, Huntington Memorial Hospital. For critical readings of our manuscripts, we thank M. Ohara.

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Perspectives on Cancer Chemoprevention Research and Drug Development Gary 1. Kelloff Chemoprevention Branch National Cancer Institute National Institutes of Health Bethesda, Maryland 20892

I. Introduction 11. Nature of Carcinogenesis A. Molecular Carcinogenesis B. Cellular Carcinogenesis C. Tissue Carcinogenesis D. Clinical Carcinogenesis III. Definition of Chemoprevention and Chemoprevention Agent Discovery A. Molecular Targets in Agent Discovery B. Mutagenesis and Mitogenesis: Empirical Targets for Chemoprevention C. Principles of Chemopreventive Agent Discovery-Mechanistic Approaches D. Representative Classes of Chemopreventive Agents E. Evaluating Chemopreventive Efficacy IV. Chemopreventive Agent Development A. Preclinical Efficacy Development Using a Balance of Molecular Target and Empirically Based Assays B. Toxicology and Pharmacology C. Importance of Intermediate Biomarkers of Carcinogenesis and Their Measurement as Surrogate End Points for Chemoprevention Studies D. Clinical Efficacy-Phase II Clinical Chemoprevention Studies V. Cancer Chemoprevention at Major Cancer Target Sites A. Prostate B. Breast C. Colon D. Lung E. Head and Neck F. Bladder G. Esophagus H. Uterine Cervix I. Skin J. Liver K. Multiple Myeloma L. Clinical Benefit in Addition to Cancer Incidence Reduction VI. Surrogate End Points in Defining Chemopreventive Efficacy-Importance of Evaluating Both Phenotypic and Genotypic Effects

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VII. Major Issues and Challenges for Cancer Chemoprevention References

I. INTRODUCTION Since the late 1960s there have been significant advances in cancer treatment and early detection. Particularly noteworthy are the decreases in mortality from childhood leukemia and increased screening for breast, colon, and prostate cancer, resulting in detection of smaller, less advanced lesions and resulting in improved treatment and, in some cases, improved outcomes. Nonetheless, during this same period overall iancer incidence is increasing, morbidity associated with surgery, radiation, and chemotherapy is still considerable, and, disappointingly, overall cancer survival has remained relatively flat (Sporn, 1996; Landis etal., 1998).During this time, however, there has been an enormous gain in our understanding of carcinogenesis, due in large part to the technology allowing exploration of molecular pathways, cancer-associatedgenes, and tissue architecture. This knowledge, in turn, has turned the focus of cancer therapies to drugs that take advantage of cellular control mechanisms to selectively eradicate cancer cells. Most importantly, knowledge of carcinogenesis has provided new and promising opportunities to prevent cancer-that is, to treat precancer or inhibit carcinogenesis (a process often involving 20-30 years in human epithelial cancers) rather than waiting to treat the cancer. Sporn (1976)coined the term chemoprevention more than 20 years ago to describe this new discipline in oncology, which is the use of drugs, biologics, or nutrients to inhibit, delay, or reverse carcinogenesis and which can be applied at any time in the process before invasive disease. Since that time, remarkable progress has been made in developing chemoprevention strategies, started by Sporn’s (e.g., 1976) and Wattenberg’s (e.g., 1978,1985) research on mechanisms of chemopreventive drugs and assays for evaluating these drugs in animal models, and spearheaded in the clinic by Hong’s early studies on prevention of head and neck carcinogenesis (Hong et al., 1986,1990). This review is intended as an overview of the progress in the field and approaches for continued successful strategies in chemoprevention. The first section describes the nature of carcinogenesis from the perspectives of the advances that have been made in the molecular, cellular, tissue, and clinical/ epidemiological sciences that are relevant to chemoprevention and that serve as a background for describing the basic and applied research needed to develop chemopreventive agents. The next section defines chemoprevention and discusses principles of molecular target-based chemopreventive drug discovery and early development of five prototypical agent classes that have already shown significant chemo-

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preventive activity, and reviews the rationale for their development. The classes include signal transduction modulators, using growth factor receptor inhibitors [epidermal growth factor receptor (EGFR)], oncogene inhibitors (Ras farnesylation inhibitors), and retinoids as examples. Steroidal hormones are strongly implicated in breast, ovary, prostate, and possibly other cancers. Antiestrogens and aromatase inhibitors are presented as examples of highly promising chemopreventive agents at these targets. Inflammation and oxidative damage are associated with carcinogenesis in most epithelial tissues, particularly colon, bladder, esophagus, head and neck, and lung. Agents with several different antiinflammatory mechanisms have shown chemopreventive activity-uonsteroidal antiinflammatory drugs (NSAIDs)which inhibit cyclooxygenase (COX-1 and COX-2); selective COX-2 inhibitors, which retain NSAID antiinflammatory activity with less toxicity than drugs that inhibit both COX-1 and COX-2; inducible nitric oxide synthase (iNOS) inhibitors; ,and lipoxygenase (LOX) inhibitors. Many antioxidants are dietary products, which could be suitable for use in the general population and which appear to have preventive potential in many diseases of aging besides cancer (particularly, cardiovascular disease, arthritis, and Alzheimer’s disease). Antimutagens have significant potential, particularly in tissues like lung and colon where there are high levels of carcinogen exposure. Inducers of enzymes involved in carcinogen detoxification [e.g., glutathione (GSH) Stransferase (GST) and NAD(P)H:quinone reductase] are described as examples of promising antimutagens. Tea polyphenols and the combination of selenium with vitamin E, which have received much attention recently, are described. This section concludes by moving from the discussion of the specific classes to the generality of molecular target- and empirical-basedscreening assays useful for discovery and early development of candidate chemopreventive agents. In the next section, a sequential drug development program is described, which is translational in nature and builds on the rational mechanism-based and empirical agent discovery approach described, acknowledginghow drug development proceeds in current practice and recapping consensus reached by the National Cancer Institute (NCI) and U.S.Food and Drug Administration (FDA) on appropriate drug development data and procedures that will lead to drug approvals for this indication. An important aspect of this program is identification and evaluation of intermediate biomarkers as surrogate end points for cancer incidence, and the rationale and methods for their development are described. The final portions of the body of the manuscript look at the need for, the progress of, and the promise of chemoprevention at major cancer targets and describe a strategy for effectively using surrogate end points in defining chemopreventive efficacy. This review concludes with a discussion of the major issues and challenges of chemoprevention drug development, and

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the tremendous opportunity it promises for reduction of the human cancer problem.

11. NATURE OF CARCINOGENESIS The understanding that carcinogenesis is progressive disorganization observed at the molecular, cellular, and tissue levels is fundamental to developing chemoprevention strategies. During carcinogenesis,molecular targets become less available, normal cell function and structure are disrupted, and aneusomy increases. Clinical cancer, the end of this chaotic process, is chargcterized by unregulated proliferation, cellular heterogeneity, and, consequently very few sites amenable to therapeutic intervention. Rapid advances in genomics, molecular and cellular biology, and tissue pathology are providing the tools for identifying and evaluating carcinogenesis. Particularly, these research findings allow characterization of early stages of carcinogenesis where cell function is still sufficiently intact and cells are sufficiently homogeneous that targets are available for cancer preventive interventions. Some of the aspects of molecular and cellular, tissue, and clinical carcinogenesis critical to prevention are described in the following.

A. Molecular Cardnogenesis Molecular events in carcinogenesis have been reviewed extensively in the literature (e.g., Wattenberg, 1985; De Flora and Ramel, 1988; Kelloff et al., 1995a; Lippmann et al., 1998) and include formation and activation of carcinogens, induction of genetic damage, and disruption of normal cell growth and differentiation regulators. Basic research in carcinogenesis has identified many genetic lesions (including many of the described oncogenes and tumor suppressors), enzymes, and other cellular constituents associated with the initiation and progression of precancers to invasive disease. It is evident that many of these activities are interrelated; for example, effects on ornithine decarboxylase (ODC), arachidonic acid (AA) metabolism, protein kinase C (PKC),insulin growth factor (1GF)-I,and transforming growth factor (TGF)Pmay be pleiotropic results of activity at single loci on signal transduction pathways. It is also clear that a single activity may not be the most important or the only one required for carcinogenesis. Significant advances have been made recently in understanding of the biochemical contrd mechanisms involved in regulating cell growth and development. Cells respond to signals from extracellular stimuli via a complicated network of highly regulated events collectively referred to as signal

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transduction pathways. Stimulation of these pathways results in changes in transcriptional activity (reviewed in Powis, 1994; Powis and Workman, 1994). While normal cells respond appropriately to extracellular stimuli, many precancerous and cancerous cells have lost this ability and display aberrant signaling (reviewed in Kelloff et al., 1995a). Numerous targets interfering with deregulated signaling pathways are potential sites for chemopreventive intervention. Key components of these pathways are the protein tyrosine kinases, which catalyze the transfer of the y-phosphate of ATP to the hydroxyl group of tyrosine on numerous proteins (Chang and Geahlen, 1992). Loss of tyrosine kinase regulatory mechanisms has been implicated in neoplastic growth; indeed, many oncogenes code for tyiosine kinases (reviewed in Kelloff et al., 1995a). Genotypic events include loss of heterozygosity (LOH) and gene amplification, either at specific gene loci (e.g., those for tumor suppressors such as p53 or tumor growth accelerators such as c-mbB-2) or at panels of microsatellite loci where mutations indicate increasing genomic instability (Califano et ul., 1996). Both phenotypic and genotypic changes during carcinogenesis may also be demonstrated by molecular events, often termed biomarkers (Kelloff et al., 1994a). For example, excess proliferation may be assessed by increased levels of cellular antigens such as proliferating cell nuclear antigen (PCNA) or Ki-67/MIB-1 or overexpression of growth factors such as epidermal growth factor (EGF), transforming growth factor (TGF)a, and IGF-I; reduced propensity to undergo apoptosis may be detected by increased expression of bcl-2. Aberrant differentiation may result in changes in G-actin, cytokeratins, and blood group antigens. Other molecular biomarkers may reflect general changes in cell growth control. These include TGFP, cyclins, p53 and other tumor suppressors, as well as mutations and overexpression of oncogenes associated with carcinogenesis such as ras and the transcription factors myc, fos, and jun. The use of these biomarkers is supported by the fact that carcinogenesis is progressive. Progression has been mapped in target tissues by the appearance of specific molecular and more general genotypic damage associated with increasingly severe dysplastic phenotypes (e.g., Kelloff et al., 1996a; Sporn, 1996; Schipper et al., 1996). In many cases early, critical steps include inactivation of tumor suppressor genes, such as APC or p53, activation of oncogenes such as rus, and damage to DNA repair mechanisms, such as by mutations in MSH and MLH genes and in BRCA. Carcinogenesis may take multiple paths, and be multifocal; not all cancers in a given tissue nor all cells in a given cancer may ultimately contain the same molecular lesions. Progression may also be influenced by factors specific to the host tissue’s environment, such as the action of hormones produced in stroma around the developing epithelial tumor (e.g., Sporn, 1996; Schipper et al., 1996). Further, carcinogenesismay not necessarily be driven by the order in which the changes

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appear; the disorganization caused by the accumulation of multiple effects may be more important. This disorganization is an obvious manifestation of carcinogenesis. Progression models that reflect increasing disorganization have been developed by Vogelstein, Sidransky, and colleagues, the seminal work being that by Fearon and Vogelstein in colon (1990). These researchers have also described carcinogenesis in brain (Sidransky et al., 1992a), bladder (Sidranskyand Messing, 1992; Sidranskyet al., 1992b; Rosin etal., 1995; Mao et al., 1996; see also Simoneau and Jones, 1994), and in head and neck (Califano et al., 1996). Lam, Gazdar, and colleagues have described early analyses of chromosomal loss correlating to grade of dysplasia and appearance of non-small-cell lung carcinoma (NSCLC) (Thiberville et al., 1995; Kishimoto et al., 1995). Also, Larson et al. (1997) described accumulating chromosomal loss at defined loci in cervical intraepithelial neoplasia (CIN). It is these genotypic and corresponding tissue and cellular histological lesions or biomarkers, when they are sufficiently stable to allow screening during carcinogenesis, that have the highest potential as measures of carcinogenesis. The specific carcinogenesis-associatedmolecular lesions identified so far, while important, may not be the most informative among those that will be discovered as research continues.

B. Cellular Cardnogenesis Experimental and epidemiological carcinogenesis studies show that >90% of cancers are associated with mutation and cellular proliferation (Henderson et al., 1992; Kelloff et al., 1995a; Ruddon, 1995). A normal functioning cell has three possible fates, which can be disrupted during carcinogenesis: (1) programmed cell death (from senescence, or in response to danger or environmental conditions such as overpopulation or hormone withdrawal); (2) maturation or differentiation; and (3)proliferation. Mutagenesis can damage the cell and destroy normal growth controls, resulting in loss of programmed cell death and maturation pathways and increased (hyper)proliferation. The specific biochemical pathways that underlie these mechanisms provide leads for identifying potential chemopreventive agents. For example, high levels of AA have been linked to increased apoptosis (Chan et al., 1998). NSAIDS inhibit AA metabolism resulting in increased AA-induced apoptosis, which is associated with their chemopreventive activity, particularly in colon. Also, polyamines are essential to cell growth and proliferation, and polyamine levels are higher in many cancer and other very rapidly proliferating cells than in normal cells. Inhibitors of polyamine synthesis have been designed to slow cell growth. One such agent is 2-difluoromethylornithine (DFMO),a specific irreversible inhibitor of ODC, a critical enzyme in polya-

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mine biosynthesis. DFMO prevents growth of tumor cells and has chemopreventive activity in numerous animal models (reviewed in Steele et af., 1994). Sporn (Hong and Sporn, 1997) has described carcinogenesis as aberrant differentiation. Several lines of evidence confirm that loss of differentiation controls is a common and critical aspect of carcinogenesis. Vitamin D and vitamin A and its analogs (retinoids) have shown high potential for restoring differentiationpathways in transformed (dedifferentiared)cells (reviewed in Kelloff et al., 1994a). These agents similarly have high chemopreventive potential, as measured in animal carcinogenesis models in breast, colon, lung, and prostate. Boone (Boone and Kelloff, 1997,1998) has described the cellular kinetics of 'carcinogenesis. Generally, the clonal evolution rate of neoplastic cells is defined as the rate of appearance in the neoplastic population of clonal variants that grow faster than surrounding cells. The proliferation rate of neoplastic cells in the population determines the production rate of genomic structural variants, because each turn through the cell cycle converts damaged DNA lesions into mutations and also subjects the genome to a greater mutagen sensitivity during S phase (Boone et al., 1992). A fraction of the genomic structural variants will be clonal variants that can grow faster. The focal expansions will add to the overall proliferation rate of neoplastic cells. The final result is a continuously accelerating kinetic cycle involving increased production rates of neoplastic cells, structural variants, and fastgrowing cellular variants, all of which are slowed by the increased production rate of apoptotic cells. The driving force of the cycle is entropic, that is, a selection pressure exists toward increasing disorder and heterogeneity as controls for maintaining homeostasis are lost. Chronic diffuse epithelial hyperplasia is a precursor to intraepithelial neoplasia ( E N ) frequently caused by growth factors and reactive oxygen species produced by the lymphocytes and macrophages of chronic inflammatory infiltrates in the subepithelial stroma. For example, in the oral mucosa, subepithelial chronic inflammatory cells were shown to induce EGF production and EGF receptors in the overlying squamous epithelium. Boone (Boone and Kelloff, 1997,1998) also cited other examples of chronic inflammation associated with the neoplastic process: ulcerative colitis, urinary bladder inflammation, gall bladder inflammation secondary to stones, and Barrett's esophagus, a condition in which esophageal inflammation secondary to gastroesophageal reflux disease (GERD) leads to hyperproliferative metaplasia of the esophageal epithelium (from squamous type to hyperproliferatingintestinal epithelial type) and then to IEN. In the skin, actinic (solar) keratosis, the commonest type of EN,is practically always associated with chronic inflammation in the subepidermis. In the larynx, subepithelial inflammation is a significant predictor of progression to carcinoma. Cigarette smoke produces chronic inflammation of

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the respiratory mucosa, inducing metaplasia of the ciliated secretory epithelium to stratified squamous type, from which E N develops. The cellular processes described result in changes in cell phenotypes that can be measured quantitatively, even before they are detected phenotypically in tissue. As will be described below, these changes include DNA ploidy and nuclear and nucleolar morphometry. The quantitative detection of such changes, such as by the use of confocal laser microscopy to evaluate aberrant crypt foci (ACF) in colorectal epithelia and computer-assisted image analysis (CAIA) to detect morphometric changes such as nuclear size and shape, will prove valuable in chemoprevention studies, in which subtle though statistically significant effects may be seen in treated samples compared with controls, and where the number o'f samples available for evaluation may be limited.

C. Tissue Cardnogenesis Intraepithelial neoplasias, such as colorectal adenomas, prostatic intraepithelial neoplasia (PIN),and CIN, are primary examples of precancerous lesions intermediate in the continuum of neoplastic progression to cancer and can be considered tissue level phenotypic biomarkers suitable for following carcinogenesis. Progression may also be influenced by factors specific to the host tissue's environment, such as the action of hormones produced in stroma around the developing epithelial tumor (e.g., Sporn, 1996; Schipper et al., 1996). One example of stromal influence is in breast. Adipocytes in breast stromal tissue are an important source of estrogen synthesis (catalyzed by steroid aromatase); estrogens, in turn, are associated with tumor progression in breast. There is evidence that higher levels of stromal estrogen synthesis are associated with the presence of breast cancer than with normal breast epithelium. Another important concept is the definition of high-risk tissue-particularly as applied to patients with previous cancers or precancers. Generally, these patients show increased risk for developing new primary lesions in tissue ihat is histologically related to tissue from which the original lesion arose. Slaughter et al. (1953) coined the term "field cancerization" to describe the early evidence of carcinogenesis found in normal-appearing mucosa of patients with previous head and neck cancers. In fact, the lifetime risk for a second primary tumor of the aerodigestive tract following a squamous cell cancer of the head or neck has been estimated at 20-40% (Benner et al., 1992). Many studies, particularly those carried out by Hong, Lippman, Hittelman, and colleagues (Benner et al., 1992; Dhingra et al., 1994; Hittelman et al., 1996), have confirmed this phenomenon. For example, Hittelman et al. (1996) used chromosomal in situ hybridization (CISH) to detect carcino-

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genesis-associated genotypic changes ( 1 3 copies of a single chromosome) in normal and precancerous tissue nearby head and neck cancers. The tissues were histologically and otherwise phenotypically distinct from cancers; hence, they were unlikely to be due to random sampling errors. In these studies, the degree of genetic change detected correlated to histologic progression of the lesion toward cancer. Very importantly, 8 of 15 patients (-53%) having premalignant lesions of the oral cavity containing high levels of genetic damage (23.5% of cells with three or more copies of chromosome 9) subsequently developed aerodigestive tract cancer compared with none among patients with lower levels. Similar results were found by Hittelman and colleagues on chromosome 9 in the lungs of previous smokers (Hittelman et al., 19’96) and on chromosome 17 in breast (Dhingra et al., 1994) and by Segers et al. (1995)on chromosome 1 among patients with various grades of CIN. The implication for clinical chemoprevention studies is that patients with previous.cancers or precancers provide cohorts at high risk for new primary cancers, who will benefit from chemoprevention and allow smaller trials to be designed in which premalignant changes (surrogate end points) can be followed both phenotypically and genotypically as predictors of eventual cancer incidence reduction. One criterion for selecting these cohorts is expectation of a high incidence of the cancer or precancer, or observable progression of the precancer, under study within a reasonable time period. For Phase II studies, study durations 5 3 years are desirable, and for Phase 111trials, 5 1 0 years (in some situations, such as preventing second primaries or progression of premalignant lesions in cancer patients, about 3 years may be feasible). The high incidence of new lesions in head and neck cancer patients was cited above. Superficial bladder cancer patients are appropriate subjects for chemoprevention studies because the recurrence rate is approximately 50% within 6-12 months (Soloway and Perito, 1992) and 60-75% within 2-5 years (Herr et al., 1990; Harris and Neal, 1992). Similar high rates of recurrence and new tumors also apply to colorectal adenomas (e.g., Winawer et al., 1993). Studies in these settings are particularly promising for the validation of surrogate end points, which may then be applied in cohorts without previous precancerskancers.

D. Clinical Carclnogenesis 1 . OBSERVATIONAL AND MOLECULAR EPIDEMIOLOGY Primary insights relating to clinical carcinogenesis risk derive in part from clinical epidemiology. Historically, this information was reported in observational findings. More recently, analytical toots have become available allowing definition and quantification of risks.

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Fearon (1997) has described cancer as a genetic disease that arises from accumulating mutations that lead to clonal selection of cells with predisposition for continued mutations and proliferation. He notes that most cancers (9899%) involve somatic mutations, that is, mutations seefi only at the cancer site not in the germline. Albeit rare and accounting for <2% of human cancers, the genetic syndromes characterized by germline lesions and extraordinarily high relative risks for cancer development have provided the leads for focusing on specific genes that will be major determinants in risk evaluation of the much larger numbers of individuals who have acquired genetic lesions at these same loci (TableI lists many of the known germline cancer risks). Another major research focus providing insight for ongoing and future prevention strategies is genetic polymorphisms in metabolic enzymes involved in activating and deactivating carcinogens. As noted by Henderson (Ross et al., 1998),although conferring much lower relative risks than the rare genetic syndromes, they will apply to much larger target populations. Howeveq the predisposing mutations in the germline are very important for understanding the molecular pathogenesis of cancers and for defining cohorts at high risk. Another way of stating this concept is that cancer risk depends on the contributions of many small relative risks which include somatic mutations (at the same and other loci as the germline mutations) and genetic polymorphisms, as well as lifestyle and environmentalexposures (Fearon, 1997; Perera, 1997). Because the role played by genetic polymorphisms in cancer risk is only now being elucidated, and since, in general, the risks associated with genetic polymorphisms are significant primarily in association with other factors such as smoking and occupational exposure to carcinogens, it is worthwhile to discuss how this risk might be used in identifying cohorts for chemoprevention and in evaluating chemopreventive efficacy. For example, Bell et al. (in M. S. Miller et al., 1997) described the association between colorectal and bladder cancer and genetic variants in NATl and NAT2 enzymes which catalyze activation of carcinogenic aromatic amines by N-or 0-acetylation. Bell’s group has studied polymorphism for these enzymes among 202 colorectal cancer patients and 112 controls in England. They found a significantly increased risk among carriers of the NATl * 10 phenotype, especially if they also carried the rapid acetylator NAT2 phenotype. Bell also reported preliminary results in bladder suggesting that the NATl * 10 phenotype was associated with higher levels of DNA adducts and that bladder cancer risk among smokers varied with NATl phenotype (including but not limited to NATl *lo). These data suggest that chemoprevention studies in bladder should consider NATl status of smokers as a stratification factor or, depending on the agent, an eligibility criterion. Glutathione S-transferasescatalyze the detoxification of many carcinogens that are activated via formation of epoxides. The relationship of GST polymorphisms to cancer risks has also been frequently documented. Eaton et al.

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(in M. S. Miller et al., 1997) recently reviewed studies on the associations of the GST M1 polymorphism and cancer susceptibility. Significant portions of the population (e.g., approximately 50% of Caucasians) carry homozygous GST M1 deletions. Increased susceptibility to carcinogenesis in smokers with the null GST M1 phenotype has been evidenced by increased urine mutagenicity, (Ames test), lymphocyte sister chromatid exchange rate, and, perhaps most significantly, lung polycyclic aromatic hydrocarbon (PAH)-DNA adducts. Multiple epidemiology studies have also suggested increased risks for lung and bladder cancers, especially in smokers. These data again suggest the wisdom of considering the GST M1 phenotype in selecting cohorts and evaluating the activity of chemopreventive agents in lung and bladder. Paiticularly, evaluation of chemopreventive agents such as the dithioldithione oltipraz, a potent inducer of GSTs, could be examined in light of GST M1 status. The agent may not be effective where GST M1 cannot be induced. Alternatively, oltipraz could induce other GST isoforms in GST M1 null subjects, thereby overcoming the GST deficiency. For example, Lin et al. (1998) showed that GST M1 null subjects exposed to broccoli which contains GST inducing agents, both dithioldithiones and isothiocyanates such as sulforaphane, were less susceptible to colorectal carcinogenesis (lower incidences of colorectal adenomas) than unexposed subjects. There is abundant evidence that steroid hormone exposure is an important risk factor for cancers in organs affected by these hormones such as breast and prostate. In particularly interesting work, Henderson and colleagues are attempting to establish a polygenic model for prostate cancer susceptibility based on polymorphisms in genes coding for regulators of androgen metabolism and activation, namely, steroid Sol-reductase type II (SRDSM), androgen receptor (AR), cytochrome P450c17a, and 3P-hydroxysteroid hydrogenase (Ross et al., 1998). Thus far, they have developed promising data showing that certain mutations in codons 89 and 49 of the SRD5A2 gene are associated with high-risk populations and are present at higher levels in cancers than in healthy patients. Functionally, the mutations are associated with high enzyme activity in subjects at high risk and in cancer patients. Also, Henderson’s group showed that the length of the (CAG), sequence on exon 1 of the AR gene correlates directly to reduced transactivation, and inversely to prostate cancer risk. In many cases, no specific carcinogenesis-associated genetic markers can be identified. This aspect of risk analysis has been addressed by molecular epidemiologists who look at measurements of “effective” carcinogen exposure. For example, Spitz et al. (1997) explored bleomycin-induced mutagen sensitivity in patients with upper aerodigestive tract cancers, alone and in conjunction with smoking, alcohol, and antioxidant intake. They concluded that mutagen sensitivity was indeed an independent risk factor; therefore, it is a good candidate for inclusion in risk models.

Table I Germline Mutations Defining High-Risk Cohorts for Chemoprevention Trialsa Lesion APC

APC

Disease Familial adenomatous polyposis (FAP) Gardner’s syndrome

Tissue

Colon Colon

Soft tissue ATM (y-induced DNA repair)

Ataxia telangectasia

Breast Hematological

BLM BRCAl

BRCA2 FACC

mcc

Bloom’s syndrome

NF-2

Adenomatous polyps, adenocarcinoma Adenomatous polyps, adenocarcinomas Osteomas, lipomas, fibromas Carcinoma

Fanconi’s anemia Lynch I, II

van Recklinghaussen’s neurofibromatosis Central neurofibromatosis

Ovary (colon, prostate) Breast Hematological Colon (endometrium,pancreas, ovary) Kidney Nervous system Nervous system

Cohort size (% target)

1%

3.8%

Leukemia, B-cell lymphoma Solid tumors

1-2% (30 % at age <40) 3Yo 0.5%

Breast

(eg., MSH2, hMLH1, hPMS1, hF’MS2) MET

NF- 1

Tumor type

Leukemia Adenomatous polyps, adenocarGnoma Hereditary papillary renal cancer Neurofibrosarcoma, brain tumors Acoustic neuromas, meningiomas

3-5 %

0.03% incidence

0.03% incidence

p16 (CDKNZ)

Dysplastic nevi, atypical moles

P53

Li-Fraumeni

F’TCH

Nevoid basal cell carcinoma syndrome (NBCCS)

PTEN

Cowden disease

RB RET

Familial retinoblastoma Multiple endocrine neoplasia ( M E N , 2)

VHL

von Hippel-Landau

WTl, wT2

Beckwith-Wiedemann Xeroderma pigmentosum

XPB, XPD, XPA (DNA repair) =See also Fearon (1997).

Skin

Melanoma

Pancreas Breast Soft tissue Hematological Brain Skin

Pancreatic cancer PCIS, comedo-type Sarcoma Leukemia

Ovary Brain Breast Thyroid Eye Thyroid

Fibroma Medulloblastoma Carcinoma Follicular carcinoma Retinoblastoms, sarcomas Medullary carcinoma

Adrenal cortex Parathyroid Kidney Nervous tissue Kidney Skin

Pheochromacytoma Adenoma, hyperplasia Renal cell carcinoma Hemangioblastomas Wilm’s tumor Malignant melanoma

<1%

Basal cell carcinoma

0.005% live births

0.002% incidence

0.01% incidence

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Lifestyle (e.g., smoking, alcohol, diet) and environmental exposure (including occupational exposures)are also critical, particularly for certain cancer targets. For example, the association of occupational exposure to aromatic amines and bladder carcindgenesis has been long established (e.g., Soloway and Perito, 1992), and much controversy surrounds the scientific data suggesting an association between DDT-like pesticides and cancer risk (Perera, 1997). Another significant concept in identifying cancer risk is that often most of the risk (exposureto carcinogens)is endogenous rather than exogenous.The breast cancer risk model as defined by Gail et al. (1989)is an example, in that the factors conferring risk are all related to endogenous estrogen production.

2. MULTIYEAR NATURE OF HUMAN CA~CINOCENESIS In most epithelial tissues, the molecular, cellular, and tissue changes leading to superficialtumors and finally to invasive disease may occur over a long time period (Table 11). For example, in the breast it is estimated that progression from atypical hyperplasia through ductal carcinoma in situ (DCIS) to adenocarcinoma may require 30 years or more (Frykberg and Bland, 1993; Page et al., 1985). Colorectal adenomas may form over a period as long as 5-20 years, and progression from adenoma to colorectal carcinoma may require another 5-15 years (Day and Morson, 1978; Bruzzi et al., 1995). PIN may develop over approximately 20 years (Bostwick, 1992). From PIN to early latent cancer may take 10 or more years, and clinically significant carcinoma may not occur until 3-15 years later (Bostwick, 1992). The prolonged time course of carcinogenesis provides the opportunity for chemoprevention-to intervene when the mutations are fewer, even before tissue level phenotypic changes are evident. However, the long latency also presents significant challenges for the clinical phase of chemopreventive drug development. These challenges and strategies to address them have been described by.us (e.g., Kelloff et al., 1996a) and by Hong and Sporn (1997). Primary prevention studies, that is, cancer incidence reduction studies in subjects at relatively low risk (asymptomatic with normal or with elevated risk based on the presence of genetic lesions such as genetic polymorphisms in carcinogen activating or deactivating enzymes) may require thousands of subjects and many years to obtain significant and definitive results. For example, the recently completed and successful trial of tamoxifen as a chemopreventive for breast cancer was carried out in women who at a minimum had a relative risk (RR)equivalent to a 60 year old (Fisheretal., 1998). Six thousand six hundred (6600)treated and an equivalent number of control subjects were required to achieve a significant (p < 0.05) treatment effect. In the finmeride prostate cancer.prevention study, 18,867 men 1 5 5 years old are being evaluated and the trial, started in 1993, is expected to require 10 years to complete (Feigl et al., 1995).

Table II Multiyear Progress from Normal Appearing Tissue to Precancer to Cancer in Major Human Cancer Targets"'b Precancer (= intraepithelial neoplasia)

Normal

Initiated

Mild

Moderate

Severe

PIN

Prostate

cancer

Latent cancer

20 years

210 years

Atypical hyperplasia

Breast

CIS

3-15 years DCIS

6-10 years

14-18 years Lung

5-20 years Lung (smokers)

20-40 pack-years Colon

Adenoma

5-20 years

5-15 years

Bladder

ns CIN III/CIS

CLN I

Cervix

9-13 years Esophagus

Barrett's

-

10-20 years Severe dysplasia

est. 5-20 years Liver

<5 years

20 years

est. 3-4 years

HBV infection

20-40 years aReferences: Prostate (Bostwick, 1992),breast (Page et al., 1985;Frykberg and Bland, 1993),lung (Mulshineet al., 1992),lung (smokers)(Peters et al., 1993;Husgaf vel-Pursiainen et al., 1995),colon (Day and Morson, 1978;B r w i et al., 1995),bladder (Cotran et al., 1989),cervix (Chanen, 1990),esophagus(Ovaska et al., 1989;Miros et al., 1991;Williamson et al., 1991;Cameron and Lomboy, 1992;Jankowskietal., 1993;Falk and Richter, 1996),liver (Grisham, 1997). bAbbreviations: CIN, cerirical intraepithelial neoplasia; CIS, cervical carcinoma in situ; DCIS, ductal carcinoma in situ; HBV, hepatitis B virus; PIN, prostatic inaaepithelial neoplasia; TIS, transitional cell carcinoma in situ.

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Secondary prevention is a useful and clinically beneficial approach to the development of chemopreventive drugs. In such studies, agents are evaluated for efficacy in subjects with previous cancers or current or previous precancers. These patients, particularly those with previous cancers, are considered to be at high risk for development of new cancers. This risk is based on the field effect described above. Most of the Phase I1 and many of the Phase I11 strategies that will be discussed in this review involve these highrisk cohorts. As Hong (Hong and Sporn, 1997) has further noted, chemopreventive treatment regimens can be matched to the subjects’ degree of risk. Patients with previous cancers with high rates of recurrence and new primaries may be treated with more potent and, hence, possibly more toxic regimens than asymptomatic subjects. Subjects with precancers, particularly with early dysplasia, will require less potent regimens, with acceptable treatments becoming more aggressive as the severity of the lesions increases. For example, subjects with previous, nonhereditary colorectal adenomas may benefit from less potent agents than would be required to significantlyreduce the incidence of adenomas in patients with familial adenomatoGs polyposis (FAP).

111. DEFINITION OF CHEMOPREVENTION

AND CHEMOPREVENTION AGENT DISCOVERY Chemoprevention is the use of agents to slow the progression of, reverse, or inhibit carcinogenesis, that is, to lower the risk of developing invasive or clinically significant disease. Chemopreventive agents modulate cancer risk at the molecular, cellular, tissue, and clinical levels. The mechanisms of these agents are defined by activities seen at the molecular and cellular levels, while chemopreventive efficacy is evaluated at the tissue and clinical levels.

A. Molecular Targets in Agent Discovery Basic research in carcinogenesis has identified many enzymes, genetic lesions, and other cellular constituents associated with the initiation and progression of precancers to invasive disease. Possible mechanisms for chemoprevention involve interfering with the expression and/or activity of these molecules; examples of the mechanisms, their possible molecular targets, and agents that act at these targets are listed in Table I11 (see also Kelloff et al., 1994a, 1995a): Many good agents have multiple chemoprevention-associated molecular activities. It is evident that many of these activities are interrelated, for example, as in the example given above, fenretinide’s effects on

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215

ODC, AA metabolism, PKC, IGF-I, and TGFP may be pleiotropic results of activity at one of these loci or at another target on signal transduction pathways. It is also clear that a single activity, even if it is the agent’s predominant pharmacological activity, may not be the most important or the only one required for chemoprevention. Such may be the case for inhibition of prostaglandin (PG)synthase by NSAIDs. These observations imply that molecular targeting, although the primary mechanistic approach to drug discovery, should not be the only approach to identifying potential chemopreventive drugs.

B.:Mutagencsis and Mitogenesis: Empirical Targets for Chemoprevention Experimental and epidemiological carcinogenesis studies showing that >90% of cancers are associated with mutagens and mitogens (Henderson et al., 1992; Kelloff et al., 1995a; Ruddon, 1995) suggest the second, very important strategy for identifying and characterizing chemopreventive agents. This empirical approach is the search for agents that inhibit or reverse these cellular processes associated with carcinogenesis. As noted above, normal functioning cells have three possible fates: (1)programmed cell death (from senescence, or in response to damage or environmental conditions such as overpopulation or hormone withdrawal); (2)maturation or differentiation; and (3) proliferation. Mutagenesis can damage the cell and disrupt normal growth controls resulting in loss of programmed cell death and maturation pathways and increased (hyper) proliferation. So the second strategy is to look for agents that block this disruption-inhibitors of mutagenesis and proliferation and inducers of apoptosis and differentiation. Table 111, despite its molecular target orientation, reflects the importance of these cellular changes, as the individual molecular activities are associated with the cellular effects. Besides these cell-based effects, there are also generalized cellular and tissue-based effects that are associated with carcinogenesis, namely, inflammation and oxidative damage. And so, agents that are antiinflammatories or antioxidants also have high potential as chemopreventives, regardless of their molecular mechanisms.

C. Principles of Chemopreventive Agent Discovery-Mechanistic Approaches In evaluating the potential efficacy of chemopreventiveagents several mechanistic parameters are weighed: ( 1) number of chemoprevention-related pharmacological activities, (2) impact of the agent on likely carcinogenesis

Table 111 Mechanism for Chemoprevention with Possible Molecular Targetsn Mechanism Antimutagenesis Inhibit carcinogen uptake Inhibit formatiodactivation of carcinogen

Deactivatddetoxify carcinogen Prevent carcinogen-DNA binding Increase level or fidelity of DNA repair AntiproliferatiodAntiprogression Modulate hormondgrowth factor activity

Inhibit oncogene activity Inhibit polyamine metabolism

Possible molecular targets

Representative agents

Bile acids (bind) Cytochromes P450 (inhibit) PG synthase hydroperoxidase, 5-lipoxygenase (inhibit) Bile acids (inhibit) GSWGST (enhance) CytochromesP450 (inhibit) Poly(ADP-ribosy1)uansferase(enhance)

Calcium PEITC, tea, indole-3-carbinol NSAIDs, COX-2 inhibitors, LOX inhibitors, iNOS inhibitors Ukxiiol Oltipraz, NAC Tea NAC, protease inhibitors

Estrogen receptor (antagonize) Androgen receptor (antagonize) Steroid aromatase (inhibit) Steroid Sa-reductase (inhibit) IGF-I (inhibit) Farnesyl protein transferase (inhibit) ODC activity (inhibit) ODC induction (inhibit)

SERMs, soy isoflavones Bicalutamide, flutamide Exemestane, vorozole, arimidex Fmasteride, epristeride SERMs, retinoids Perillyl alcohol, limonene, DHEA, FIT-276 DFMO Retinoids

Induce terminal differentiation Restore immune response Increase intercellular communication Restore tumor suppressor function Induce apoptosis

Inhibit angiogenesis Correct DNA methylation imbalances Inhibit basement membrane degradation Inhibit DNA synthesis

TGFp (induce) COX (inhibit) T, NK lymphocytes (enhance) Langherans cells (enhance) Connexin 43 (enhance) p53 (inhibit HPV E6 protein) TGFp (induce) RAS farnesylation (inhibit) Telomerase (inhibit) Arachidonic acid (enhance) Caspase (activate) FGF receptor (inhibit tyrosine kinase) Thrombomodulin (inhibit) CpG island methylation (enhance) Type IV collagenase (inhibit) Glucose 6-phosphate dehydrogenase (inhibit)

Retinoids, vitamin D, SERMs NSAIDs Selenium, tea Vitamin E Carotenoids, retinoids

-

Retinoids, SERMs, vitamin D FeriUyl alcohol, limonene, DHEA, FT1-276 Retinoic acid NSAIDs, COX-2 inhibitors, LOX inhibitors Retinoids Soy isoflavones, COX-2 inhibitors Retinoids Folic acid Protease inhibitors DHEA, fluasterone

nAbbreviations: COX-2, cyclooxygenased; CpG, cytosine-guanosine; DFMO,2dinuoromethylornithine;DHEA,dehydroepiandrostenedione;FGF, fibroblast growth factor; Fn,farnesyl transferase inhibitor; GSH,glutarhione; GST, GSH S-transferase;HPV,human papilloma virus; IGF-I. insulin-like growth factor I; LOX,lipoxygenase; NAC, N-acetylcysteine;NOS, inducible nitric oxide synthetase; NSAIDs, nonsteroidal antiintlammatory drugs; ODC, ornithine decarboxylase; PEITC, phenethylisothiocyanate; PG, prostaglandin; S E W , selective estrogen receptor modulators; TGFp, transforming growth factor p.

218

Gary I.

