Molecular pathology of the pituitary

Molecular Pathology of the Pituitary Frontiers of Hormone Research Vol. 32 Series Editor Ashley B. Grossman London...

Author: George Kontogeorgos

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Molecular Pathology of the Pituitary

Frontiers of Hormone Research Vol. 32

Series Editor

Ashley B. Grossman

London

Molecular Pathology of the Pituitary

Volume Editors

George Kontogeorgos Athens Kalman Kovacs Toronto

70 figures, 1 in color, and 12 tables, 2004

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Singapore · Tokyo · Sydney

George Kontogeorgos, MD, PhD Department of Pathology General Hospital of Athens G. Gennimatas Athens, Greece

Kalman Kovacs, MD, PhD, DSc Department of Laboratory Medicine and Pathobiology St. Michael’s Hospital University of Toronto Toronto, Canada

Library of Congress Cataloging-in-Publication Data Molecular pathology of the pituitary / volume editors, George Kontogeorgos, Kalman Kovacs. p. ; cm. – (Frontiers of hormone research, ISSN 0301–3073 ; v. 32) Includes bibliographical references and indexes. ISBN 3–8055–7740–0 (hard cover) 1. Pituitary gland–Cancer–Molecular aspects. 2. Pituitary gland–Diseases–Molecular aspects. I. Kontogeorgos, George. II. Kovacs, Kalman. III. Series. [DNLM: 1. Pituitary Neoplasms–pathology. 2. Pituitary Gland–pathology. 3. Pituitary Neoplasms–genetics. WK 585 M718 2004] RC280.P5M656 2004 616.99⬘447–dc22 2004042743

Bibliographic Indices. This publication is listed in bibliographic services. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2004 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 0301–3073 ISBN 3–8055–7740–0

Contents

VII Foreword Melmed, S. (Los Angeles, Calif.) IX Preface Kontogeorgos, G. (Athens); Kovacs, K. (Toronto) 1 Molecular Basis of Pituitary Development and Cytogenesis Asa, S.L.; Ezzat, S. (Toronto) 20 Molecular Pathology of the Pituitary. Development and Functional Differentiation of Pituitary Adenomas Osamura, R.Y.; Egashira, N.; Miyai, S. (Kanagawa); Yamazaki, M. (Tokyo); Takekoshi, S. (Kanagawa); Sanno, N.; Teramoto, A. (Tokyo) 34 Cell Cycle Dysregulation in Pituitary Oncogenesis Mus¸at, M. (Bucharest/London); Vax, V.V.; Borboli, N.; Gueorguiev, M.; Bonner, S.; Korbonits, M.; Grossman, A.B. (London) 63 Role of Regulatory Factors in Pituitary Tumour Formation Korbonits, M.; Morris, D.G.; Nanzer, A.; Kola, B.; Grossman, A.B. (London) 96 Growth Factors and Cytokines: Function and Molecular Regulation in Pituitary Adenomas Renner, U.; Paez-Pereda, M.; Arzt, E.; Stalla, G.K. (Munich) 110 Proliferation Markers and Cell Cycle Inhibitors in Pituitary Adenomas Saeger, W. (Hamburg)

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127 Down-Regulation of E-Cadherin and Catenins in Human Pituitary Growth Hormone-Producing Adenomas Sano, T.; Rong, Q.Z.; Kagawa, N. (Tokushima); Yamada, S. (Tokyo) 133 Morphologic Changes and Molecular Regulation of Angiogenesis in Pituitary Adenomas de la Torre, N.G.; Wass, J.A.H.; Turner, H.E. (Oxford) 146 Advances in Pituitary Pathology: Use of Novel Techniques Lloyd, R.V. (Rochester, Minn.) 175 Pituitary Tumor Transforming Gene: An Update Yu, R.; Melmed, S. (Los Angeles, Calif.) 186 Pituitary Tumour Clonality Revisited Clayton, R.N.; Farrell, W.E. (Staffordshire) 205 Molecular Cytogenetics of Pituitary Adenomas, Assessed by FISH Technique Kontogeorgos, G. (Athens) 217 Morphology, Molecular Regulation and Significance of Apoptosis in Pituitary Adenomas Kapranos, N. (Athens); Kontogeorgos, G. (Athens/Toronto); Horvath, E.; Kovacs, K. (Toronto) 235 Somatostatin Receptors in Pituitary Function, Diagnosis and Therapy Hofland, L.J.; Lamberts, S.W.J. (Rotterdam) 253 Pathology and Molecular Genetics of the Pituitary Gland in Patients with the ‘Complex of Spotty Skin Pigmentation, Myxomas, Endocrine Overactivity and Schwannomas’ (Carney Complex) Stratakis, C.A.; Matyakhina, L.; Courkoutsakis, N.; Patronas, N.; Voutetakis, A.; Stergiopoulos, S.; Bossis, I. (Bethesda, Md.); Carney, J.A. (Rochester, Minn.) 265 Recent Advances in MEN1 Gene Study for Pituitary Tumor Pathogenesis Kameya, T. (Shizuoka); Tsukada, T. (Tokyo); Yamaguchi, K. (Shizuoka) 292 Author Index 293 Subject Index