Kelloff

pathways to the targeted cancer, (3)pharmacodynamics, and (4) specificity for chemopreventive activity compared with interference with normal cellular function (Table IV). Mechanistic data are important throughout the development process for chemopreventive drugs, and they are particularly important in the earlier phases of identifying promising candidate agents and characterizing efficacy. In vitro mechanistic assays are a first step in evaluating chemopreventive potential. Mechanistic considerations are also useful in defining animal efficacy models and in interpreting the results of assays in these models. Systematic evaluation of classes of agents acting at molecular targets is an important strategy for identifying and characterizing new potential chemopreventive agents (Kelloff et al., 1996b, 1997a). In the following paragraphs, the rationale and development strategies for prototypical classes of chemopreventive agents are reviewed: (1)signal transduction modulators, including those targeting growth factor, oncogene, and tumor suppressor-mediated activities [examples are EGFR inhibitors, Ras farnesyl protein transferase (FPT)inhibitors, and retinoids]; (2) hormone modulators such as antiestrogens and aromatase inhibitors; (3) antiinflammatories including several classes of compounds interfering with arachidonic acid metabolism such as the NSAIDs, selective inhibitors of COX-2, and lipoxygenase inhibitors, as well as iNOS inhibitors; (4) antimutagens such as phase I1 enzyme inducers; and ( 5 )antioxidants such as the combination of selenium with vitamin E and tea compounds. As noted above, these classes of agents and the activities are not independent, but are closely interrelated and overlapping. Hormones, for example, are critical components of signal transduction pathways and are affected by other modulators of these pathways. Likewise, many agents that are antioxidants have antiinflammatory activity that is mediated via inhibition of the AA cascade. Both antiinflammatories and antioxidants affect sites on signal transduction pathways, presumably at least partially due to modulation of AA metabolism at the membrane level. Also, there is significant cross talk among different but related targets in signal transduction, such as between hormones and their receptors and retinoids and their receptors. Many more agents than those represented by the classes described below have been evaluated on the basis of mechanistic leads. As suggested above, agents with anticarcinogenic effects at the cellular level are prime candidates for further evaluation, whether or not specific molecular targets for their activities have yet been identified. For example, the empirically determined promotion of differentiation by vitamin A and its analogs were important in assessing the chemopreventive potential of these agents long before their interactions with retinoid receptors were elucidated. For each of the representative classes below, the general pharmacological characteristics of its member agents are described along with the agents’ roles

Table N Approaches to Chemopreventive Agent Discovery and Development-Representative Dnnloaocnt SDXJ

S i Pansdumon mod&tom

Definition of d a s s assocation with carcinogenesis

Deregulated expressiodmutation of target signal s e n in neoplasia (e.g., EGFR overexpressionin bladder, breast; rm mutations in pancreas, bladder, colon; loss of RARp in lung and head and neck)

Determine chemopreventivc/ mechanistic activity of candidate agents in uitro

Characterize potency of candidates against cell-free target (e.g., inhibition of A431 human epidumoid cell EGFR-catalyzed ATP incorporation into peptide and autophosphorylation; inhibition of FFP ' incopmation hy recombinant or isolated FIT)

Deteruune speciSuty/ Compare activity of candidates among related cell-frec targets selectivity of (e.g., compare activity of candidate chemopreventivd EGFR inhibitors against various mechanistic activity tyrosine kinases such as EGFR, of candidate agents c-erbB-2, v-src, PDGFR; compare in vino activity of candidate FPT inhibitions against FPT and GGTass; evaluate specificity of retinoids for activation of retinoid receptor isoforms)

(continues)

Hormone modillatom

Classes

Antiinanmmptav npenfo

.. Antimuupars

AntiOXidaUts

Many compounds which demonStrong correhtion of choNc strate antimutagenic activity inflammation and cardnogenesis in uitro have chemoprwentive (e.g., in colon and lung). Human evidence that antiidammatory aqiyity in animal models at agents reduce (pre)cancerincidence multiple targets. Particularly, or cause regression of precancers antimutagens which induce (e.g.. sulindac causes regression phase lI,merabolic enzymes of adenomas in FAP patients, have demonmated potent epidemiologicalnudies show chemopreventiveactivity reduced incidences of colorectal (e.g., diallyl sulfides, cancers among aspirm users) dichiolthiones) For hormone synthesisand metabolism Test activity of candiate agents Evaluate inhihition of mutageniagainst isolated or recombinant enzymes, test modulating activity of city in well-recognized mutacandidace agents against cell-free enzymes (e.g., COX-IICOX-2, genicity ScIeens (e.g., the Ames target (e.g., inhibition of aromatase 5-LO) SalmoneL assay using isolated fromhuman placental or appropriate panels of tester PMSGstimulated rat ovary strains and carcinogen) microsoma)

A large body of epidemiological data has associated dietary an.tioxidants (e.g., vitamin At caroteuoids, vitamin E, selenium, vitamin C, tea polyphenols)with reduced risk for cancer. Also, activated oxygen has teen found to induce DNA damage

-

-

Carcinogensis is hormone-dependent in hormone-responsivetissues (i.e., breast and prostate). Lowering hormone exposure reduces risk of Cancer

Antioxidants are likely to have both andmutagenic and antiproliferative activity. Becaux of the involvement of reactive oxygen species in in8ammation, antioxidants are also likely to be good anfiinflammatory agents. A fir* step in characterizing candidate agents might be to evaluate them in mutagenicity jnst as for other antimuragens. Antimutagenicity assays might also prove useful in idennfymg active components of complex natural produns (e.g., tea

emam)

Determine differential inhibitory activity against different AA metabolism enzyme isoformsas appropriate. Particularly, evaluate selectivity for COX-2 over COX-1 (implicationsfor toxicity as well as efficacy)and for NOS over eNOS

See below, under Determine activity in intact cells

N N

0

Table IV (continued) Development step

S i d uansduction modulators

Hormone mndulams

Determine chemopreventivd mechanistic activity in intacf cells

Evaluate effect of candidates on target in intact cells where activities measured can he correlated to mga inhibition (e.g., inhibition of EGFR aurophosphorylation, transformation and proliferation in A431 or Swiss 3T3 fibroblasts stimulated wirh growth hcfor; inhibition of morphological transformation in Met-18b-2 cells using FIT inhibitors1

Determine mechanistic activity in uivo

Evaluate inhibitory effect of candidate Evaluate candidate agents in agents on growth of targetdependent same tissues as form vino tumors (e.g., inhibition of EGFRassays, but administer agents dependent tumors in A431 xenoto animals and then isolate grafts or Ras-dependenttumors in cells nude mice injected with transformed cells bearing mutated rm)

Characterize hormone receptor interaction or enzyme activity in hormone-responsivecells (e.g., charanerizc aromame inhibition by candidate agents in MCF-7 cells or human genital fibroblasts; characterize ER binding and expression and TGFB induction activity of candidate antiestrogens in MCF-7 cells)

AntiinBammamIy agents

-

AntiUlIItPgenS

As appropriate, determine ability to induce phase

n metabolic

enzymes in whole cells [measure

induction of GSHJGST or use NAD(P)H:quinone reductax as a surrogate for phase II enzymes]. Determinespecikityfot . modulating phase II enzymes (cf. cytochrome P450s involved in carcinogen activation). Also, d $ + n e ability of agents to inhibit carcinogen-DNA adduct formation [e.g., B(o)P-DNA adducts in human BEAS2-B cells]

Evaluate candidates in standard animal screens for antiidamatory activity (e. g., carrageenan-induced rat paw edema). Use assays to distinguish agents on bioavailabiliry and toxicity, as well as potency

-

~UOXibntS Deternune abhty to u h b a free radrcals (e.g., by itdubinon of TPA-mnducuon of acuvated oxygen s p e s UIHL-60 cells) and carmogen-DNA adduct formanon [e.g., B(a)P-DNA adducts m human BEAS2-B cells]

Determine chemoprcventive activity in viuo

N N L

Evaluate candidate agents in Evaluate candidate agents in carcinogen-inducedanimal models carcinogen-inducedor uansgemic susceptibleto carcinogenesisat rodents susceptibleto carcinogenesis hormone-sensitivetargets (i.e., rat at appropriate targets (e.g., evaluate mammary gland or prostate) EGFR inhibitors in hamster buccal much. rat mammary eland. or mouse idadd&; evaluate FEk-inhiditors in m o w lung or rat d o n ; retinoids in rat mammary gland)

Evaluate candidate agents in carcinogen-induced or transgenic animals at appropriate targets (i.e., evaluate NSADs and COX4 inhibitors in rat colon, mow bladder, and &bly mouse l u n s evaluate NOS lnhtbimrs ut rat colon; evaluate LOX mhibitors ut mouse lung and rat prostate)

Th-,agents may be expecud to Antioxidants are expmed to have a broad spectrum of have a broad spenrum of activity. Initial screening for cbemopreventiveactivity, but they are of especiglly &gh @terest cbemopreventive activity in vivo might be most easily for use against smoking-related accomplished by topical cancers (becauseof E ~ I O U ~ C exposure to caranogens from application to skin unless absorption and h i b u t i o n tobacco smoke). Therefore mouse characteristicsof the agent luag and bladder arc pnmary weU-known. Testing in rat targets colon models is also possible. Bioavailability m a y be a major issue in the evaluation and development of antioddants. Many (particularly the plyphenols and some carotenoids) are poorly absorbed. Determinationof distribution to target tissues might be considered for promising agents before extensive development work is undenalen. Many antioxidants are redox effectors, having the ability to form as well as reduce active oxygen, with the mode of action depending on tissue concentration. It may be worthwhile to determine an activityltoxicityprofile for such bimodal agents (e.g., carotenoids and plyphenols)

222

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in inhibiting carcinogenesis (including cancer targets), and specific strategies for evaluating the chemopreventive potential and developing candidates in the classes for use in the clinic are described (see Table V in Section III,E,l). The same general strategies may be applied to the evaluation and development of any class of agent or single agent.

D. Representative Classes of Chemopreventive Agents I . SIGNAL TRANSDUCTION MODULATORS Many sites on deregulated signaling pathways are possible targets for chemopreventive intervention. For example, tyrosine kinases, which catalyze the transfer of the y-phosphate of ATP to the hydroxyl group of protein tyrosine, are critical components of these pathways (Chang and Geahlen, 1992), and deregulated tyrosine kinases have been associated with carcinogenesis (e.g., Powis, 1994; Powis and Workman, 1994), suggesting tyrosine kinase inhibitors as a potential chemopreventive mechanism. EGFR is one of the tyrosine kinases which are membrane-associated growth factor receptors implicated in carcinogenesis. Many tyrosine kinases on signal transduction pathways are oncogenes, such as mutated rus. EGFR and YUS farnesyl protein transferase (FPT) inhibitors are presented below as examples of classes of chemopreventive agents that affect signal transduction tyrosine kinases. The mechanisms of the two classes of inhibitors reviewed are different: the EGFR inhibitors affect the activity of EGFR; the YUS FPT inhibitors affect the activation of Ras tyrosine kinase by preventing a required posttranslational modification of the protein. Nuclear transcription pathways are also possible sites for chemopreventive intervention. Retinoids may selectively bind to and activate retinoid receptors, thereby affecting signal transduction leading to cellular maturation and differentiation along these pathways. These three classes of chemopreventive agents are described in the following.

a. Epidermal Growth Factor Receptor Inhibitors Od the basis of the redundancy of growth factor networks, selective inhibition of signaling pathways activated in precancerous and cancerous cells should be possible. Proliferation of normal cells is dependent on more than one growth factor, and one growth factor activates multiple intracellular signaling pathways. Gene knockout experiments have established that if a particular growth factor signaling pathway is inactivated, an alternative pathway takes over (Levitski, 1994). Because overexpression or mutation can lead to constitutive activation of a single signaling pathway, inhibition of this specific pathway should not disturb other pathways necessary for normal cell function. Thus, inhibiting a specific locus activated in target tissues should

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result in fewer effects on the growth of normal compared with precancerous or cancerous cells (Levitski, 1994; Kelloff et al., 1996b). As noted above, many specific tyrosine kinases associated with cancers of various organs have been identified. EGFR, the product of the c-erbB-1 protooncogene, is one of the most extensively studied, and the available evidence suggests that disrupting the EGFR-mediated signaling pathway would be a useful approach for cancer prevention. We have reviewed this evidence and discussed strategies for evaluating these agents as chemopreventive agents (Kelloff et al., 1996b). These observations are summarized here; references to the primary research may be found in our review. The EGF receptor is a 170-kDa transmembrane protein that is a member of a family of related receptor protein kinases that also includes the proteins encoded by the c-erbB-2 (neu), c-erbB-3, and c-erbB-4 genes. Currently recognized EGFR ligands are EGF, TGFa, AR, betacellulin, and heparin binding-EGF. Some ligands including AR, EGF, and TGFa are capable of acting as inhibitors, as well as stimulators, of cell proliferation depending on the specific circumstances. The importance of EGF and TGFa in controlling cell growth has been well established; the importance of other ligands is only beginning to be explored. Activation of EGFR can occur via autocrine, paracrine, or juxtacrine mechanisms. When bound to ligand, EGFR dimerizes with neighboring receptors and is autophosphorylated at three major tyrosine residues. Subsequently, the receptor interacts with a number of proteins that are elements of signal transduction pathways including phospholipase Cy, phosphatidylinositol-3’ [PI(3)K],growth factor receptor binding protein 2 (Grb2), Src family kinases, and components of the Jak/STAT pathway. Association of EGFR with Carcinogenesis. Deregulated expression of EGFR and its ligands has been associated with the development of neoplasia in both animals and humans. Aberrant EGFR signaling can occur with mutated receptor or, more commonly, with receptor overexpression. The most frequently identified mutant, EGFR vIII, has lost amino acids 6-273, resulting in ligand-independent tyrosine kinase activity, altered subcellular location, increased stability, and enhanced tumorigenicity. It has been detected in up to 57% of high-grade and 86% of low-grade glial tumors, 78% of breast cancers, 73% of ovarian cancers, and 16% of NSCLC, but not in any normal tissues examined to date. Overexpression of EGFR does not usually involve mutations, although sometimes amplification and rearrangement are observed. The level of EGFR appears to determine the signal transduction components which are activated. At physiological levels, EGF binding to EGFR results in phosphorylation of Src homology 2 domain-containing proteins and a normal mitogenic response. At higher levels other substrates, such gs phospholipase Cy and RasGAP,are phosphorylated. This tendency to phosphorylated oncogenes or

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oncogene itimulators at higher levels may explain, at least in part, how EGFR overexpression contributes to carcinogenesis. Increased expression of the EGFR ligands, particularly TGFa, has also been linked to tumorigenesii. Transgenic mice overexpressing TGFa develop benign skin lesions and malignancies of the liver and mammary gland. TGFa may combine with both viral and cellular oncogenes in the induction of neoplasia, and available evidence supports a role for TGFa in transformation independent of its stimulatory effects on cell growth. EGF has also been associated with carcinogenesis both in vitro and in vivo. Also, ras oncogene activation may be mediated in part by the EGFR signaling pathway, TGFa, and other ligands. Abnormal EGFR signaling has been implicated in the devdopment of many cancers, although in most tissues the precise role remains unclear. Much available clinical data have been generated from studies of established cancers. These investigations have shown that in some organs EGFR is overexpressed in a subset of cancers (overexpression may or may not be associated with poor prognosis). Targets for chemopreventive intervention are tissues where aberrant EGFR-mediated signal transduction occurs at a relatively early time point during tumor development-bladder, breast, cervix, colon, esophagus, head and neck, lung, and prostate. Two patterns have emerged from studies of EGFR signaling during carcinogenesis. The first is increased expression of EGFR during the early stages of carcinogenesis followed by receptor downregulation, often subsequent to increased ligand production. This pattern has been observed, for example, in lung, cervix, and prostate. The second recurring pattern is expansion of receptor and/or ligand expression from a subset of cells in normal tissue (usually in the basal layer) to extended cellular layers during progression (e.g., head and neck, bladder, cervix). These changes in cellular distribution are usually detected using immunohistochemical (IHC) techniques which allow the maintenance of tissue architecture. Approach to Developing EGFR Tyrosine Kinase Inhibitors as Chemopreventive Agents. Briefly, the requirements for potentially useful EGFR inhibitbrs are specificity for EGFR compared with other tyrosine kinases and classes of protein kinases (e.g., serinekhreonine protein kinases such as PKC, histidine protein kinases), high potency, activity in contact cells, and activity in vivo. Inhibitors can inhibit autophosphorylation or act on downstream targets; autophosphorylation is the preferred site because it should result in total blockade of the signaling pathway. Inhibitors that compete with substrate rather than at the ATP binding site are also preferred because they are not so likely to inhibit other ATP-utilizing cellular enzymes. Additionally, the high ATP concentrations in the cell may render inhibitors that are quite effective against the isolated enzymes inactive in intact cells. A possible testing

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and agent development strategy is outlined below, with priorities for further development based on results at each step in the order following. Determine EGFR tyrosine kinase inhibitory activity. Inhibition of EGFRcatalyzed incorporation of [32P]ATPinto various substrate peptides can be assessed, using EGFR from A43 1human epidermoid carcinoma cells or the * should be demonstrated. Evaluate EGFR specificity/selectivity.An extensive screening battery examining the selectivity of potential inhibitors on several tyrosine kinases besides p60v-srcplatelet-derived growth factor receptor EGFR, including p l 8Sc-erbB-2, (PDGFR),colony stimulating factor-lr (CSF-lr), and insulin receptor (INS-r), has been described as has a method for evaluating specificity among a panel 6f protein kinases using a single substrate. Determine inhibition of EGFR-mediated effects in intact cells. Selectiveinhibition of autophosphorylation and effects on EGFR-mediated proliferation and transformation can be examined in whole cells, and particularly in A431 carcinoma cells, which express high levels of EGFR. Swiss 3T3 fibroblasts stimulated with various growth factors can be used to determine inhibitor selectivity on phosphorylation reactions. Determine inhibition of EGFR-mediated effects in vivo. Minimal requirements for testing EGFR inhibitors in vivo include deregulation of the specific protein tyrosine kinase in the target tissue and demonstration that tumor growth depends on the targeted signalingpathway. In this case, EGFR should be known to be overexpressed in the tumor and tumor growth should be dependent on its expression. Xenografts of A431 cells in nude mice overexpress EGFR, and their growth can be inhibited by treatment with anti-EGFR monoclonal antibodies; this model has been used to demonstrate activity of EGFR kinase inhibitors in a chemotherapeutic setting. Determine chemopreventive eficacy in vivo. 7,12-Dimethylbenz[a]anthracene (DMBA)-inducedcancer in the hamster buccal pouch has been suggested as a model for human head and neck cancers. Overexpression of EGFR in this model has been observed in both dysplastic lesions and squamous cell carcinomas. Pending validation of the dependence of these lesions on EGFR for tumor growth, this model may be especially useful to assess the chemopreventive effects of EGFR kinase inhibitors. Tests for chemopreventive efficacy in other established animal tumor models where high levels of proliferation are particularly important to carcinogenesis-for example, rat bladder, colon, and mammary gland and mouse bladder-may logically follow (see Table VI, in Section III,E,3,a).

b. ras Famesylation Inhibitors The ras genes code for 21-kDa proteins that belong to a large superfamily of GTP-binding proteins which cycle between an active GTP-bound and an inactive guanosine diphosphate (GDP)-bound state. Four human rus

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genes have been identified: H-ras, K-ras-4A, K-ras-4B, and N-ras (Lowy and Willumsen, 1993). Normal Ras proteins serve as molecular switches in the mitogenic signal transduction pathway and regulate many physiological functions including cell growth and differentiation. Activating mutations in ras genes result in proteins which are “stuck” in the active GTP-bound state and constitutively transmit growth signals (Lowy and Willumsen, 1993; Khosravi-Far and Der, 1994). Oncogenic activity can also result from overexpression of normal Ras proteins (Barbacid, 1987). Oncogenic mutations in ras genes have been identified in a variety of human tumors (Bos, 1989). Because Ras proteins are central connectors between signals generated at the plasma membrane and nuclear effectors, disrupting Ras signaling has significant potential as a cancer chemopreventive straiegy. Possible methods include lising antisense oligonucleotides that block mutant ras genes or disrupting interactions of Ras with downstream effector molecules (Manne et al., 1995). Another strategy is based on the observation that Ras proteins require posttranslational modification with a farnesyl moiety for oncogenic activity (Khosravi-Far et al., 1992; Schafer and Rine, 1992; Newman and Magee, 1993). Inhibitors of the enzyme that catalyzes this reaction, FPT, should therefore inhibit Ras-dependent proliferative activity in cancerous and precancerous lesions (Gibbs et al., 1994). As for EGFR inhibitors, we have reviewed the rationale for using Ras antagonists as chemopreventive agents, analysis of potential clinically relevant target organs, and identification of inhibitors with specificity toward FPT (Kelloff et al,. 1997b). These are summarized in the following, and primary references to the data cited can be found in our review. Ras proteins can be activated by a variety of signals, including receptor tyrosine kinases. The most extensively studied pathway is activation by EGFR. Several potential downstream targets of Ras have been identified, most significantly the protein Raf. Activated Ras recruits Raf to the plasma membrane resulting in a series of phosphorylation and activation events along the mitogen-activated protein kinase (MAPK)cascade and leading ultimately to activation of several transcription factors and their target genes. However, Raf-mediated responses cannot account for all the consequences observed in Rk-activated cells; other possible Ras target proteins include MEKKl (MAPK/ERK kinase), PI(3)K, pl20GAP, RalGDS, and PKCS. Similarly to the potential chemopreventive effects described for EGFR, Ras signaling inhibition in precancerous or cancerous cells constitutively overexpressing the ras transduction pathway should have greater effects on cell growth and proliferation than in normal cells. Association of Deregulated Ras Activity with Carcinogenesis. Activated ras genes have been implicated in carcinogenesis in both animals and humans. Activation is generally associated with mutations at codons 12, 13, and 61. Although overexpression of normal Ras proteins also leads to transforming activity, activating mutations have been observed more consistent-

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ly in human cancers, and interpretation of studies examining overexpression has been complicated by methodological problems leading to inconclusive results. Patterns of YUS mutations may vary across both species and target organs. For example, rus mutations are rare occurrences (<5%) in human breast cancer; however, -90% of N-methyl-N-nitrosourea (MNU)-induced rat mammary cancers carry codon 12 H-rus mutations and -50% also harbor codon 12 K-rus mutations. Azoxymethane (A0M)-induced rat colon tumors, N-nitrosobis(2-oxopropy1)amine(BOP)-induced hamster pancreatic carcinomas, and human cancers at both of these target sites carry a high percentage of K-rus mutations. Similarly, lung adenocarcinomas in humans and sikilar carcinogen-induced lesions in mice have high frequencies of K-rus mutations. H-rus oncogenes are activated in many animal cancers but in only a few human tumors. Different target organs show varied patterns of individual IUS oncogene activation. In humans, most carcinomas (colon, pancreas, lung) harbor activated K-rus genes, whereas N-rus mutations have been associated with myeloid leukemia. Only -15% of total human cancers harbor oncogenic YUS mutations, but higher mutation rates have been observed in specific human neoplasms. Targets for chemopreventive intervention include tissues in which rus activation, generally via mutation, occurs prior to invasion. It has been noted that the timing of these mutations may vary during the carcinogenic process in the same tissue. For example, in the colorectum, activating K-rus mutations often occur early during malignant progression but may also be acquired during the later stages. It has been shown that the various rus gene products (H-, N-, Ka-, Kb-) display distinct affinities for FPT and are inhibited to different degrees by inhibitors of the enzyme. Therefore, for chemopreventive drug development, it is particularly important to note the specific YUS gene(s) activated in the target cancers. Target Organs for Rus Processing Inhibitors: Aberrations in Elements Vpstream of Rus in the Signal Trunsduction Pathway. Additional target organs for Ras-based chemopreventive strategies can also be envisioned based on the central role of Ras proteins in the signal transduction pathway. Activation of an upstream element(s), such as growth factors and their receptors, could lead to deregulated signaling via Ras proteins. Blocking signals at the level of Ras protein may provide a viable means of inhibiting such deregulated upstream elements (e.g., cancers associated with deregulated signaling via EGFR in lung, cervix, and prostate, p185c-eybB-2 in breast and ovary, and PDGFR in glioblastomas). Importantly, based on this hypothesis some of the major target organs that do not harbor rus mutations, such as breast cancers, may be amenable to chemopreventive strategies that block Ras activity. Furnesyl Protein Trunsferuse and Related Prenyl Trunsferuses. Two types

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of cellular prenyl group transfers are the most common and involve transfer of a C,, farnesyl or a C,, geranylgeranyl moiety to a cysteine residue via a thioether linkage. Prenylated proteins share characteristic C-terminal sequences, which include the C & X , XXCC, and XCXC motifs (where C is cysteine, A is usually an aliphatic amino acid, and X is another amino acid). Three enzymes that catalyze protein prenylation have been identified: FPT, geranylgeranyl transferase (GGTase) I, and GGTase 11 (also called Rab GGTase). GGTase 11modifies proteins ending in XXCC and XCXC. Until recently, the CAAX tetrapeptide was believed to be the minimum region required for interaction of protein substrates with FPT or GGTase I, with the last residue of the CAAX motif directing enzyme specificity. However; newer studies suggest that enzyme specificity is more complex. Both enzymes form stable noncovalent complexes with farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) when a protein acceptor is not present. FPT only transfers the farnesyl moiety, whereas GGTase will transfer either the farnesyl or geranylgeranyl moiety depending on which protein acceptor is present. The four cellular Ras proteins are substrates for FPT. In addition, at least eight other cellular proteins undergo farnesylation: nuclear lamins a and b, the Ras-related proteins Rap2 and RhoB, phosphorylase kinase, rhodopsin kinase, cyclic GMP phosphodiesterase a,and the y subunit of transducin. The latter three proteins are involved in vision. GGTase I substrates include the y subunit of mammalian G proteins, Rapl, and CDC42. GGTase I1 prenylates many Rab proteins, which are involved in protein secretion and endocytosis. Requirements for Specific Ras Farnesylution Inhibitors. Requirements for Ras farnesylation inhibitors include the following: specificity for FPT compared with GGTases, particularly, GGTase I; specificity for FPT compared with other FPP-utilizing enzymes; ability to specifically inhibit processing of mutant K-rus (the most commonly mutated ras gene in human cancers); high potency; selective activity in intact cells; activity in vivo; and lack of toxicity. FPT inhibitors may be classified on the basis of proposed mechanisms of action: (1)FPP competitive inhibitors; (2) CAAX competitive inhibitors; (3) bisubstrate inhibitors; and (4) inhibitors with unknown mechanism(s). On the bHsis of their activity in in vitro and in vivo screening assays, some may have chemopreventivepotential. It should be noted that FPT inhibitors which do not meet all the requirements set forth above may still be viable chemopreventive drugs under appropriate circumstances. Most currently available animal data have been obtained in models relevant for establishing chemotherapeutic effectiveness, that is, tumor cell growth inhibition in vivo. Because changes in Ras-mediated signaling occur during the process of carcinogenesis, (in)effectiveness toward established tumors may not accurately predict (in)activity toward precancerous lesion growth and development. This differential effectiveness has been demonstrated, for

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example, with retinoids, which are well-known chemopreventive agents but have generally been ineffectual against established tumors in animal models. Approach to Developing Ras Famesylation Inhibitors as Chemopreventive Agents. Several in vitro and in vivo assays would appear to be useful in evaluating the potential of Ras farnesylation inhibitors as chemopreventive agents. On the basis of differences in the affinities of the Ras proteins for FPT, it is particularly important to establish inhibitory activity toward KRas, the form of Ras most often mutated in human cancers. A possible testing strategy is outlined below. Priorities for further development would be based on results at each step in the order following. Determine inhibition of FPT activity. Inhibitory activity can be determined by measuring incorporation of [3H]FPPinto Ras proteins or Ras-related peptides in a reaction catalyzed by isolated or recombinant FPT. Evaluate selectivity for FPT. The selectivity of the inhibitor toward FPT relative to GGTases can be measured in vitro using GGTases I and I1 isolated from several sources such as bovine brain; recombinant human GGTase I can also be used. Inhibition of GGTase I and II activity can be measured via incorporation of [3H]GGT into Ras-CAIL (Cys-Ala-Ile-Leu) and YptGGCC, respectively. If the drug is competitive with respect to FPP, selectivity toward FPT relative to squalene synthase should also be determined. Determine inhibition o f Ras-mediated effects in intact cells. Selective inhibition of Ras processing and effects on Ras-mediated proliferation and transformation should be examined in whole cells. Specificity for FPT can be established by comparing prenylation inhibition of farnesylated and geranylgeranylated proteins in the presence of [3H]mevalonate. Often these experiments are performed in the presence of a P-hydroxy-P-methylglutarylcoenzyme A [HMG-CoA] reductase inhibitor to prevent isotopic dilution of [3H]mevalonate. Because cells are relatively impermeable to mevalonate, Met-18b-2 cell (CHO cells with efficient mevalonate uptake) or cells transfected with cloned mutant Mev cDNA, which facilitates cellular uptake, can be used. Inhibition of ras-mediated cellular effects, such as inhibition of anchorage-independent and -dependent growth, and reversal of morphological transformation should be established. Determine inhibition of Ras-mediated effects in vivo. Activity of FPT inhibitors on the growth of Ras-dependent tumors can be evaluated in nude mice injected with transformed rodent or human cells carrying mutant ras genes. Determine chemopreventive eficacy in vivo. Potentially, mouse lung is the most efficient model for evaluating FPT inhibitors. In A/J (or A/J F1) mice, virtually all chemically induced tumors have mutated K-ras. Using Nnitrosonornicotine ( N N K )as the carcinogen, >90% of lung tumors have Kras and -50% of liver tumors have Ha-ras mutations. The relatively small size of the mice and high tumor multiplicity allow testing with small amounts I

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of drug. Alternative models are AOM-induced rat colon tumors and BOPinduced hamster pancreatic carcinomas. Both are considered good models for human cancers at these target sites and carry high percentages of K-ras mutations. Further, K-ras mutations have clearly been associated with the development of human cancers at these target sites. A caveat is that K-ras is preferentially activated by geranylgeranylation, not farnesylation. Tests for chemopreventive efficacy in other established animal tumor models where high levels of proliferation are particularly important to carcinogenesis, for example, rat and mouse bladder, may logically follow. c. Retinoids Vitamin A (comprising retinol and its esters,'with fatty acids, 3,4-didehydroretinol, and retinal) is required for growth and bone development, vision, reproduction, epithelial tissue differentiation, immune system integrity, and biochemical reactions such as mucopolysaccharide synthesis, cholesterol synthesis, and hydroxysteroid metabolism (McEvoy and McQuarrie, 1994). Vitamin A is not synthesized by humans but is supplied in diet or supplements, for example, as retinyl esters in animal foods such as eggs, whole milk, butter, meat, and fish liver oils or 3,4-didehydroretinol (i.e., vitamin A2) in freshwater fish (McEvoy and McQuarrie, 1994; Futoryan and Gilchrest, 1994). Also, dietary provitamin A carotenoids (e.g., a-carotene, @carotene, cryptoxanthin) found in green and yellow vegetables and fruits are intestinally converted to retinal (Kelloff et al., 1994b). Vitamin A and its analogs (retinoids) have shown activity against many carcinogenesis-associatedmolecular targets, primarily in signal transduction pathways. At the cellular level, the retinoids act primarily by inhibiting proliferation (Verma, 1991) and inducing differentiation (e.g., Mehta et al., 1989; Ioaniddes et al., 1990). Many of these effects are likely mediated by interaction with retinoid receptors belonging to the superfamily of steroid and-hormone receptors (Gudas et al., 1994). All these receptors function as transcription factors that regulate the expression of specific genes. Changes in gene transcription have been associated with retinoid chemopreventive functions: modulation of intercellular (connexin) (Hossain et al., 1993; Bex et al.; 1995) and intracellular signaling (PKC, ODC); growth factor (TGFP, TGFa, EGF, IGF-I), receptor (EGFR) (Gudas et al,. 1994), and oncogene expression (e.g., Prasad and Edwards-Prasad, 1990); modulation of hormones (progesterone) and immune response (Ross and Hammerling, 1994); altered extracellular matrix (collagen, angiogenesis) and proteolytic enzymes (Gudas et al., 1994); and inhibition of viral replication (Gudas et al., 1994). The differences in activity among the retinoids may result from differences in binding affinitieszo receptors. Retinoid receptors are classified into two subfamilies, RARs (a,p, y ) and RXRs (a, p, y), on the basis of differences in primary structure, sensitivity to synthetic retinoid ligands, and ability to reg-

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ulate expression of different target genes (Heyman et al., 1992).The known retinoid receptors have little affinity for retinol, but specific subfamilies bind 9-cis-retinoicacid (RARs, RXRs), all-trans-retinoic acid (RARs),and 1 3 4 s retinoic acid (RARs). For example, all-trans-retinoic acid is a more potent ligand for RARy than the other two isoforms, and it has been shown specifically to induce secretion of TGFP in normal and human papillomavirus (HF’V)-immortalizedcells (Batova et al., 1992) and to decrease expression of c-myc and c-erbB (Prasad and Edwards-Prasad, 1990). All-tram-retinoic acid binds with high affinity to, and transcriptionally activates, RARs (Mangelsdorf et al., 1994). However, although all-trans-RA can activate RXRs to regulate expression of target genes, it is not a ligand f i r the RXR subfamily of receptors (Mangelsdorf et al., 1994). The RAR transcriptional profile for 13-cis-retinoicacid was found to be similar to that of all-trans-retinoic acid, with potency for RARy being 10-fold greater than the other two isoforms (Dawson et al., 1993). However, specific effects in vivo are also related to differences in receptor distribution between tissues and the pharmacokinetics of the retinoid. Rat tissues with the highest R A R y expression include trachea, lung, bladder, and skin (Verma and Denning, 1995). In humans, most of the RAR receptors in skin are RARy; significant expression is also found in lung (Mangelsdorf, 1994; Futoryan and Gilchrest, 1994). The association of RARs with carcinogenesis has been described by Hong (e.g., Hong and Sporn, 1997). In fact, retinoids such as 13-cis-retinoic acid, which have high affinity for RARy, show chemopreventive activity in these targets. RARP expression is often lost in lung and head and neck cancers, and Hong has shown that 13-cis-retinoic acid can restore the expression of RARP,and induce regression of oral leukoplakia (Lotan et al., 1995). Unfortunately, the typical retinoid toxicities have also been associated with binding to RARs. More recently, 9-cis-retinoic acid was identified as a high affinity ligand for the RXRs (Heyman et al., 1992); it is up to 40 times more potent than all-trans-retinoic acid in transactivating RXRs. 9-cis-Retinoic acid also binds to and transactivates RARs, serving as a “bifunctionalyyligand (Mangelsdorf et al., 1994). The RXRs can form homodimers and are capable of acting independently; however, they also form stable heterodimers with vitamin D, thyroid hormone, and peroxisome proliferation-activating receptors (PPAR) (Tate et al., 1994), as well as RARs. These interactions result in positive or negative regulation of transcription. The ability of RXRs to heterodimerize with receptors responsive to several ligands suggests a central role for RXRs in hormonal signaling, and a retinoid signaling pathway distinct from that mediated by the RARs (Mangelsdorf et al., 1994). Together with the observation that the tissue distribution of RXRs and RARs differs (Mangelsdorf et al., 1994), this suggested that 9-cis-retinoic acid, as the high-affinity lig-

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and for the RXRs, might have distinct chemopreventive properties. That is, since 9-cis-retinoic acid can be delivered exogenously as well as being formed metabolically from all-trans-retinoic acid, interconversion between 9-cisretinoic acid and all-trans-retinoic acid could afford a novel method for differential, cell-specific regulation of the two retinoid signaling pathways. LG100268 and targretin bind and activate all three RXRs, but they have essentially no affinity to the RARs. These agents are being developed with the intent of taking advantage of the chemopreventive activity afforded by the RXR-mediated pathway, while avoiding toxicity possibly related to the RAR pathway (e.g., Hong and Sporn, 1997). Fenretinide is a synthetic amide of all-trans-retinoic acid. As a retinoid, fenretinide is an antiproliferative and differentiation-inducing agent when administered during the promotion and progression phases of carcinogenesis (e.g., Delia et al., 1993), although so far it has not been shown to bind retinoic acid receptors. It has been specifically shown to inhibit ODC activity induction and PG synthesis, as well as to enhance immune responses, modulate protein kinase activity and cytoskeletal organization, decrease circulating IGF-I, and induce apoptosis (reviewed in Kelloff et al., 1994~). Rationale for Development of Retinoids as ChemopreventiveAgents. The naturally occurring retinoids described above, which have varying vitamin A activity and are produced during metabolism of retinol (all-trans-retinoic acid, 9-cis-retinoic acid, and 13-cis-retinoicacid) have shown chemopreventive efficacy in animal models, and have shown efficacy or are on test in clinical chemoprevention studies (reviewed in Kelloff et al., 1996c,d,e). For example, 9-cis-retinoic acid has inhibited rat mammary gland (see also Gottardis et al., 1996)and prostate carcinogenesis, all-trans-retinoic acid has shown clinical efficacy in treating CIN, and 13-cis-retinoic acid has prevented second primary cancers in patients treated for head and neck cancers. Hundreds of synthetic retinoids have been made with the objective of retaining efficacy while eliminating characteristic retinoid toxicities (skin dryness, cheilitis, hypertriglyceridemia, conjunctivitis). Fenretinide has demonstrated chemopreventive activity in animal models in bladder, skin, breast, lung, oral cavity, and prostate, and it is now being evaluated in Phase 11and III ilinical trials at these cancer targets (Kelloff et al., 1994~).In breast, preliminary data show that it inhibits second primary tumors in premenopausal patients treated for breast cancer, and results suggest that it inhibits ovarian cancer (De Palo et al., 1996). An acyclic retinoid (E-5166) has shown clinical chemopreventive activity in liver cancer (Muto et al., 1996), and LG100268 and its congener targretin have prevented rat mammary gland cancer (Hong and Sporn, 1997). Retinoic acid metabolism blocking agents (RAMBAs) are also being considered for evaluation as chemopreventive agents, particularly in prostate. Retinoids (fenretinide, 9-cis-retinoic acid) have shown synergistic chemopreventive efficacy with antiestrogens

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(tamoxifen, raloxifene) in carcinogen-induced rat mammary gland and prostate carcinogenesis (Moon et al., 1992; Anzano et al., 1994, 1996; Lucia et al., 1995). Overall, the epidemiological evidence that retinoids inhibit human carcinogenesis is compelling, although it is not clear-cut since it comes largely from uncontrolled studies (reviewedin Kelloff et al., 1996f, and summarized here). Vitamin A deficiency is well known to cause carcinogenesis-associated histological changes (squamous metaplasia, hyperkeratinization) in many epithelial tissues (respiratory, gastrointestinal, genitourinary). Epidemiological studies to test this effect showed direct relationships between low dietary and serum P-carotene (provitamin A) and development of cancers of the lung, cervix, ovary, esophagus, larynx, oral cavity, and nasopharynx. Low dietary intake of preformed retinol has been associated with increased risk for breast and colon cancer in prospective studies and with lung and prostate cancer in retrospective studies. Serum retinol has been inversely associated with risk for gastrointestinal cancer in prospective studies and with sarcoma and lung, prostate, bladder, and ovarian cancer in retrospective studies. It should be noted that some studies are inconsistent with these results, maybe resulting from the fact that serum retinol does not reflect dietaryhpplement intake in well-nourished populations where it is stored in the liver. Approach to Developing Retinoids as Chemopreventive Agents. Several factors are important in selecting retinoids for development. Determine specificity for retinoid receptor isoforms. As described above, binding and activation of RARs and RXRs may be useful in determining chemopreventive efficacy and toxicity and thus, would be a first step in evaluating and prioritizing novel retinoids. Currently, very potent activators of RARs (RARP) and specific activators of RXRs would appear to have the highest promise for use as chemopreventive drugs. Determine profile o f activity against molecular biomarkers of carcinogenesis. Retinoids are pleiotropic modulators of signal transduction pathways, and they also may block carcinogen activation. A panel of cell-based assays measuring effects against specific molecular markers, inhibition of malignant transformation, and modulation of cell-based effects (proliferation, apoptosis, and differentiation) should provide sufficient background for selecting retinoids for further evaluation in animal carcinogenesis models. A panel of tests in tumor cells (HL60, F9) has been used for more than 20 years to determine the potency of retinoids in inducing differentiation. Determine chemopreventive eficacy in vivo. Those retinoids appearing most effective and least toxic in vitro would be evaluated in animal carcinogenesis models. To some extent, specificity could be estimated from the receptor and in vitro testing profiles compared with previously tested retinoids. Typically, since most of the retinoids have shown some chemopreventive activity in breast cancer models, rat mammary gland assays would be a first

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choice. On the basis of the available clinical studies, retinoids that modulate RARP would be tested in animal lung models. Conduct special toxicity studies. Previous data (probably predictable from the potential for cross talk with other members of the steroid receptor family) suggest that retinoids should be evaluated for teratogenicity early in their development. 2. HORMONE MODULATORS

Epidemiologicaland experimental evidence strongly supports a role for estrogens in the development and growth of breast tumors. A role for estrogen in prostate neoplasia has also been postulated. Therefore, one chemopreventive strategy for breast and prostate cancers is to decrease estrogen production and activity, particularly in these tissues. Association of Estrogens with Carctnogenesis in Breast. The most consistently documented risk factors for breast cancer-early age at menarche, late age at menopause, late age at first full-term pregnancy, and postmenopausal weight gain-all increase cumulative endogenous estrogen exposure (Henderson et al., 1988). Although early studies were unable to establish a link between breast cancer risk and endogenous hormone levels, more recent investigations have found a strong relationship (see Feigelson and Henderson, 1996). In vivo and in vitro experimental data also clearly indicate a role for estrogens in breast cancer development. Animal studies have shown that estrogens can support initial development and promote the growth of mammary cancers in rodents (Feigelsonand Henderson, 1996). The proliferative effects of estrogen on breast cancer cells are firmly established and have been associated with upregulation of various growth factors (e.g., TGFol, amphiregulin, and IGF-11) and growth factor receptors(e.g., EGFR, IGF-I receptor) and downregulation of TGFP (Feigelsonand Henderson, 1996). Estrogens also modulate expression of proliferation-associated oncogenes such as c-myc, c-fos, and c-jun in breast cancer cells (reviewed in Feigelson and Henderson, 1996). Although the available data are not conclusive, there is alsd evidence that estrogens may be directly genotoxic, for example, by promoting DNA alkylation or oxidation leading to free radicals, which in turn bind to and damage DNA (e.g., Lemieux and Fuqua, 1996). Some evidence suggests that upregulation of the estrogen receptor (ER) may be a risk marker for breast cancer. Normal breast epithelial cells generally express low ER levels (dependent on the phase of the menstrual cycle) (reviewed in King, 1993). ER levels are higher in precancerous breast tissue adjacent to breast cancers than in breast tissue from benign breast disease controls (Khan et al., 1994). Several studies also suggest that ER expression increases early in carcinogenesis then diminishes during later stages. For ex-

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ample, increased ER expression has also been observed in atypical ductal hyperplasia (ADH) compared with more advanced stages of breast carcinogenesis (Barnesand Masood, 1990),whereas high-grade (grade I11 nuclei and necrosis) DCIS express lower levels of ER than low-grade (nonnecrotic) lesions (Lagios, 1996). Increased ER expression may facilitate carcinogenesis by making breast epithelium susceptible to estrogen’s proliferative effects, thereby increasing its susceptibility to mutagenicity (Khan et al,. 1994; Lemieux and Fuqua, 1996). Although further supporting data are needed, loss of ER expression during tumor progression suggests that early precancers may be more sensitive to the chemopreventive activity of antiestrogens than later stages of carciiogenesis when lower levels of ER bring less estrogen responsiveness. Association of Estrogens with Carcinogenesis in Prostate. Estrogens have also been postulated to play a role in the development of prostate cancer. High-affinity ERs (like those found in the breast) have been identified in normal, benign, and cancerous human prostatic tissues. Most studies have localized the ER to the stromal compartment, but estrogen binding sites have also been reportedly found in epithelial cells (Carruba et al., 1996). In dogs, estrogens synergize with androgens and result in complex stromal and glandular hyperplasia. This effect appears to be due to an estrogen-mediated increase in stromal and epithelial androgen receptor levels (Trachtenberg et al., 1980), although the induction of benign prostatic hyperplasia (BPH)as a result of injury by estrogen metabolites, followed by Sa-dihydrotestosteronestimulated growth of altered prostatic cells, has also been postulated (Winter and Liehr, 1996). Increased levels of estrogen have been found in BPH stroma compared with BPH epithelium and normal prostate epithelium and stroma (Trachtenberg et al., 1980). In both normal prostate and BPH, stroma1 estrogen levels increase with age, resulting in an increased estrogen:androgen ratio. These results suggest that estrogen is involved in the development of BPH, but clinical trials of aromatase inhibitors in BPH patients have not provided clear-cut evidence (Etreby and Habenicht, 1994; Gingell et al., 1995). In the Noble rat, prostate dysplasia can be induced by simultaneous treatment with testosterone and estradiol for 16 weeks, but not by treatment with testosterone or Sol-dihydrotestosterone alone (Leav et al., 1988). Long-term treatment of Noble rats with testosterone induces a low incidence of adenocarcinomas in the dorsolateral prostate (Noble, 1977), whereas treatment with testosterone and estrogen significantly increases carcinoma incidence and decreases tumor latency. The mechanism of estrogen action on the rat prostate is unknown. No effect of estrogen on prostate androgen receptor levels was observed in estradiol- and testosterone-treated Wistar rats (Suzuki et al., 1995). In Wistar rats, estrogen treatment increased nuclear Sol-reductase activity, although microsomal reductase activity was decreased

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(Suzuki et al., 1994).Treatment of Noble rats with estradiol and testosterone was shown to result in a unique DNA adduct in the dorsolateral prostate (the tissue where carcinomas originate in this model), coincident with presumed preneoplastic lesions, but the structure and mechanism of its formation are unknown (Han et al., 1995). This adduct may play a role in carcinogenesis in this model. Interestingly, Spencer et.al. (1995) also identified DNA adducts in normal human prostate and prostate tumor biopsies. Estrogens have also been associated with increased expression of ras protooncogenes (Yu et d., 1993) and upregulation of ER (as a proliferation marker) (Ho and Yu, 1993) in dysplastic dorsolateral prostatic tissue in rats. It has been proposed that normal controls on prostatic cell proliferation can be overcome by androgen-supported, estiogen-enhanced stimulation of prostatic epithelium (see Carruba et al., 1996). Estradiol has been shown to stimulate the growth of an androgen-responsive human prostate cancer cell line (LNCaP) and to inhibit the growth of an androgen nonresponsive line (PC3) in vitro. The detection of ERs in the androgen-responsive, human prostate cancer LNCaP cell line is paralleled by a highly intense staining for progesterone receptors, suggesting that these ER are functional (Castagnetta et al., 1995). In these cells, estradiol significantly increases growth, as well as expression of prostate specific antigen (PSA) (Castagnetta et al., 1995). However, estradiol inhibits the growth of the androgen-nonresponsive PC3 cell line, which may partially explain the therapeutic effects of estrogens in advanced prostate cancer. a. Antiestrogens

Antiestrogens compete with estrogen for ER binding. These agents, particularly the nonsteroidal triphenylethylene tamoxifen, have successfully been used for treatment of breast cancer, and recently tamoxifen has demonstrated chemopreventiveactivity against breast cancer (i.e., to reduce the risk of breast cancer in women at high risk) (Fisher et al., 1998). The observation that nonsteroidal antiestrogens selectively inhibit or mimic estrogen action in a tissue-specific manner has established the search for additional selective estrogen receptor modulators (SERMs) that act as estrogen antagonis’ts in the breast and uterus and agonists in the skeletal and cardiovascular systems (Grese and Dodge, 1996). Estrogen receptor is a member of the large superfamily of steroid nuclear receptors that act as ligand-activated transcription factors (reviewedin Katzenellenbogen, 1996). Estrogen binding results in a conformational change in the ER, receptor dimerization, and subsequent interaction with the estrogen response element (ERE) in DNA. One explanation of the activity of antiestrogens is-that they compete with estrogen for binding to the ER but do not cause gene transcription. However, many nonsteroidal antiestrogens, such as tamoxifen, have agonistic activity in some tissues, suggesting that the

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mechanisms involved are much more complex. For example, site-directed mutagenesis and deletion experiments have shown that the ER has six functional domains, at least two of which (AF-1 and AF-2) are directly involved in estrogen-inducible gene transcription. Transactivation of AF-1 is hormone-independent, whereas that of AF-2 is hormone-dependent. The tamoxifen-ER complex binds to the ERE as a dimer and blocks hormonedependent transcriptional activation by the AF-2 region but has little effect on AF-1. The response to partial agonists in various cell types appears to be due, at least in part, to the degree to which that response is mediated by AF2. The mechanisms involved in the actions of the pure steroidal antiestrogens, such as ICI 164,383, are quite different and include inhibition of rece$tor dimerization, inability to activate either AF-1 or AF-2, degradation of the ER, and interference with nucleocytoplasmic ER shuttling. In addition to the ERE, other ER responsive elements have been identified that may also contribute to the tissue-selective actions of both estrogens and antiestrogens. Binding to the ER of certain estrogen metabolites or the benzothiophene-type antiestrogen raloxifene activatestranscription of the TGFP3 gene, an important regulator of bone remodeling. These effects are not mediated by the ERE but by another DNA sequence called the raloxifene response element (RRE). Activation of the RRE by raloxifene may contribute to the drug’s bone preserving effects (Yang et al., 1996). The AP-1 transcription site is also involved in gene regulation by estrogens (Parker, 1995). It is activated by both estrogen and tamoxifen in uterine tissue, but not raloxifene, and may help explain why tamoxifen stimulates proliferation of the uterus while raloxifene does not (Yang et al., 1996). Gustafsson (Couse et af,. 1997) has isolated and cloned a second ER subtype, ERP, which is not found in mouse or rat mammary glands or human breast but is seen in prostate and many other tissues affected by estrogenovary, testis, and the cardiovascular and central nervous systems. ERP has high affinity for estradiols and thus probably mediates many estrogenic effects in these targets. This receptor subtype differs from classic ER (now called ERa) in sequences in both the C-terminal ligand and the N-terminal transactivation domain. Thus, besides different tissue distribution, ERP shows different ligand binding affinities for various SERMs, leading Gustafsson and colleagues to suggest that ERP-specific agents may be useful in treatment of postmenopausal symptoms, and breast and prostate cancer, and may be without many of the side effects associated with ERa-specific agents. Increasing evidence suggests cross talk between ER-mediated and other signal transduction pathways. The transcriptional response can be regulated by factors such as CAMP, growth factors, agents that affect protein kinases and phosphorylation (Katzenellenbogen, 1996), and retinoids (Gottardis etaf., 1996).The influence of antiestrogens on the ER phosphorylation status may also contribute to their tissue-selective effects (Katzenellenbogen,

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1996). Tamoxifen displays therapeutic activity in about 10% of ER negative breast cancer patients (Tonetti and Jordan, 1996) suggesting that non-ERmediated targets of antiestgogens may exist. Pharmacological actions of tamoxifen and/or other nonsteroidal antiestrogens that appear to be ER-independent include inhibition of PKC, calmodulin antagonism, changes in membrane structure/function, antioxidant activity, and inhibition of cholesterol biosynthesis (reviewed in Kelloff et al., 1994d; Tonetti and Jordan, 1996). Approach to Developing Antiestrogens as Chemopreventive Agents. The major classes of synthetic antiestrogens include triphenylethylenes (e.g., tamoxifen, toremifene), benzothiophene derivatives (e.g., raloxifene, SERM3), and substituted estradiol derivatives (ejg., ICI 182,780). As indicated *above, the nonsteroidal antiestrogens have repeatedly demonstrated both antagonist and agonist activity that is both species- and tissue-specific.Those SERMs that are antagonists in breast and uterus but agonists in bone and cardiovascular system have been suggested to be ideal candidates for hormone replacement therapy which might also protect against breast cancer. Also as described above, steroidal antiestrogens (e.g., ICI 164,384, ICI 182,780, EM 800) are generally considered to be pure antiestrogens, that is, lacking any estrogenic activity (Wakeling, 1995). Steroidal derivatives have limited oral bioavailability, and because their activity is not tissue-specific, negative impact on bones and cardiovascular system are of particular concern. Determine estrogen receptor modulation in vitro. The ER+ human breast cancer cell line MCF-7 is the most commonly used cell culture system for screening antiestrogens (e.g., Kitawaki et al., 1993).Assays for assessing ER binding, ER-dependent transcriptional expression, and reproductive tract response to estrogens have been reviewed (Reel et al., 1996). Induction of TGFPl by antiestrogens has been proposed to contribute to their antiprolifemive effects in the early stages of tumorigenesis (Wakefield et al., 1995), and to the beneficial effects on osteoporosis, atherosclerosis, and autoimmune diseases. Screening for TGFP induction should be included in evaluation of new antiestrogens. Determine chemopreventive eficacy in vivo. Models for hormone-dependent premenopausal breast cancer include the MNU- and DMBA-induced Sprague-Dawley rat mammary models (see Table VI). Both systems have been used to screen antiestrogens, but MNU-induced tumors better model human breast cancer. Older (120 day) rats may be more useful models for postmenopausal breast cancer. Models for prostate cancer are the testosterone- and estradiol-treated Noble rat, and the male Wistar rats treated with cyproterone acetate, MNU, and testosterone, described above. A third model which is being developed is the Rao model, in which Noble rats are treated with testosterone prior to MNU, and then with testosterone and estradi-

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01 for the duration of the study. The biology and histology of this new model are less well defined than the other two models, but the induction of tumors in accessory sex glands is more rapid. Other considerations. In evaluating antiestrogens as candidates for chemoprevention studies, emphasis is placed on their ability to inhibit cancer development, to positively affect the skeletal and cardiovascular systems, and to avoid the negative factors associated with tamoxifen treatment (e.g., hepatocarcinogenicity, uterotrophic effects). Tamoxifen now sets the standard for chemopreventive antiestrogens, and both therapeutic effects (preclinical and clinical) and toxicity profiles of new antiestrogens should be compared with tamoxifen in making decisions for development. Cross talk between signaling pathways suggests that combinations of agents may be particularly effective for controlling cellular growth. The synergistic activity of retinoids with antiestrogens was discussed above. Protein kinase activators enhance ER transcriptional activity and influence the agonist/antagonist actions of some antiestrogens. The observation that alterations in cellular phosphorylation may be important determinants of the activity of antiestrogens suggests that combinations of agents which affect phosphorylation status may lead to enhanced efficacy. The tissue-selective action of antiestrogens provides the rationale for development of SERMs that would both prevent breast cancer and serve as hormone replacement. An ideal agent for this use should exhibit estrogenic effects in the liver (cardiovascular effects), bone, and central nervous system (no hot flashes) and antiestrogenic effects in the breast and uterus. Clearly, the successful development of S E W S will depend on a better understanding of the molecular mechanisms involved in regulation of the response to both estrogens and antiestrogens in different cell types. Estrogen activation at the AP-1 site and the RRE, in addition to the ERE, the presence of various ER-interacting proteins, selective mediation of response by AF-2 transactivation, cross talk between signal transduction pathways, and the presence of ER variants suggest multiple and intricate layers of regulation. The potential for non-ER-mediated biological actions of antiestrogens to influence pharmacological response adds additional layers of complexity.

b. Arornatase Inhibitors Estrogen production can be decreased by inhibiting aromatase, the cytochrome P450 catalyzing the fmal, rate-limiting step in estrogen biosynthesis (Cole and Robinson, 1990). The use of aromatase inhibitors is of clinical interest for cancer therapy, and selective, potent aromatase inhibitors have been developed. As for EGFR and YUS FPT inhibitors, we have recently reviewed the rationale for and development of aromatase inhibitors as chemopreventive agents (Kelloff et al., 1998). These subjects are summarized in the following, and primary references to the data cited can be found in our review.

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Association of Aromatase Activity with Carcinogenesis in Breast. Aromatase is expressed in several tissues in women. In premenopausal women, the granulosa cells of ovarian fpllicles produce the majority of circulating estrogen, primarily in the form of estradiol. Estrogen is also produced extragonadally in liver, muscle, and fat by aromatization of adrenal androgens. After menopause, adipose tissue is the major source of circulating estrogens. Extragonadal production of estrogen primarily involves aromatization of adrenal androstenedione, resulting in estrone, a weaker estrogen than estradiol. The estrogen that stimulates tumor growth can be derived from extratumoral or tumor sources, and the relative importance of each source is controversial. Within the breast, adipose tissue isthe major extratumoral source .of aromatase, although aromatase was also detected immunocytochemically in normal breast epithelial cells in one study. Aromatase activity is higher in adipose tissue from breast cancer patients than in,those with benign breast disease. In breast cancer, aromatase expression and activity are higher in quadrants bearing tumors than those without tumors. The exact cellular localization of aromatase expression in breast cancer tissue is also somewhat controversial. Immunocytochemical studies have detected aromatase in breast carcinoma cells and in stromal spindle cells in breast tumors. Thus, it appears that both adipose and breast tumor cells may contribute to locally high estrogen production. Evidence supporting the use of aromatase inhibitors in breast cancer prevention comes from clinical studies in which aromatase inhibitors cause tumor regression in postmenopausal breast cancer patients. In experimental mammary cancer models, aromatase inhibitors both induce regression of established tumors and, more importantly, prevent cancer development. Association of Aromatase Activity with Carcinogenesisin Prostate. In men, 10-25% of estrogen is synthesized locally in the testes and 75-90% arises from extraglandular aromatization of testosterone and androstenedione. The estrogedandrogen ratio increases with age, presumably due to greater estrogen synthesis accompanied by unchanged or decreased androgen production. Whether the prostate is a source of estrogen is controversial. Some studies have reported an absence of aromatase in normal prostates and BPH. Others have variously reported aromatase activity in normal prostate and BPH, and in both BPH and prostate cancer cells. Aromatase Activity and Regulation. Aromatase is an enzyme complex localized in the endoplasmic reticulum that consists of a specific cytochrome P450 heme protein and a flavoprotein NADPH cytochrome P450 reductase. It catalyzes the synthesis of estradiol from testosterone and estrone from androstenedione. -Three separate hydroxylation steps are catalyzed by the enzyme, which requires three moles of molecular oxygen for each mole of C!, androgen converted to C,, estrogen. Importantly, because aromatization IS

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the last step in steroid biosynthesis, selective inhibition of the enzyme should not disrupt production of other steroids such as adrenal corticoids. Although the translated region of the aromatase gene is identical among different tissues, the untranslated regions and regulatory control appear to be tissue-specific. At least four major promoter sites have been identified which respond to gonadotropins, glucocorticoids, growth factors, and cytokines. A unique transcriptional promoter of aromatase gene expression, 1.4, has been identified in breast adipose tissue. Aromatase Inhibitors. Both nonsteroidal and steroidal aromatase inhibitors have been developed. Nonsteroidal inhibitors (e.g., aminoglutethimide, fadrozole, anastrozole, letrozole, vorozole) act by binding to a prosthetic henie group on the enzyme. However, since this heme group is present on all members of the cytochrome P450 superfamily, these inhibitors may lack specificity and thus inactivate other steroidogenic enzymes. Unlike steroidal inhibitors, nonsteroidal inhibitors lack hormonal agonist or antagonist activity and are more likely to be orally absorbed. Steroidal inhibitors (formestane, exemestane, atamestane, plomestane) bind very tightly or irreversibly to the active site of the enzyme and are suicide inhibitors, competing with androstenedione and testosterone for the active site of aromatase. They are then converted to reactive alkylating species by the enzyme, which form covalent bonds at or near the active site, thereby irreversibly inactivating aromatase. Recovery of enzymatic activity depends on the rate of de novo enzyme synthesis. The potential advantages of using suicide inhibitors include potent and sustained inhibitory activity with the possibility of intermittent dosing schedules. Disadvantages include unwanted hormonal agonist (particularly androgen) or antagonist effects. Approaches to Developing Aromatase Inhibitors as Chemopreventive Agents. Goss and Gwyn (1994) have reviewed the model systems used for testing the efficacy of aromatase inhibitors. Characterize aromatase inhibition in vitro. In vitro cell-free studies can be carried out with microsomal aromatase preparations from human placenta or pregnant mare serum gonadotropin (PMSG)-stimulatedrat ovaries. In vitro cell culture systems for screening aromatase inhibitors include the hormone-dependent human breast cancer cell line MCF-7 and human genital skin fibroblasts. Two models using MCF-7 cells transfected with the aromatase gene have also been reported. Characterize aromatase inhibition in vivo. In vivo models for screening inhibitors are similar to in vitro assays; however, compounds are administered to animals rather than incubated with microsomal preparations. Ovarian microsomes isolated from PMSG-primed female rats treated with the drug are generally used to measure radiolabeled water released after in vitro incubation with tritiated androgens. Serum estrogen levels of animals treated with aromatase inhibitors can be ascertained using radioimmunoassays ( U s ) .

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Characterize chemopreventive efficacy in vivo. Testing in chemoprevention models in vivo will be important for evaluating this class of compounds, and the same test systems used for antiestrogens are appropriate for breast and prostate.

3. ANTIINFLAMMATORY AGENTS Many pharmacological, including chemopreventive, effects of antiinflammatories are mediated via inhibition of AA metabolism. The inhibition of COX and LOX pathways have been implicated in chemoprevention. More recently, the accumulation of AA, leading to increased ceramide production and apoptosis, has been implicated as a chemopreventive mechanism for antiinflammatories (Chan et al., 1998). AA accumulation could result from inhibition of COX or LOX, as well as from activation of the phospholipases (e.g., PLA, which is encoded by moml) (Taketo, 1998a,b).

a. Nonsteroidal Antiinflammatory Drugs We and others have reviewed the role of AA metabolism in carcinogenesis (Marnett, 1992; Zenser and Davis, 1992; Kelloff et al., 1995a). Essentially, AA is metabolized to PGs, thromboxanes, leukotrienes, and hydroxyeicosatetraenoic acids (HETEs) via oxidative enzymes. Activated oxygen species and alkylperoxy species are formed throughout this process; AA metabolism is increased during inflammation. There are two components of AA metabolism that have been associated strongly with carcinogenicity. Both can be inhibited by antioxidants and antiinflammatory agents. The first is the PG synthetic pathway and involves the enzyme prostaglandin H (PGH) synthase. This enzyme has two activities: COX, which catalyzes the formation of PGG, from AA, and hydroperoxidase, which catalyzes the reduction of PGG, to PGH,. To return to its native state, the hydroperoxidase requires a reducing cosubstrate, and carcinogens such as aromatic amines have been found to be appropriate substrates. According to the model proposed, during catalysis, the carcinogens produce free radicals and electrophiles that can form adducts with DNA and initiate carcinogenesis. This process can be stopped four ways: (1)at formation of PGG, via inhibition of COX, (2) by inhibition of peroxidase activity, (3)by prevention of formation of reactive intermediates, and (4) by scavenging reactive intermediates (e.g., by GSH conjugation). Relevant to these potential mechanisms, COX inhibitors such as NSAIDS (e.g., aspirin, ibuprofen, indomethacin, piroxicam) and certain antioxidants [e.g., nordihydroguaiaretic acid (NDGA)] are effective inhibitors of carcinogenesis. Additionally, PGH, itself breaks down to form a known direct-acting mutagen, malondialdehyde. Thus, inhibition of COX may directly prevent the formation of a potential carcinogen, as demonstrated by aspirin inhibition of nitrofuran-induced rat bladder carcinogenesis.

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There is also evidence that PG biosynthesis is linked to cell growth control. For example, PGF,a stimulates mitogenesis in Swiss 3T3 fibroblasts and reverses indomethacin inhibition of EGF-dependent proliferation in BALB/ c 3T3 fibroblasts. Additionally, PGF,a stimulates the proliferation of osteoblastic MC3T3-El cells, possibly by enhancing synthesis IGF-I receptors. Evidence shows that PGs are involved in phorbol ester-stimulatedtumor promotion; the phorbols release AA and induce synthesis of PGs. PGE, and PGE, can overcome NSAID inhibition of phorbol ester induction of ODC and mitogenesis. Cyclooxygenase inhibitors may also reduce tumorigenesis by modulating immune response. For example, they may slow tumor growth at an early stage by attenuating the immune suppression that results from PGE, inhibition of blastogenesis of T cells and the cytotoxicity of natural killer (NK) cells. Besides COX inhibition (which appears to correlate best to carcinogenesis inhibition), NSAIDs also have other potentially chemopreventive activities. They can scavenge reactive oxygen species involved in tumor initiation or promotion, and prevent the activation of procarcinogens by inhibiting cytochrome P450 monooxygenases. They might inhibit tumor promotion by inhibiting ODC induction. Use of COX inhibitors such as the NSAIDs sulindac and aspirin has a particularly strong association with prevention of gastrointestinal cancers. In case studies and limited intervention studies patients with FAP or Gardner’s syndrome, sulindac consistently caused regression of existing colonic polyps and prevented formation of new polyps. Epidemiology studies of aspirin use provide evidence of an association with reduced risk of gastrointestinal (esophagus, stomach, colon, and rectum) cancer and colon cancer mortality. In animal studies with NSAIDs, tumor inhibition has been seen with aspirin (colon, bladder), ibuprofen (colon, mammary glands, bladder), indomethacin (bladder), ketoprofen (colon, bladder), piroxicam (colon, skin, bladder), and sulindac (colon, bladder). Despite the potent chemopreventive activity of NSAIDs, their promise for extended chronic use is limited by toxicity. The NSAIDs currently in use all cause adverse effects in a significant number of patients. Most common are gastrointestinal effects such as bleeding and ulcers, which may occur in 30% or more of treated patients (e.g., Singh et al., 1994) and have been attributed to reduced PGE, and, hence, less protective mucus secretion in the gut. Several strategies have been proposed to limit the impact of these side effects, including combinations with other agents such as DFMO to allow use of lower doses [the combination of DFMO with piroxicam has shown potent synergistic activity in rat colon (e.g., Reddy et al., 1990)l. Another is administering a second drug that inhibits associated gastrointestinal toxicity (e.g., the PGE, analog misoprostol). A third is the development of chemical structural or pharmacological

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derivatives of NSAIDs that retain chemopreventive activity while minimizing the side effects. Examples are the sulfone metabolite of sulindac and, as discussed below, selective inhibitors of COX-2.

b. Cyclooxygenase-2 Inhibitors As is now well known, there are at least two isoforms of the COX enzyme, COX-1 and COX-2. These proteins are encoded by separate genes (Yokoyama and Tanabe, 1989; Fletcher et al., 1992; Kraemer et al., 1992; Miller et al., 1994), but they are similar in molecular weight, are approximately 60% homologous at the amino acid level, and those residues important for catalytic activity in COX-1 are conserved in COX-2 (Smith, 1992).The two enzymes have similar K, values for AA and appear to have similar VmaXvalues (Meade et al., 1993a,b). COX-1 is expressed constitutively in most tissues, producing PGs that regulate normal cellular processes such as vascular homeostasis, regulation of renal blood flow, ,maintenance of glomerular filtration rate, and gastrointestinal functions, usually in response to circulating hormones (Klein et al., 1994). In contrast, COX-2 is inducible, and appears to be expressed primarily during inflammation and/or mitogenesis stimulated by cytokines, growth factors, mitogens, and endotoxins (Mitchell et al,. 1993). In addition, COX-2 can be downregulated by glucocorticoids such as dexamethasone (Simmons et al., 1993). Because of its early induction following stimulation by growth factors, COX-2 is grouped in the immediate early gene family (Herschman, 1991; Ryseck et al., 1992), a group of proteins thought to initiate the ordered cascade of processes required for mitogenesis; thus, PGs produced by COX-2 may also be involved in regulating cellular proliferation (Smith et al., 1994). Most NSAIDs inhibit both COX-1 and COX-2. Taketo (1998a,b) reviewed the pharmacology of COX inhibition and the evidence supporting the use of COX-2 inhibitors as chemopreventive agents, particularly in colon. These data are summarized briefly in the following paragraphs. The mRNA levels of COX-1 and COX-2 have been quantified in normal human tissues by reverse transcription-polymerase chain reaction (RT-PCR); both COX-1 and COX-2 mRNAs are normally coexpressed at low but deteitable levels in most normal human tissues (Smith, 1992). The highest levels of COX mRNAs were detected in the prostate, where approximately equal levels of COX-1 and COX-2 transcripts were present. In the lung, higher levels of COX-2 mRNA were observed; COX-1 mRNA levels were about 2-fold lower. An intermediate level of expression of both COX-1 and -2 mRNAs has been observed in mammary gland, stomach, small intestine, and uterus. The lowest levels of both COX-1 and COX-2 mRNAs were found in the testis, pancreas, kidney, liver, thymus, and brain. The study did not examine levels of COX mRNAs after, for example, hormonal or inflammatory stimulation. COX-1 mRNA is present in many cell lines and in ex-

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tracts of virtually all mammalian tissues. With the exception of Rat-1 cells and mitogen-stimulated mouse 3T3 fibroblasts, COX-2 mRNA is not found in common cell lines. The potential for selective COX-2 inhibitors in chemoprevention of colorectal cancer was first suggested by the observation that COX-2 levels appear to. be elevated in colorectal tumors compared with normal tissue. For example, O’Neill and Ford-Hutchinson (1993) and Kargman et al. (1995) examined the expression of COX-1 and COX-2 in normal colon and colorectal tumors from 25 colon cancer patients. COX-1 was detected in both tumor and nontumor tissues; however, the level of COX-1 was reduced in 21 of 25 tumor samples compared to nontumor tissues from the same patiehts. In contrast, COX-2 was not detected in 23 of 25 nontumor samples but was detected in 19 of 25 colon tumors (a highly significant difference), showing that COX-2 is induced in the majority of tumor samples. Numerous studies have demonstrated that COX can be induced by growth factors (e.g., EGF), cytokines [e.g., interleukin-1 (IL-1)], tumor promoters [e.g., 12-0-tetradecanoylphorbol-13-acetate(TPA)], and immune modulators [e.g., lipopolysaccharides (LPS)] (reviewed in Eberhart et al., 1994). It was eventually shown that the inducible enzyme (COX-2) is a distinct isozyme form that expressed constitutively (COX-1) and that the two forms are expressed and regulated differentially (Eberhart et al., 1994). It now appears that COX-1 is expressed in tissues such as the gastrointestinal tract and kidney where it is required for normal physiological functions, whereas COX-2 is induced by mediators of inflammation. In several studies, selective inhibitors of COX-2 were shown to eliminate the inflammatory response while having no effect on the production of COX-1 or its normal function in the stomach. Most of these NSAIDs are not selective in their inhibition of the constitutive (COX-1) and inducible (COX-2) forms of cyclooxygenase, or they show selectivity for COX-1. However, selective COX-2 inhibitors have been shown to retain antiinflammatory activity (e.g., in carrageenan-induced rat paw edema, pleurisy, or air pouch inflammation models), while having no effect on PG synthesis in the stomach, and are not ulcerogenic (seeTaketo, 1998a). Selective COX-2 inhibitors may be either steroidal or nonsteroidal. The steroidal antiinflammatories (e.g., dexamethasone), also known as glucocorticoids, have been shown to inhibit both COX-2 and PLA, without affecting COX-1. They are some of the most efficacious antiinflammatory drugs, but they exhibit serious adverse effects because of their glycogenic activity. Several nonsteroidal antiinflammatories with specificity for COX-2 inhibition have been identified: Nabumetone (relafen, approved by the FDA for use in the United States), nimesulide, and meloxicam (marketed in Europe) are examples. NS-398 and DuP 697 are widely studied antiinflammatories

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that are highly selective for COX-2 (ratios of the COX-2 to COX-1 IC,, values were in the range of 0.001-0.05), and they are effective at doses that will not inhibit PG synthesis in tissues requiring it for their normal maintenance. Very recently (December 1-998), a highly selective COX-2 inhibitor, celecoxib (Celebrex), has been approved by the FDA as a treatment for osteoand rheumatoid arthritis, with recognition of its lesser gastrointestinal toxicity. Most importantly, there is strong evidence that COX-2 inhibitors will be chemopreventive. Efficacy has been observed in AOM-treated rat colon (celecoxib, Reddy et al., 1996) or mouse colon (nimesulide, Fukutake et al., 1998), the Min mouse colorectal adenoma model (MF tricyclic, Oshima et al., 1996), N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN)-induced mouse bladder (nimesulide, Okajima et al., 1998), benza[a]pyrene (B[a]P)induced mouse lung (NS-398, Rioux and Castonguay, 1998), and human prostate cancer cells (LNCaP, NS-398, Liu et al., 1998). Approach to Developing Selective COX-2 Inhibitors as Chemopreventive Agents. Testing for chemopreventive efficacy of COX-2 inhibitors should proceed from a simple in vitro screen to determine differential COX inhibitory activity, through a short in vivo antiinflammation assay, to tests to measure inhibition of tumor induction in selected target tissues. Although more agents can be tested in the short-term assays, the results of these tests should not be the sole criterion for selecting agents for the longer term tests. Superior in vivo performance might result from better absorption, longer half-life, lower first-pass clearance, and/or differentialdistribution. These factors will need to be considered when selecting agents for long-term testing. Determine specificityfor COX-2 relative to COX-1. The initial screen to identify the most potent COX-2 inhibitors would likely be one that determines the relative inhibitory activity of the agent for COX-1 and COX-2, using recombinant enzymes or purified enzymes. Determine antiinflammatorypotency. The second level screen could be one of .the in vivo antiinflammation models. The carrageenan-induced rat paw edema assay appears to be most widely used, as are carrageenan-induced pleurisy in rats and air pouch inflammation. Determine chemopreventive eficacy in animal tumor and biomarker models.’Appropriate target tissues for evaluation of efficacy of COX-2 inhibitors in biomarker and tumor induction assays would be colon and bladder as these are the target organs where efficacy has been seen in humans and animals with the current NSAIDs. Biomarkers might include inhibition of AOMinduced ACF in rat colon. Other rat colon markers to consider are PGs [e.g., PGE,, 6-keto-PGFIa, and thromboxane B, (TXB,)] as drug effect markers, oncogene expression (e.g., c-Ha-ras, ras p21 level, myc, p53), proliferation markers (e.g., BrdU incorporation, PCNA, [3H]thymidine incorporation), histological lesions (e.g., GST-ITpositive foci, hyperplasia), and differentiation markers (eg., cytokeratins, lectin SBA, sialylated lex). In rat bladder

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suitable biomarkers might include proliferation markers (e.g., EGFR), histological lesions (e.g., dysplasia, GGT-positive foci), and genomic instability (e.g., LOH, gene amplification, microsatellite instability). In carcinogenesis models such as AOM-induced rat colon and OH-BBNinduced mouse bladder, inhibition of tumor induction by the COX-2 inhibitors can be measured. In addition, studies examining the expression of COX mRNAs in normal, premalignant, and tumor tissues are useful. Lung may also be a target of interest. NSAIDs have been shown to inhibit lung ttlmorigenesis in the strain A mouse lung adenoma model, a close inverse relationship between plasma levels of PGE, and chemopreventive efficacy was observed in one study, and a selective COX-2 inhibitor (NS-398) has been shown to inhibit mouse lung tumors.

c. Inducible Nitric Oxide Synthase Inhibitors Nitric oxide (NO), which is produced by constitutive and inducible forms of nitric oxide synthase (NOS), mediates many physiological and pathological processes, including blood flow, platelet aggregation, neurotransmission, and memory, and is also involved in defense against pathogens and elimination of tumor cells (reviewed in Kerwin et al., 1995). NO deficiency is observed in diseases including arteriosclerosis, susceptibility to infection, hypertension, and impotence, while increased NO is seen in septic shock, acute and chronic inflammation, immune-type diabetes, neurodegenerative diseases, transplant rejection, and hypotension (Kerwin et al., 1995). Unlike other signal transduction messengers, NO diffuses through cell membranes and so does not require membrane receptors. NO is synthesized by oxidation of L-arginine to L-citrulline. Two constitutive forms of NOS, endothelial (eNOS, ecNOS, NOS-111) and neuronal (nNOS, ncNOS, bNOS, NOSI), and one inducible form (iNOS, mNOS, ncNOS, NOS-11) have been characterized. All forms require NADPH, L-arginine, tetrahydrobiopterin, thiol, and flavins. The constitutively expressed forms produce “puffs” of NO in a highly controlled and transient manner and are regulated by exogenous Ca2+ and calmodulin. iNOS is not dependent on exogenous Ca2+/calmodulin and is regulated primarily at the transcriptional level; once activated it produces NO for hours to days, at 100- to 1000-fold greater levels (Pfeilschifter et al., 1996). iNOS activity is also regulated via feedback inhibition by NO (Assreuyet al., 1993) and possibly by endogenously produced inhibitors. Like other redox effectors, NO plays a complex, at least dual role in carcinogenesis depending on local NO concentration, presence of reactive oxygen species (ROS) and other redox components, and susceptibility of individual cell types. Current evidence suggests that at low levels NO supports tumor growth by mechanisms such as inhibiting apoptosis and promoting angiogenesis. Moreover, as a free radical, NO is mutagenic itself and thus

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cannot only cause initiating mutagenic events, but can also support malignant progression of surviving clones by accelerating genetic damage (Harris, 1991). On the other hand, continuous exposure to high levels of NO produced by stimulated immune cells, or by increased expression of iNOS in tumor cells, is cytostatic/cytotoxic to the tumor cells themselves (Jenkins et al., 1995). As can be expected from their involvement in redox, the NO and ROS systems can interact. Concurrent local production of high ROS levels, for example, by immune cells, can generate even more reactive compounds such as ONOO- (reviewed in Pryor and Squadrito, 1995). Taken together, these observations suggest that the balance between the NO and ROS systems may play a key role in regulating cell growth and integrity, and imply that manipulating the redox status of the cell may be an effective way to control NO. Excess, continuous NO production by iNOS under nonphysiological conditions can lead to toxicities associated with suppressed glycolysis, mitochondrial respiration, and DNA repair and replication (reviewed in DarleyUsmar et al., 1995).These effects can be mediated directly by NO, or other reactive nitrogen species (RNS) such as ONOO- (Pryor and Squadrito, 1995). NO/H,O,:mediated mechanisms of cellular toxicity also appear to be important (Farias-Eisner et al., 1996). On the other hand, increasing evidence indicates that NO can protect cells from oxidative damage. Antioxidant effects have been associated with scavenging or preventing the formation of hydroperoxyl or alkyl peroxyl radicals (Wink et d.,1994, 1995), as well as other mechanisms, such as nitrosylation of heme and nonheme iron (Gorbunov et al., 1997). A number of agents alone or in combination induce iNOS expression inducing US, inflammatory cytokines [e.g., IL-1, IL-2, TNFa, interferon-y (IFN-y)], phorbol esters, adenosine 3’,5’-cyclic monophosphate (CAMP), CAMP-elevating agents, ultraviolet light, and ozone (reviewed in Pfeilschifter et sl., 1996). Appropriate stimulation leads to expression of iNOS in many cell types including macrophages, vascular endothelium, smooth muscle, chondrocytes, myocardium, and various cancer cells (Szabo and Thiemermann, 1995; Griffith and Stuehr, 1997). iNOS has also been found in some apparently unstimulated human tissues such as fetal and adult lung (reviewed in Marletta, 1993).NO production varies considerably among cell types and species, and it has been hypothesized that iNOS can be expressed in all nucleated cell types on appropriate stimulation (Griffith and Stuehr, 1997). The cellular products of NO differ depending on the biochemical milieu. In aqueous solution NO and 0, form nitrite (NO;); however, in some cells both NO,- and nitrate (NO;) are produced. NO reacts with oxyhemoglobin or oxymyoglobin to form NO; and methemoglobin or metmyoglobin, respectively. This likely serves as an important mechanism to control NO levels in vivo. Nitrite does not significantly accumulate in blood or urine, either

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because it is not formed, or because it is readily oxidized to NO; (Feldman et al., 1993). Nitric oxide reacts with many cellular molecules including protein thiols and DNA, as well as small molecules such as oxygen and superoxide (0;).Many physiological effects of NO are mediated via activation of the heme-containing enzyme guanylate cyclase, in turn increasing intracellular cGMP levels and the activity of cGMP-dependent protein kinases, cGMP-regulated cyclic nucleotide phosphodiesterases, and cGMP-gated ion channels (McDonald and Murad, 1995).NO also modulates other enzymes, mostly decreasing their activity [e.g., aconitase, catalase, cytochrome c oxidase, cytochromes P450, glucose-6-phosphate dehydrogenase (G6PDH), PKC, and superoxide dismutase (SOD)](reviewedin Feldman et al., 1993). COX activity is either increased or decreased depending on NO concentration (Swierkoszet af., 1995). NO or its derivatives react with protein thiols and GSH to form nitrosothiols, some of which serve as NO carriers (Feldman et al., 1993) that may have important biological activities. For example, the antimicrobial activity of S-nitrosoglutathione and S-nitrosocysteine appears to far exceed that of NO itself. Association of the NO/NOS System with Carcinogenests. The effects of the NO/NOS system on carcinogenesis are complex and incompletely understood. NO or other RNS affect DNA integrity, influence tumor cell growth and development, and are associated with inflammatory and infectious diseases that increase cancer risk in humans. However, both positive and negative effects on a number of these processes have been observed. The most convincing evidence of NO involvement in human cancer stems from the upregulation of iNOS activity/expression in precancerous inflammatory conditions in the colon. The finding that NO is an endogenous regulator of COX-2, and the specific link between iNOS and COX-2, supports a role for NO/iNOS-mediated pathways in human colon cancer development. The feasibility of suppressing tumor growth by diminishing iNOS activity in tumor cells was recently demonstrated using the highly specificiNOS inhibitor 1400W (Thomsen et af., 1997). Calcium-dependent, presumably constitutive forms of NOS have also been associated with human cancers of the breast (Thomsen et af,. 1995), female genital tract (Thomsen et al., 1994), and central nervous system (Cobbs et af., 1995). The association of constitutive forms of the enzyme with hormone-responsive tumors is interesting in light of the observation that estrogens, which, as described above, are well-established promoters of breast cancer, can upregulate eNOS expression in vascular endothelium. This activity has been hypothesized to play a role in estrogen’s cardioprotective action (Kauser and Rubanyi, 1997). A Ca2+-dependent inducible isoform of NOS has been identified in human hepatocytes (Geller et al., 1993), and so it is possible that Ca2+-dependent NOS activity observed in human cancers may actually be of an inducible form of the enzyme.