Contents

VI

Foreword

Our understanding of fundamental mechanisms leading to pituitary tumorigenesis has largely been driven by the remarkable contributions of our pathology colleagues over the past 30 years. Since the modern advent of accurate pituitary immunostaining leading to a functional cytologic classification of pituitary adenomas, there have been enormous advances in unraveling pituitary pathogenetic events at the molecular level. The editors, themselves pioneers of modern pituitary pathology, have assembled a coterie of expert authors who now provide a cutting-edge comprehensive text which encompasses all the recent advances in the field. Thanks to application of sensitive molecular and cytologic techniques, pituitary disease can now be classified according to functional defects at the subcellular level. Unraveling of these pathogenetic events is now possible by expert pituitary pathologists, allowing for accurate diagnoses, reliable prognostic indicators and ultimately provide prospective indicators for therapeutic outcomes. The rich range of topics from gene mutations, cell cycle defects, structural cell biology, protein expression, peptide diagnostic imaging, and familial and acquired syndromes, underlie the fundamental contributions of pituitary pathology to etiology, pathogenesis and diagnosis of pituitary disorders. The elegant work of the applied modern pathologist is appropriately reflected in this volume as the intellectual and functional underpinning of pituitary medicine. Shlomo Melmed Los Angeles, Calif.

VII

Preface

In his beautifully illustrated, outstanding book entitled ‘De Humani corporis fabrica’, Andreas Vesalius (1514–1564) expressed the view that ‘the pituita or phlegm of the brain distills into the underlying pituitary gland and escapes into the nasal passages’. This mistake was not challenged for many years. Now it is common knowledge that the pituitary secretes several hormones into the blood which affect practically every function of the body. The pituitary is the conductor of the endocrine orchestra and its importance cannot be overemphasized. In 1886, Pierre Marie, the French neurologist, described the clinical features of growth hormone excess and introduced the term ‘acromegaly’. Pierre Marie’s contribution created great interest, and 1886 is regarded as the birth date of pituitary endocrinology. The pituitary shifted to the center of endocrine research and progress accelerated. Every year brought provocative discoveries. Despite unprecedented advances, many questions remained unanswered. Thanks to the development of new technology, a dramatic revolution occurred and molecular endocrine pathology emerged. Several new methods were introduced and many sensational novel results were published. The literature explosion resulted in not only a deeper insight into the mechanisms and causation of pituitary diseases, but also created controversies. Despite significant breakthroughs, we are still in the stage of transition. Due to the myriad of new data, it is difficult to be sure which information is valid and which is important. The aim of this book is to summarize current knowledge and stimulate further research on molecular pituitary pathology. The best internationally wellknown and respected experts were invited to participate. Their contributions, in

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a critical way, unravel several mysteries of molecular pituitary pathology and will lead to a better understanding of pituitary structure and function in health and disease. We wish to express our sincere thanks and appreciation to Dr. Ashley Grossman for inviting us to edit this book. We are very much indebted to the contributors, who should receive the credit if this book succeeds. We are grateful to Dr. Shlomo Melmed for writing the Foreword and to the Publishers; they were very helpful, supportive and did an excellent job. For us, it was a creative work, a stimulating and exciting exercise. George Kontogeorgos, Athens Kalman Kovacs, Toronto

Preface

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Kontogeorgos G, Kovacs K (eds): Molecular Pathology of the Pituitary. Front Horm Res. Basel, Karger, 2004, vol 32, pp 1–19

Molecular Basis of Pituitary Development and Cytogenesis Sylvia L. Asaa,c,e, Shereen Ezzatb,d,e a

Laboratory Medicine and Pathobiology and bMedicine, University of Toronto; cDepartment of Pathology, University Health Network/Toronto Medical Laboratories; dDepartment of Medicine, Mount Sinai Hospital and University Health Network; eFreeman Centre for Endocrine Oncology, Mount Sinai and Princess Margaret Hospitals, Toronto, Ont., Canada

Abstract The factors that play a role in pituitary development have been identified as transcription factors and growth factors that regulate cell migration, proliferation and differentiation. These factors are expressed in a tightly regulated fashion to ensure the dorso-ventral migration of Rathke’s pouch, the proliferation of cells in a correct temporal and spatial fashion, and the cytodifferentiation of the hormone-producing cell types of the mature gland. Dysregulation of these processes results in congenital abnormalities and hormone insufficiency syndromes. Understanding these normal processes of differentiation and proliferation sheds new light on the factors implicated in disordered structure and function of the mature gland during pituitary tumorigenesis. Copyright © 2004 S. Karger AG, Basel