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Many human tumor cell lines, including those derived from cancers of the colon (Jenkins et al., 1994), cervix (Werner-Felmayer et al., 1993), and breast (Zeillinger et al., 1996), produce NO either constitutively or in response to exogenous stimuIi. NOS activity ( Ca2+-dependent and -independent) and levels of NO, (NO;/NO;) were higher in primary breast cancers than in benign lesions or normal breast tissue; increased activity correlated positively with tumor grade (Thomsen et al., 1995). Increased Ca2+-dependent NOS expression and/or activity in gynecological (Thomsen et al., 1994) and central nervous system (Cobbs et al., 1995) tumors also correlated with tumor grade. In head and neck cancers iNOS was extensively expressed, whereas it showed much more limited expression in normal squamous epithelium (Thomsen et al., 1994; Rosbe et ali, 1995), and NO, levels were higher in the bronchoalveolar lavage fluid of lung cancer patients than in controls (Edwards et al., 1996). Several studies have reported higher iNOS activity in human colon cancers compared with nearby tissue (cited in Vecchini et ul., 1997). Increased NO formation and/or increased iNOS activity/expression have been observed in the colonic mucosa of patients with inflammatory bowel disease. These patients are at increased risk for colon cancer. Studies in animal models also suggest that iNOS can play a role in tumor development. Human colon cancer cells transfected with murine iNOS cDNA grew faster in vivo and were much more vascularized than wild-type cells (Jenkins et al., 1995). Likewise, murine breast cancer cells stimulated with LPSIIFN-y to produce NO grew faster and were more metastatic than control cells on injection into syngenic mice (Edwards et al., 1996). In a murine solid tumor model, increased NO levels and tumor mass were associated with increased iNOS expression (Doi et al., 1996), and in carcinogen-induced rat colon cancers, iNOS and eNOS were expressed in carcinoma epithelial cells, although they were barely detectable in normal colonic epithelium (Takahashi et al., 1997). A number of activities may contribute to the activity of NO in tumorigenesis. NO is mutagenic in both bacterial and mammalian systems. It can induce DNA damage via deamination, strand breakage, oxidation of DNA basks, and formation of carcinogenic N-nitrosoamines (e.g., Liu and Hotchkiss, 1995; Tamir and Tannenbaum, 1996; Keefer and Wink, 1996).NO also inhibits several enzymes involved in DNA synthesis (e.g., ribonucleotide reductase) and repair (e.g., O6-alkylguanine-DNA-alky1transferase,DNA ligase) (Graziewicz et al., 1996). Furthermore, augmented production of endogenous NO increases mutation frequency in mice (Gal and Wogan, 1996). NO-induced DNA damage results in p53 accumulation and subsequent downregulation of NO synthesis through p53-mediated repression of the iNOS promoter (Forrester et al., 1996). Besides DNA damage, the carcinogenic activities of NO have been associ-

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ated with its ability to increase angiogenesis (Jenkins et al., 1995), enhance vascular permeability (Doi et al., 19969), suppress the immune system (Lejeune et al., 1994), enhance metastasis (Edwards et al., 1996), and prevent apoptotic cell death (e.g., Mannick et al., 1994; Vecchini et al., 1997). Studies with NOS inhibitors also support a role for NO in tumor growth and development. Under circumstances that increase iNOS activity in cell culture, these compounds suppress neoplastic transformation (Mordan et al., 195)3),decrease angiogenesis (Leibovich et al., 1994), and enhance apoptosis (Mannick et al., 1994). In animal models they reduce mutation frequency (Gal and Wogan, 1996), inhibit tumor growth and metastasis (e.g., Edwards et al., 1996), reduce vascular permeability (Doi et al., 1996), and dicrease tumor blood flow (Tozer et al., 1997). The inhibitors used in the aforementioned studies were not very selective for iNOS compared with constitutive isoforms. However, the highly specific iNOS inhibitor 1400W suppresses the growth of solid tumors that express iNOS within tumor cells, clearly supporting a role for intratumorally produced NO in carcinogenesis (Thomsen et al., 1997). On the other hand, continuous, high levels of NO produced by macrophages or endothelial cells are cytotoxickytostatic toward tumor cells (Farias-Eisner et al., 1994). Also, high NO levels produced in tumor cells themselves by stimulation of iNOS activity or via transfection with iNOS genes are associated with decreased tumor growth and metastasis in vivo (e.g., Xie et al., 1997a). The negative effects of NO on tumorigenesis have been associated with inhibition of angiogenesis (Pipli-Synetos et al., 1994), differentiation (Magrinat et al., 1992), and apoptosis (e.g., Martin-Sanz et al., 1996). NO also protects against DNA damage under some circumstances and can have antioxidant effects (e.g., Wink et al., 1994). This apparent paradox in which NO both enhances and inhibits tumorigenesis may be associated with several factors including local NO concentrations, cell type, and redox status. Low levels of NO appear to increase its tumor promoting effects, while high levels are cytostatickytotoxic. iNOS activity in genetically engineered human colon cells with increased growth and angiogenic potential was at least one to two orders of magnitude lower than that associated with antitumor actions such as cytotoxicity and apoptosis (Jenkins et al., 1995).NOS activity in these cells was similar to that observed in human breast cancers (Thomsen et al., 1995). This biphasic effect on cell growth has been observed in primary hepatocyte cultures that are protected against TGFP-induced apoptotic cell death by low nanomolar NO concentrations, while higher concentrations promote both apoptotic and necrotic cell death (Martin-Sam et al., 1996). Also, N O activates the p2lras protooncogene in human T cells in a concentration-dependent manner: low concentrations activate, while higher concentrations significantly reduce response (Lander et al., 1995).

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The redox state of the cell may also affect NO action. For example, NO can prevent apoptosis via upregulation of intracellular antioxidant systems (e.g., Nicotera et al., 1997). NO/NOS and Znflarnrnation. Enhanced NO formation is associated with inflammatory diseases which increase cancer risk in humans (e.g., BoughtonSmith et al., 1993; Rachmilewitz et al., 1995),and iNOS has specificallybeen implicated in these pathological conditions. Increased Ca2+-independent NOS activity has been found in the colonic mucosa of patients with inflammatory bowel disease compared with controls (e.g., Boughton-Smith et al., 1993).iNOS expression and nitrotyrosine labeling (an indicator of ONOOformation) were observed in the inflamed colonic epithelium of such patients, but not in uninflamed epithelium or in cbntrols (e.g., Singer et d.,1996). NO/NOS and COX Enzymes. A number of studies have demonstrated cross talk between the NO and PG biosynthetic pathways. NO activates both COX-1 and COX-2 and enhances production of PGs; NOS inhibitors suppress PG formation in vitro and in vivo (e.g., Salvemini et al., 1995; Di Rosa et al., 1996). The effects of NO on COX activity appear to be mediated by ONOO-, which is an efficient substrate on both COX-1 and COX-2. Several lines of evidence indicate a specific association between iNOS and COX-2. For example, inflammatory cytokines induce both iNOS and COX2 in several cells types, and these isozymes are concurrently induced in inflammatory tissues during the acute phases in vivo. Glucocorticoids, which inhibit induction of COX-2 without affecting COX-1, also specifically suppress iNOS induction (reviewed in Di Rosa et al., 1996). Potential Strategies for Znhibiting iNOS.The production of excessive NO by iNOS can theoretically be diminished in a number of ways. These include limiting availability of L-arginine or cofactors necessary for NOS activity, inhibiting iNOS induction, or decreasing enzyme activity. The harmful effects of NO might also be quenched by scavenging NO. As opposed to constitutive forms of the enzyme, iNOS is regulated primarily at the transcriptional level, suggesting that agents which inhibit induction will be specific for this isoform. Because high NO levels can have deleterious effects, it is not surprising that iNOS induction is inhibited by numerous endogenous compounds such as glucocorticoids, IL-4, IL-10, TGFP, PDGF, IGF-I, thrombin (reviewed in Wu, 1995), taurine chloramine (Marcinkiewicz et af., 1995), and ursodiol (Hattori et al., 1996). A number of other agents also suppress induction including retinoids (Becherel et al., 1996), tyrosine kinase inhibitors (e.g., genistein) (D. R. Miller et al., 1997), curcumin (Brouet and Ohshima, 1995),and epigallocatechin gallate (Lin and Lin, 1997). These agents have demonstrated cancer chemopreventive activity, which hypothetically may be associated with their ability to suppress iNOS induction. Direct scavenging of NO by flavonoids and curcuminoids may also contribute to the chemopreventive actions of these compounds.

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Because the signal transduction pathways involved in iNOS induction are incompletely understood and vary among cells and species, targeted inhibition of iNOS expression is difficult. Direct inhibition of enzyme activity appears at present to be the most useful and specific approach for regulating NO production. Approach to Developing iNOS Inhibitors as Chemopreventive Agents. Important considerations in evaluating iNOS inhibitors include source and purity of enzymes, potency and selectivity toward iNOS both in vitro and in vivo, and effects on non-NOS-mediated systems. The following battery of tests might be employed to screen potential iNOS inhibitors. Determine potency and selectivity activity against isolated enzymes. Inhibitors can be screened using purified or partially purified enzymes from animals or humans. Recombinant human iNOS, eNOS, and nNOS can also be utilized. Determine activity in cultured cells. The cells that have been most widely employed for this purpose are stimulated mouse macrophages. iNOS can be induced with Escherichia coli LPS. Determine specificity for iNOS versus eNOS in vascular tissue. Nonselective, tissue-permeable NOS inhibitors induce contraction of endotheliumintact rat aortic rings (demonstrating eNOS inhibition), as well as of endothelium-removed, LPS-treated tissue (demonstrating iNOS inhibition) with similar concentration curves. On the other hand, selective iNOS inhibitors are active in endothelium-removed, LPS-treated tissue, but not endothelium-intact rat aortic rings (Garvey et al., 1997). Determine activity in in vivo models. Inhibitors of iNOS have beneficial effects in animal models of septic shock (reverse or prevent hypotension in LPS-treated animals) and inflammation (reviewed in Moncada and Higgs, 1995).The specificity of an inhibitor for iNOS over eNOS can be monitored in vivo by measuring effects on blood pressure. Selective inhibitors of iNOS should not affect blood pressure in normal animals, while those that also inhibit eNOS will increase blood pressure (pressor activity). Determine chemopreventive activity in animal carcinogenesis models. Increased expression of iNOS and eNOS has been observed in AOM-induced rat colon tumors (Takahashi et al., 1997). Given the association of iNOS with precancerous conditions in the human colon, this would appear to be an appropriate model to test iNOS inhibitors for potential chemopreventive activity. In clinical settings it will be important to employ inhibitors with a high degree of selectivity toward iNOS. Nonselective inhibitors can cause hypertension, diminish cardiac output, and affect renal hemodynamics by interfering with constitutive forms of the enzyme. Additionally, compensatory increases in NO, which appear to be due to elevated iNOS expression, have been observed in experimental studies where nonselective inhibitors have

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been administered via nonacute protocols. Given the important homeostatic functions of NO, it is not surprising that tissues stringently maintain adequate NO levels.

d. Lipoxygenase Inhibitors Although PGs and other COX-derived metabolites have been studied more extensively, there is also evidence that lipoxygenase (LOX)-catalyzed products, the leukotrienes (LTs)and HETEs, are involved in the development and progression of human cancers. For example, 12-LOX mRNA expression has been well documented in many types of solid tumor cells, including those of prostate, colon, and epidermoid carcinoma (Honn et al., 1994a; Chen et al., 1994). Additionally, the production of 12(S)-HETE by some tumor cells, including prostate cells, has been positively correlated to their metastatic potential (Tang and Hong, 1994). Also, studies show that 12(S)-HETEis a critical intracellular signaling molecule that stimulates PKC and elicits the biological actions of many growth factors and c);tokines which regulate transcription factor activation and induction of oncogenes or other gene products needed for neoplastic cell growth (e.g., Timar et al., 1996), including EGF, FGF, PDGF, tumor necrosis factor (TNF),granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-1 and IL-3. PKC activation by 12(S)-HETEmediates the release and secretion of cathepsin B, a cysteine protease involved in tumor metastasis and invasion, particularly in colon cancer cells (Honn et al., 1994a). Furthermore, tumor cell synthesis of 12(S)HETE stimulates adhesion by increasing the surface expression of integrin receptors (Tang et al., 1993). Besides 12(S)-HETE, other LOX metabolites, particularly the 5-LOX products (5-HETEs), have been implicated in cancer development. For example, published data have shown that 5-HETE directly stimulates prostate cancer cell growth (Ghosh and Myers, 1997). Like 12(S)-HETE, these molecules are capable of exerting pleiotropic effects on.normal and malignant cells through autocrine- and paracrine-mediated mechanisms. On the basis of this information, pharmaceutical agents that directly interfere with the production of LOX metabolites or antagonize the signaling functions of LOX products may be effective chemopreventives. Over the past 10 years, pharmacological agents that specifically inhibit the LOX metabolic pathway have been developed to treat inflammatory diseases such as asthma, ulcerativecolitis, arthritis, and psoriasis. These include 5-LOX inhibitors, agents that interact with the 5-LOX activating protein (FLAP),and leukotriene B, (LTB,) receptor antagonists. Compounds demonstrating 12LOX inhibitory activity may show promise as antiproliferative agents. The lipoxygenases comprise a family of nonheme iron-containing dioxygenases that catalyze the stereospecific oxygenation of the 5-, 12-, or 15-carbon atoms of AA (e.g., Lewis et al., 1990). In cells, AA is esterified to membrane phospholipids in the sn-2 position. The reaction begins with the

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intracellular release of arachidonic acid, mediated either by PLA, or by the combined actions of phospholipase C (PLC) and diacylglycerol kinase or phospholipase D (PLD) and PLA,. In leukocytes, cytokines including IL-1 and TNF can’activate PLA, by stimulating a phospholipase-activating protein. Once released, AA is either converted by the catalytic action of 5-, 12-, or 15-LOX into the corresponding HETEs, or is metabolized into LTs or lipoxins through additional sequential reactions, which depend on the biosynthetic capacity of each specific cell type (see below). Production of LTs proceeds via 5-LOX, which catalyzes the first two steps in the metabolic cascade. First, 5-LOX in the presence of FLAP catalyzes the oxygenation of arachidonic acid into 5-hydroperoxyeicosatetraenoicacid (WETE), followed by a second reaction in which 5-HPETE is dehydrated to form the epoxide LTA,. Once formed, LTA, is further metabolized either to LTB, via stereoselective hydration by LTA, hydrolase or to LTC, through glutathione conjugation catalyzed by LTC, synthase. Sequential metabolic reactions, catalyzed by y-glutamyltransferase and a specific membranebound dipeptidase, convert LTC, into LTD, and LTE,, respectively. These three sulfidopeptide LTs are commonly referred to as the slow-reacting substances of anaphylaxis. In the lung, sulfidopeptide LTs are known to act on a single high-affinity, smooth muscle receptor, the cys-LT, receptor (Coleman et al., 1995), resulting in bronchoconstruction and alterations in vascular permeability and mucus secretion in this tissue (O’Byrne, 1997). Important cellular sources of these LTs include eosinophils, mast cells, and basophils (Drazen and Austen, 1987). Metabolic products of the LOX biosynthetic pathways modulate the growth of several normal human cells types, including T lymphocytes (Atluru and Goodwin, 1986), skin fibroblasts (Baud et al., 1987), epidermal keratinocytes (Kragballe eta l., 1985), and glomerular epithelial cells (Baud et al., 1985). Both LTB, and LTC, increase in vitro growth of arterial smooth muscle cells (Palmberg et al., 1991), airway epithelial cells (Leikauf et al., 1989), and mitogen-stimulated lymphocytes (Yamaoka et al., 1989). Besides these cells, Snyder and others showed that LTs play important roles in regulating both human and murine hematopoiesis (e.g., Snyder, 1991). Specific factors that regulate LOX gene expression have been identified. Glucose, EGF, and angiotensin I1 stimulate 12-LOX mRNA expression and production of HETEs and hydroxyoctadecadienoic acids (HODEs) (Chang et al., 1992,1993), and both IL-4 and IL-13 are positive regulators of monocyte 15-LOX gene expression (Conrad et al., 1992). Experiments show that transgenic mice with defective 5-LOX genes are not hematologically abnormal; fertility and survival are not affected in knockout mice lacking one of the LOX genes (Funk, 1996). However, whether eliminating LOX biosynthetic capacity renders these animals less susceptible to carcinogenesis remains to be determined.

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Lipoxygenase Metabolites in Cancer and Other Human Diseases. Lipoxygenase-derived metabolites play important roles in regulating diverse biological and pathological processes (Samuelsson, 1983). As inflammatory mediators, LTs elicit a numbe; of reactions, including vessel wall adhesion, smooth muscle contraction, granulocyte degradation, chemotaxis, and increased mucus secretion and vascular permeability (Samuelsson et al., 1987). LTs are known to be involved in pathological disorders such as allergic rhinitis, colitis, inflammatory bowel disease, bronchial asthma, rheumatoid arthritis, glomerulonephritis, and psoriasis. Besides involvement in inflammatory disorders, elevated levels of LOX metabolites have been documented in some human cancers. For example, notably higher levels of LOX products have bken recovered from malignant human breast tumors than from normal or benign breast tissue (Kort et al., 1992). In squamous epithelial carcinomas of the head and neck, 12- and 15HETE are major arachidonic acid metabolites (El Attar et al., 1985). Also, 12(S)-HETE is the predominant metabolic product of metastatic B16 melanoma cells (Liu et al., 1994a). Additionally, excess LT production, specifically LTC,, has been documented in cells from patients with both acute and chronic leukemias (e.g., Anderson et al., 1993, 1996). Adding 5LOX inhibitors to these cells reduced DNA labeling and decreased cell numbers within 72 hr. Likewise, growth inhibition with LOX inhibitors has been demonstrated in several malignant human hematopoietic cell lines; the COX inhibitor indomethacin lacked a suppressive effect (Snyder, 1991). These data imply that LOX products are essential for the in vitro growth of malignant hematopoietic cells. There are data suggesting the role of LOX in several cancer targets including breast, colon, skin, prostate, and lung. The data in prostate are of particular interest. Association of lipoxygenase metabolites with carcinogenesis in prostate. Altered eicosanoid biosynthesis in relation to prostate cancer development has been documented. Initial studies found dramatically reduced levels of AA; 10-fold greater turnover in malignant versus benign prostatic tissue suggested a possible increase in metabolism via the LOX and COX pathways in this tissue (Chaudry et al., 1991,1994). In experiments conducted in human prostate cancer cells, linoleic acid stimulated cell growth while indomethacin, esculetin, and piroxicam inhibited it, substantiating the involvement of eicosanoids in prostate cancer cell proliferation. Although attention has focused on COX-derived products, particularly PGE,, the COX inhibitors indomethacin and aspirin failed to reduce human prostate PC-3 cell DNA synthesis while the AA antagonist eicosatetraynoic acid (ETYA) did reduce synthesis (Ablin-and Shaw, 1986; Anderson et al., 1988), suggesting that LOX products are essential in modulating prostate DNA synthesis. However, until 5-LOX products are recovered from prostate tissue and their syn-

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thesis directly shown to be inhibited by 5-LOX inhibitory agents, other possible mechanisms cannot be ruled out. Additional studies by Anderson and co-workers (1996)show reduced DNA synthesis and growth inhibition of prostate cancer cells with specific 5-LOX inhibitors, further supporting involvement of the LOX metabolic pathway in prostate cancer growth. Likewise, work by Ghosh and Myers (1997)using PC3 cells provides convincing evidence that the 5-LOX metabolic pathway stimulates prostate cancer cell growth. More specifically, 5-HETE, particularly the 5-0x0-eicosatetraenoic form (5-0x0-ETE), stimulates PC-3 cell growth similarly to AA; LTs had no effect. 5-HETEs also effectively reversed growth inhibition produced by a FLAP inhibitoc Both a 5-LOX and a FLAP inhibitor effekively blocked prostate tumor proliferation induced by AA, whereas the COX inhibitor ibuprofen and 12-LOX inhibitors were ineffective. Besides 5-LOX products, other LOX metabolites have been implicated in prostate, tumor growth. The 12-LOX metabolite 12-HETE plays a critical role in prostate tumor metastasis and invasion (Honn et al., 1994b). In 122 matched normal and cancerous prostate tissues, 12-LOX mRNA expression was confined to prostate epithelial cells and elevated in malignant cells (Gao et al., 1995). Also, elevated levels of 12-HETE mRNA significantly correlated with advanced stage, poor differentiation, and invasive potential of prostate cancer cells. Additional biochemical evidence demonstrated that 12HETE stimulates secretion of cathepsin B, which is involved in tumor metastases and integrin expression in other tumor cells (Honn et al., 1994b), providing more support for the importance of LOX products in the development and spread of prostate and other human cancers. Liu et al. (1994b) have shown that enhancement of prostate tumor cell invasion by 12(S)-HETEinvolves selective activation of membrane-associated PKCa. Furthermore, the ability of the PKC inhibitor calphostin C to block the 12-HETE-stimulated release of cathepsin B lends credence to the observation that 12(S)-HETE acts via activation of PKC (Liu et al., 1991). Among several possible mechanisms by which LOX inhibition may reduce prostate PC-3 cell proliferation is by altering the concentration of second messengers needed for continued cell growth by shunting other AA metabolites into alternate pathways that are linked to signal transduction mechanisms (Crooke et al,. 1989). For example, agents that inhibit 12-LOX and block 12-HETE production may result in widespread interference in signal transduction by preventing the activation of PKC. LOX inhibition may further affect the expression of protooncogenes or their products, including EGG, FGF, TGFa and -p, N- and K-ras, c-myc, int-2, IGF, and the retinoblastoma gene, also directly or indirectly associated with normal or transformed prostate epithelial cell growth (Thompson, 1990; Ware, 1993). Whether antagonists of LOX biosynthesis can influence, expression of these factors, however, has not yet been determined.

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Association of lipoxygenase metabolites with carcinogenesis in lung. Evidence links LOX metabolites with lung cancer cell growth. Studies conducted by Avis et al. (1996)in seyeral human lung cancer cell lines (small cell and non-small cell) found that 5-LOX is stimulated by two autocrine growth factors, gastrin-releasing peptide (GRP) and IGF, both of which stimulate production of 5-HETE. 5-HETE stimulated the growth of lung cancer cells, whereas cells treated with 5-LOX inhibitors decreased proliferation; the COX inhibitor aspirin had little effect. Expression of 5-LOX, and FLAP mRNA by lung cancer cell lines was confirmed using RT-PCR, and the presence of 5-LOX mRNA was identified in samples of primary lung cancer tissue, including both small cell and non-small cell lung carcinomas. Also relevant to lung cancer development are studies demonstrating that lipoxygenases mediate oxidation of potent carcinogens such as benzidine, 0dianisidine, and others; this activation can be blocked by adding the LOX inhibitors NDGA and esculetin (Kulkarni et al., 1992). Rat lung LOX also oxidizes benzo[a]pyrene (Nemoto and Takayama, 1984), and lipoxygenases have been found in human lung tissue. Chemopreventive Mechanisms. The antiproliferative effects of LOX inhibitors have not yet been clearly defined, but the large number of LOX metabolites and their ability to exert diverse biological activities suggest several molecular mechanisms. The first and most obvious hypothesis is that LOX inhibition prevents DNA synthesis. Substantial experimental evidence demonstrates that LOX products, particularly 12-HETE, can influence the production or activities of various second messengers such as tyrosine kinases, CAMP,and PKC. Additionally, growth factors such as EGF, TGF, TNF, PDGF, FGF, IGF, and colony-stimulating factors can either activate or are linked to LOX-mediated pathways. Blocking LOX catalytic activity, therefore, may reduce cell proliferation by interfering with these pathways or other growth factor signaling events needed for cell transformation. Antioxidation. Because lipoxygenases are multifunctional enzymes capable of generating free radicals via dioxygenase and hydroperoxidase activities (Ford-Hutchinson et al., 1994), another plausible chemopreventivemechanism involves preventing lipid peroxidation and radical production. Ample expe'rimental data indicate that free radicals play significant roles in carcinogenesis, inducing oxidative damage to cellular DNA by producing strand breaks that can ultimately result in chromosomal rearrangements and deletions. As tumor promoters, radicals can also modify the transcriptional activation of early response genes such as c-fos, c-myc, and c-jun, which are associated with cell growth and proliferation (Crawford et al., 1988). In tissues such as the lung, LOX mediates oxidation and activation of potent xenobiotics as well as lipid peroxidation (Kulkarni et al., 1992) and may serve as an alternate enzyme to the cytochrome P450 isoenzymes. Many compounds that inhibit LOX metabolic activity also possess antioxidant

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properties (Brooks and Summers, 1996);these agents may be effective in preventing cancer development by blocking radical-induced genetic damage or by interfering with the metabolic activation of carcinogens. Apoptosis induction. Alternatively, inhibiting LOX biosynthetic pathways may reduce cell proliferation by increasing apoptotic cell death and restoring growth regulation in transformed cells where apoptotic cell death is reduced (eg., Bedi et al., 1995). LOX products and factors that are affected by LOX metabolism (oncogenes, cations, and radicals and other ROS) have been implicated in apoptosis. Antiangiogenesis/antimetastasis.In addition to its role in apoptotic cell death, 12-HETE is involved in multiple steps of the metastatic cascade, from increasing tumor cell motility, adherence to endothelial matrix, and extravasation to cell proliferation in secondary sites. 12-LOX mRNA and protein expression have been documented in a variety of human tumors including Clone A colon cells (Chen et al., 1994), erythroleukemia cells (Tang and Honn, 1996), epidermoid carcinoma cells (Chang et al., 1993),and human prostate cells (Gao et al., 1995), as well as in murine Lewis lung carcinoma and rat Walker W256 carcinosarcoma cells (Chen et al., 1994). Moreover, the ability of human prostate tumor cells (Gao et al., 1995), K-1735 melanoma cells, and others to synthesize 12-HETE correlates with their metastatic potential (Tang and Honn, 1994). On the basis of the above data, blocking production of 12(S)-HETEmay be effective in preventing the spread of already formed cancers by inhibiting tumor cell motility, attachment, and invasion through basement membrane. With respect to chemoprevention, 12-LOX inhibition may prevent the spread of early cancer cells by affecting angiogenesis factors such as PDGF and FGF, thereby interfering in early metastatic events. However, further studies will be needed to support the concept that 12(S)-HETEor other LOX metabolites are an important means of eliciting angiogenic events. Antiinflammation. The association of inflammation and carcinogenesis has been described above. The LOX biosynthetic pathway plays a prominent role in evoking inflammatory reactions. As polymorphonucleocyte (PMN) chemoattractants (Ford-Hutchinson et al., 1980), LTs, particularly LTB,, stimulate neutrophil-mediated increases in vascular permeability (Wedmore and Williams, 1981). Studies conducted in both human and rat models of ulcerative colitis confirm that, once activated, PMNs stimulate the release of a host of enzymes and other substances such as oxygen radicals, resulting in tissue damage (Keshavarzian et al,. 1992). LTB, also modulates the immune system by influencing the activity of suppressor and helper T lymphocytes (Ford-Hutchinson, 1995). Because LTs are associated with inflammatory processes, drugs have been developed using 5-LOX inhibitory activity as a therapeutic target to treat asthma, inflammatory bowel disease, psoriasis, rheumatoid arthritis, and other diseases of chronic inflammation.

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Approach to Developing Lipoxygenase Inhibitors as Chemopreventive Agents. To assess potency and enzyme specificity, pharmacological profiles of each agent are established based on screening data obtained from both cell-free assays and in vitro model systems. For this purpose, each compound is subjected to a series of enzymatic screens to determine 5-, 12-, and 15LOX inhibitory activities. 5-LOX measurement is typically conducted in cell lysates from rat (RBL-1)cells (Carter et al., 1991). 12-LOX can be assessed in lysates of human platelets or in partially purified preparations available through commercial sources. Likewise, purified forms of rabbit reticulocyte or soybean 15-LOX can be obtained through local suppliers. Conduct cell proliferation and D N A synthesis assays. Once inhibitory potency has been established in whole cell models, each agent is further subjetted to a series of antiproliferative screens using established cell culture cancer assay systems. These assays should include representative human cell lines for breast, colon, prostate, and lung cancer. Experiments determine LTor HETE-mediated growth stimulation and concentration-dependent inhibition of cell proliferation and DNA synthesis for each agent. Evaluate chemopreventive efficacy in vivo. The chemopreventive efficacy of select inhibitory agents can be further tested in the well-established in vivo carcinogen models as described for other agent classes. Because of the activity of LOX inhibitors as antiasthmatics, the lung has a high priority for future chemoprevention studies and applications of LOX inhibitors. The strain A/J mouse lung adenoma model would be appropriate for evaluating the chemopreventive activity of LOX inhibitors in lung. Although approved pharmaceuticals in this class are bioavailable orally, it could be beneficial to explore the use of inhalant formulations, which would deliver agent locally to the lung, potentially reducing toxicity and allowing higher, more efficacious doses. The prostate is also a cancer target of high interest based on the high rate of AA metabolism and antiproliferative activity of LOX inhibitors in prostate cancer cells. 4. ANTIMUTAGENS-PHASE

I1 ENZYME INDUCERS

Cakinogen deactivation and detoxification are generally regarded as a very important mechanism of carcinogenesis inhibition (Wattenberg, 1985, 1992a,b; Kelloff et al., 1995a); enhancement of this detoxification may prove to be an important strategy for chemoprevention. Two metabolic pathways are critical. The first is the introduction or exposure of polar groups (e.g., hydroxyl groups) on xenobiotic compounds via the phase I metabolic enzymes, which are primarily the microsomal mixed-function oxidases. In many cases, the polar groups become substrates for conjugation. The second pathway is via the phase I1 metabolic enzymes responsible for conjugation

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and the formation of glucuronides, GSH conjugates, and sulfates. Chemopreventive agents that can act via this mechanism have been divided into two groups. Type A compounds are those that primarily enhance the activity of phase I1 enzymes, particularly GSTs and also UDP-glucoronyltransferase. Type B inhibitors induce increases in microsomal mixed-function oxidase activities,.while also increasing the activity of the major phase I1 enzymes. Type A compounds are now considered more promising chemopreventive agents, since the enzymes in the microsomal mixed-function oxidase system induced by Type B agents are more likely to increase carcinogen activation. Glutathione is a prototypical carcinogen scavenger (seealso under the more general mechanism of electrophile scavenging below, Section III,D,5). It reacts spbntaneously or via catalysis by GSTs with numerous activated carcinogens including N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), aflatoxin B, (AFB,), and B[u]P-diolepoxideand other activated PAHs. Likewise, GSH protects against mouse skin tumors induced by DMBA/TPA, rat forestomach tumQrsinduced by MNNG, and rat liver tumors induced by AFB, . A number of promising chemopreventive agents are potent inducers of GSH and GSTs. Prominent among these compounds are the allylic sulfides, natural products found in onion, garlic, and other members of the genus Allium. Oltipraz [5-(2-pyrazinyl)-4-rnethyl-l,2-dithiol-3-thione] is a potent GST inducer with a wide spectrum of chemopreventive activity that may be related to phase I1 enzyme induction. N-Acetyl-L-cysteine (NAC) is essentially a precursor of GSH. NAC shows inhibitory activity in rat colon against dimethylhydrazine(DMH)-and AOM-induced tumors, in mouse lung against urethane-induced tumors, in rat mammary glands against MNU-induced tumors, and in mouse bladder against OH-BBN-induced tumors.

5. ANTIOXIDANTS Chemopreventive antioxidant activities include scavenging reactive electrophiles (GSH-enhancing agents), scavengingoxygen radicals (polyphenols, selenium, vitamin E), and inhibiting AA metabolism (glycyrrhetinic acid, NAC, NSAIDs, polyphenols, tamoxifen). Two examples of antioxidant agent regimens with chemopreventive potential are the combination of selenium with vitamin E and tea polyphenol extracts. These are both interesting in that they are examples of diet-derived chemopreventives, either as food substances or supplements or trace minerals. As such they potentially may find use as chemopreventives in normal risk populations. Much epidemiological and preclinical efficacy data have shown the association between dietary substances or extracts, such as tea, herbs (e.g., curcumin), cruciferous vegetables (e.g., sulforafan), and flavonoids (including isoflavone phytoestrogens such as genistein), and lowered riskhncidence of cancers.

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a. Vitamin E and Selenium Rationale for Developing Vitamin E plus Selenium as a Cbemopreventive Regimen. In animal studies, pharmacological levels of inorganic or organic selenium compounds have inhibited chemically and virally induced tumors in mammary glands, colon, skin, lung, trachea, liver, stomach, and pancreas, as well as the development of transplanted tumors.(Medina and Morrison, 1988; Whanger, 1992). Published animal chemoprevention studies with all selenium compounds indicate that selenium inhibits the postinitiation phase of mammary carcinogenesis and the initiation phase of colon carcinogenesis; the inhibitory effect is reversible, and chemopreventive doses approach toxic levels (Medina and Morrison, 1988; Ip and Hayes, 1989). L-Selenomethionine, specifically, was ineffective in mouse colon and lung carcinogenesis models; however, racemic or unspecified enantiomers inhibited mammary tumor development in several rat models. Further, the combination of selenium with vitamin E was more efficacious in inhibiting MNU-induced rat mammary gland carcinogenesis than selenium alone (Ip and White, 1987). The only well-defined function for selenium in animals is as a constituent of the selenium-dependent form of glutathione peroxidase (GSH-Px) (Wendel, 1980), where it is incorporated into the active site of the enzyme as the substituted amino acid, selenocysteine. GSH-Px is a cytosolic enzyme that reduces both hydrogen peroxide and organic hydroperoxides, providing potential antioxidant activity; however, other chemopreventive mechanisms may exist, since GSH-Px activity plateaus in blood and plasma and does not always correlate to tissue selenium levels at cancer inhibitory doses. Besides the antioxidant activity of GSH-Px, selenium compounds have other antiinitiation effects through altered carcinogen metabolism (by affecting heme metabolism), as well as antiproliferative effects resulting from inhibition of DNA (Arthur and Beckett, 1994) and protein synthesis (Sunde, 1990; Kohrle, 1994),and altered immune function (Taylor, 1995; Roy et al., 1995). We have previously reviewed the evidence of the potential of vitamin E as a chemopreventive agent (Kelloff et al., 1994e), which is summarized in the following. Vitamin E is lipid-soluble and is generally considered an essential nutrient for higher animals, including humans, due to its function as the major antioxidant present in all cell membranes. Eight related naturally occurring substances have vitamin E activity, including a-p-, &,and y-tocopherols (saturated side chains) and tocotrienols (unsaturated side chains). In animal chemoprevention studies, d-a-tocopherol or the acetate inhibited tumorigenesis in preclinical oral cavity (hamster buccal pouch and tongue), skin (mouse), mammary gland (rat), liver (rat), colon (mouse), and small intestine (rat) models; Other enantiomers decreased tumor formation in the pancreas (hamster), esophagus (mouse), lung (rat), and ear duct (rat). Vitamin E reacts with a variety of oxy-radicals and singlet oxygen; thus,

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one of its main antioxidant (and anticarcinogenesis) functions is to prevent the peroxidation of polyunsaturated membrane lipids. Although animal studies have demonstrated that vitamin E supplementation inhibits carcinogenesis, antioxidant activity is not a sufficient explanatory mechanism in some models (e.g., DMBA-induced rat mammary glands). Other properties that may contribute to the observed chemopreventive activity include membranerelated effects (physicochemical stabilization of membranes, protection of normal cytochrome P450 metabolism), immunostimulation, differentiation induction, promotion of gap junctional intercellular communication, antiproliferation, AA metabolism inhibition, prevention of nitrosamine formation, and ODC inhibition. Although epidemiological studies have shown lower serum vitamin E levels in people who subsequently developed cancer (especially in pancreas, stomach, bladder, lung, and other smoking-related tissues) compared with controls, it is not clear that pharmacological doses of vitamin E can be of chemopreventive or therapeutic value. The effect may be limited to increasing deficient or marginally normal serum vitamin levels to the normal range. However, there are animal data showing that vitamin E may potentiate the efficacy of more toxic agents such as that cited above for selenium in rat mammary gland. Also, several epidemiological studies found that higher risk for cancer development at several sites correlated to the combination of low serum vitamin E with low selenium status. Prostate cancer may be an important target for chemopreventive intervention with the combination of selenium with vitamin E, in that there is evidence for each of selenium and vitamin E alone in the prevention of prostate cancer. Clark reported on his study of 3120 subjects administered selenium (selenized yeast) for 5 years (Clark etal., 1996,1998). In this study he found lower RRs for lung (17:31, RR = 0.54) and prostate (13:35, RR = 0.37) cancer compared with placebo. Vitamin E acetate decreased the incidence of prostate and colorectal cancer in a study of over 29,000 Finnish male smokers; the 32% reduction in prostate cancer incidence was particularly noteworthy (Heinonen et al., 1998). Results of a study on prevention of esophageal cancer suggest that the combination of selenium with vitamin E has promise for clinical chemoprevention. In this trial, the combination of vitamin E, selenium, and @-carotenereduced the overall mortality rate in Linxian, China; a reduced risk for esophageal cancer was a contributing factor, although it was not statistically significant (Blot et al., 1993; Taylor et al., 1994).

b. Natural Polyphenols-Tea

Polyphenols

Tea is a beverage made from the leaves of the species Camellia sinensis of the family Theaceae. This beverage is one of the most ancient and, next to water, the most widely consumed liquid in the world. Tea leaves are primarily manufactured as green, black, or oolong, with black tea representing approximately 80% of tea products consumed. Green tea is the nonoxidized/

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nonfermented product and contains several polyphenolic components such as epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate (EGCG). EGCG is the major green tea polyphenol (GTP) (>40% dry weight). The major components of black tea (the fermented product) are theaflavins (1-2% dry weight) and thearubigins (10-20% dry weight), which have shown chemopreventive activity. These studies are reviewed in Kelloff et ul. (1996f) and are summarized below. Although not conclusive, epidemiological studies have suggested a protective effect of black or green tea consumption against human cancers of the breast, colon and rectum, gall bladder, liver, lung, nasopharynx, pancreas, stomach, and uterus. In contrast, a number of other ecological, cohort, and case-control studies have associated an increased risk of cancer of the breast, c'olorectum, esophagus, kidney, lung, pancreas, and stomach with tea intake. These inconsistenciesmay be attributed to consumption of salted or very hot tea (esophagus) or to geographical location, as observed with stomach cancer (e.g., inhibition of endogenous formation of nitroso compounds which are a major cause of gastric cancer in some areas). Other confounding factors and variables may include the use of tobacco and alcohol and lack of information on the type of tea consumed (e.g., black or green). In published preclinical studies, tea, GTPs, and EGCG have demonstrated antimicrobial, antimutagenic, and anticarcinogenic activities. In experimental animals, chemopreventive efficacy has been observed in a number of target organs including colon and large intestine, duodenum, esophagus, forestomach, liver, lung, mammary glands, and skin. Additionally, modulation of intermediate biomarkers by tea compounds have been reported; epicatechin complex (a combination of four major catechins found in green tea), green and black tea extracts, and GTPs inhibited carcinogen-induced precancerous lesions in rat esophagus, GGT-positive foci in rat liver, and GSTwpositive foci in rat liver, respectively. Epidermal hyperplasia induced by such tumor promoters as TPA was also reduced by GTP administration. Further, GTPs inhibited aberrant hyperproliferation (an in vitro cellular biomarker for premalignant transformation) induced in mammary epithelial cell !ines by carcinogens and oncogenes (YUS or myc). Topical application of EGCG to mouse skin and oral administration of green tea extracts in drinking water were shown to inhibit TPA-induced ODC, PKC, and c-myc expression and NNK-induced lung oncogene (c-myc, C-H-YUS, and c-ruf) expression, respectively. Nearly all tea components tested have shown inhibitory activity in the rat tracheal epithelial (RTE) cell transformation, mouse mammary organ culture, and human lung tumor A427 cell assays. Significant inhibitory activity was also observed in in vitro assays measuring formation of DNA adducts and free radicals, and enhancement of GSH levels and GST, ODC, and NAD(P)H:quinone reductase activities. The antimutagenic activity of tea, GTPs, and EGCG has also been shown in stan-

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dard assays in Salmonella typhimurium (Ames assay), Escherichia coli, Bacillus subtilis, and V-79 cells. Inhibitory effects on the formation and mutagenicity of food mutagens, as well as formation of endogenous nitrosation products and cell transformation, have also been reported. Recently, green tea extracts were demonstrated to protect against DNA oxidative damage in rat colon measured by inhibition of DMH-induced 8-hydroxy-deoxyguanosine (8-OHdG) formation. In addition, several in vivo studies have demonstrated the inhibitory effects of tea, GTPs, and EGCG on tumor growth, as well as invasion and metastasis. Several mechanisms may be responsible for the antiinitiation and antipromotion properties of tea compounds. These include inhibition of radiationarid chemical-induced lipid peroxidation and free radical formation (antioxidant activity), inhibition of radiation- and TPA-induced epidermal ODC, COX, and LOX inhibition, PKC and cellular proliferation inhibition, inhibition of carcinogen-DNA binding and adduct formation, inhibition of inflammation including edema and IL-la mRNA and protein expression, inhibition of type 1 Sa-reductase activity, modulation of cytochrome P450 activity, enhancement of phase I1 (GST) as well as GSH-Px, catalase, and NAD(P)H:quinone reductase activities, and enhancement of gap junction intercellular communication. Additionally, ECGC has a so-called sealing effect; it inhibits interaction of tumor promoters, hormones, and various growth factors with their receptors. Approach to Developing Tea as a Chemopreventive Agent-Characterization ofActive Component. A significant effort in the development of dietary substances such as the tea components is standardization and characterization of the type, source, and manufacturing and storage techniques, since these factors will no doubt greatly impact the anticancer properties of such compounds. Part of the approach throughout the development work is parallel studies with a specific purified and known efficacious (based on animal carcinogenicity inhibition) component of tea, EGCG. Clinical studies with tea extracts are now in progress. Two Phase I trials of tea polyphenol extracts are in progress; one of these studies is also evaluating purified EGCG. A Phase 11 study of a tea polyphenol ointment in prevention of actinic keratosis is also in progress. Future trials may evaluate the safety and efficacy of the tea compounds in colon, esophagus, and lung.

E. Evaluating Chemopreventive Efficacy In developing chemopreventive agents a tiered approach is used to evaluate potential efficacy-starting with in vitro and cell-based mechanistic assays and efficacy screens, then screens in vivo in animal carcinogenesis models with cancers and precancerous lesions as end points. Finally, the most

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promising agents are characterized more fully in animal carcinogenesis models (e.g., dose-response and dosing regimens are evaluated, and combinations with other agents are Jested). 1. MECHANISTIC ASSAYS

Most often, agents not previously tested will be put first into mechanistic assays to determine their potential range of chemopreventive activities. Many of the assays are described above in relation to characterizing various classes of chemopreventive agents. The battery of assays used is continually evolving and is designed to address various specific activities associated with general categories of chemopreventive activity-antimutagenicity, antiprofiferation, and antioxidation (see Table V). As the molecular bases of carcinogenesis become better known, additional mechanistic activities to be explored for chemoprevention are identified. Examples of those in the early stages of investigation are inhibition of cell cyclins, telomerase, and angiogenesis, as well as binding to peroxisome proliferator-activated receptors (PPARs); the expansion of leads to mechanisms cited above such as RXRactivating retinoids and COX-2 inhibitors would also likely be fruitful. 2. CELL-BASED ASSAYS

Selected cell-based assays have been used routinely to screen the efficacy of potential chemopreventive agents. Examples are (1)inhibition of B[a]Pinduced morphological transformation in RTE cells, (2)inhibition of DMBAinduced hyperplastic-nodule formation in mouse mammary organ cultures (MMOC),and (3) inhibition of anchorage independence in human lung tumor (A427) cells. Initial criteria for selecting the in vitro tests include (1)efficiency in terms of time and cost, (2) sensitivity and ease of quantification, (3)-controlledtest conditions, (4)relevance to organ systems of interest, ( 5 ) use of epithelial cells, and (6)use, if possible, of human cells. The three current in vitro assays all use epithelial cells. The human lung tumor A427 cell assay primarily detects agents blocking postinitiation stages of carcinogenesis, 'whereas the MMOC can detect both antimutagens and antiproliferatives, depending on the treatment conditions (e.g., with DMBA alone or with DMBA and TPA). In each assay, the agents are tested over a wide range of concentrations, and IC,, values are determined (Steele et al., 1996). More recently, in vitro efficacy testing has been expanded, with the intent to incorporate new cell and organ culture technologies and newer information on genetic susceptibility to cancer. For example, the use of raft cultures which allow evaluation of stromal-epithelial interactions, cells from transgenic mice, and cells from subjects carrying known cancer-predisposing genes (e.g., Li-Fraumeni syndrome, APC mutations) are being explored.

Table V Representative Assays of Chemopreventive Mechanismsa Assays Antimutagenesis B[u]P-DNA adduct formation (inhibition) NAD(P)H:quinone reductase (induction) GSH S-transferase (induction) GSH synthesis and GSSG reduction (induction) Antiproliferation TPA-induced ODC (inhibition) Normal epithelial cell proliferation (inhibition) Poly(ADP-rib0se)polymerase(inhibition) Calmodulin-regulated phosphodiesterase (inhibition) TPA-induced tyrosine kinase (inhibition) EGFR (inhibition) rus Farnesylation (inhibition) HMGCoA reductase (inhibition) Steroid aromatase (inhibition) Estrogen receptor (antagonism of binding and expression) Sa-Reductase (inhibition) Cellular differentiation characteristics (modulation) DNA fragmentation (induction) Antioxidant/antiinflammatoryactivity AA metabolism: micronuclei in keratinocytes (inhibition) TPA-induced active oxygen (inhibition) COX-2 (inhibition) LOX (inhibition) %ee Sharma eta!. (1994).

CeU substrate

Mechanism measured

Human bronchial epithelial cells (BEASZ-B) Human (Chang) liver cells Human (Chang) liver cells Buffalo rat liver (BRL-3A)cells

DNA damage inhibition Carcinogen detoxification Carcinogen detoxification Carcinogen detoxification

Rat tracheal epithelial cells (2CS cell l i e ) Primary human keratinocytes Primary human fibroblasts Human leukemia (HL60)cells

Antiproliferative activity Antiproliferative activity Error-prone DNA repair inhibition (DNA damage inhibition) Signal transduction regulation

Human leukemia (HL60) cells Human A431 and mouse 3T3 cells Rat brain farnesyltransferase Rat liver HMG-CoA reductase PMSG-stimulated rat ovary aromatase MCF-7 cells

Signal transduction regulation Signal transduction regulation Signal transduction regulation Signal transduction regulation Antiestrogenic activity Antiestrogenic activity

Rat prostate Sa-reductase Human leukenia (HL60) cells

Antiandrogenic activity Differentiation

Human leukemia (HL60)or U937 cells

Apoptosis

P388 macrophageslhuman keratinocytes (macrophage-generatedperoxides induce micronuclei in keratinocytes) Human leukemia (HL60) Cells Sheep placenta COX-2 Rat RBL-1 cell LOX (for S-LOX)

Antiinflammatory activity Free radical scavenging Antiinflammatory activity Antiinflammatory activity

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3. INHIBITION OF ANIMAL CARCINOGENESIS

a. Prevention of Cancers in Carcinogen-Induced Animals Numerous animal models-are used to study inhibition of chemical carcinogenesis (see Table VI) (Steele et al., 1994; Kelloff et al., 1995a). Most studies are carried out in rats, mice, or hamsters. Typically, a carcinogen is administered to the animal at a dose level high enough to induce a significant incidence of tumors in a specific target tissue. The carcinogen dose and treatment schedule usually are selected to ensure that the tumor incidence is not so high as to mask the potential of the inhibitor to reduce tumorigenicity. The inhibitor is administered before, at the same time, or after, or in any combination of these times relative to the administration of the carcinogen. The relative timing of the administration of the carcinogen and the inhibitor is useful in interpreting the mechanism of inhibition. For example, a compound that inhibits when it is administered before the carcinogen, but not when it is given after carcinogen treatment is completed, most likely inhibits the initiation phase of carcinogenesis. Studies typically last as long as required for the carcinogen to induce a high tumor incidence. Because the activity of most of the carcinogens used is well known, these tests are usually shorter than chronic carcinogenicity studies. Most often, they last 6 months to 1 year. Inhibition is usually measured as the percentage by which the inhibitor lowers the incidence, multiplicity, or total number of tumors, or increases the latency of tumor induction. Sometimes such factors as tumor size and degree of invasiveness are considered. Results usually are determined by histopathological evaluation of the target tissues, although gross pathology also may be used. For example, rat mammary tumors often are detected by palpation, and mouse skin tumors are determined visually. Some general guidelines have been suggested for interpreting the results of testing a potential inhibitor of carcinogenesis (Kelloff et al., 1995a). For a test to indicate an inhibitory effect, the chemical must cause a statistically significant (p < 0.05) decrease in tumor incidence, multiplicity, size, or invasiveness, or a statistically significant increase in tumor latency compared with karcinogen controls. Tumor latency is measured as the time to appearance of the first tumor or the time to 50% tumor incidence. In the absence of statistics, at least a 2-fold decrease in incidence, multiplicity, size, or invasiveness, or a similar increase in latency, should be observed to confirm an inhibitory effect. A result is considered suggestive if no statistical analyses are performed but the inhibition ranges from 35 to 50%. These criteria are for a single dose of a chemical. If at least three doses are tested and a doseresponse inhibition is observed, the result may be considered positive even if the effect is not statistically significant or of 2-fold magnitude at any dose tested.

Table W Carcinogen-Lnduced Animal Models for Chemoprevention Efficacy Studiesu.’ Organ model

species

Carcinogen

End point: inhibition of

Buccal pouch Colon

Hamster Mouse Rat Rat Mouse Rat Mouse Rat Mouse Hamster

DMBA AOM, DMH, MAM AOM, DMH, MAM, MNU Nitrosamines BbIP AOM, DMH Various AAF, DEN, DMN, Me-DAB B[a]P, DMBA, NNK,urethane DEN, MNU (trachea)

Mouse Rat Hamster Rat Mouse

Rat

DMBA DMBA, MNU BOP LAzaserine W radiation, B[a]P, B[n]PTTPA, DMBA, DMBAITPA, MCA MNNG

Squamous cell carcinoma, papilloma Adenocarcinoma, adenoma, aberrant crypt foci Adenocarcinoma, adenoma Squamous cell carcinoma, papilloma Squamous cell carcinoma, papilloma Adenocarcinoma, adenoma Hepatocellular carcinoma, adenoma Hepatocellular carcinoma, adenoma Adenoma Squamous cell carcinoma, adenosquamous carcinoma Menocarcinoma, adenoma Adenwarcinoma, adenoma Ductal adenwarcinoma, adenoma Acinar cell carcinoma Carcinoma, papilloma

Mouse Rat

OH-BBN MNU, OH-BBN

Esophagus Forestomach Intestines (NOS) Liver Lung

Mammary glands Pancreas

Skin Stomach (and glandular stomach) Urinary bladder

Adenocarcinoma Transitional cell carcinoma Transitional cell carcinoma

%ee Steele et ul. (1994) and Kelloff et a/.(199Sa). bAbbreviations: AAF, acetylaminotluorene; AOM. amxymethane; B[u]P, benzo[u]pyrene; BOP, N-nitrosobis(2-oxopropyl)amine;DEN, N,N-diethylnitrosamine; DMBA, 7,12dimethylbenz[a]anthracene; DMH, dimethylhydrazine; DMN, N,N-dimethylnitrosamimine;MAM, methylazoxymethanol; MCA, methylcholanthrene; Me-DAB, methyl-N,N-dimethyl-4-aminoamknzene; MNNG, N-methyl-N’-nitro-N-nitrosoguanidine; MNU, N-methyl-N-ninosourea; NNK, N-nitrosonornicotine; OH-BBN, N-butyl-N-(4-hydroxybutyl)Ntrosamine; P A , 12-0-teaadecanoylphorbol-13-acetate.

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Regardless of the magnitude of the effect observed, other factors are considered in determining that a test is an adequate measure of inhibition. First, the numbers of animals in the treatment and control groups should be sufficient to demonstrate statistical significance. Also, survival in both test and control groups should be adequate to allow statistical evaluation, that is, the toxicities observed due to carcinogen or inhibitor treatment should not be so severe as to compromise the results of the study. Evidence of carcinogenicity should be established in concurrent carcinogen-treated control animals. Examples of specific conditions that call the adequacy of a test into question are as follows: statistically significant lower body weights in animals treated with inhibitor compared with carcinogen controls; a relatively srpaller number of animals used; or a lower than expected tumor incidence in the carcinogen control group. Body weight is a particularly meaningful and confounding factor in inhibition experiments, since decreased or delayed weight gain can be a measure of slowed growth. Slowed growth alone can depress tumorigenicity without other specific effects of an inhibitor.

b. Inhibition of Carcinogenesis in Transgenic and Gene Knockout Mice Targeted research using animal models that mimic specific characteristics of human carcinogenesis is valuable in fully evaluating chemopreventive efficacy and in determining appropriate biomarkers for measuring chemopreventive activity (see Table VII). Transgenic and gene knockout mice that carry well-characterized genetic lesions predisposing to carcinogenesis are appropriate models. For example, the multiple intestinal neoplasia (Min) mouse (Su et al., 1992) and other strains carrying lesions in the Apc gene (Edelmann et al., 1999) may be the best developed. The Min mouse has an Apc mutation qualitatively similar to that in human FAP patients, which prediseoses the mice to developing colorectal adenomas and carcinomas. Jacoby et al. (1996) have found a strong correlation between inhibition of PG synthesis and adenoma formation in this strain. Also, an HPV-infected (K14HPVl6 heterozygote), estradiol-treated mouse develops cervical squamous carcinomas that result from progression of CIN-like lesions. These lesions can be inhibited by DFMO (Arbeit et al., 1996). Closer approximations to human carcinogenesis may be possible by manipulating two or more carcinogenesis-associated genes, including modifier genes, in a single animal. For example, it might be feasible to knock out p53 in an animal that already carries another tumor suppressor defect (e.g., Apc or p16). Studies are now being done on Min and Apc1638 (de Wind et al., 1998; Edelmann et al., 1999) mice also carrying genes allowing errorprone DNA repair (MSH2 and MLH1, respectively). A key contribution to future development of such animal models will be identification of specific cancer-related genes (e.g., in the Cancer Gene Anatomy Project) that can be

Table VII Representative TransgenidGene Knockout Mouse Models for Chemoprevention Studies Transgenic mouse model

Target

Genetic lesions

Min APC MLHlIApcl638 MSH2lMin pim TG.AC TSG-pS3 A/JXTSG-p53 A/JXUL53 TGFpl v-Ha-rus

Colon Colon Colon Colon Lymphatic system Skin Skin Lung Lung Live4 lung Skin

Heterozygous Apc2549 HeterozygousApc1638 Heterozygous MLHl and Apc1638 Heterozygous MSH2 and Apc2549 Amplified pim-1 H a m s mutation Heterozygous pS3 deficient Heterozygous p53 deficient Heterozygous p53 mutant Heterozygous TGFpl mutant Ha-rus + human keratin K-1

K14-HPV16

Skin

HPV-infected (K14-HFVl6 heterozygote), estradiol-treated + SV40 T-antigen HPV-infected (K14-HPV16 heterozygote), estradiol-treated + SV40 T-antigen Heterozygous rat prostatic steroid binding gene [C3(1)] + SV40 T-antigen Heterozygous rat prostatic steroid binding gene [C3(1)] + SV40 T-antigen

K14-HPV16

C3(1)-SV40

Prostate

C3(1)-sv40

Mammary glands

Histological lesions Adenomas, adenocarcinomas, some CIS Adenomas, adenocarcinomas Adenomas, carcinomas Adenomas carcinomas T-cell lymphomas Papillomas, possible carcinomas Papillomas, possible carcinomas Adenomas Adenomas Adenomas, carcinomas Hyperplasia, hyperkeratoses, squamous papillomas Papillomas, condylomas

Cervical dysplasia

Dysplasia, adenoma, adenocarcinoma Adenocarcinoma

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applied to the construction of animal models for evaluating chemopreventive efficacy. The treatment of transgenic and gene knockout mice with carcinogens may prove to be particularly effective as a strategy for modeling human carcinogenesisat specific cancer targets. For example, You and colleagues (Matzinger et al., 1995) are evaluating chemopreventive effects of various agents in p53 mutant and gene knockout mice also treated with carcinogens (e.g., B[a]P or NNK to induce lung tumors, or dimethylhydrazine to induce colon tumors).

IV. CHEMOPREVENTIVEAGENT DEVELOPMENT The National Cancer Institute (NCI) and the U.S. Food and Drug Administration (FDA) have collaborated to provide conceptual and practical guidance in developing cancer chemopreventive drugs (see Table VIII) (Kelloff et al., 1995b).The general strategy (see also Kelloff etal., 1994f, 1996a, 1997a) is to first characterize the efficacy of candidate drugs using in uitro transformation modulation, chemoprevention-related mechanistic assays, and animal tumor modulation models of carcinogenesis, as just described. Agents that appear most efficacious are evaluated for preclinical toxicity and pharmacokinetics as needed. Clinical development is then planned and implemented for those agents that meet the criteria for acceptable toxicity as well as efficacy. Often, additional efficacy and toxicity testing is done to test alternative routes of agent delivery, dosage regimens, new target tissues, and combinations of agents for increased efficacy and decreased toxicity, and to evaluate toxicities seen in early clinical studies. Clinical development of chemopreventiveagents, as for other pharmaceuticals, is carried out primarily in Phase I, 11, and III trials. Phase I clinical trials are safety and pharmacokinetics studies. These trials include single dose studies in both fasting and nonfasting normal subjects to characterize single dose pharmacokinetics and acute toxicity. Also, repeated daily dose studies to assess, multiple dose pharmacokinetics and chronic toxicity are conducted using multiple dose levels for a period of 1-3 months in normal subjects or up to 12 months in subjects at increased risk of cancer(s), for which the drug demonstrates efficacy in preclinical evaluation. Participation of normal subjects for more than 1month is considered based on available information (toxicity,clinical experience, etc.) for each drug on a case-by-case basis. In most cases, the Phase I studies evaluate agent effects as well as agent serum (and sometimes, tissue) levels. Agent effects measured are those believed to be potentially associated with chemopreventive activity. For example, in studies of NSAIDs, serum and tissue levels of PGEz would be measured. In studies with the irreversible ODC inhibitor DFMO, tissue levels of polyamines are measured.

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Phase I1 trials are initial efficacy studies. These randomized, double-blind, placebo-controlled trials emphasize the evaluation of intermediate biomarkers that are highly correlated to cancer incidence and may serve as surrogate end points for cancer incidence reduction. Phase 111studies are randomized, blinded, placebo-controlled clinical efficacy trials. These studies are typically large and have the objectives of demonstrating a significant reduction in incidence or delay in occurrence of cancer, validating surrogate end points, further assessing drug toxicity, and further characterizing the relationship of dose and/or pharmacokinetics to efficacy and toxicity. Because cancer has a long latency, reduced incidence is an impractical end point for clinical evaluation of chemopreventive agents. Thus, intermediate biomarkers of carcinogenesis are being evaluated and validated as surrogate end points for chemoprevention trials. These biomarkers are addressed in both preclinical and clinical studies. The criteria for surrogate end point biomarkers are that they fit expected biological mechanisms (i.e., differential expression in normal and high-risk tissue, on or closely linked to the causal pathway for the cancer, modulated by chemopreventive agents, and short latency compared with cancer), may be assayed reliably and quantitatively, may be measured easily, and correlate to decreased cancer incidence (Kelloff et al., 1994a). They must occur in sufficient incidence to allow their biological and statistical evaluation relevant to cancer. The rationale for the testing done during chemopreventive agent development is described in the following sections.

A. Preclinical Efficacy Development Using a Balance

of Molecular Target and Empirically Based Assays The discussion above on specific prototypical chemopreventive agent classes focused on mechanism-based approached to identifying and evaluating the relative efficacy of new chemopreventive agents within a class. Another, equally useful approach described in the previous section employs a combination of molecular target and empirically based screening for candidate agents for which less is known about their potential mechanisms of action. Screening starts with testing in a battery of the mechanistic assays (see Table V) representing a wide range of chemopreventive activities. Positive agents may then be screened in a battery of in vitro cell-based assays. Positive agents are then moved on to animal models (TablesVI and VII). Typically, the models are selected based on clues provided by the mechanistic and cell-based assays. Besides the mechanism-based and screening approaches, there is also a need for more translational research, directed at testing specific hypotheses (e.g., new model development, agent delivery mechanisms, potential synergy of efficacy and safety by the use of agent combinations, and rational ap-

N 4

P

Table Vlll FDA/NCI Consensus on Approaches to the Development of Chemopreventive Drugsa I. Preclinical efficacy studies recommended for initiation of Phase Vn clinical trials for chemopreventive investigational drugs are (1) + (7),(2)+ (4 or 5) + (8), (3)+ (4 or 5) + (8), (6): (1) In vivo tumor modulation with (statisticallysignificant)reduced tumor incidence or multiplicity or increased latency (2) In vivo tumor modulation with (statisticallynonsignificant but dose-associated positive trend) reduced tumor incidence or multiplicity or increased tumor latency (3) In uivo surrogate end point modulation (statisticallysignificant) (4) In vitro transformation modulation ( 5 ) In vitro chemoprevention-relatedmechanistic studies (6) Epidemiologicalstudy demonstrating a cancer-inhibitory effect of the specific agent in the target issue (7) In vivo concentration-effect relationship (8) In vitro concentration-effect relationship 11. Preclinical safety studies for initiation of Phase uD[ clinical triais for chemopreventive investigationaldrugs are the following: (1)General toxicity studies conducted in two species, rodent and nonrodent, of equal or greater duration than the proposed clinical trial or up to 6 months in rodents and 12 months in dogs; route of administration should be equivalent to the intended clinical route, and drug substance should be that prepared for clinical trials (preferablythe clinical formulation) (2) Genotoxicity assessed in a battery of assays [Ames test in SalmonelIa typhirnurium, gene mutation in mammalian cells in vitro (either L5178Y mouse TK+’-lymphoma cells or another cell line with an autosomal locus with documented sensitivity to mutagenic chemicals, such as Chinese hamster ovary AS52 cells), and cytogenetic damage in vivo (mouse bone marrow micronucleus and/or mouse or rat chromosomal aberration tests)] (3) Segment I reproductive performancdfertility in rat and Segment II teratology in rat.and rabbit.should be conducied-asearly as possible, prior to large clinical trials or trials of long duration, and in accordance with the International Conference on Harmonization (ICH) and the Guideline for the Study and Evaluation of Gender Differences in the Clinical Evaluation of Drugs (4) Combinations of chemopreventive drugs should be evaluated in at least one general toxicity study of appropriate duration in the most appropriate species for interactions in pharmacokinetics,toxicity, enzyme effects, or other relevant parameters (Pharmacokineticsand metabolite profiles should be examined in conjunction with toxicity studies to aid in interpretation of findings and evaluation of relevance to humans) Required before clinical studies > I year’s duration (large Phase I1 and Phase 111) (1) Completion of general chronic toxicity studies in two species (6months in rodent, 12 months in nonrodent) (2) All special toxicity studies (assessingneurotoxicity, cardiotoxocity, etc., as appropriate) before Phase III (3) Initiation, preferably completion, of at least one of the rodent carcinogenicity bioassays prior to initiation of large Phase ITI studies

Required for New Drug Application (1)Segment 111perinatal and postnatal development study in rats (2) Completion of two rodent carcinogenicity bioassays ID. Phase I-111 clinical studies for chemopreventive investigational drugs Required Phase I (1)Single dose studies in both fasting and nonfasting normal subjects to characterize single dose pharmacokinetics (i.e., absorption, distribution, metabolism, elimination) and acute toxicity (2) Repeated daily dose studies using multiple dose levels for a period of 1-3 months in normal subjects or up to 12 months in subjects at increased risk to cancer(s), for which the drug demonstrates efficacy in preclinical evaluation, to assess multiple dose pharmacokinetics and chronic toxicity; participation of normal subjects for more than 1 month is considered based on available information (toxicity, clinical experience, etc.) for each drug on a case-by-case basis Recommended Phase I Include placebo control and pharmacodynamicevaluation of dose response for modulation of selected drug effect or surrogate end point biomarkers; subject follow-up on completion of treatment will include evaluation of modulation of marker status Phase I1 (1)Phase IIa:In the event that a clearly defined and standardized surrogate end point biomarker is not identified, then a randomized, blinded, parallel dose-response chronic dosing study will be conducted for 3 months or more in subjects at high risk for cancer at the site of investigation using dosing levels shown to be safe in prior Phase I studies; as a basis for the 2b study, the objectives are to evaluate measurements of candidate biomarkers (drug effect and/or surrogate end point) and the dose-response relationship of biomarker modulation and tolerance to modulation, to standardize assays and quality control procedures, and to characterize chronic dosing toxicity (2) Phase IIb: Randomized, blinded, piacebo-controlled chronic dosing study for 3 months or more in subjects at high risk for cancer at the site of investigation at one or more dosing levels shown to be safe and effective in modulating biomarkers; study objectives are to establish dose-surrogate end point marker response and chronic dosing toxicity and to select a safe and effective dose based on surrogate end point marker response and chronic dosing toxicity Phase I l l Randomized, blinded, placebo-controlled clinical trials with the following objectives: (1)Demonstrate a sigdcant reduction in incidence or delay in occurrence of cancer (2) Validate surrogate end points (3) Assess drug toxicity (4) Characterize the relationship of dose and/or pharmacokinetics to efficacy and toxicity ( 5 ) In case of formulation differences, establish the bioequivalence between the to-be-marketed formulation and the formulation used in pivotal clinical trials ~~

'Adapted from Kelloff et al. (1995b).

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proaches to the development of defined mixtures as the best means to further test and verify hypotheses being generated by epidemiology data on diet and cancer).

I . NEW MODEL DEVELOPMENT Translational research directed at evaluating new hypotheses will do much to spur the development of chemoprevention. Some hypothesis may be applied to constructing new carcinogenesis models as described above or evaluating new classes of agents. Research to develop new cell and organ culture as well as whole animal models that approximate human carcinogenesis is still in its early stages and will grow in importance to chemoprevention over the next few years. Models combining carcinogen administration and gene manipulation hold great promise. However, patent issues, which now preclude the extensive use of transgenic models for testing novel agents, will need amelioration. Other hypotheses may be directly related to developing agents for clinical use, for example, investigating new delivery systems or pharmacodynamic modeling. A recent example confirmed the chemopreventive potential of aerosolized steroids (Wattenberg et al., 1997). In this study Wattenberg used aerosolized budesonide in B[aJP-treated mice to establish the principle of topical delivery to precancerous epithelia with multifocal (field defect) damage as a chemoprevention strategy. It has particular promise for the lung but is applicable to several target organs, the primary advantage being to improve therapeutic index (in the case of corticosteroids about 1/30).Retinoids formulated and delivered by this means could well improve efficacy without toxicity, being applicable eventually to large populations. A pilot study of one of the currently approved aerosolized steroids is in progress in patients with precancerous lesions in the bronchus (visualized by the LIFE scopeto locate the best areas to sample). This study should provide the proof of principle for allocating resources to develop some of the more obvious drugs.

2. AGENT COMBINATIONS One strategy to improve efficacy and lessen toxicity is using combinations of agents. In some combinations of two agents with different presumed mechanisms of activity, synergistic or additive activity may be seen. Such improved activity may allow either or both the agents to be administered at lower doses, thereby reducing potential toxicity. For example, as cited above, synergisticactivity has been observed in rat colon studies with combinations of DFMO and the NSAID piroxicam and in rat mammary gland with combinations of retinoids and antiestrogens, and these strategies are now being tested clinically. Another example now being evaluated in a Phase I clinical

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study is the combination of the GST-enhancer oltipraz with the GSH precursor NAC; the hope is that the increased level of GSH produced by NAC will maximize the effect of oltipraz-induced GST. The identification and evaluation of potentially effective agent combinations will be an ongoing research effort for chemoprevention. Another combination strategy is the use of a second agent to counter the toxicity of a known effective chemopreventive agent. An example, also cited above, is coadrninistration of the PGE, analog misoprostol to counter the gastrointestinal toxicity associated with administration of NSAIDs.

3.. DEVELOPMENT OF DIETARY COMPONENTS Dietary components with chemopreventive activity typically start as complex mixtures. The preparation and characterization of optimal standardized mixtures and purification of the active substance are challenges for the development of dietary components. For example, the preclinical efficacy of curcumin has been determined primarily with food-grade agent, which is a mixture of curcuminoids, ranging from 40 to 85% curcumin. A purified curcumin, micronized for increased bioavailability, is now being evaluated. It is possible that this preparation will enhance both efficacy and toxicity. Two soy isoflavone mixtures containing genistein, other isoflavones (primarily daidzein), fat, and carbohydrate are being developed. One is nearly “pure,” containing 90% genistein; the second more closely resembles a natural soy product, containing less than 50% genistein. Similarly, as described above, tea polyphenol extracts have been well characterized for evaluation in preclinical studies, and EGCG, which appears to be a primary active component, is being developed in parallel. The effort to confirm dietary leads is expected to burgeon over the next few years. For example, the FDA has proposed guidelines for the identification and evaluation of heterogeneous botanicals such as the tea and isoflavone mixtures, and the number of publications on chemopreventive effects of characterized dietary components is increasing [e.g., many on tea polyphenols, curcuminoids, selenized garlic/selenomethylcystine, and broccoli compounds (sulforaphan)]. It is expected that the increasing level of sophistication in the analysis of epidemiological data will lead to many more new chemopreventive hypotheses regarding dietary components.

B. Toxicology and Pharmacology As for other pharmaceuticals, the FDA requires sufficient preclinical toxicity and Phase I clinical safety and pharmacokinetics testing to ensure that an investigational chemopreventive agent will not jeopardize the health of

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patients in efficacy trials. Because chemopreventive agents are intended for chronic use in relatively well subjects, the safety criteria are more stringent for chemopreventives than for many other classes of pharmaceuticals. The consensus guidance of the N C I and FDA includes recommendations for preclinical toxicity and Phase I clinical studies. These recommendations are summarized in Table VIII and recounted below. 1 . PRECLINICAL TOXICITY AND PHARMACOKINETICS Preclinical safety studies for chemopreventive drugs are generally the same as for other drugs and include investigations at relevant doses and schedules of acute and subchronic toxicity (incorporating pharmacokinetic measurements), reproductive performance, and genotoxicity. Generally included are a single dose, acute toxicity study and an absorption-elimination study in rats, and subchronic repeated daily dosing studies in rodents and dogs. Combinations of chemopreventive drugs are evaluated in the species most closely related to humans in terms of metabolism in at least ofie study of appropriate duration (generally, studies >90 days are not needed) to determine interactions in pharmacokinetics, toxicity, enzyme effects, or other relevant parameters. Preclinical efficacy studies also should incorporate limited toxicity that may help identify appropriate doses for the formal toxicity studies. For example, most of the animal efficacy screens include a preliminary 2-6 week study to determine the maximum tolerated dose (MTD) of the test agents. Blood levels of test agent are usually obtained during animal studies designed to more fully characterize the efficacy of an agent or agent combination. The pharmacokinetics of the test agent is an important recommended component of preclinical safety studies. Pharmacokinetic data can help in the development of a Phase I clinical dose escalation strategy. Absorption-elimination studies in rats are used to develop analytical methods for drug monitoring, which can be standardized and used in the clinic. These studies also provide other agent behavior information, including protein binding. Sinqle dose pharmacokinetics are also assessed at the initiation of the repeated daily dosing studies in dogs, and measurements of plasma drug levels at steady state are performed in these studies in rodents and dogs (pharmacokinetic studies using radioactive drug to quantify tissue distribution and metabolism are performed later in development). The information developed at this stage ( Cm,,, t,,,,. Cmin,AUC, C,,, etc.) is evaluated with information from efficacy studies in order to provide a dose-concentrationeffect profile of the test agent and to estimate a margin of safety; the relationship of dose to effectiveness and toxicity is then used to refine dosing strategies and regimens. For example, if a promising chemopreventive agent

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has slight toxicity with daily dosing it might be evaluated in further Phase I trials using intermittent dosing schedules chosen to induce or inhibit a drugeffect enzyme over the whole treatment period while not reaching a C,,, that is toxic or maintaining a C,, that may cause side effects. As needed, a battery of three genotoxicity tests is performed: (a) mutation in Salmonella typhimurium, (b)mutation in mammalian cells in vitro (either L5178Y mouse TK+’-lymphoma cells or another cell line with an autosoma1 locus with documented sensitivity to mutagenic chemicals, such as Chinese hamster ovary AS52 cells), and (c) cytogenetic damage in vivo (mouse bone marrow micronucleus and/or mouse or rat chromosomal aberration tests). Chronic toxicity, carcinogenicity and segment 111reproductive tests are undertaken later in development, prior to or during Phase III development. Special toxicity studies are also undertaken, as appropriate, in response to safety issues arising on clinical use of the agent. One example of special studies recently pursued is for DFMO, which is now in Phase II clinical trials. In previous clinical studies, this agent had shown significant ototoxicity. The mechanism appeared to be destruction of cochlear cilia. Thirteen-week studies in dogs were undertaken to evaluate the effect quantitatively. At doses of 25 or 100 mg/kg/day no effects were seen on cochlear hair cell measurements, brainstem auditory evoked responses (including histology of auditory nuclei), or observed response to auditory stimuli such as clapping or yelling. For most investigational drugs used chronically to treat disease states, carcinogenicity studies are required prior to submission of a New Drug Application (NDA). Generally one rodent carcinogenicity study is initiated prior to initiation of large Phase 111clinical studies. However, for drugs under development for cancer prevention, where the agent is to be used prophylactically in essentially well people, completion of one carcinogenicity study prior to conduct of sizable long-term trials is considered based on the expected toxicity of the drug, the population, the planned clinical trial duration, the trial design, and other factors.

2. PHASE I CLINICAL SAFETY AND PHARMACOKINETICS Phase I single dose studies in humans are designed to characterize agent pharmacokinetics and tolerability. The dose and schedule of administration are based initially on preclinical toxicity and efficacy and are selected to achieve safe and effective plasma agent levels in humans. As is typical for other pharmaceuticals, the maximum initial dose in humans is a milligram per kilogram dose that is the lower of one-tenth the highest no observed adverse effect dose (NOAEL) in rodents or one-sixth the highest NOAEL (in mg/kg) in nonrodents. The NOAEL is based on toxicity studies of equal or

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greater duration than the proposed clinical trial. The in vitvo inhibitory concentration and in vivo plasma drug levels from efficacy testing may be used as a relative guide to the needed concentration, that is, within an order of magnitude, but are not easih quantitatively extrapolated due to the conditions used in screening tests (i.e., the high dose of carcinogen). Ordinarily the human dose (usually in mg/kg) is not escalated above the animal NOAEL, but this could depend on the nature of the adverse effect. Higher human doses may be justified on the basis of pharmacokinetic or pharmacodynamic differences between humans and animals or clinical experience at lower doses. Where possible the dose escalation strategy uses pharmacokinetic parame, Cs,,AUC) to guide dosing and predict toxicters across species (e.g., C ity and efficacy. After a cautious initial dose, further escalation is based on , and AUC and its relation to the toxic , C and AUC in aniubserved C mals. Differences in the pharmacokinetic profile after acute and chronic dosing are also evaluated. Dose selection is, of course, ultimately controlled by emphasis on empirical clinical safety and toxicity observations. Consistent with current FDA regulatory practice, normal subjects are used in studies 1-3 months in duration; participation of normal subjects for more than 1month is considered based on available information (toxicity, clinical experience, etc.) for each drug on a case-by-case basis. When longer Phase I studies are undertaken, up to 12 months, then subjects at increased risk for cancer(s) are enrolled. Longer studies are designed not only to obtain pharmacokinetic and safety information after chronic administration but also to develop and evaluate biomarkers. During Phase I studies these may include agent effect as well as potential surrogate end point biomarkers. Agent effect biomarkers are tissue, plasma, and urine indicators of the presence of active agent. These measurements correlate to an effective tissue, plasma, or urine concentration of agendmetabolite and reflect a biochemical activity that is relevant to the pharmacological action of the agent. For example, DFMO inhibition of ODC, which is induced during proliferation, will decrease ODC activity levels and alter levels of the product putrescine and the downstream metabolites spermidine and spermine. Low levels of these polyamines cause slowed progression through the cell cycle. Measurements of ODC activity and polyamine levels have been made in target tissue (e.g., colorectal mucosa), blood, urine, and skin as potential agent effect biomarkers of proliferation. Agent effect biomarkers include, as a subset, agent exposure biomarkers. These are defined as the pharmacokinetically monitored tissue, plasma, or urine concentration of the agent or its metabolites. Local concentrations of the agent do not usually give evidence of an agent’s effect; however, in the case of covalent modification or adduct formation, the agent’s effect and exposure may be measured together.

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C. Importance of Intermediate Biomarkers of Carcinogenesis and Their Measurement as Surrogate End Points for Chemoprevention Studies Intermediate biomarkers of cancer are the phenotypic, genotypic, and molecular changes that occur during carcinogenesis. The development of intermediate biomarkers as surrogate end points for chemoprevention clinical trials is important to a viable chemopreventive agent development program. Because of the shorter latency to intermediate biomarker end points and the smaller cohorts required for treatment, this effort is critical to the progress of. chemoprevention and the potential for cost-effective development of chemopreventive agents. Westra and Sidransky (1998)and Mao et al. (1998)have underscored the need to evaluate genotypic changes in normal-appearing epithelia surrounding dysplastic lesions with the finding that the phenotype of the whole tissues does not always reflect underlying genotypic changes that contribute to new primary tumors. Progressive genome instability as measured by LOH or amplification at specific microsatellite loci was used by Sidransky and colleagues (e.g., Califano et d,1996) to characterize head and neck carcinogenesis. These biomarkers are potential surrogate end points in head and neck, and may also prove useful in other tissues where microsatellite instability is a predominant feature of carcinogenesis, for example, in hereditary nonpolyposis colorectal cancer (HNPCC) and some sporadic colorectal cancers. For all the biomarkers, it is highly desirable to measure modulation quantitatively as the difference (A) between the biomarker value at the end of treatment and baseline. Thus, baseline biopsies or other baseline tissue measurements are important. New technology such as computer-assisted pathology, high-volume gene chip-based assays and improved diagnostic tools such as the confocal microscope, the LIFE scope for visualizing bronchial tissue, and the magndying endoscope for colorectal monitoring will be critical to assuring the adequate development of surrogate end point biomarkers for chemoprevention studies.

I . QUANTITATIVE END POINTS-COMPUTER-ASSISTED IMAGE ANALYSIS The quantitative evaluation of surrogate end points is important, since it is very likely that qualitative measures will be too crude and lack the reproducibility to detect carcinogenesis-associated changes in small samples. Biomarkers measured by CAIA, including both nuclear and nucleolar morphometry and cytophotometry, should prove valuable in this regard. Ade-

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quate performance ensures that a small trial with limited tissue availability will produce meaningful results. Quantitative nuclear and nucleolar morphometric changes (i.e., changes in size, shape, and texture of nuclear material) may be used to describe the histopathology that characterizes progression of IEN. Their promise is based on gradient changes associated with increasing E N severity. Several computer-assisted imaging systems are commercially available. These systems essentially consist of a light microscope, light sensor, digitizer to convert the light to computer-readable form, and a computer with appropriate software to analyze the tissue measurements. Examples of these measurements are nuclear size, shape, texture, and pleomorphism and nucleolar number, size, shape, yosition, and pleomorphism. CAIA is also useful for cytometry including measurements of cellular prolifer'ation and DNA ploidy that typify IEN histopathology. 2. GENE CHIP TECHNOLOGY, IMMUNOCHEMISTRY, AND QUANTIFYING EFFECTS AT MOLECULAR TARGETS The supporting technology as well as the data generated from the Human Genome Project and the Cancer Gene Anatomy Project have provided the means to look quantitatively at general genetic damage as well as specific genetic changes at the molecular level. Much of the work in this area has involved gene sequence comparisons. For example, the measurement of microsatellite instability using RT-PCR at predefined markers has been described above for detection of gene expression changes at loci relevant to carcinogenesis. Microsatellite instability is evidenced by amplification or LOH at these loci. Sidransky (Califano etal., 1996) has used this technique in the development of a molecular progression model for head and neck cancer. However, although quantitative, this technique looks only at DNA sequences and not at specific gene functions. Comparative gene hybiidization (CGH)is being used to approach the evaluation of functional changes. In this technique, gene chips with specific gene sequences (cDNA) and mutations are made (e.g., wild-type and well-characterized mutations in p5.3). Corresponding changes in genes in tissue undergoing carcinogenesis can be evaluated by hybridizing the tissue DNA to the specialized chips. Many different commercial gene chip packages are now made. More importantly, the capability for making individualized gene chips with specific genes is now available. Besides evaluating changes in tissues undergoing carcinogenesis, these chips may be designed to evaluate subjects at risk, for example, those carrying specific germline mutations and genetic polymorphisms. Fluorescence in situ hybridization and particularly CISH are also powerful techniques to quantify potential functional changes in carcinogenesis-related genes. Both techniques involve labeling specific gene products related

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to carcinogenesis. FISH applies a fluorescent label to a gene product of interest. CISH applies different labels (different colors) to wild and mutant gene products, allowing the comparison of different relative amounts at different stages of carcinogenesis and before and after treatment with chemopreventive agents. Hittelman et al. (1996)applied CISH to head and neck as described above. I

3. VALUE OF ANIMAL MODELS FOR VALIDATION

OF SURROGATE END POINTS As suggested above, animal models, particularly in transgenic and genehockout mice, that mimic specific characteristics of human carcinogenesis are useful in evaluating biomarkers as surrogate end points for cancer incidence. Particularly, the correlation of surrogate end point modulation to effects on cancer incidence in such models can provide strong evidence for validating the surrogate end point. This correlation can strengthen efficacy claims prior to definitive clinical validation. As described above, transgenic and knockout mice that carry well-characterized genetic lesions predisposing to carcinogenesis are proving to be good models for biomarker evaluation. For example, the inhibition of adenomas in the Min mouse was described above. A key contribution to future development of animal models will be identification of specific cancer related genes (e.g., in the Cancer Gene Anatomy Project) that can be applied to the construction of animal models for evaluating surrogate end points. Extensive biomarker research and development are also being carried out in carcinogen-induced animals, as well as in carcinogen-induced transgenic mice. For example, Bacus (Boone et al., 1997) has described use of CAIA morphometric measures to follow skin carcinogenesisin B[a]P-induced SENCAR mice and esophageal cancer in nitrosamine-induced rats. They also have shown that the chemopreventive agents DFMO and phenethylisothiocyanate (PEITC),respectively, inhibit early lesions as well as cancers in these models.