The anterior pituitary is a complex gland composed of several cell types that are responsible for the production of many hormones that are critical for growth, development, homeostasis and reproduction. These actions are carried out by six hormone-producing cells types that are admixed in varying geographic distributions within acini of the adenohypophysis. Corticotrophs elaborate proopiomelanocortin (POMC) and its cleavage products including adrenocorticotropin (ACTH), somatotrophs produce growth hormone (GH), lactotrophs produce prolactin (PRL), mammosomatotrophs are bihormonal cells that contain GH and PRL, thyrotrophs synthesize thyrotropin (TSH), and the gonadotrophs produce follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The functions of the gland are regulated by

hypothalamic hormones and by positive and negative feedback by target organ hormones [1]. The adenohypophysis derives from Rathke’s pouch, an invagination of the oral ectoderm [2]. The dorsal portion of Rathke’s pouch comes into direct contact with the midline ventral diencephalon, which evaginates and forms the neurohypophysis. The dorsal or posterior limb of Rathke’s cleft gives rise to the intermediate lobe of the pituitary. The anterior limb proliferates to form the anterior lobe, and a small portion of the most superior portion of this component wraps itself around the hypophysial stalk to form the pars tuberalis. These three components comprise the adenohypophysis. Our understanding of development has been expanded by the identification of the molecular mechanisms that specify cell determination and localization [3]. These include extrinsic and intrinsic signals, provided by growth and differentiation factors. The process of adenohypophysial development and cell differentiation follows a highly specific pattern and temporal sequence which differs in various species [2, 4–7]. Insights into the molecular basis of cell differentiation and phenotype expression have been advanced by the recognition of a number of transcription factors that are necessary for tissue-specific gene expression. Several transcription-regulating proteins have been identified in the adenohypophysis and have been implicated in the definition of cell-specific phenotypes and the regulation of hormone gene expression. Advances in our recognition of the factors that regulate cell differentiation in the adenohypophysis have led to a new classification of adenohypophysial cell types and a more sophisticated understanding of the mechanisms that determine the patterns of hormone production in pituitary adenomas [1]. The factors that govern cell differentiation in the pituitary probably also play a role in determining the hormonal activity and cytodifferentiation of pituitary adenomas.

Early Pituitary Development

Early pituitary development is regulated by extrinsic patterning signals and endogenous gene expression. The initial extrinsic signals that seem to be most important arise from the ventral diencephalon and include members of three protein families, bone morphogenic protein (BMP), fibroblast growth factor (FGF) and Wnt [7–9]. The oral ectoderm provides signaling in the form of Sonic hedgehog (Shh) [7]. BMP signaling by BMP4 is required for normal pituitary development [7]. Targeted expression of the BMP2/4 antagonist Noggin results in arrest of Rathke’s pouch after invagination, in mice at about embryonic day 10 (e10), and

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deletion of BMP4 causes embryonic death at e10 with complete failure of pouch invagination [10]. BMP2 is initially expressed in a ventral-dorsal gradient, and there is evidence that this gradient is required for positional determination of cell differentiation, but by e12.5 BMP2 is expressed throughout the gland. Inhibition of BMP signaling results in loss of differentiation of cells in the Pit-1 lineage and of gonadotrophs. Ultimately, however, attenuation of BMP signaling is required for terminal differentiation [7]. Shh is expressed throughout the oral ectoderm at e8 but expression is extinguished in the invaginating epithelium of Rathke’s pouch. This would suggest that Shh is not required for pituitary development, but in other studies, expression of an inhibitor hedgehog interacting protein (HIP) in mice or mutation of the zebrafish homolog of the Shh mediator Gli [7] result in deficient pituitary development after day e10. Overexpression of Shh causes hyperproliferation of some differentiated cells, mainly thyrotrophs and gonadotrophs [7]. FGF signaling through several FGF receptors plays a role in pituitary development after initial invagination of Rathke’s pouch. Dissection of the roles played by this complex group of more than 20 ligands and numerous isoforms of the 4 members of the receptor family is complicated; despite a wealth of experimental data implicating FGF signaling in various developmental processes, genetic inactivation of individual genes encoding specific FGFs or FGFRs has generally failed to demonstrate their role in vertebrate organogenesis due to early embryonic lethality or functional redundancy. Deletion of FGF10, one ligand, or the FGFR2 IIIb isoform that is an FGF10 receptor, results in failure to proliferate and apoptosis after formation of the primordial pituitary gland [11]. Mid-gestational expression of a novel secreted kinase-deficient receptor that acts as a soluble dominant negative ligand for FGF signaling causes agenesis or severe dysgenesis of kidney, lung, specific cutaneous structures, exocrine and endocrine glands, including pituitary aplasia, and craniofacial and limb abnormalities reminiscent of human skeletal disorders associated with FGFR mutations [12]. A dorsal to ventral FGF8/10 gradient seems to be required for appropriate positional differentiation of specific cell types. In contrast, overexpression of FGF8 results in overproliferation that prevents terminal differentiation [7]. Expression of the Wnt family is also required for normal pituitary development. The Wnt/Dvl/␤-catenin pathway plays a role in regulating expression of the transcription factor Ptx2 (see below) and is required for effective cell-type-specific proliferation by directly activating specific growth-regulating genes [13]. Novel transcription factors that play a role in anterior primordial development are being identified at a rapid pace. Many of these are implicated in early pituitary organogenesis.