D. Clinical Efficacy-Phase I1 Clinical Chemoprevention Studies With the development of intermediate biomarkers, clinical trials that support claims of chemopreventiveefficacy can be designed using biomarkers as surrogate end points. At each major target site, three critical aspects govern the design and conduct of these trials-well-characterized agents, reliable biomarkers for measuring efficacy, and suitable cohorts (Kelloff et al., 1994a,f, 1996a, 1997a).

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I . AGENTS The first criterion is evidence that the agent to be studied is efficacious, with high likelihood that theagent will be active in preventing cancer at the target site. Because chemopreventive agents may be given to relatively healthy subjects for long periods of time, a high margin of safety is also necessary to warrant consideration of an agent for clinical evaluation. This second criterion implies that sufficient prior clinical u6e or preclinical efficacy, toxicity, and pharmacodynamics data are available to allow estimation of an efficacykafety ratio. Often, dose-titration studies to determine the optimal dose and dosing regimen will be performed as part of the Phase II chemoprevention trials. .The third criterion is that there is a logical, presumed mechanism of chemopreventive activity of the agent. Such mechanisms guide the selection of both cohorts and end points for clinical trials. For example, an antiproliferative agent like DFMO may be most effective ,against cancers with a pronounced proliferative component, such as the development of colon cancers from hyperproliferative tissue and adenomas. Also, an antiestrogen would be expected to be effective against hormone-responsive breast lesions. An antimutagenic agent like oltipraz may be more effectively evaluated in a cohort such as smokers, who are constantly exposed to the DNAdamaging effects of carcinogens. Likewise, indicators of proliferation such as S-phase fraction and PCNA may prove to be more reliable and easily quantified measures of the effects of antiproliferative agents than would be the identification of specific mutations. In other words, the intermediate end point and ultimately the cancer should be modulatable by the chemopreventive agent. 2. lNTERMEDlATE BIOMARKERS AS CANDIDATE SURROGATE END POINTS It is important to evaluate intermediate biomarkers as surrogates for cancer in the context of carcinogenesis at the target site. The assurance that the chemopreventive agent can modulate the biomarker(s) chosen as the surrogate end point is clearly critical. IEN is modulatable. For example, Meyskens et al. (1983) demonstrated the regression of CIN I1 with all-trans-retinoic acid. Hyperproliferation is another example of a biomarker that can be modulated. Lipkin and colleagues have described reduction of proliferation by calcium as measured by [3H]thymidine uptake in patients at high risk for colon cancer. Although they will more often be risk biomarkers, genetic lesions or their encoded products can in certain circumstances provide modulatable biomarkers. Although acquired genetic lesions would not be excised by a given agent, one can assume that some agents will confer a selective ad-

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vantage for cells not carrying the lesion over those that do. The biomarker in this instance would be the quantitative reduction at the tissue level of genetically altered cells. The surrogate end point should have short latency compared with cancer incidence-ideally, months or a few years compared with the many years and decades required for cancers to develop. To ensure the design of efficient trials, a balance may be struck between short latency to occurrence of the biomarker and its temporal closeness to cancer. Some cohorts may be available for too short a period of time to allow modulation of the lesions most closely related to cancer. For example, treatment of presurgical cohorts in breast primarily may explore changes in proliferation indices rather than actual progression of histological lesions. Despite the brevity of the associated trials, studies in these cohorts may be expected to provide valuable information on modulation of selected biomarkers that are proven to be associated with cancer risk. 3. COHORTS

The first criterion for a cohort is that it be matched to the chemopreventive agent being evaluated. A chemopreventive agent is likely to be most effective in subjects whose disease or risk of disease can be modulated by the presumed mechanism of the agent within the relatively short duration of Phase 11trials (1month to 3 years). There are cohorts at high risk for cancer who are not good candidates for Phase 11chemoprevention trials. An example is subjects at risk because of germline mutations but who do not have any premalignant lesions (histological or molecular changes associated with carcinogenesis).Practically, the chemopreventive effect should also be easily measurable in the subject population. Tissues that are more accessible and that can be monitored relatively noninvasively should provide better sites for definitive efficacy trials than less accessible tissues. This is not to say that chemopreventive agents will not be effective in more difficult settings, but that initial demonstration of chemopreventive activity may be best carried out where fewer obstacles to measurement exist. As technology for tissue visualization improves, the location of the lesion will be less of a problem. Often, the cohorts in chemoprevention clinical trials will be cancer patients who have undergone previous treatment. These patients are constantly monitored for possible recurrences. Many times the recommended treatment is excision of a lesion. It is important that chemoprevention trials work within the constraints of standard treatment so that patients are not at unusual risk. In early Phase II trials where the goal is identification and standardization of biomarkers as end points, standard treatment may also lead to very short-term trials prior to surgery in patients who are scheduled for excision of cancers or high-risk tissue, for example, early prostate cancers or DCIS in breast.

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Generally, the Phase I1 cohorts provide accessible IEN lesions that can be used for rigorous dose-response studies establishing efficacy. With additional developmental work, results showing reversion, slowed progression, or inhibition of recurrence 03 the target lesions can be obtained within 3-24 months in such patients. Besides direct correlation of their modulation by chemopreventive agents to decreased cancer, major considerations in validation of biomarkers as surrogate end points are that the biomarkers are expressed differentially in normal and high-risk tissue, and that a temporal progression can be shown from normal tissue to intermediate biomarkers to cancer. The sensitivityand specificity of the biomarkers as measures of cancer are also important. They should appear with high frequency in precancerous or high-risk tissue. They should be specific for cancer in that they are expressed in high-risk tissue but not in response to other diseases or to conditions such as normal growth or wound healing.

V. CANCER CHEMOPREVENTION AT MAJOR CANCER TARGET SITES The promise of chemoprevention is evidenced by the increasing number of clinical strategies and studies at most of the major cancer target organs. At this point in time, cancers in at least 11 organ systems have been evaluated for development of chemopreventive agents-prostate, breast, colon, lung, head and neck, bladder, esophagus, cervix, skin, liver, and multiple myeloma. This section contains brief summaries of the current state of chemoprevention in these targets (see also Kelloff et al., 1994f, 1996a). In each organ system cancer is associated with earlier, well-defined lesions that may serve as surrogate end points. For each site, chemoprevention trial protocols have been developed that use the surrogate end points. These protocols are described in the following discussion, along with epidemiology, etiology and risk factors, chemopreventive mechanisms, possible intermediate biomarkers, 2nd potential chemopreventive agents under consideration (Table IX summarizes this information). To indicate the breadth and level of clinical research and development in chemoprevention, Table X lists representative ongoing Phase II and In studies by cancer target and cohort.

A. Prostate Prostate cancer is the most common cancer in U.S. males, accounting for

184,OO (29%)of all new cancers and 39,200 (13%)of cancer deaths in males

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(Landis et al., 1998). Etiological and risk factors for prostate cancer include age >50 years, family history, high serum testosterone, high fat diet, prostatis, and geographical background (prevalence being highest in the United States, Canada, and northwest Europe). Besides clinically evident disease, microscopic foci of adenocarcinoma have been found at autopsy in prostates of men who died from other causes. The frequency of such “latent” tumors has been shown to increase with each decade of life from the 50s (5.3-14% incidence) to the 90s (40-80%) (e.g., Kelloff et al., 1992). Prostate cancer is slow growing, often requiring decades to appear as a clinical tumor. Exogenous risk factors seem to contribute more to the disparity between latent and clinical prostate cancer than hereditary factors. For example, the incidence of latent prostate adenocarcinoma does not vary widely among populations (cited in Kelloff et al., 1992). In one study, the prevalence of microscopic lesions at autopsy was 20.6,28.8, and 36.9 per 100,000 in Japanese, Germans, and African-Americans, respectively. On the other hand, rates of clinical cancer were 2.7, 21.1, and 67.1 per 100,000, respectively. Further, within two generations after immigrating to the United States, Japanese men have a rate of clinical cancer approaching that of U.S. Caucasians. These data suggest the presence of a baseline incidence of early microscopic prostatic neoplasia that is subsequently accelerated to different degrees depending on exogenous environmental influences. Promising chemopreventive agents in prostate include antiandrogens and antiestrogens (e.g., flutamide, toremifene, raloxifene, SEW-3, and steroid aromatase inhibitors), retinoids (e.g., fenretinide and 9-cis-retinoic acid), RAMBAs (retinoic acid metabolic blocking agents), vitamin E, organoselenium, lycopene, soy isoflavones (e.g., genistein), 2-difluoromethylornithine (DFMO), steroid 5a-reductase inhibitors (e.g., finasteride, dual type 1and 2 inhibitors), apoptosis inducers (e.g., perillyl alcohol), and differentiation agents (e.g., vitamin D analogs). As described above, data also suggest that antiinflammatories such as LOX inhibitors and selective COX-2 inhibitors have promise as prostate cancer chemopreventives, as does the combination of selenium with vitamin E. A large Phase III clinical trial (18,000 subjects) of finasteride (Proscar);which inhibits the enzyme testosterone Sa-reductase, is in progress (Feigl et al., 1995). Early experimental data suggested that retinoids such as fenretinide will be chemopreventive in the prostate, provided sufficient tissue levels are achieved (e.g., Pollard et al., 1991). ODC is found at high levels in the prostate and prostate neoplasms (Kadmon, 1992), so that DFMO appears to be a promising chemopreventive agent. As described above, selenium in the form of selenized brewer’s yeast (200 Fg Se/ day) was associated with a 63% reduction in prostate cancer in a cohort with prior nonmelanoma skin cancer compared with placebo-treated controls (Clark et al., 1996, 1998). In the study of Finnish smokers (also described

N

00 00

Table IX Aspects of Chemopreventionat Major Cancer Target Sites Aspect Cancer burden

Prostate In US., most common cancer in men: 29% (184,500) of total new

cancer cases in men (estimated 1998); 13% (39,200) of cancer deaths in men (estimated 1998)

Risk factors markers

Promising agents

Age >50 years; familial history of prostate cancer; high serum testosterone; high far diethigh red meat consumption; populatiodgeograpbical background (highestincidences in Canada and northwest Europe); prostatitis, genetic polymorpbisms (e.g., in SRD5A2, gene for steroid 5a-reductase);low micronutrient levels (e.g., selenium, erotenoids, vitamin D) Steroid 5.-reductase inhibitors (e.g., finastetide);retinoids (e.g., 9 4 s retinoic acid); RAMBA (retinoic acid metabolism blocking agents); antiproliferatives(e.g., DFMO, DHEA analogs);differentiating agents (e.g., vitamin D analogs); antioxidants (e.g., vitamin E, selenium, lycopene); GSH-enhancing agents (e.g., oltipraz); antiestre gens (e.g., totemifene, tamoxifen,

Breast In US., most common cancer in women 30% (180,300) of total new cancer cases; 16% (43,500) of cancer deaths (estimated 1998)

Colon In US.,rhird most common cancer in men, women 11%

(134,900) total new cases; 10% (28,100) of cancer deaths in men, 11%(28,900) in women (estimated 1998)

Age 250 years; familial history of breast cancer or genetic syndrome (e.g., Li-Fraumeni, BRCAI), previous breast, endomcuial, or ovarian cancer, atypical byperplasia, DUS, LCIS; esuogen cxposure (e.g., early menarche, late menopause, late age at first full-term pregnancy); lifestyle factors (e.g., diet)

High fadlow fiber diet, low fresh fruit, vegetable intake, low calcium and vitamin D intake; familial history of generic syndrome (e.g., FAP, HNPCC); familiaUpast history of colorectalcancer or adenomatous polyps; past history of breast or endometrial cancer; inflammatory bowel disease

Antiestrogens (eg., tamoxifen, raloxifene, and other SERMs); aromatase inhibitors (e.g., exemestane. vorozole); antiproliferatives(e.g., DFMO); soy isoflavones; fluasterone (DHFA analog 8354); retinoids (e.g., fenretinide, 9-cis-retinoic acid); monotcrpenes(e.g., limonene, perillyl alcohol)

Antiiinflammatories (e.g., sulindac, piroxicam, aspirin, selective COX-2 inhibitors, curcumin, iNOS inhibitors, ASA derivatives); antiprolieratives (e.g., calcium, DFMO, ursodiol)

Lung In U.S., second most common cancer and leading cause of cancer deaths in men and women; 15% (91,400) of total new cases in men, 13% (81,100) in women; 32% (93,100) of cancer deaths in men, 25% (67,000) in women (estimated 1998) Tobacco use (smoking, chewing); alcohol consumption,especially combined with tobacco use; occupationalexposure (e.g., asbestos, nickel, copper); cytochrome P450 genetic polymorphism (.g., CYPlAl, GSTMZ); low fruit, vegetable consumption; previous oral, laryngeal, lung cancer

Retinoids (e.g., vitamin A, 13-cis- . retinoic add, fenretinide, all-transretinoic acid); antimuragens(e.g., oltipraz, anethok mthione, PFJTC); andinnammatories (e.g., aerosolized corticosteroids. LOX inhibitors)

Head and neck In US.,5% (29,600) of total new cases in men, 2% (11,800) in women; 3% (8,700) of cancer deaths in men, 1% (3,600) in women (estimated 1998) 1

Tobacco use (smoking, chewing); alcohol, especially combined with robacco use; males, 50-70 years

Ret@oi&/carotenoids (e.g., vitamin A, 13-5s-retinoic acid, fenrednide, p-camnne); anrjinflammatories (e.g., tea, curcumin)

raloxifene, and other SERMs); arornatase inhibitors (e.g., vorowle); antiandrogens (eg., leuprolide, flutamide);angiogenesis inhibitors (e.g., linomide);signal transduction regulators (e.g., soy isoflavones),antiinflammatories (e.g., LOX inhibitors, selective COX-2 inhibitors) Intermediate Histological: PIN (nuclearmorphobiomarkers merry, nucleolar morphometry, nuclear texture, DNA ploidy); proliferation: loss of high molecular weight cytokeratins (SO44 kDa), altered blood group antigens (e.g., Lewisy antigen), vimentin; genetid regulatory: c-erbB-2, TGFa, P53, bc/-2/bax, pc-1 chromosomalloss or gain (e.g., Sp, 9p, and 16q), TGFB, IGF-I; biochemical: F'SA levels, PAP levels; angiogenisis: microvessel density, vWF, VEGF Clinical cohorts: Patients scheduled for radical prostatectomy; patients with PIN; Phase n patients with cancer on biopsy, treated by watchful waiting; patients at high risk for biocbemical failure or rising PSA postradical prostatecmny; subjects with positive family history Clinical cohom: HGPIP; men at high risk (e.g., PSA Phase U >4 nglml and negative biopsy); mert fromgeneral population, age 8 5 5 years, normal PSA and DRE

Atypical hyperplasia, DCIS, nuclear morpbometry,ploidy, c-nbD-2 amplification, p53 mutation, IGF-I

Adenomas (recurrence,regression); Ollular atypia in sputum, bronchial ACF; nuclear and nucleolar atypical metaplasialdysplasia increased cytokeratin 19 expresmorpbometry;apoptosis; sion, PCNA, blood group-related proliferation indices (PCNA, antigens, p53 mutation; RARB Ki-67); crypt proliferation induction kinetics; differentiation indices (Lewisblood group antigens, sialyl-Tn antigen)

Leukoplakia with dysplasia, erythroplakia, GGT, keratins, c-n6B-1 amplification; LOH; proliferation indices (PCNA, Ki-67)

Patients scheduled for breast cancer surgery, patients with LCIS or mammographically detected calciIications/DClS, high risk with multiple biomarker abnormalities

Patients with previous colon cancer or adenomatous polyps, FAP patients; HNPCC patientdcarriers

Patients with recently resected stage I lung or laryngeal cancer, chronic smokers with squamous metaplasial dysplasia

Patients with dysplastic kukoplakia; patients with previous head and neck cancers

Women age 8 6 0 or 35-59 years old with risk factors for 60 years old, patients with previous breast cancer

Patients with previous colon cancer or adenomatous polyps, FAP patienu; HNPCC patiendcarriers

Men exposed to asbestos or patients with asbestosis; chronic or heavy cigarettesmokers; patients with previous lung, bead, or neck cancer

Patients with previously treated head and neck cancer; subjects at high risk (e.g., smokers, tobacco chewers) (continues)

N OD 9

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9

0

Table IX (continued) Aspect Cancer burden

Bladder In US., fourth most common cancer in men: 6% (35,500) of total new cases, 2% (7100)of cancer deaths; sixth mwt common cancer in women 2% (14,900). 2% (4500) (estimated 1998)

Risk factors markers

Males, age >55 years, cigarette, pipe, and cigar smoking; occupational exposures to aromatic amines; metabolic polymorphism slow N-acetyltransferase phenotype; chronic cystitis or urinary tract infections; coffee drinking; chlorinated tap water

Promising agents

AnriiQammatories (e.g., sulindac, piroxicam, aspirin, ibuprofen); antiproliferatives(e.g., DFMO); retinoids (e.g., fenretinide)

Esophagus

Cervix

In US.,55,000 cases of CIS, In U.S., represents 1%(12,300) of all cases and 2% (11,900) 13,700 cases of invasive cancer, 4900 deaths (estimated 1998) of cancer-related deaths, third shortest 5-year survival rate among all cancers (behind pancreas and liver). Trearment involves high morbidity and significantly lower quality of lie (i.e., esophagectomy with associated difficulties in eating and speaking) HPV infection; early age at first Alcohol and tobacco use; p a r diet (e.g., lacking fresh fruit intercourse; multiple sexual and vegetables);chronic gastropartners; oral contraceptiveuse; esophageal reilux disease immuncdepression; smoking (GERD)for the cancer, as well as for Barrens esophagus; genetic syndrome (e.g., tylosis); Barren's esophagus Antiproliferatives (e.g., DFMO, BBI, selenium);antioxidants (e.g., tea polypheuols, PEITC, BHA, diallyl sulfide); antiinflammatories (e.g., NSAIDs, selective COX-2 inhibitors); vitamins (e.g., vitamin E, nicotinic acid, riboflavin)

Retinoids (e.g., vitamin A, fenretinide, 9-cis-retinoicacid); antiproliferatives(e.g., DFMO); folic acid

Skin

Liver

In US.,-1,000,000 cases BCC and In US., 1% (13,900) of all SCC with low mortality (esticancers and rising and 13,000 mated 1998); cutaneous melanoma deaths; much more important continues to rise (41,600 cases of worldwide (estimated 1998) melanoma and 21,100 cases of I melanoma in ritu expected in 1998)

Dermatologic factors (e.g., fair skin, Alcohol consumption;smoking, HBV and HCV infection freckling); genetic susceptibility (e.g., xeroderma pigmentosum, basal cen'nevus syndrome, albinism, epidermodysplasia verruciformis); environmental . exposures (e.g., W radiation, cigarette smoke, tanning booths, PAH);lupus, immunosuppression Antiinflammatories (e.g., piroxicami Antimutagens (e.g., oltip rzz); retinoids (e.g., polycurcumin, selective COX-2 inhihiprenoic acid) tors); antimutagens(e.g., oltipraz, BHA/BHT, diaUyl sulfide); antiproliferatives(e.g.. DFMO); retinoids (e.g., fenretinide, 13-CISretinoic acid, retinyl palmitate, vitamin A); antioxidants (e.g., tea polyphenols, selenium, carotenoids)

Intermediate biomarken

TIS, dysplasia, DNA content, LOH, Rh, blood group-relatedantigens, Fand G-actins, integrins

Barren’s esophagus(area and grade of dysplasia);nuclear1 nudmlar polymorphism; DNA ploidy; proliferation indices (Ki-67);apaptosis; pS3; EGFR,EGF, TGFa; LOH (e.g., chromosome 17); microsatellite instability; iNOS expression Patients with low grade, intestinal type Barrett’s esophagus with or without dyspIasia

Clinical cohom: Patients with previous resected superficial transitional cell carcinoma Phase Il (TaKl with or without TIS), patients with previous resected superiicial transitional ceU carcinoma treated with BCG Clinical cohorts: Subjects at high risk (e.g., occupational Patients at high risk for Phase UI exposure to aromatic amines) esophageal cancer (e.g., GERD,smokers, geographid ethnic, such as Linxian, China)

CIN (grade);aneuploidy; nuclear polymorphism; proliferation indices (e.g., PCNA, EGFR, TGFa, TGFB); differentiation markers (e.g., involucrin)rasoncogene expression

Actinic keratosis; proliferation indices &., PCNA, IGF-VIGFR, EGFR, cycli D1, ODC); TGFB; differentiationindices (e.g., integrins); genetidregulatory hiomarkers (e.g., c-fos, c-myc, c-jun)

Carcinogen-DNA adducts

.

HPV-negative patients with CIN LII Patients with actinic keratosis

Suhims with environmental exposure (e.g., carcinogen or HPB)

Patients with CIN I, 11; patients with HPV infection

Patients with previous hepatoma

Patients with previous BCC or SCC; subjects with previous intense chroNc or episodic sun exposure; patients with preexisting dermatologicdisorders; patients with actinic keratosis; subjects with dysplastic nevi

Table X Representative Phase IVIII Clinical ChemopreventionTrials with Biomarker Endpoints Target site Prostate

Breast

Colon

AgedW DFMO Fenretinide Fmasteride Flutamide Flutamide + finasteride Flutamide + leuprolide Toremifene Soy isoflavones Flutamide Selenomethionine Soy protein DFMO Exemestane Fenretinide Tamoxifen Fenretinide + tamoxifen SERM SERM Aspirin + calcium Sulindac Calcium Calcitriol Vitamin D, Sulidac sulfone Selective COX-2 inhibitor

Cohort (treatment period) Scheduled for prostate cancer surgery (2-8 weeks)

Representative end pointdother biomarkers Histopathology (PIN grade, nuclear/nucleolar polymorphism, ploidy), proliferation biomarkers' (e.g., PCNA, Ki-67, IGF-I), differentiation biomarkers (e.g., LewisYantigen), genetidregulatory biomarkers (e.g., TGFa, pS3, bcl-2, pc-1,chromosome 8p loss)

HGPIN (1-3 years) Mammographic lesion requiring biopsy (DCIS) ( 2 4 weeks)

High risk for breast cancer with biomarker abnormalities (6 months) Previous colorectal adenomas (6 months) Previous adenomas (resected within past 2 years) or colon cancers (6 months) FAF' patients (6 months)

Histopathology (DCISgrade, nuclear polymorphism, ploidy), proliferation biomarkers (e.g., PCNA, Ki-67, S-phase fraction, IGF-I, c-erbB-2), p53

Proliferation biomarkers (PCNA),PGE, levels Histopathology (nuclear polymorphism), proliferation biomarkers (DNA labeling index, crypt proliferation pattern-PCNA), differentiation biomarkers, genetidregulatory biomarkers (pS3, bcl-2) Adenoma size and number, proliferation biomarkers (PCNA), apoptosis

Lung

Selective COX-2 inhibitor Aspirin Folic acid Aspirin + folic acid Calcium Selective COX-2 Inhibitor Calcium + vitamin D,

HNPCC subjects (6 months) Previous colorectal adenomas (1-3 years)

Adenoma ske and number, proliferation biomarkers, ACF, apoptosis, LOH, microsatellite instability Adenoma size and number, other biomarkers

Colorectal adenomas <6 mm diameter (3 years)

Sulindac

Colorectal adenomas, left-side, 5-9 mm diameter (1 year) Chronic smokers with bronchial squamous metaplasia (index 2 15%) or dysplasia (6 months) Chronic smokers, or prior resected carcinoma of respiratory tract (6 months)

Adenoma size and number, histopathology (nuclear/ nucleolar polymorphism, ploidy), proliferation biomarkers (crypt proliferation pattern-PCNA) Adenoma size and number, proliferation biomarkers (PCNA)

Budesonide Vitamin A Fenretinide Oltipraz

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9 W

Head and neck

DFMO Fenretinide 13-cis-retinoicacid

Dysplastic oral leukoplakia (6 months)

Bladder

DFMO Selective COX-2 inhibitor Fenretinide

Previous superficial bladder cancer (Ta, T1 disease without TIS) (12 months)

Histopathology (dysplasia regression, ploidy), proliferation biomarkers (PCNA),genetidregulatory biomarkers (p53, EGFR), mutagen sensitivity, micronucleated cell frequency Histopathology (nuclear polymorphism, ploidy), proliferation biomarkers (MiB-1), genetidregulatory biomarkers (p53), agent specific (GSTp phenotype, GST activity in lymphocytes, bronchial cells) Recurrence, histopathology (dysplasia/.leukoplakia grade, nuclear polymorphism, ploidy), native cellular fluorescence, proliferation biomarkers (PCNA, Ki-67, S-phase fraction), differentiation biomarkers (cytokeratin 19, blood group antigens), genetid regulatory biomarkers (TGFP) Histopathology, proliferation biomarkers (Ki-67), differentiation biomarkers (Lewisx antigen), genetid regulatory biomarkers (EGF, EGFR, p53, PKC isotypes), agent specific (ODC activity, polyamine levels)

Table X (continued) Target site

A g e d s)

Fenretinide

Cohort (treatment period) Previous superficial bladder cancer (Ta, T1 disease with TIS, treated with BCG) (12 months) Dysplastidmetaplastic Barren’s esophagus (6 months)

Esophagus

DFMO Selective COX-2 inhibitor

Cervix

DFMO Fenretinide 9-cis-retinoic acid

CIN I1 or III (3-6 months)

Skin

Fenretinide Selective COX-2 inhibitor Tea polyphenols Oltipraz

Actinic keratosis (6 months)

DHEA Biaxin Selective COX-2 inhibitor

Monoclonal gammopathy of unknown significance (MGUS) (6 months)

Liver

Multiple Myeloma

Aflatoxin exposure (Qidong, China) (1year)

Representative end pointdother biomarkers Recurrence, histopathology (ploidy), proliferation biomarkers (Ki-67, DD23, M-344), differentiation biomarkers (G-actin) Histopathology (nuclear/nucleolarpolymorphism, ploidy), proliferation biomarkers (Ki-67), genetid regulatory biomarkers (pS3, TGFa, EGFR, microsatellite instability) Histopathology (CIN grade, nuclear polymorphism, ploidy), proliferation biomarkers (PCNA),differentiation biomarkers (keratins, involucrin, transglutaminase), genetidregulatory biomarkers (ras, EGFR, TGFa) agent specific (e.g., ODC activity, polyamine levels, RAR) Histopathology (lesion grade), proliferation biomarkers (PCNA),genetidregulatory biomarkers (EGFR, TGFB) .. Urinary aflatoxin-DNA adducts, serum aflatoxinalbumin adducts; phase I1 enzyme activities, mRNA transcripts, and genotypes in lymphocytes Bone marrow plasmacytosis, serudurine M-protein or Bence Jones protein, nuclear pleornorphism index, ploidy, proliferative index (digital image analysis), ThlKh2 ratios (flow cytometry), serum Il-6 and SIL-6r (ELISA),differentiation antigedadhesion molecule expression (flow cytometry), cytokine expression, FISH, circulating monoclonal plasma cells (flow cytometry), plasma cell apoptosis, bone markers (osteoblast and osteoclast)

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above), a significant reduction in prostate cancer incidence of 32% (95%CI = 12-47%) was seen in those taking vitamin E (50 mg/day) compared with placebo or P-carotene (Heinonen et al., 1998).Because testosterone and estrogen are believed to be involved in prostate carcinogenesis, antiestrogens and antiandrogens (Aquilina et al., 1997) may have potent chemopreventive activity. individually, and in combination they may reduce undesirable side effects of antiandrogens such as gynecomastia. Epidemiological studies suggest that increased serum levels of lycopene, the most abundant serum carotenoid, ate associated with a decreased relative risk of prostate cancer (Giovannucci et al., 1995). The presence of vitamin D receptors in prostate cancers and low levels of vitamin D in the sera of prostate cancer patients (Cbrder et al,. 1993) suggest vitamin D analogs as potential chemopreventive agents in men at risk. Serum PSA is well established as a biomarker of prostate cancer. However, PSA is not specific to neoplasia, and the data do not suggest that the level is directly related to degree of neoplastic progression. Most data indicate that other measurements of PSA, especially density and velocity of PSA rise (Crawford and DeAntoni, 1997), may correlate better to progression than serum level alone. The validation of PSA as an intermediate biomarker awaits further data, some of which may be obtained in the large Prostate, Lung, Colorectal, and Ovary Cancer Screening Trial in which PSA is being monitored over several years in more than 30,000 men. Even without further refinement, PSA may prove useful in identifying clinical cohorts at risk as subjects for chemoprevention studies. The evidence that PIN is a precursor of prostatic adenocarcinoma has been summarized by Bostwick (1992).It includes ( 1 ) morphology and cytology similar to malignant lesions; (2)presence of carcinoma in foci of PIN; (3)frequent location in the peripheral zone of the prostate, the site at which 70% of prostatic carcinomas occur; (4)proliferative activity similar to that of carcinoma (3-fold that of benign tissue); ( 5 ) cytokeratin immunoreactivity, lectin binding, and loss of blood group antigen similar to carcinoma; (6)foci found in 82% of prostates with carcinoma, but in only 43% of normal prostates; and (7) significant increase in the incidence of prostatic carcinoma in patients with PIN. The prevalence of PIN and temporal association with prostatic carcinoma is demonstrated by the 40-50% PIN incidence seen in men aged 40-60, progressing to 40-50% cancer incidence in men aged 280. A cohort for a clinical intervention study using high-grade PIN as the surrogate end point is individuals with PIN but without demonstrable prostatic carcinoma. These subjects are treated with a chemopreventive agent for approximately 2 years and then evaluated by transrectal ultrasound (TRUS)directed biopsy every 3-6 months to determine the modulation of PIN, changes in proliferation indices, and nuclear abnormalities.

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Another potential cohort for short-term trials of chemopreventive agents involves patients with newly diagnosed stage B,/B, cancers. Such patients are usually not scheduled for prostatectomy until 3-8 weeks after diagnosis. At diagnosis, treatment with a chemopreventive agent begins and continues until prostatectomy. At surgery, the removed prostate gland is analyzed for PIN modulation and other potential biomarkers. Although not fully satisfactory, the analysis of PIN is made more specific by taking sextant TRUSdirected biopsies at diagnosis, primarily from hypoechoic areas, to capture the PIN lesions. These biopsy sites are marked with fluorescein dye or India ink (the markings will survive 3-4 months) and examined after surgery. Detection of PIN underscores sampling issues that must be addressed in chemoprevention studies using surrogate end pbints. In men aged 2 5 0 years, hlgh-grade PIN (HGPIN) incidence is 50%. However, of all sextant prostate biopsies taken in this subpopulation for any reason, when no cancer is present, only 5% HGPIN incidence is detected. In the general population, <1% HGPIN incidence is detected in sextant prostate biopsies. These discrepancies are most probably due to an inability to ensure adequate tissue sampling in the prostate and call for standardization of measurement methods. The number and location of samples from invasive cancer, HGPIN, and adjacent normal-appearing tissue, as well as the thicknesdnumber of histologic sections processed and scored, are important parameters that affect variability, accuracy, and reproducibility. Current approaches for measuring effects on the prevalence and extent of PIN involve calculating the percent of patients with PIN, mapping area and volume of PIN, and digital imaging of the prostate whole mount.

B. Breast Breast cancer is by far the most common cancer in U.S. females, causing 30% (180,300)of total new cancer cases, and is the second highest cancer killer in women, accounting for 16% (43,500)of all cancer deaths estimated for 1998 (Landis et al., 1998). The risk factors are well known and includk family history of breast cancer, Li-Fraumeni syndrome, past history of breast, endometrial, or ovarian cancer, atypical hyperplasia of the breast, nulliparity or late age at first full-term pregnancy, early menarche and late menopause, and obesity. Antiproliferation is an important chemopreventive mechanism in breast cancer, demonstrated by agents such as DFMO, retinoids, and antiestrogens. As discussed above, the promise of the antiestrogen tamoxifen is widely known based 06 its success in reducing the risk of breast cancer in women at high risk (Fisher et al., 1998). The second generation SERM raloxifene,

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already approved in prevention of osteoporosis and without endometrial toxicity, will be compared with tamoxifen as a breast cancer chemopreventive in postmenopausal women. A third generation SERM with greater efficacy than raloxifene in animal studies will be evaluated in a Phase I1 study in patients scheduled for breast surgery and in subjects at high risk with multiple biomarker abnormalities. Fenretinide is being evaluated in a Phase III clinical trial to prevent recurrence in women previously treated for breast cancer, and it appears to be effective in premenopausal women; the incidence of ovarian cancers also looks to be reduced (De Palo et al., 1996). In this regard, studies are in progress to evaluate fenretinide in prevention of ovarian cancers (Kelloff et al., 1996g). Moreover, as discussed above, synergistic activity in animal studies indicates that the combination of antiestrogen (e.g., tamoxifen or raloxifene) with retinoid (e.g., fenretinide or 9-cis-retinoic acid) has potential. Phase I1 studies on the combination of fenretinide with tamoxifen are now ongoing. In one study, patients scheduled for breast surgery on the basis of mammographically detected lesions are being treated for 1421 days between diagnosis and surgery; intermediate biomarkers, primarily measures of antiproliferation, are being followed. In the second study, low dose ( 5 mg/day) tamoxifen is being evaluated. d-Limonene has demonstrated activity against carcinogen-induced mammary tumors in rats (Kelloff et al., 1996h). Agents, such as d-limonene and a more potent related compound perillyl alcohol (Kelloff et al., 1996h), that inhibit YUS protein isoprenylation are candidates for clinical evaluation. Soy isoflavones, which have some antiestrogenic activity and are inhibitors of growth factor-stimulated signal transduction, are also being evaluated in clinical chemoprevention studies. As noted above, aromatase inhibitors may be chemopreventive agents in breast, particularly in postmenopausal subjects. DFMO is being evaluated in a presurgical cohort and in women at risk on the basis of the presence of biomarker abnormalities (e.g., atypical ductal hyperplasia, EGFR overexpression, and mutated p53; see Fabian et al., 1996). Much has been accomplished toward understanding the genetic factors that contribute to breast cancer etiology, but reliable precancerous lesions that could serve as end points for chemoprevention trials have not yet been identified. For example, atypical hyperplasia is certainly a valid biomarker. It confers a 4-5 relative risk; however, it is detected in only about 7% of women with proliferative breast disease (Page and Dupont, 1992). Even when found, it is extremely difficult to follow or sample serially. Animal models may help to identify early breast lesions. Gould and associates (Wang et al., 1991; Zhai et al., 1993) have described a very interesting model in which neu oncogene is transfected into rat mammary glands. The tumors that form with high frequency and short latency closely resemble human ductal carcinoma. Also, progression from carcinoma in situ (anal-

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ogous to human DCIS) to invasive carcinoma is observed, suggesting that this model could be useful to study the effects of chemopreventive intervention on this progression. Patients with CIS, either DCIS or lobular carcinoma in it^ (LCIS),are potential cohorts for breast chemoprevention trials. DCIS is considered to be a direct precursor of invasive carcinoma (reviewed in Swain, 1992) and accounts for approximately 70% of breast CIS (Posner and Wolmark, 1992). Frequently occurring histologic subtypes of DCIS are micropapillary, cribriform, papillary, solid, and comedo carcinoma (Swain, 1992). Comedo-type DCIS exhibits high rates of local recurrence, with progression and microinvasion. In studies summarized by Page and Dupont (1990,1992),recurrence of DCIS was 50% within 3 years after treatment by local excision. The absolute risk of developing invasive cancer for women with non-comedo DCIS is approximately 25-30% within 15 years (Page and Dupont, 1992). Traditionally DCIS, like invasive cancer, is treated by surgery (Page and Dupont, 1992; Swain, 1992). Lobular CIS is a risk marker for both ductal and lobular carcinoma occurring anywhere in either breast (e.g., Page and Dupont, 1992; Posner and Wolmark, 1992). The relative risk of LCIS patients for developingcancer has been estimated to be 7-9 times that of the normal population (Page and Dupont, 1992), with an absolute lifetime risk of approximately 20% (Grooff et al., 1993). LCIS cohorts may be more desirable than DCIS cohorts for chemoprevention trials, because the risk is not related to the original biopsy site, but rather to cancer occurring in any location in either breast (Page and Dupont, 1990). Further, LCIS is treated less often by mastectomy or other definitive surgery than is DCIS. It has been recommended that LCIS patients be monitored regularly by mammography and physical examination (Posner and Wolmark, 1992). This monitoring period would allow for intervention with and evaluation of chemopreventive agents, providing that minimally invasive techniques for biomarker evaluation were available (e.g., serum IGF-I and possibly fine needle aspirate cytology, as described by Fabian et al., 1996). Another possibility, as in the trial designed for presurgical prostate cancer patients, is to administer chemopreventives to patients newly diagnosed with break cancer and scheduled for surgery. Breast tissue taken at surgery is used to evaluate the effects of the chemopreventive agent on a variety of molecular and cellular biomarkers and lesions, including hyperplasia and atypical hyperplasia. Fabian and co-workers (1996)have defined a risk-based cohort that is now being used in short-term chemoprevention studies of a SERM. In the study defining the cohort, 213 women at high risk for breast cancer were selected on the basis of having first-degree relatives with breast cancer (73%),prior biopsy indicating premalignant disease (26%), history of breast cancer

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(13%),or a combination of these factors (11%).Fine needle aspirates (FNA) from these women and 30 low-risk women were analyzed for cytological abnormalities and other biomarkers (aneuploidy, EGFR, ER, p53, and erbB-2) and compared. The results suggested that the presence of multiple biomarker abnormalities exclusive of cytology could be used to refine the selection of high-risk subjects. Thirty-one percent of the high-risk subjects had two or more biomarker abnormalities, whereas none of the low-risk group had more than one such abnormality. The presence of multiple biomarker abnormalities increased directly with cytologic atypia, ranging from 16% of subjects with normal cytology to 29% of those with hyperplasia to 60% of those with atypical hyperplasia. No significant differences in the number ofbiomarker abnormalities or abnormal cytology were seen among the original risk groupings (i.e., first-degree relatives, prior positive biopsy, history of breast cancer, or multiple factors). Because of the association of multiple biomarkers with cytological evidence of dysplasia, the investigators have suggested that changes in the pattern of biomarker abnormalities in the FNA (particularly, p53 and EGFR), as well as atypical hyperplasia, can be explored as end points in a chemoprevention study in this cohort.

C. Colon Colorectal cancer is the third most common cancer and cause of cancerrelated deaths in both males and females in the United States. For 1998, such cancers were estimated to account for 11% (134,900) of all new cancers, 11% (28,900) of cancer deaths in females, and 10% (28,100) of cancer deaths in males (Landis et al., 1998). Risk factors for colorectal cancer include dietary factors such as high fat, low fiber, low fresh fruit and vegetables, as well as low calcium and vitamin D intake, and the presence of other disease (e.g., inflammatory bowel disease) that is associated with high rates of cell proliferation in the colonic epithelium (see Kelloff et al., 1994f ). Genetic predisposition is notable and includes patients with FA€' or family history of colorectal cancer (e.g., HNPCC) or adenomatous polyps. Past histories of colorectal cancer, colorectal polyps, or breast or endometrial cancers are also significant risk factors. The developmental path for most colorectal cancer is well documented. Histopathologically, it starts with hyperproliferation in colon mucosa, formation of adenomas with varying degrees of malignant potential, and finally adenocarcinoma (Muto et al,. 1975; Hamilton, 1992; Lipkin, 1992). Adenomas are prototypic IEN. Overall, the incidence of adenomas is high. In Western countries, they have been found in 30-40% of autopsies of patients over 60 years old (Winawer et al,. 1990).

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The malignant potential of adenomas appears to correlate to histological growth pattern, size, and degree of epithelial dysplasia (e.g., Hamilton, 1992). The rate of progression of tubular adenomas to adenocarcinomas is low, ranging from 2 to 5%, but that for villous adenomas is much higher, ranging from 20 to 55%. The rate of conversion for the intermediate tubulovillous lesions is estimated to be 22%. Also, risk of malignancy correlates with size; it is negligible in adenomas 11 cm in diameter and increases at larger diameters. Of the 70% of adenomatous polyps that are mildly dysplastic, only 5% progress to cancers. A much higher fraction of the 10% of adenomas that are severely dysplastic become cancerous. In one study, onethird of severely dysplastic adenomas contained invasive carcinoma (Hamilton, 1992). Furthermore, severe dysplasia is found most commonly in larger adenomas with villous histology (Morson, 1983). Aberrant crypt foci have been implicated as precursors to colorectal adenomas and carcinomas on the basis of studies in AOM-induced mice and rats. They have also been detected in human colorectal epithelium and, on the basis of surgical and autopsy specimens taken for various reasons, are more prevalent in patients with colorectal cancer than in those with noncancerous lesions. Recently, Takayama et al. (1998) studied ACF by magnifying endoscopy in normal subjects and patients with colorectal adenomas and carcinomas. They found significant direct correlations of the number, size, and dysplasia of ACF in a subject and the number of adenomas. Further, this study provided a model for evaluating chemopreventive intervention. In 11 subjects treated with 100 mg sulindac three times per day for 8-12 months, the number of ACF decreased (and disappeared in seven subjects) compared with no decrease among nine untreated controls (p < 0.001 for the difference between the groups). This well-documented histopathology along with the accessibility of all stages of colon carcinogenesis facilitates the evaluation of chemopreventive activity in colon (reviewedin Kelloff et al., 1994f, 1996a). For example, the association of colon cancer with hyperproliferation in colonic epithelial mucosa led to the investigation of proliferation indices such as DNA labeling, PCNA, and Ki-67 as intermediate biomarkers by correlating their levels with increising severity of dysplasia in adenomas. Apoptosis is receiving increasing attention as a mechanism for control of cell growth and proliferation, and in particular the overall kinetics of cell growth, proliferation, and apoptosis in colon crypts is being evaluated. The expression of differentiation markers, such as Lewis blood group antigens and sialyl-Tn antigen, has also been suggested as a measure of chemopreventive activity. Abnormal expression of the Lewis and sialyl-Tn antigens is seen in adenomas and cancers; the pattern of expression corresponds to the size and degree of dysplasia in adenomas. Further, as noted above, sequences and combinations of genetic events seen

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in adenomatous polyps and colon cancer also have been described elegantly by Vogelstein and co-workers (e.g., Fearon and Vogelstein, 1990). Alterations in the long arm of chromosome 5 (AF'C, MCC genes), DNA hypomethylation, and rus mutations appear to be early lesions, c-K-rus gene expression may be an important intermediate biomarker. Mutated c-K-rus oncogenes have been identified in 50% of colorectal carcinomas and adenomas >1 cm in size; increased frequency of these mutations in adenomas has been associated with increasing degree of dysplasia and lesion size. Moreover, rus gene mutations have been found in the very early stages of abnormal proliferation in colon mucosa, that is, in aberrant crypts in rats and in normal-appearing colon mucosa of rats 15 weeks after treatment with DMH. Not surprisingly, preclinical and clinical studies indicate that antiinflammatories and other agents that slow proliferative activity have potential as chemopreventive agents in colon (reviewed in Kelloff et uf., 1994f, 1996a). Agents showing chemopreventive activity in animal models-primarily against AOM- or DMH-induced colon cancers in rats and mice-include the NSAIDs sulindac, piroxicam, aspirin, and ibuprofen, as well as compounds such as DFMO, calcium, curcumin, and ursodiol. The antiproliferative activity of DFMO was noted above. Calcium inhibits colonic mucosa hyperproliferation. Curcumin has antioxidant and antiinflammatory activity, and ursodiol competes with tumor-promoting bile acids, particularly deoxycholic acid. Preliminary clinical studies also support the potential activity of sulindac and aspirin. Sulindac has shown dramatic effects in causing the total or almost total regression of colorectal adenomatous polyps in patients with FAP and Gardner's syndrome. In a prospective mortality study, aspirin use was found to reduce the relative risk of death from colon cancer, and, in patients with previous colorectal adenomas, it reduced the risk of new adenomas. The agent combination of DFMO and sulindac is being evaluated in colon on the basis of the synergism of DFMO and piroxicam observed in animal efficacy screens. Patients with a history of adenomatous polyps provide an obvious and feasible cohort for clinical chemoprevention studies, since their risk of developing new adenomas is high. In several studies, new adenomas were seen at rates ranging from 37 to 60% within 1-4 years following polypectomy (Winaweret ul., 1990). In the National Polyp Study, a recurrence rate of 2935% was seen in patients after removal of all synchronous adenomas. Patients with FAF' are a special subset of this high-risk group, since they develop many adenomas, some of which inevitably progress to adenocarcinoma. Several chemoprevention trials with colorectal adenoma recurrence and regression as the end point are currently in progress in this cohon; the agents being evaluated are sulindac, sulindac sulfone, DFMO, and the combination of p-carotene, vitamin C, and vitamin E. A very promising positive result

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was recently obtained with a selective COX-2 inhibitor. A Phase II/III chemoprevention trial measuring adenoma recurrence is estimated to require 3-6 years and 700-1500 patients.-The high incidence of adenomas in the population suggests that adequate accrual for such studies is possible. One complicating factor in estimating sample size required for an adenoma prevention trial arises because of the profound effects of NSAIDs and other antiinflammatories in colon and the widespread use of aspirin as a cardioprotective in the target population. From the standpoint of chemoprevention, adenoma development is relatively slow; trials measuring adenoma development and progression are of longer duration than is ultimately desirable in chemopreyention studies. From the patient’s viewpoint, adenomas are difficult to monitor because they reiuire endoscopy, and from the clinician’s viewpoint, endoscopic measurements are difficult to standardize (although high resolution video endoscopes are becoming available and may help remove this obstacle). These drawbacks are more than balanced by the knowledge that the adenoma is on the causal pathway to colorectal carcinoma and can be used as a standard against which to evaluate other biomarkers. Rigorous attempts are now being made to validate and standardize the measurement of earlier biomarkers of colon cancer. These efforts, though promising, will require several more years of research before their success can be evaluated.

D. Lung Lung cancer is the second most common cancer and the leading cause of cancer-related deaths in both males and females in the United States (Landis et al., 1998).For 1998,91,400 cases (15% of all cancers) were estimated for males, and 81,100 cases (13% of all cancers) were estimated for females. Thirty-two percent (93,100) of all U.S. cancer deaths in men and 25% (67,000) of those in women were attributed to lung cancer. Lung cancer in heavy smokers exhibits high rates of local recurrence or the associated occurrence of second primary tumors in the upper aerodigestive tract exposed to carcinogens in tobacco smoke. The basis for the high incidence of second primary tumors has been attributed to “field cancerization” (Slaughter et al., 1953)-the multiple foci of IEN throughout exposed respiratory tissue caused by the irritation and chronic carcinogen insult from smoking. Tobacco use is by far the greatest risk factor in the development of lung cancer, and chronic smokers are a primary target for chemopreventive strategies in lung (reviewed in Kelloff et al., 1994f). Vitamin A and other antioxidant retinoiddcarotenoids have been looked at on the basis of epidemiological data showing inverse correlation of vitamin A blood levels to lung cancer and the role of vitamin A in maintaining differentiation in squamous

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tissues. However, results with these agents have been disappointing, with several studies showing increased risk of lung cancer in retinoid/carotenoid treatment groups [e.g., the a-Tocopherol p-Carotene study in male Finnish smokers (Albanes et al., 1996) and the CARET study in smokers and asbestos-exposed smokers (Omenn et al., 1996)l. It has been suggested that these effects are due to biphasic redox activity of the agents; at some concentrations the agents exhibit prooxidant rather than antioxidant activity. Dose-titration and mechanistic studies are needed to fully evaluate chemopreventive strategies for these agents. Other potential lung chemopreventives-NAC, oltipraz, and PEITC-have pronounced antimutagenic activity against tobacco carcinogens. PEITC is an analog and potent inhibitor of the metabolic activation of NNK. Mutated p53 is observed in >50% of lung cancers; the tobacco carcinogens B[a]P and nitrosamines such as NNK are inhibited by oltipraz. On the basis of animal studies, other antioxidandantiinflammatory agents, such as LOX inhibitors, also have potential as lung cancer chemopreventives. Another promising strategy in lung is local, topical administration of agent by aerosol delivery, which minimizes systemic toxicity and circumvents bioavailability problems. As described above, Wattenberg has demonstrated the chemopreventive activity of aerosolized corticosteroids (budesonide) in B[u]P-induced mouse lung (Wattenberg et al., 1997), and budesonide is now being evaluated for prevention and regression of bronchial dysplasia in chronic smokers. The concept of local delivery of chemopreventive agents is also likely to find application in other target tissues such as colon and the upper aerodigestive tract, and, of course, topical application of sunscreen has long set the precedent for local delivery of chemopreventives to skin. Bronchial dysplasia is IEN in lung. Chemoprevention trials using dysplasia as a surrogate end point in patients with a history of prior laryngeal carcinoma or stage I lung cancer who have undergone resection are reviewed in Kelloff et al. (1949f). Such patients are at high risk for the presence of dysplasia, and have second primary tumors at the rate of 10-15% over 5 years. This cohort is studied by multiple brushings and biopsies at predetermined sites within the bronchi. Similarly, as implied above, chronic smokers (current and former) have high incidences of bronchial dysplasia and provide good targets for chemopreventive intervention. Quality control of tissue sampling and marker measurement is especially important in these studies because of concern that biopsy-induced regression may confound results. Improved ability to visualize lungs and bronchus (e.g., using the LIFE scope), elucidation of genetic progression pathways such as described by Kishimoto et al. (1995), and definition of molecular activities required for chemopreventive effect such as retinoid activation of RARp (Hong and Sporn, 1997) should allow more accurate and quantitative evaluations to be made in lung.

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E. Head and Neck Cancers of the oral cavity, pharynx, and larynx have been estimated to account for 5% (29,600) of total new cancer cases in U.S. males and 2% (11,800) of cases in U.S. females (Landis et al., 1998). These cancers result in an estimated 3% (8700) of cancer deaths in males and 1%(3600) in females (Landis et d., 1998). The oral cavity, like the lung, is subject to field cancerization; thus, these statistics underestimate the severity of the disease, which is reflected by high lifetime recurrence and local second primary tumor rates of 20-40% (Benner et al., 1992). Also, extensive morbidity is associated with recurrence and new tumors. The most significant risk factors for disease are tobacco use and tobacco use kombined with alcohol consumption (Benner et al., 1992). Again, control of cellular proliferation and preservation of normal differentiation have been important strategies in selecting potential chemopreventive agents. Both P-carotene (Garewal et al., 1990) and the retinoid 13-cis-retinoic acid (Hong et al., 1986) have been shown to reverse oral leukoplakia, which is associated with increased risk of oral cancer and is considered to be a premalignant lesion when dysplasia is present. Oral leukoplakia with dysplasia is a possible end point for rapid evaluation of chemopreventive agents in short-term clinical trials. It is easily accessible, and, as noted above, it fits the definition of a potential surrogate end point biomarker and has been used successfully in chemoprevention trials. The cohort is patients with biopsy-proven dysplastic leukoplakia, but no oral malignancy. Treatment with putative chemopreventive agents for 6 months should be sufficient to determine the ability of the agents to cause regression of the leukoplakia. Because tissue sampling is easy, the patients can be followed closely so that any lesion that progressed could be immediately removed surgically. Also, the lesions and surrounding normal tissues can be monitored for modulation of other, possibly earlier, intermediate biomarkers such as those associated with cell proliferation (e.g., EGF and EGFR, PCNA, TGFP) and differentiation (e.g., involucrin). One difficulty is that it is common practice and prudent for the patient’s dentist or oral surgeon to remoire the visible dysplastic lesion on diagnosis. Thus evaluation of regression may rely on modulation of early molecular targets in surrounding, normal-appearing tissue (see Hittelman et al., 1996). 13-cis-Retinoic acid is the most widely studied and effective retinoid in cancer chemoprevention trials in head and neck. A vanguard randomized, placebo-controlled chemoprevention trial was undertaken by Hong and associates (1990) and demonstrated a significant decrease in second primary tumors after high-dose adjuvant 13-cis-retinoic acid (-4.1-8.2 pmol/kg body weightlday). As suggested in the discussion of oral leukoplakia, the elucidation of

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genetic and molecular progression models in carcinogenesis will greatly benefit the discovery of appropriate molecular targets and the evaluation of chemoprevention strategies. Significant progress in this regard has been made in head and neck, especially by Sidransky and colleagues, who showed the correlation of LOH frequencies at ten specific microsatellite loci and increasing numbers of affected loci to severity of dysplasia (Califano et al., 1996).

F. Bladder gladder cancer is the fourth most common cancer in U.S. males, estimated for 1998 at 6% (35,500) of new cancer cases and 2% (7100) of cancerrelated deaths (Landis et al., 1998). In U.S. females, the incidence is lower2% (14,900) of new cases and 2% (4500) of cancer deaths. Of the 98% of these cancers that are confirmed histologically, 93 % are transitional cell carcinoma (TCC) (Silverman et d,. 1992). The high recurrence rate (50%within 6-12 months and 60-75% within 2-5 years) was described above. The recurrent lesions are highly unpredictable. Although they may be of the same type as the initial lesions, the subsequent cancers may have progressed in grade or type. The association with environmental and lifestyle factors suggests that bladder cancer incidence may be modulated by chemopreventive agents. Bladder cancers have been attributed to several such factors including tobacco use and occupational exposure to aromatic amines. Chronic inflammation and infection are also important etiological/risk factors. As in other epithelial mucosa, abnormal proliferation is observed in bladder cancers, and so antiproliferatives such as DFMO and differentiating agents such as retinoids-specifically, fenretinide-have been found to have chemopreventiveactivity in animal studies. The agents thus far found to have the most profound chemopreventive activity in animal bladder cancer models are NSAIDs. Piroxicam and sulindac are particularly effective in reducing the incidence of OH-BBN-induced TCC in mice (Moon et al., 1993; Rao et al., 1996). As in colon, the combination of DFMO and an NSAID is interesting because of possible synergistic activity. As noted above, nimesulide, an antiinflammatory which shows preferential inhibition of COX-2 over COX-1, also potently inhibited OH-BBN-induced bladder cancers in rats (Okajima et al., 1998), suggesting the potential for selective COX-2 inhibitors at this target. Phase I1 chemoprevention trials are in progress with fenretinide, DFMO, and a selective COX-2 inhibitor. Recurrence and progression of superficial bladder neoplasia provide important opportunities for evaluating the effects of chemopreventive agents. There are two pathways of neoplastic progression in the bladder (reviewed in Kelloff et al., 1994f). One leads to papillary lesions designated by the no-

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tation Ta [intraepithelial) and T1 (superficiallyinvasive). Initially, 7 0 4 0 % of TCC present as these superficial tumors with limited potential for invasion (Harris and Neal, 1992). The other pathway does not involve papillomas, has a high likelihood-of invasion, and leads to transitional cell carcinoma in situ (TIS). Both intraepithelial lesions could be suitable end points for chemopreventive studies. Other intermediate biomarkers have been implicated (see Kelloff et al., 1994f). Changes in EGFR expression and EGF excretion may precede detectable TCC (Messing and Reznikoff, 1992). As described above, cells may develop abnormal differentiation patterns during carcinogenesis, resulting in associated changes in cell components. Such effects have been observed in bladder, including altered expression of Lewis" blood group antigen, integrins, and F- and G-actins. Genetic effects have also been observed, including changes in DNA content, LOH, and loss of Rb gene function. As cited above, Sidransky and colleagues have made significant progress in establishing a genetic progression model for bladder carcinogenesis (Sidransky and Messing, 1992; Mao et al., 1996), which should identify additional specific molecular and genetic biomarkers for chemoprevention studies. One likely candidate is chromosomal loss at 9q. Bladder tumor recurrence and progression suggest using two clinical cohorts to allow relatively short-term Phase 11trials of chemopreventive agents in bladder. One cohort is patients with stage Ta/T1 disease with TIS. This group of patients receives normal treatment, namely, resection followed by Bacillus calmette-Guerin (BCG). After BCG treatment, the patients are randomized to chemopreventive treatment and placebo groups and monitored for cancer recurrence. The other cohort is patients with stage Ta/T1 disease without TIS. Normally, such patients are followed but receive no treatment unless the disease progresses. These patients are treated with chemopreventive agent and monitored for disease progression. In both cohorts, as many potential intermediate markers as possible are followed by urine cytology in search of good, validated surrogate end points for further trials. As noted for studies in lung, standardization of analytical techniques is critical to establishing intermediate biomarkers.

G. Esophagus Although esophageal cancer accounts for only 1% (12,300) of all cases and 2% (11,900) of cancer-related deaths in the United States (Landis et al., 1998), it has the third shortest 5-year survival rate among all cancers (behind pancreas and liver). Also, treatment involves high morbidity and significantly lower quality of life (i.e., esophagectomy with associated difficulties in eating and speaking).

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As for other cancers of the aerodigestive tract, risk factors include alcohol and tobacco use, as well as poor diet (e.g., lacking fresh fruit and vegetables). As noted earlier in this review, chronic GERD is a risk factor for the cancer, as well as for Barrett’s esophagus, which appears to be the most reliable precursor to esophageal cancer, and a logical intermediate biomarker for esophageal cancer. Barrett’s esophagus with intestinal-type metaplasia is seen in 10-15% of patients who have endoscopy for GERD (or up to 0.4% of the general population) (Falk and Richter, 1996; Roth et al., 1997).The standard of care for Barrett’s patients with moderate dysplasia is regular endoscopy, which allows surveillance during chemopreventive intervention. The standard for severe disease is esophagectomy,which provides a rationale for’chemopreventive intervention in Barrett’s esophagus. Chemoprevention trials are now being conducted in patients with intestinal type Barrett’s esophagus using DFMO and a selective COX-2 inhibitor. A pilot study with DFMO previously showed its potential effectiveness (Garewal et al., 1988). The population of Linxian Province in China has a high incidence of esophageal and gastric cancers, as well as endogenously low dietary levels of some nutrients, including selenium. Following daily treatment of subjects with 50 Fg selenium-enriched brewer’s yeast plus 15 mg P-carotene and 30 mg vitamin E or placebo for 5.25 years, the total mortality rate was significantly decreased (9%) in 29,584 subjects from the general population, primarily the result of lower total and stomach cancer rates. A 42% reduction in esophageal cancer risk in a subset of the subjects was noted although it was not statistically significant (Blot et al., 1993). Several models exist for evaluating the potential of a chemopreventiveagent for inhibiting esophageal cancer, including rats and hamsters treated with carcinogen; the most reliable and widely used model is nitrosamineinduced rats. In fact, a recent study showed that the pathology of nitrosamineinduced carcinogenesis in rat esophagus could be followed quantitatively by changes in nuclear morphometry, and, most importantly, that its inhibition by a chemopreventive agent (DFMO) could be demonstrated by reduced changes in nuclear morphometry (Boone et al., 1997). Other biomarkers that have been associated with esophageal cancer and are promising for measuring chemopreventive effects include changes in p53 (including LOH at chromosome 17) and EGF expression, and microsatellite instability, along with the more general biomarkers of proliferation and apoptosis.

H. UterineCervix Cervical cancer, despite its high incidence-more than 50,000 new cases of carcinoma in situ, 13,700 new cases of invasive carcinoma, and 4900 related deaths estimated for 1998 (Landis et al., 1998)-probably does not

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present a suitable opportunity for chemoprevention. Early detection and surgical excision have produced a high cure rate for cervical carcinoma except for those very aggressive lesions associated with HPV infection and those that are undetected because-of poor medical care. However, as noted above, CIN, the premalignant lesion in cervix, is the prototypical IEN, and CIN 111 is a suitable intermediate end point for studies of chemoprevention. Results obtained in this tissue-for example, demonstration of agent efficacy and identification of intermediate biomarkers-could be generalized to other cancer sites. Moreover, the cervix is easily, and relatively painlessly, accessible for biopsy by speculum and for visualization of tissues by colposcopy. The risk factors for cervical neoplasia are well known. Besides HPV infection, they include early age at first intercotirse, multiple sexual partners, race, immunodepression, oral contraceptive use, and smoking. As for the other major cancers, the association of incidence with environmental factors indicates that the course of the disease may be modulated. There is also epidemiological evidence suggesting that vitamin deficiencies may play a role in CIN progression. Vitamin A, carotenoids, folic acid, and vitamin C were thought to prevent progression of CIN to CIS; P-carotene has been observed to have a protective effect against invasive cervical carcinoma. These epidemiological data on vitamin A suggest that retinoids may be good candidates for chemoprevention in cervix. Further, since the epidemiological data show that CIN progression may be inhibited, other antiproliferatives, such as DFMO, have potential. In fact vitamin A has shown ability to cause regression of CIN 11, and, in a preliminary study, DFMO showed ability to reduce PCNA levels and cause clinically observable regression of CIN I11 (Boiko et ul., 1997; Hu et al., 1997). The retinoids fenretinide and 9-cisretinoic acid are currently on test. The cohorts in short-term chemoprevention trials are patients with CIN II11 without HPV infection. Modulation of CIN is expected within 3-6 months of treatment. Both normal and dysplastic tissue are monitored throughout the course of treatment, and at the end of treatment or at any sign of progression, the lesions could be treated definitively by excision. As in the other cancers, multiple intermediate biomarkers are monitored, including proliferation markers (e.g., PCNA, EGFR, TGFu, TGFP), differentiation markers (e.g., involucrin), and genetic markers (DNA, content, rus, oncogene expression).

I. Skin Nonmelanoma skin cancer [basal cell (BCC) and squamous cell carcinoma (SCC)] has a high rate of occurrence-approximately 1,000,000 cases were expected in 1998-but low associated mortality (Landis et al., 1998).

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Nonetheless, its high incidence and prevalence among various segments of the population (e.g., fair-skinned, sun-exposed, smokers) as well as the morbidity associated with surgical removal make it an appropriate target for chemoprevention. Many classes of agents show promise in animal models for preventing skin cancers, and, currently, retinoids, antiinflammatories, and tea polypbenols are of high interest. Actinic keratosis is the benign precursor of BCC and SCC that has been used most frequently to define cohorts and serve as a surrogate end point in chemoprevention trials. Other cohorts that may benefit from chemopreventive treatment are subjects with previous BCC or SCC, those with intense chronic or episodic sun exposure, as well as those with preexisting skin disorders, immunosuppression, or genetic predisposition (e.g,, xeroderma pigmentosum). For example, in the SKICAP-AK trial, subjects (n = 2800) with a history of >10 actinic keratoses and 5 2 previous SCC/BCC were randomized to 25,000 I(J retinol/day (-0.4 pmol/kg body weight/day) or placebo for 5 years. Retinoid-treated participants had a significantly lower incidence of new SCC, but no change in BCC (Moon et al., 1997). In contrast, in the three-arm SKICAP-S/Btrial no reduction in the incidence of new skin cancers in patients (n = 525) with prior multiple SCC or BCC was found after 3 years of treatment with retinol (25,000 IU/day, or -0.4 pmol/kg body weighdday), 13-cis-retinoic acid, or placebo (Levine et al., 19978~). Several chemoprevention trials with other agents are ongoing in subjects with actinic keratoses, including studies with topically applied tea polyphenols and those with orally administered DFMO, fenretinide, and a selective COX-2 inhibitor. The incidence of the more deadly cutaneous melanoma has continued to rise [41,600 cases of melanoma and 21,100 cases of melanoma in situ expected in 1998 (Landis et al., 1998)], calling for increased research to develop chemopreventive strategies in this cohort. In preliminary studies, dysplastic nevi, precursors of melanoma and considered histological intermediate biomarkers, have responded to topical all-trans-retinoic acid treatment (Meyskenset al., 1994).A small placebo-controlled study of 21 patients (six nevi/patient) demonstrated significant improvement in both clinical and histological parameters; 47% (7/15) of nevi treated with 0.05% all-transretinoic acid of unknown volume daily for 4 months reverted to benign nevi or disappeared completely (Halpern et al., 1994). Recently, melanoma research has focused on developing animal models showing progression of dysplastic nevi, and evaluating surrogate end points for testing chemopreventive efficacy in melanoma-susceptible cohorts. For example, the human homolog of the Drosophila patched (PTCH) gene at chromosome 9q22.3 appears to code for a tumor suppressor of familial BCC (Xie et al., 1997b)and also is mutated in subjects with nevoid basal cell carcinoma syndrome (Wicking et al., 1997).

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J. Liver Liver cancer worldwide isa major health risk. Although the U.S. incidence is relatively low [13,900 cases or -1% of cases expected in 1998 (Landis et al., 1998)Jit has been rising steadily and is associated with risk factors shared by large segments of the population, including alcohol consumption, smoking, and hepatitis B and C infection. Also, mortality is extremely high (13,000 liver cancer-related deaths expected in 1998) considering the incidence (Landis et d.,1998). Several classes of agents have potential efficacy against liver cancer, with most supporting data obtained with antimutagens and retinoids. Oltipraz is a potent inducer of phase I1 metabolic enzymes such as GST, ind its chemopreventive activity has been attributed to its ability to enhance these enzymes, resulting in conjugation and excretion of carcinogens. The rationale for its study as a chemopreventive agent in,liver has been reviewed (Greenwald and Kelloff, 1996) and is summarized briefly here. In studies in carcinogen-induced animals, lower amounts of effective carcinogens are seen as measured, for example, by carcinogen-DNA adducts; particularly, lower levels of aflatoxin B, (AFB,)-DNA adducts were observed. Thus, it was thought possible that the cancer risk associated with AFB,, a carcinogenic contaminant of foods and feeds worldwide, could be reduced by oltipraz. Experimental studies had already suggested that oltipraz can modify the risk for AFB,-induced hepatocarcinogenesis, as shown by inhibition of hepatoxicity and aflatoxin-DNA adduct formation in AFB,-exposed rats. Inclusion of oltipraz in the diet, beginning 1 week before AFB, exposure, resulted in lower levels of AFB,-DNA adducts throughout the exposure period. Epidemiological studies have shown a strong association between estimated aflatoxin intake and primary liver cancer. High levels of aflatoxins, produced by Aspergillus species, have been found in groundnuts and maize in Africa, southeast Asia, and southern China, where these foods are dietary staples. In the United States, contamination of commodities such as corn, cottonseed, and peanuts occurs at high levels during some years, but contamination general!y is much lower than in underdeveloped countries. Oltipraz is now being tested in a Phase I1 clinical chemoprevention trial in Chinese subjects at high risk for liver cancer, based on their exposure to aflatoxins as well as smoking history and hepatitis exposure. This study is evaluating the modulation of urinary and serum AFB,-DNA and serum AFB1-albumin adducts. Also, urine samples from subjects in this trial will be screened for the effect of oltipraz on urinary mutagens. Results from the Phase IIa portion of this study, in which the same biomarkers were measured, are promising (Kensler et al., 1998). Patients with treated (resected and ethanol injection) hepatocellular carcinoma are at high risk for recurrent and second primary tumors and provide

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another cohort for evaluation of chemopreventive strategies. Muto et al. (1996) described a placebo-controlled trial of an acyclic retinoid (600 mg/ day polyprenoic acid, also known as E-5166, for 12 months) to treat patients with previous primary hepatocellular carcinoma. This study showed that in the group of 89 patients, those treated with polyprenoic acid had a relative risk for B second primary of 0.31 compared with placebo controls. This result is sufficiently promising to warrant further investigation of the acyclic retinoid.

K. Multiple Myeloma Monoclonal gammopathy [including monoclonal gammopathy of undetermined significance (MGUS)] is associated with older populations; a fraction of these patients progresses to multiple myeloma. The incidence of both is quite high. Although the exact mechanism of disease progression is unclear, increased levels of the cytokine IL-6 appear to be important in the process. Agents such as dehydroepiandrostenedione (DHEA) may be able to alter the ratio of T-helper (Th)cell subsets (increasingT h l relative to Th2), thereby causing a decrease in Th2-related cytokines (IL-4, IL-5, IL-6, and IL10) and boosting cellular immunity. Feeding DHEA to aged laboratory animals leads to a decrease of age-related increases of IL-6 production. Thus, administration of DHEA may help to lower circulating levels of IL-6 and prove beneficial to patients with monoclonal gammopathy and prevent the onset of multiple myeloma. Besides DHEA other agents have potential for reducing MGUS; these include bisphosphonates and antiinflammatories.

L. Clinical Benefit in Addition to Cancer Incidence Reduction Besides cancer prevention, there are several situations in which chemoprevention can be used as a definitive treatment of precancerous lesions, thereby providing additional clinical benefit. These benefits include reduced morbidity, enhanced quality of life, delayed surgery, and increased intervals for surveillance requiring invasive procedures. Examples include the following: Prevention of precancers in subjects at high risk associated with genetic predisposition (e.g., prevention of colorectal adenomas in patients with FAP). Familial adenomatous polyposis is characterized by germline mutations in the APC tumor suppressor gene. Usually starting when they are teenagers, patients with FAP develop hundreds of colorectal adenomatous

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polyps. If untreated, FAP patients will almost certainly develop colorectal cancer by age 50; they are also at risk for developing other lesions, particularly duodenal polyps-and cancers. Once adenomas begin to appear, these patients are monitored by periodic colonoscopy (at approximately 6-month intervals), removal of existing polyps, and cancer screening. When the polyp burden becomes unmanageable, most patients have partial or total colectomies. Thereafter, they continue to be monitored. Agents that prevent or slow the progression of the adenomas could benefit these patients by delaying the need for colectomy and increasing the intervals between surveillance colonscopies and cancer screenings. Prevention o f precancers for which organ removal or other major surgery with high morbidity is standard of care (e.g., Barrett’s esophagus, superficial bladder cancers). Current treatment for Barrett’s esophagus, a precursor of esophageal cancer, almost always involves partial or total esophagectomy (Roth et af., 1997). Because of the high rate of their recurrence and potential for progression, treatment for superficial bladder cancers includes periodic surveillance (every 3 months) and removal of new lesions, and may include cystectomy (Linehan et al., 1997). In both diseases, treatment has profound detrimental effects on quality of life. Both are examples of situations in which preventive agents could provide clinical benefit by reducing the need for these surgeries. Prevention of precancers in patients at risk for recurrence (e.g., sporadic colorectal adenomas). As noted above, new adenomas occur within 1-3 years postresection in approximately 30% of patients with sporadic colonrectal adenomas or cancers. These patients are screened routinely at 1-to 5year intervals, receiving colonoscopies with removal of new lesions. Preventive treatment could potentially provide benefit by increasing the screening interval, thereby decreasing associated morbidity and lowering health care costs.

VI. SURROGATE END POINTS IN DEFINING I

CHEMOPREVENTIVE EFFICACY-IMPORTANCE OF EVALUATING BOTH PHENOTYPIC AND GENOTYPIC EFFECTS The use of surrogate end points, particularly precancerous lesions, rather than cancer incidence requires that efficacy be carefully defined. An important issue is determining when less than complete phenotypic response of a precancerous lesion constitutes prevention. A related issue is distinguishing chemoprevention from regression of existing disease. To establish chemopreventive efficacy solely on the basis of phenotypic regression essentially

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would require that 100% of existing lesions regress. Similarly, phenotypic chemoprevention could only be demonstrated rigorously by 100% inhibition of new lesions. With less than 100% efficacy, it is possible that the remaining lesions are those that will continue to progress to cancer. As technology advances and genetic and molecular progression models are fully elucidated, genotypic biomarkers should contribute to evaluating chemopreventive efficacy. For instance, if a posttreatment genotype showed decreased incidence of cancer-related changes (either in specific genes or in more general measures of genomic instability) compared with baseline, <100% regression could be considered prevention. Also,
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ment group than from the placebo group, treatment may only have inhibited less severe dysplasia, which would be less likely to develop into carcinoma. This result would not be a convincing demonstration of chemopreventive efficacy. The genetic progression model for head and neck cancer described by Sidransky and colleagues (Califano et al., 1996) ,shows the correlation of LOH frequencies at 10 specific microsatellite loci and increasing numbers of affected loci to severity of dysplasia. In the hypothetical study of regression of oral leukoplakia, a chemopreventive agent or placebo is administered to patients with existing oral dysplasia. At baseline these lesions are measured and are biopsied along with normal-appearing adjacent tissue. Biopsies of both the dysplastic lesions and normal-appearing tissues are also taken at the end of treatment. The same rigorous attention to adequate and representative sampling described for the colorectal adenoma example is applied to these assessments, and significant regression of the dysplastic lesions is observed. Fewer lesions remain in the treatment group than in the control group; lesions in the treatment group are also significantly smaller. LOH at the 10 important loci is analyzed in cells from the lesions and nearby, normal-appearing tissue. The LOH frequency distribution is similar in the dysplasia from the treated and control groups. Also, LOH frequency distributions in normal-appearing mucosa are comparable in all groups. Hence, assuming that the 10 loci analyzed are informative biomarkers, no genetic changes are seen that would suggest that the lesions in the treated patients are likely to progress faster than those in controls, providing supporting evidence that the reduced incidence of dysplasia is a true chemopreventive effect. No chemopreventive effect would have been demonstrated if the average frequencies of LOH were significantly higher in lesions from the treated patients than the controls, indicating that the lesions in the treated patients are likely to progress more quickly than those in the controls, and that the chemopreventive intervention is only preventing lesions less likely to progress. Note that the reliability of genotypic assessments will be determined by knowledge of the important genetic lesions. Continued research defining genotypic progression of cancers will make these evaluations possible.

VII. MAJOR ISSUES AND CHALLENGES FOR CANCER CHEMOPREVENTION This review discusses in some detail the major progress in the field of chemoprevention. The nature of carcinogenesis, and therefore the potential for its inhibition, is the subject of intense experimental and clinical research

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by scientists from various disciplines focused on the molecular, cellular, tissue, and clinical aspects of this process. The understanding of the nature of this process has made possible the identification of candidate chemoprevention drugs that are being developed to hit key molecular targets. Carcinogenesis at the cellular and tissue levels is characterized by accelerating hypermutagenesis and hyperproliferation, and drug development strategies modulating these measurable events are also proving successful. The rapid sequencing of the human genome, estimated now to contain about 100,000 genes, and the progress in functional genomics deriving from this will lead to the identification of a few hundred genes that are the major etiological players in human carcinogenesis. Rare genetic syndromes have already provided the experimental leads to identifying oncogenes and tumor suppressor genes to which are continually being added the more subtle conveyors of risk such as the modifier genes. The intensive study of genetic polymorphisms are defining genes that contribute a much smaller absolute risk of an individual developing cancer, but are of paramount importance in terms of the high attributable risk of cancer for the whole human population. Notable examples include genetic polymorphisms of the 50 or more genes that control estrogen and androgen metabolism and therefore breast, prostate, and other cancers, as well as polymorphisms of enzymes that metabolize endogenous and exogenous human carcinogens. Human epithelial carcinogenesis, which accounts for more than 80% of the human cancer burden, is a multiyear (sometimes decades long) process of clonal selection and evolution of genetically damaged cells, which provide the abnormal phenotype of precancer the eventually leads to invasive cancer. The genetic progression models continually being defined for major human cancers, for example, colon (Fearon and Vogelstein, 1990) and head and neck (Califano et al., 1996), reveal that the sequence of genetic damage is multiple choice, multiple path, and by nature stochastic. Therefore, integration of the science of genomics along with tissue histomorphometry and imaging technology not only provide the best means of defining human risk of later cancer development, but also provide measurable parameters that when modulated by drugs provide compelling evidence that the drug will result in cancer incidence reduction. Excellent and efficient transgenic animal models of human carcinogenesis becoming available allow validation of these phenotypic and genotypic surrogate end points of cancer incidence by conduct of chemoprevention experiments comparing drug versus placebo modulation of these end points and its correlation to cancer incidence reduction. The science of chemoprevention drug development is now solidly based on inhibition of carcinogenesis at all levels. This review provides five prototypical examples of classes of chemopreventive drugs, of which there are many more. These examples, especially the antioxidants and antiinflammatories, reveal that strategies and drug classes for chemoprevention are relevant to

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prevention of other chronic diseases such as cardiovascular, neurodegenerative, and other chronic degenerative diseases. The recent definitive cancer incidence reduction shown by tamoxifen and vitamin E for breast and prostate, respectively, show the tre-mendous public health impact possible from chemoprevention as better drugs are developed. At present more than 50 candidate chemoprevention drugs are being evaluated in Phase I1 trials, and these will provide a few high priority drugs for definitive Phase 111cancer incidence reduction trials as the progress in surrogate end point development continues. The NCI and FDA have defined the process for incremental accumulation of drug effectiveness and safety needed to secure chemoprevention drug marketing approvals. As more data are accumulated additional definitive guidance may be provided regarding the use of surrogate end points of cancer incidence as a rapid basis for drug approval. Proof of the effectiveness of chemopreventive drugs will be less of a challenge than proving chronic safety, as once approved these drugs may be prescribed to large populations at relatively low absolute risk of developing cancer. Notwithstanding the progress, there remain major issues and challenges for cancer chemoprevention looking forward into the next decade. Noteworthy examples include the following: Carcinogenesis as a process of progressive disorganization-the need for early intervention. The dismal record of standard therapy in improving survival from cancer of the major epithelial target organs over the last 20 years is well documented (see Landis et al., 1998).The human genetic progression models are suggesting clearly that this failure is more due to the nature of the end point of clinical carcinogenesis, human cancer, characterized by aneusomy and heterogeneity, statistically leaving most bonafide molecular targets unavailable to rational therapy, than to any failure of omission or effort by the intensive therapeutic preclinical and clinical research effort. There remains the possibility of customizing therapy to an individual once a molecular genotype can be effectively determined and is amenable to yet to be defined dominant pathways for inducing cell death. In the meantime, rational mechanistic thinking, compelling efficacy data of candidate drugs in relevant animal model systems, and a few human efficacy examples tell us that early intervention provides profound hope for the future, and the complexities of chemoprevention drug evaluation and approval will not be determined by failure of efficacy but by intolerance of even minor toxicities given the large target populations for eventual intervention. The etiology of most human cancers involves small relative risks-the limitations of human epidemiological data and the need to measure individual absolute risks. With the obvious exception of the rare genetic syndromes where the relative risk of an individual developing cancer can be very high (subjects with FAP, Li-Fraumeni, BRCA, etc.), measurable risk factors defin-

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ing any population most often confer relative risks of developing cancer in the units or tens. Examples to put this in perspective include fairly good agreement from three large studies of the risk of tobacco smoking showing relative risks of 30 for 30 pack-years and the presence of atypical ductal hyperplasia in the breast of adult females conferring a risk of 5-6 of developing breast cancer. These small relative risks, along with the multifactorial etiology of most human cancer, leaves investigators with the inability to prove the effectivenessof cancer prevention interventions except through randomized intervention trials where the variable under question can be evaluated and the confounding variables can be controlled. The impracticality of numerous large human intervention trials to test various hypotheses emphasizes the importance of more accurate prediction of an individual’s absolute risk and the methods to monitor that risk, both of which are being developed in the dynamic fields of functional genomics, histomorphometry, and imaging. These technologies will allow both the planning of interventions and the evaluation of data deriving therefrom to make informed public health recommendations on the basis of knowledge of risk benefit. Precancer as a focal disease currently requires biopsies for detection-the need for noninvasive measurement o f cancer risk. Clinical cancer is the end stage of a process, proceeded by precancer, sometimes characterized by changes detectable by the pathologist with standard histopathology techniques and sometimes, in a field that’s developing rapidly, with more subtle histomorphometric measurements made by computer-assisted image analysis. Progress in molecular pathology indicates that genotypic abnormalities with molecular products, in the absence of detectable abnormal morphology, will provide more sensitive detection of incipient neoplasia. Notwithstanding this tremendous progress, the precancer to cancer disease process is a tissue-based clonal evolution and, at present, with few exceptions requires biopsies and cannot be found by less invasive techniques. Advances in in vivo imaging modalities provide promise for less invasive early detection. The challenge for early detection by these technologies differs among key human cancer target organs. Access by direct visualization of colon, upper aerodigestive tract/head and neck/lung, bladder, cervix/endometrium, and skin provides advantage over less accessible targets such as breast, prostate, pancreas, ovary, and liver. In some cases, the amount of abnormal tissue can be quantified; therefore, not only can risk estimates be improved but, since statistical sampling is not so much of an issue, the extent of modulation by chemopreventive drugs can also be quantified. Advances in the specificity and sensitivity of detecting moieties present in the serum from microscopic disease are becoming more likely and practical from the rapid advances occurring in genomics and proteomics. Cancer is a complex biological system requirilrg very sophisticated analysis of high volumes of data coming from genomics and proteomics. Genom-

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ic chip arrays being produced for analyzing specific target organs number in some cases in the thousands of genes, and the volume of data being produced will require very sophisticated data analysis to define and quantify contributors to risk. To this end, proven cases from archival specimens from properly designed tissue banks will help provide the end points and validation needed. Further, the use of specimens from chemoprevention interventions both preclinically and clinically from known efiective drugs compared to patterns from placebo controls may provide very specific etiologic insights as to which single moieties or patterns in this pleiotropic process are the most important. Importance of molecular target-based mechanistic approaches and more empirically based cellular approaches to making progress in chemoprevenmtive drug development. Comprehensive evaluations of the processes involved in carcinogenesis have yielded numerous simultaneously expressed biological activities, with unknown and possibly interwoven cause and effect relationships, many of which suggest themselves as molecular targets for effective chemopreventive intervention. This dynamic and interdependent carcinogenesis process makes it more difficult to predict whether a drug designed to inhibit a specific molecular target will indeed be efficacious. Further, many chemopreventive drugs known to be active in experimental systems have modulated many biological activities, often making it difficult to conclude which activities best predict chemopreventive potential. Since carcinogenesis at the cell and tissue level is usually characterized by hyperproliferation, decreased apoptosis, or hypermutagenesis, cell-based assays using normal or precancer cell substrates, in which these parameters are measured, are a valuable part of chemoprevention drug development. The importance of safe chemopreventive drugs-how safe is safe and the importance of risk benefit. The science of toxicity evaluation of drugs developed for chronic human use is well established, and guidance specific for chemopreventive agent development has been published (Kelloff et al., 1995b).Volumes of animal and human data as well as multiyear experience with drugs given chronically to humans over four plus decades have provided few unanticipated safety problems. The preclinical toxicology evaluation of Landidate drugs involves chronic administration of 10-fold the starting doses in human safety evaluations, which are pushed to doses creating detectable preclinical toxicities, so that the profile of expected problems are identified. In addition to this, carcinogenic risk is evaluated by essentially lifetime administration of drug to rodents, and specialized (e.g., reproductive) toxicity studies are done routinely. Safety evaluation in humans is performed incrementally and often begins in cohorts at high risk for disease, and thus at greater potential for benefit. The decision for a drug approval for a given indication is made on a case-by-case basis after a rigorous analysis of efficacy and safety and the risk benefit profile of the intended recipients. The

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public health impact of chemopreventive drug intervention will develop incrementally, as drugs are found effective in higher risk cohorts and, in the absence of safety problems, are justifiably qualified for use in lower risk populations. Postmarketing surveillance will continue to find the few unanticipated safety problems that may emerge, and availability of this process itself makes possible the approval of drugs where the weight of the clinical evidence has clearly determined that the drug will make an impact on the reduction of human cancer risk. The rapid progress being made in detection of genetic polymorphisms as it relates to drug metabolizing enzymes, as well as more sophisticated evaluation of risk as described above, will define subsets of rhe population most likely to benefit from the drug intervention. Regulatory issues-the need for approval of chemoprevention drugs based on surrogate end points of cancer incidence and on improvements in quality of life. Proof of chemopreventive drug efficacy based on cancer incidence reduction trials can require up to 45,000 subjects and more than a decade depending on the risk of the population under study. New technologies that allow risk evaluation or trials in already known high-risk cohorts can reduce the size and duration of some cancer incidence reduction trials to as few as 500-1000 patients and 3 years. The number of candidate reduction trials, the scarcity of eligible and willing subjects, and the opportunity provided by the wealth of scientific data and technology make the safe approval of chemopreventive drugs for selected populations on the basis of well-characterized surrogate end poinrs a desirable and practical necessity. A challenge for the future will be to define this process. In addition to reduction of the cancer burden, chemoprevention drugs could provide a sound basis for improvement of quality of life in some clinical situations. For example, as described above for the specific instances of high-risk subjects with precancers, improved quality of life could result from delaying the need for and reducing the morbidity due to surgical treatments and invasive screening procedures. Societal issues-the need for widespread education efforts that cancer is preventable and the need for insurance coverage for proactive prevention. The past two decades have seen a tremendous increase in media coverage, publications, and educational programs on disease prevention, which are directed to the general population. Most of the formal programs provide information on cancer prevention, usually not exclusively, but in concert with other chronic diseases (particularly, cardiovascular disease). A common theme is behavior modification to create a healthy lifestyle, such as by smoking cessation (ASSIST)or general dietary improvement (Five-A-Day). 0 th ers advocate surveillance and early detection (e.g., guidelines for mammographic screening). A future challenge is educating the population on the potential additional benefits of active intervention, whether it is by prescribed drug or specific dietary substances. Important aspects of the educa-

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tional process will be the methods for fully characterizing an individual’s risk and incorporating such risk estimates into early detection programs, so that the most appropriate intervention can be planned. Although the information needed to calculate individial risk is for the most part only now beginning to emerge, the “risk disk” recently developed by the National Cancer Institute for evaluation of breast cancer risk is a prototype for this effort. To ensure that the benefits of prevention are available widely, a further challenge must be met-that is, gaining recognition for proactive prevention as a standard of care. Preventive care in the general population, such as early detection screening (e.g., annual mammograms), has only recently begun to have limited insurance coverage. Widespread coverage will be required so that all who could benefit have access to such care. ’ High-quality basic and applied prevention research defining the genotypic and phenotypic (functional and histological) changes in the progression of normal tissue to precancer to cancer in humans (and experimental animals), the risk conferred by these changes, and the modulation of these changes in preclinical experimentation and randomized clinical trials by active intervention with chemopreventive drugs, dietary agents and regimens, and treatments resulting from early detection provide the main scientific focus for a cancer prevention effort. The key elements of this research effort are too numerous to list in detail but should include comprehensive basic and translational tissue; cell; and serum-based risk evaluation programs; chemopreventive and dietary agent drug discovery and development; further development and testing of transgenic animal models; required safety and pharmacology studies; Phase I, 11, and I11 chemoprevention and appropriate nutriceutical and diet-based studies with rigorous criteria established for prioritizing large studies and proactive planning to ensure accrual; and much expanded early detection programs. The promise of prevention is here, and the growing number of research programs in chemoprevention sciences ensures that this promise will continue to be realized in the next decades.

ACKNOWLEDGMENTS Many thanks to Peter Vogt and Robert Huebner for giving me my start in cancer research and Stephen Orozlan, Masakazu Hatanaka, and Ray Gilden for valuable postdoctoral training. My appreciation goes to Mike Sporn and Lee Wattenberg for their pioneering work in chemoprevention and to Ki Hong and Bernie Fisher for making it a clinical reality. I would like also forecognize my valuable colleagues in the NCI, Chemoprevention Branch, including Charles Boone, Caroline Sigman, Winfred Malone, Vernon Steele, James Crowell, Ronald Lubet, Ernest Hawk, Ronald Lieberman, Julia Lawrence, Levy Kopelovich, Iqbal Mi, and Jaye Viner.

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I also want to acknowledge all my other colleagueswho have and are contributing to the field of chemoprevention research. They are too numerous to name, but many come to mind: Denn i s Ahnen, David Alberts, John Alexander, James Bacus, John Baron, Samuel Beenken, Steven Benner, Monica Bertagnolli, Guido Biasco, David Bostwick, Dean Brenner, Thomas Budd, Julie Buring, Paul Carbone, Margie Clapper, Donald Coffey, Alan Conney, Charles Coltman, Alberto Costa, Dave Crawford, Patrick Creaven, Andrea Decensi, Silvio DeFlora, Kapil Dhingra, Nikolay Dimitrov, Ray DuBois, Paul Engstrom, Carol Fabian, Leslie Ford, Adi Gazdar, Gary Goodman, Gary Gordon, Michael Gould, Robert Greenberg, William Grizzle, Clinton Grubbs, Steve Hecht, George Hemstreet, Brian Henderson, Charles Hennekens, Walter Hittelman, Aryeh Hurwin, Karen Johnson, Thomas Kensler, Bruce Kimler, Barry Kramer, Stephen Lam, Bernard Levin, Martin Lipkin, Scott Lippman, Reuben Lotan, David McCormick, Frank Meyskens, John Minna, Michele Mitchell, Rodolfo Montironi, Richard Moon, James Mulshine, Harold Newmark, Hoyoku Nishino, Gilbert Omenn, Michael Osborne, Joyce O’Shaughnessy, Branko Palcic, Ugo Pastorino, Michael Pereira, John Pezzutto, John Potter, Bandaru Reddy, Wael Sakr, Sheela Sharma, David Sidransky,Gary Stoner, Paul Talalay, Phillip Taylor, Robert Temple, Henry Thompson, Umberto Veronesi, I. Bernard Weinstein, Michael Wargovich, John Weisburger, and Ming You. Finally, I wohld like to acknowledge and thank Peter Greenwald for maintaining focus on the public health importance of cancer prevention research and Richard Klausner for directing and making the scientific investments that will help further the field of chemoprevention research.

REFERENCES Ablin, R. J., and Shaw, M. W. (1986).Anticancer Res. 6,327-328. Albanes, D., Heinonen, 0. P., Taylor, P. R., Virtamo, J., Edwards, B. K., Rautalahti, M., Hartman, A. M., Palmgren, J., Freedman, L. s., Haapakoski, J., Barrett, M. J., Pietinen, P., Malila, N., Tala, E., Liippo, K., Salomaa, E.-R., Tangrea, J. A., Teppo, L., Askin, F. B., Taskinenen, E., Erozan, Y., Greenwald, P., and Huttunen, J. K. (1996).J. Nutl. Cancer Inst. 88, 15601570. Anderson, K. M., Wygodny, J. B., Ondrey, F., and Harris, J. (1988).Prostate 12,3-12. Anderson, K. M., Levin, J., Jajeh, A., Seed, T., and Harris, J. E. (1993). Fatty Acids 48, 323326. Anderson, K. M., Seed, T., Jajeh, A., Dudeja, P., Byun, T., Meng, J., Ou, D., Bonomi, P., and ’ Harris, J. E. (1996).Anticancer Res. 16,2589-2599. Anzano, M. A., Byers, S. W., Smith, J. M., Peer, C. W., Mullen, L. T., Brown, C. C., Roberts, A. B., and Sporn, M. B. (1994). Cancer Res. 54,4614-4617. Anzano, M. A., Peer, C. W., Smith, J. M., Mullen, L. T., Shrader, M. W., Logsdon, D. L., Driver, C. L., Brown, C. C., Roberts, A. B., and Sporn, M. B. (1996).1.Nuti Cancer Inst. 88, 123-125. Aquilina, J. W., Lipsky, J. J., and Bostwick, D. G. (1997).J. Nutl. Cancer Inst. 89, 689-696. Arbeit, J. M., Howley, P. M., and Hanahan, D. (1996). Proc. Natl. Acad. Sci. U.S.A. 93,29302935. Arthur, J. R., and Beckett, G. J. (1994).Proc. Nutr. SOC. 53,61. Assreuy, J., Cunha, F. Q., Liew, F. Y., andMoncada, S. (1993).B x ] . Pburmucol. 108,833-837. Atluru, D,. and Goodwin, J. S. (1986). Cell. Immunol. 99,444-452. Avis, I. M., Jett, M., Boyle, T., Vos, M. D., Moody, T., Treston,’A. M., Martinez, A., and Mulshine, J. L. (1996).1.ch. Invest. 97, 806-813.

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.

Index

A Aberrant crypt foci, 300 N-Aceyl-L-cysteine,261,277, 303 Actin, 50,58-59, 87, 306 proteins, modifying, 63-65,68-70 Actinomycin D, 59, 71, 182, 184-185, 186 , Adhesion and cell motility, 41-42, 51-53,55-57, 73-74 and E7,13 and EGF, 79-80 and invasiveness, 81-82, 86 and plasminogen, 125-131,132 and rho, 69 Adipose tissue, 240 Adrenal cancer, 211 Adriamycin, 182 Aerosols, 276,303 Aflatoxins, 261,310 Agent development biomarkers, 283 combinations, 276-277 discovery principles, 215-222 pharmacokinetics, 272,276,278,280 Phase I studies, 272,275,279-280 Phase I1 studies, 275,284,285-286,289, 291 Phase I11 studies, 275 safety clinical, 279-280 preclinical, 272,274-275,278-279 in vitro assays, 266-267,274 Allografts, 37-38 Aminoglutethimide, 241 Amphiregulin, 72 Anastrozole, 241 Angiogenesis, see also Neoangiogenesis and Kaposi’s sarcoma, 166 and LOX inhibitor, 259 and MMP, 40,136

and nitric oxide, 250-251 and plasminogen, 109,145-146 and VEGF, 40 Annexin 11, 109 Anogenital cancer, 2 Antiandrogens, 295 Antiestrogens, 236-239,276,287; see also specific antiestrogens Anti-inflammatory agents, 219-221,267, 311; see also specific agents; specific categories Antimutagens, 219-221,260-261,267, 284,303 Antioxidants, 219-221,258-259,261-265, 267 Antiproliferation assays, 267 AP-1, 10, 14, 87 APO-~LITRAIL,186-187, 190 Apoptosis and colon cancer, 300 and E7,16-17,20,21 Fas-mediated, 184-1 86 in Kaposi’s sarcoma, 181-188 and LOX products, 259 and nitric oxide, 251 Arachidonic acid, 204,242,260,261 Aromatase inhibitors, 239-242,287,297 Aspirin, 243,293,301 Autocrine growth factors, 45-46,72 Autotaxin, 45,60

B Bak, 5 Barrett’s esophagus, 307,312 BCG, 306 B~1-2~183, 185,187-188,203 Benzidine, 258 Biaxin, 294 Biomarkers, see also specific cancers and breast cancer, 297,299

335

336

Index

Biomarkers (continued) carcinogenicity,203-204,281-283,284285,288-289 and colon cancer, 300 Biphosphonates, 311 Bladder cancer animal models, 269 biomarkers, 291,293,305-306 and carcinogens, 212 chemoprevention trials, 291,293-294, 306 and COX inhibitors, 246-247 and EGF, 224,306 genetics, 208 invasiveness, 36,37,60,78-79 latency, 213 and NSAIDs, 242,243 promising preventive agents, 290,305 quality of life, 312 recurrence, 207 and retinoids, 233 risk factors, 208,209,290 Breast cancer, see also Mammary carcinoma and antiproliferation agents, 296 aromatase activity, 240 biomarkers, 289,291,297,299 chemopreventiontrials, 212,289,292, 298-299 clinical cohorts, 289,298-299 ductal and lobular, 298 and EGF, 42,224,299 and estrogen, 234-235,238 germline mutations, 210,211 metastatic spread, 35 and motility suppressors, 82-83 and nitric oxide, 250, 251 and ~ 5 3 , 2 9 9 PI3-kinase effect, 67 and PLCy, 60 promising preventive agents, 288,297 ras, 227 retinoids, 232,233 risk factors, 288-289,298-299, 320 and tea, 264 time course, 212-214 Broccoli, 277

-

.

C Cadherins, 45,54 Calcitriol, 292

Calcium and colon cancer, 292,293,301 and invasiveness, 65-66,74, 76, 86 and plasminogen, 140 Calcium-bindingprotein, 5 Calpain, 65-66,76 Capillaries, 34 Carbohydrate metabolism, 15-16, 18-19 Carcinogenesis cell level, 204-206 clinical level, 207-214 and EGF, 205-206,223-224 , and estrogen, 234-235 ' and LOX, 2S4,256-258 molecular level, 202-204 and nitric oxide, 247-248,249-252 Ras, 226-227 and cetinoids, 303 time course, 212-214 tissue level, 206-207 Carcinogenicity,205-206,233,279; see also Biomarkers p-carotene and cervical cancer, 308 and colon cancer, 301 and esophagus cancer, 263,307 and head and neck cancers, 304 and prostate cancer, 295 Carotenoids, 303,308 Catenins, 45 Cathepsin D, 78 Caveolin, 134, 140 CD9,82-83 CD44,42,49,66 Cdk, see Cyclinlcdk network Celecoxib, 246 Cell cycle, E7 effect, 9-13 Cell membrane, 263; see also Membrane ru fling Cell motility, see also Invasiveness; Signaling into acellular areas, 45-46,48 and actins, 58-59,63 cell changes, 49-54 definition, 40-41 directionality, 43,44,51,57 external determinants, 54-57 measurement, 46-48 persistence, 44,48, 51 rate, 48,51,57,60, 78-81 suppressors, 82-83

Index Cervical cancer biomarkers, 291,294,308 chemoprevention trials, 291,294,308 and E6,4 and EGF, 224 gene knockout model, 271 and HW,2 latency, 213 and nitric oxide, 250 promising preventive agents, 290, 308 and Ras, 227 risk factors, 290, 308 C-fos, S, 14,258 Chemokines, C-X-C family, 75-76 Chemokinesis, 41-45,48,52,68-69 Chemoprevention,see also Agent development antiestrogens, 236-239 anti-inflammatory agents, 242-244,244247,247-254,254-260 antioxidants, 219-221,258-259,261265 aromatase inhibitors, 239-242 bladder cancer, 290-291,293,305-306 breast cancer, 288-289,292-293,296299 cervical cancer, 290-291,294,307-308 clinical trials, 288-294,316, 319 colon cancer, 288-289,292-293,299302,313 combination approach, 276-277 delivery systems, 276, 303 diet, 277 efficacy, 283-285,288-291,292-294, 313-314 esophagus cancer, 290-291,294,306307 evaluation guidelines, 268-272 and genotype, 215,312-314 head and neck cancer, 288-289,293, 304-305,314 hormone modulators, 234-242 insurance coverage, 3 19,320 liver cancer, 290-291,294,310-311 lung cancer, 288-289,293,302-303 molecular targets, 214-215 multiple myeloma, 294,311 phase II enzyme inducers, 260-261,265 promising agents, 288-291 prostate cancer, 287-296,292-293 quality of life, 311-312, 319

337 regulatory issues, 319 retinoids, 230-234 and risk factors, 288-291,316-318,320 safety, 272,274,316,318-319 signal transduction modulators EGF receptor inhibitors, 222-225 ras farnesylation inhibitors, 225-230 skin cancer, 290-291,294,308-309 timing, 268,285 in vitro, 38-40,165-166,266-267,274 Chemotaxis, 41-46,48,50,52,68-69 Chloramine, 252 Chromosomes, 20,21,204 Cisplatinurn, 182 C-jun, 14,143,258 C-myc, 5,143,258,264 Cofilin, 63 Collagen, 72,137 Colon cancer and aberrant crypt foci, 300 animal models, 269,270 gene knockout, 271 biomarkers, 281,289,292,300 cell motility, 76, 82-83 chemoprevention trials, 292-293,299, 313-314 combination chemoprevention, 276 and COX enzymes, 245 and DFMO, 284 differentiation markers, 300 and EGF, 224 gene knockout model, 271 genetics, 208,210,313-314 hereditary nonpolyposis, 281 latency, 212,213 and NAC, 261 and nitric oxide, 250,251,253 and NSAIDs, 204,243 promising preventive agents, 288,301 and ras, 227,230 and retinoids, 233 risk factors, 288,299-300 screening, 312 and selenium, 262 and tea, 264,265 and Vitamin E, 262,263,301 Colony scattering assay, 48 Comparative gene hybridization, 282 Cortactin, 70 COX inhibitors, see Cyclooxygenaseenzymes

338 Curcumin, 2;7,301 C-X-C family, 75-76 Cyclin A, 7-8, 10,12, 17 Cyclinlcdk network, 9, 10,13-14;17-19, 177 Cyclin D, 177 Cyclin-dependent kinases, See Cyclinlcdk network Cyclin E, 7-8, 10, 12, 17, 177-178 Cycloheximide, 182 Cyclooxygenaseenzymes as chemopreventive agent, 246-247 COX-1 and COX-2,244-245 COX-2 inhibition and bladder cancer, 293,305 and colon cancer, 292-293,301-302 and esophagus cancer, 294,307 and multiple myeloma, 294 and prostate cancer, 287 and skin cancer, 294,309 and growth factors, 245 and immune response, 243 and nitric oxide, 252 Cytokines and cell motility, 41-42 and Kaposi's sarcoma, 166, 181 and MMPs, 119 and plasminogen, 108-109 Cytoskeleton, 58-59, 64, 87

.

D Desmosomes, 54 Dexamethasone, 171-173,176, 188,244 DFMO a d biomarkers, 280 and bladder cancer, 293, 305 and breast cancer, 292,296,297 and cervical cancer, 294,308 and colon cancer, 284,301 and esophagus cancer, 294,307 and head and neck cancers, 293 with NSAIDs, 305 and piroxicam, 276 and polyamines, 204-205 and prostate cancer, 287,292 and skin cancer, 309 toxicity, 279 DHEA, 294,311 Diacylglycerol, 140 Diaphragm, 38

Index Dictyostelium, 43, 68 Differentiation, 205,300, 302-303,305, 306 DuP 697,245-246

E Education, 319 ES protein, 3-4 EGCG, see Epigallocatechin gallate EGF, see Epidermal growth factor Embryogenesis, 89 Endocrine signaling, 44 Entactin, 56 Epidermal growth factor and bladder cancer, 224,306 and breast cancer, 42,224,299 and calcium, (i5,74 and carcinogenesis, 205-206,223-224 and cell changes, 49,50,51,52-53 motility, 43,44-46,47 chemokine effect, 75-76 and chemoprevention, 222-225,227 and ES protein, 3-4 and focal adhesion, 41 and matrix-derived signals, 71, 72 and metalloproteinase, 78 and PU-kinase, 67 and PLCy, 61,SO-81,87-89 in prostate carcinoma, 75 and Ras protein, 227 and stromal-epithelial interaction, 54-55 as therapeutic target, 87 and tumor invasiveness, 33,36,60, 7881 Epigallocatechin gallate, 264 Epithelial cells, SO, 52,54-55, 73 erk MAP kinases and invasiveness, 65, 70-71, 75 and Kaposi's sarcoma, 174-176,184 and plasminogen, 138 and Ras proteins, 226 E7 protein and apoptosis, 16-17,20,21 and carbohydrate metabolism, 15-16, 18 cell cycle targets, 9-11 and cell proliferation, 17-19 C-terminus, 8 and cyclin-dependent kinases, 13-14 E6 coexpression, 19 and immortalization, 19-22

Index and metastases, 6 oncogenic domains, 6-7 and p53,13,14,17 with ras, 5 and smooth musck, 15 subcellular localization, 7 target proteins, 10 and telomeres, 20 viral gene expression, 14-15 and zinc, 8-9 E6 protein, 4-5, 19,20 E6TP1,5 Esophagus cancer animal models, 269,283 and aspirin, 243 biomarkers, 291,294 chemoprevention trials, 291,294 and EGF, 224 latency, 2 13 and ~ 5 3 , 3 0 7 promising preventive agents, 290,307 risk factors, 290,306-307 and tea, 264 and Vitamin E, 262,263 Estrogen; 76,235-236,240,249, see also Antiestrogens Etoposide, 182 Extracellular matrices, see also Integrins; Matrix metalloproteinases; Plasminogen activators and adhesion, 53 and cell motility, 55-57,71-73, 77-82 and intracellular signaling, 137-142 and invasiveness, 38-39 proteinase degradation, 122-125, 143146 as therapeutic target, 85-88,146

F Fadrozole, 241 Familial adenomatous polyposis, 243,301, 311-312 Farnesyl protein transferase, 227-230 Fas system, 184-1 86 Fenretinide and bladder cancer, 293-294,305 and breast cancer, 297 and cervical cancer, 294,308 and head and neck cancer, 293 and lung cancer, 293

339 mechanism of action, 232 and ovarian cancer, 297 and prostate cancer, 292 and skin cancer, 294,309 FGF, see Fibroblastic growth factor Fibrin, 134 Fibroblastic growth factor, 61-62, 108-109, 166-169 Fibroblasts, 50-51, 52,54, 56,59 Fibronectin, 106,110, 115 Field cancerization, 206, 302, 304 Finasteride, 212,287,292 Flutamide, 292 Focal adhesion kinase, 139 Folic acid, 293, 308 Free radicals, 258

G GAG, see Glycosaminoglycans Gall bladder cancer, 264 GAP protein, 5 Gardner’s syndrome, 243,301 Gastric cancer animal models, 269 invasiveness, 78-79 and selenium, 262,307 and tea, 264 Gastrointestinal cancers, 243,262 Gelsolin, 63,64, 70 Gene knockout mice, 270-272 Genes cell cycle regulatory, 12-13 and colon cancer, 313-314 comparative hybridization, 282 and E7,14-15,21 and head and neck cancer, 314 and invasiveness, 33-34,36-37 and LOX,255 polymorphisms, 208,209,315 Glioblastoma and growth factors, 60,78-79, 81 as invasiveness model, 35,85 and ras, 227 Glioma, 38, 78-79 Glucocorticoids, 119,164,171-174,252; see also Dexamethasone Glucose transporters, 140 Glutathione,,261 Glutathione S-transferase, 208-209,261, 277

340 Glycosaminoglycans,104 Glycosylphosphatidylinositol-anchoredproteins, 104 GM-CSF, 166 gp-130,172-174,188 GPI-anchored proteins, 104 GTPases, 69-70, 87

H Haptokinesis and CD9,82 versus chemokinesis, 41-42 and focal adhesion, 52,53-54 .and PU-kinase, 68 and PLCy, 61 Haptotaxis, 41-42,47,48,51 hDLG, 5 Head and neck cancers animal models, 269 biomarkers, 288,289,293 chemoprevention trials, 289,293, 304 and EGF, 224 genetics, 3 14 and HETE, 256 as invasiveness model, 37, 76, 83 latency, 213 and LOX, 256-257,259,260 , molecular progression model, 282 and nitric oxide, 250 promising preventive agents, 287,288289,293,304 risk factors, 288 second primaries, 206-207,232 and steroids, 209 and tea, 264 and Vitamin E, 262 Hematological malignancy, 210,211,227, 256 Heparin, 46, 110 Hepatocyte growth factor and angiogenesis, 40 and cell motility, 44,45,49, 50,55 and PI3-kinase, 61,67-68 and plasminogen activators, 106-107 Hepatoma, 70; see also Liver cancer HETEs, see Hydroxyeicosatetraenoic acids HGF, see Hepatocyte growth factor High risk tissue, 206

Index Hormone modulators, 219-221,234-236 antiestrogens, 236-239,276,287 aromatase inhibitors, 239-242,287,297 Hormone replacement therapy, 238 HPV, see Papillomavirus Hyaluronan, 57 Hydroperoxidase, 242 Hydroxyeicosatetraenoicacids, 242,254, 259 Hyman nephritis autoantigen, 110

I Ibuprofen, 243 Immortalization, 19-22 Indomethacin, 243 Inflammation, see also Antiinflammatory , agents and carcinogenicity, 39,205-206 and cell motility, 33, 88-89 and COX, 245 and LOX, 259 and nitric oxide, 252 Insulin-like growth factor, 60, 87-88,252 Insurance, 319,320 Integrins and bladder cancer, 306 and cell motility, 42,49, 52,58 and growth factors, 72,73,81-82 and mammary carcinoma, 82 and MAP kinase, 71 and MMF's, 135-137 and PI3-kinase, 68 and plasminogen, 132,133-134 as therapeutic target, 86 Interferon regulatory factor, 5 Interleukin-lp, 118, 164, 166, 174 Interleukin4,252,255 Interleukin-6, 166, 168, 170-172, 181, 311 Interleukin-8, 166 InterIeukin-l0,252 Interleukin-l3,255 Invasiveness, see also Cell motility animal models, 37-38,40 p-carotene effect, 308 genetic factors, 33-34,36 versus growth, 76-77 and growth factors, 35,40,45,67 host contribution, 37,39,40,51 versus metastases, 35-36

Index and MMPs, 38,40,56,77-78 and motility, 77-82,88-89 steps, 35, 38 therapy, 84-89,8$-88,88-89 in vitro models, 38-40 IP-10, 76

K Kaposi's sarcoma and Apo-2L/TRAIL, 186-187, 190 and Bcl-2,183,185,187-188 cell surface markers, 162 and Fas system, 184-186 and FGF, 176-179 and glucocorticoids, 164, 171-274 histology, 161-162 and immunosuppression, 160, 163 and interleukin-6, 170-171 metastases, 165 nude mice model, 166 and oncostatin M, 168-169 pathogenesis, 167 ploidy, 162-163,166 resistance, 181-182 symptoms, 163 TNFa, 164,174-176,183-184 types, 160 and virus, 161,173-174,179-181,187188 in vitro, 165-166 Keratinocytes, 40 Ketoprofen, 243 Kidney cancer, 211,264 Kringle domains, 106

34 1 Liver cancer animal models, 269 gene knockout, 271 biomarkers, 291,294 chemoprevention trials, 291,294 latency, 213 promising preventive agents, 290,310,311 risk factors, 290, 310 second primaries, 310 and tea, 264 and Vitamin E, 262 LOX inhibitors, see Lipoxygenase inhibitors Lung cancer biomarkers, 289, 293 chemoprevention trials, 289,293 and COX inhibitors, 247 and EGF, 224 gene knockout model, 271 latency, 213 local delivery, 276,303 and LOX metabolites, 258,260 and NAC, 261 and nitric oxide, 250 and ~ 5 3 , 3 0 3 promising preventive agents, 288,303 and Ras inhibition, 227,229 and retinoids, 232,233 risk factors, 288, 302-303 and second primaries, 303 and selenium, 262 and tea, 264 and vitamins, 263, 302-303 Lycopene, 295 Lymphangiogenesis, 34,35 Lymph vessels, 34,35

L

M

Lamellipodia, 49-51, 64 Laminin, 72,81-82, 110 Letrozole, 241 Leukemia, 227,256 Leukemia-inhibitory factor, 166, 168-169 Leukoplakia, 304 Leukorrienes, 254, 256, 259 Leuprolide, 292 Lewis blood group antigens, 300,306 Lifestyle, 212, 319 d-limonene, 297 Lipoxygenase inhibitors, 254-260,287, 303

Macrophages, 134, 143 Malondialdehyde, 242 Mammary carcinoma, see also Breast cancer and adhesion, 52 animal models, 269 gene knockout, 271 and EGF, 43,45 and integrins, 82 and message transcription, 75 and NSAIDs, 243 and retinoids, 233-234,276 and selenium, 262 and Vitamin E, 262

342 MAP kinases, 65,66,69, 70-71 Maspin, 83 Matrix, see Extracellular matrices Matrix metalloproteinases and angiogenesis, 40,136 and collagen, 137 inhibitors, 120-122 and integrins, 135-137 and intracellular signaling, 141-142 and invasiveness, 38,40,56, 77-78 and PKC, 119 and plasminogen activators, 122 regulation, 118-120 structure, 114-116 substrate specificity, 111-1 14, 116-118, 124 and TGF, 119 as therapy, 145-146 Melanoma and germline mutation, 21 1 and LOX metabolites, 256,259 and MMPs, 136 and plasminogen activators, 141 and retinoic acid, 309 Meloxicam, 245 Membrane ruffling, 49-50,68,69 Mesenchymal transition, 35, 45 Metalloproteinases,see Matrix metalloproteinases; Tissue inhibitors of metalloproteinases Metastases induction, 6 versus invasion, 35-36 in Kaposi's sarcoma, 165 and LOX metabolites, 259 and nitric oxide, 251 Mice, gene-knockout, 270-272 Microinvasiveness,34 Microsomal mixed-function oxidases, 260261 Misoprostol, 277 Mitogen-activatedprotein kinases, see Erk MAP kinases; MAP kinases Mitogenesis, 59 : MMP, see Matrix metalloproteinases Motility, see Cell motility MRP/CD9, 82 M2-PK, 10,lS-16,21 Multiple myeloma, 294, 311 Muscle, smooth, 15 Mutagens, 242,250; see also Antimutagens

Index Mutations, 209,215,227; see also Antiinflammatory agents Myeloid leukemia, 227 Myosins, 53,65,70 ,

N Nabumetone, 245 NAC, see N-Acetyl-L-cysteine N-Acetyl-L-cysteine, 261, 277, 303 NAT enzymes, 208 NDGA, 242,258 Neoangiogenesis, 34,35,86 Neu, 297' Neurological cancers, 210,211 Neutrophils, 54,134 Nimesulide, 245, 305 Nitric oxide synthase inhibitors, 247254 Nitrogen species, reactive, 248 NOS inhibitors, see Nitric oxide synthase inhibitors NS-398,245-246 NSAIDs chemopreventive activity, 204,215,242244,305 with DFMO, 305 toxicity, 243-244,277

0 6-actin, 15 Oltipraz, 277,284,294,303,310 Oncostatin M, 168-170 Organogenesis, 33 Orthografts, 37-38 Osteoblasts, 56 Ovarian cancer germline mutations, 210,211 and ras, 227 and retinoids, 232,233 Oxidative damage, 258-259

P Paclitaxel, 182

PAI-1,40 Pancreatic cancer animal models, 269 germline mutations, 21 1 and ras, 227,230

343

Index and selenium, 262 and tea, 264 Papillomavirus bovine, 3 human, 2-3,5,6 Parathyroid cancer, 21 1 Paxillin, 5 PDGF, see Platelet-derived growth factor PEITC, 303 Perillyl alcohol, 287,297 P53 and breast cancer, 299 andE6,4 and E7,13,14,17 and esophagus cancer, 307 and lung cancer, 303 Phase I1 enzymes, 260-261,265 Phosphatidylinositoltransfer protein, 62 Phospholipase C-y, 60,61-62, 80-81,8789 Phospholipids, 68 Phosphorylation, 3, 178-179,239 PI3-kinase, 61, 67-69 PI4-kinase, 62 PIP,, 63-64,68 Piroxicam, 243,276,305 PITP, see Phosphatidylinositoltransfer protein PKC, see Protein kinase C 9, 10, 13, 17 Plasminogen activators and fibronectin, 106 and gene expression, 142-143 inhibitors, 110-111, 122-123,133 and intracellular signaling, 137-141 and MMPs, 122 regulation, 107-1 08 tissue type, 105-107,109-110 urokinase type, 105-107,108-109,125131,132-135,138-143 Platelet-derived growth factor and chemoprevention, 227 and ES protein, 3 and integrin, 72 and invasiveness, 39,40,60 and Kaposi's sarcoma, 166 and nitric oxide, 252 and PI3-kinase, 61,67-68 and PLC-y, 87-88 PLC-y, see Phospholipase C-y Ploidy, 162-163, 166,299

Polyamine synthesis, 204 Polyphenols, 263-265,277,294,309 Polyphosphoinositides, 50 Polyprenoic acid, 311 p107 protein, 7-8,10,12 p130 protein, 7, 10 Profilin, 63 Promoters, 5 Prostaglandins, 242,243,252,270 Prostate cancer and actin, 63 and aromatase, 240-241 biomarkers, 289,292,295 chemoprevention trials, 289,292,295296 and EGF, 42,45,60,75,79-81,224 and estrogen, 235-236 and finasteride, 7 gene knockout model, 270,271 germiine mutations, 2 10 HETE role, 254 latency, 213 and motility suppressors, 83 promising preventive agents, 287-296 and Ras, 227 and retinoids, 232-233 and skin cancer, 295 and vitamins, 263,287,295 Prostate intraepithelial neoplasia, 295-296 Proteases, 77-78,79-80 Proteasome activity, 10, 12 Protein kinase C, 66-67,74, 119 p16INK4a gene, 19-20 p2lWAF-', 9, 10,13-14, 17 Public health, 314-320 Puromycin, 59

0 Quality of life, 311-312, 319

R Rac, 69-70 Raloxifene, 233,237,287,297 Ras (gene),5-6,225-227,264 Ras (protein) activation, 226 and breast.cancer, 297 and cervical cancer, 227 and colon cancer, 301

344 Ras (continued) farnesylation, 227-230 inhibition, organ targets, 227 and ovarian cancer, 227 Reactive nitrogen species, 248 Regulatory issues, 319 Relafen, 245 Resonance energy transfer, 132 Retinoblastoma, 211 Retinoblastoma protein, 7-8, 9,10,12, 14, 17,19-20 Retinoic acid, 232,294,304-305,308,309 Retinoids, see also Fenretinide with antiestrogens, 276 .mechanism of action, 230-234 and skin cancer, 309 toxicity, 232,234,303 Rho, 69 Risk disk, 320 Risk factors, 288-291,316-319

S Selective estrogen receptor modulators, 236, 238,239,292; see also specific agents Selenium, 262-263,277 and colon cancer, 262 and esophagus cancer, 307 and mammary carcinoma, 262 and prostate cancer, 263,287-295,292 and skin cancer, 262 with Vitamin E, 262-263 Serine proteinases, see Plasminogen activators SERM, see Selective estrogen receptor moduI'ators Serpin, 83 SF, see Hepatocyte growth factor Signaling and8actin modifiers, 63 adhesion-derived, 73-74 autocrine, 54-55 calcium-related, 65-66 chemotactic venus chemokinetic, 44-46 death-inducing, 183-184 de novo transcription, 74-75 growth factorlgrowth factor, 59-60 haptokinesis versus chemokinesis, 41-42 intracellular, and pioteinase, 137-142 MAP kinases, 65,66,69, 70-71 matrix-derived, 56,71-73

Index PI3-kinase, 67-69 PKC, 66 and PLC-y, 60,61-63 rac and rho GTPases, 69-70 ras-mediated, 228-229 sequestrine, 46 Signal transduction mgdulators, 219-221 EGF receptor inhibitors, 222-225 Skin cancer, see also Melanoma agent efficacy evaluation, 283 animal models, 269 gene knockout, 271 biomarkers, 291,294 chemobrevention trials, 291,294,309 gene knockout model, 271 germline mutations, 211 and NSAIDs, 243 promising preventive agents, 290 and prostate cancer, 295 risk factors, 290 and selenium, 262 and Vitamin E, 262 Smokers, 263,284,295,302 Soft tissue cancers, 210,211 Soy isoflavones, 277,287,292,297 SPARC, 53 Sulforaphan, 277 Sulindac and bladder cancer, 243,305 and colon cancer, 292,300,301 and gastrointestinal cancer, 243 Syndecans, 49,52

T Tamoxifen chemoprevention trials, 212,292,297 and ER status, 238 and fenretinide, 297 mechanism of action, 236-237 with retinoids, 232-233 Tea, 263-265,277,294,309 Telomeres, 20,21 Tenascin, 53,56,72 TGF, see Transforming growth factor T helper cells, 311 Thrombin, 104,252 Thyroid cancer, 21 1 Thyroid cells, 40 TIMPs, see Tissue inhibitors of metalloproteinases

Index Tissue inhibitors of metalloproteinases, 120122,142,144 T lymphocytes, 133-134,255 TNF, See Tumor necrosis factor Topical delivery, 276,303,309 Toremifene,,292 Toxicity of NOS inhibitors, 253-254 of NSAIDs, 243-244,277 of retinoids, 232,234,303 and risk versus benefit, 316,318-319 safety studies, 272,274 Trachea cancer, 262 Transcription, 74-75 Transforminggrowth factor and antiestrogens, 238 and EGF, 224 and invasiveness, 40,45 and Kaposi's sarcoma, 166 and MMPs, 119,122 and nitric oxide, 251,252 and TIMPs, 122 and tumor progression, 76 Tumor necrosis factor, 118,164, 189 Tyrosine kinase, 55,119,203,224-225 Tyrosine phosphorylation, 3, 178-179

345 and invasiveness, 35,67 and Kaposi's sarcoma, 166 and plasminogen, 108-109 VEGF, see Vascular endothelial growth factor Viruses, 14-15 Vitamin A, see also Retinoids and cervical cancer, 308 deficiency, 233 and differentiation, 205,218 and lung cancer, 302-303 Vitamin C, 301,308 Vitamin D, 205,292,293,295 Vitamin E and colon cancer, 301 and esophagus cancer, 263,306 and head and neck cancers, 262 and lung cancer, 263 mechanism of action, 262-263 and prostate cancer, 263,287,295 with selenium, 63 and skin cancer, 262 Vitronectin, 108,125, 133, 134,142,144 Vorozole, 241

W

U

Wound healing, 35,45-46,47-48,57, 89

Urogenital carcinoma, 45,46 Urogenital epithelia, 54-55 Ursodiol, 252,301

X Xenografts, 37-38,40,60

V Vascular endothelial growth factor and angiogenesis, 40

Z Zinc, 8-9,119

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