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The bicoid-related pituitary homeobox factor (Ptx) 1, initially proposed as an activator of POMC gene expression [14], is now known to be an early determinant of brain and facial development that precedes pituitary development [15]. Ptx1 is expressed in the developing pituitary and is subsequently expressed in all adenohypophysial cell types [16]. Targeted disruption of Ptx1 results in reduction of thyrotrophs and gonadotrophs and increased POMC gene expression [17, 18] suggesting that it is not required for pituitary development but that it plays a role in ensuring production of factors that regulate expansion of specific adenohypophysial cells. The structurally related pituitary homeobox factor 2 (Ptx2) has two alternatively spliced mRNA products that encode two proteins of 271 and 317 amino acids. Ptx2 is expressed in the developing and mature pituitary as well as in eye and brain tissue [19]. It likely plays a key role in pituitary development since in Ptx2⫺/⫺ mice, the pituitary does not develop past the stage of the e10.5 primordium [20, 21]. P-LIM, a member of the LIM family of homeobox protein transcription factors, is selectively expressed in the pituitary with highest levels at the early stages of Rathke’s pouch development [22]. It appears to be expressed in all pituitary cell types, however, and therefore is not a likely candidate for regulation of terminal cytodifferentiation. Isl-1, another LIM family member, is required for proliferation after invagination of the pouch [23]. Two other members of the LIM homeobox gene family, the Lhx genes, have been implicated in early pituitary development [24]. Lhx3 and Lhx4 appear to play an important role in directing the invagination of the oral ectoderm to form Rathke’s pouch. Null mutation of either Lhx3 or Lhx4 does not prevent formation of Rathke’s pouch, but animals devoid of both genes develop only a rudimentary pouch. Subsequent expression of Lhx3 is identified throughout the gland whereas Lhx4 develops a pattern of expression restricted to the anterior lobe. Targeted disruption of Lhx3 alone prevents further differentiation of all adenohypophysial cells; lack of Lhx4 alone results in defective but not absent gonadotroph differentiation. Another early marker of pituitary differentiation is the Rathke’s pouch homeobox (Rpx) protein, also known as HesX-1, which is identified in the pituitary primordium prior to the onset of known pituitary hormone production [25]. The expression pattern appears to be more generalized than the area destined to become the adenohypophysis alone; it is likely, therefore, that Rpx/HesX-1 is involved in the initial determination of the anterior region of the embryo. This factor is subsequently extinguished in other areas and becomes restricted to Rathke’s pouch. Downregulation of Rpx/HesX-1 occurs at the time of onset of other pituitary-specific transcription factors. Mutations of Rpx/HesX-1 have been reported to result in septo-optic dysplasia, also known as de Morsier’s syndrome,

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a complex developmental disorder with variable manifestations of aplasia of the septum pellucidum, hypoplasia of the optic nerves, and endocrine dysfunction due to hypopituitarism [26–30]. The Prophet of Pit-1 (PROP-1) is a paired-like homeodomain protein that is expressed early in pituitary development. It induces Pit-1 expression and plays a role in the downregulation of Rpx [31, 32]. Inactivating mutations of PROP-1 have been identified as the cause of Pit-1 deficiency in Ames dwarf mice [31] and in humans with combined pituitary hormone deficiency [33, 34]. Prop1 mediates the dorsally-restricted expression of a groucho-related gene, transducin-like enhancer of split 3 (TLE3), which encodes a transcriptional co-repressor. Deficiency of a related gene, amino terminal enhancer of split (AES), causes pituitary anomalies and growth insufficiency. TLE3 and AES have been shown to interact with TCF family members in cell culture systems. In the absence of TCF4, Prop1 levels are elevated, pituitary hyperplasia ensues and palate closure is abnormal. Thus, TCF4 and AES influence pituitary growth and development as targets of PROP-1 [35]. Pax 6 is expressed in the developing pituitary with a dorsal-ventral gradient of expression levels. Pax 6 mutant mice exhibit abnormal relationships and relative numbers of the differentiated pituitary cell types [36–38]. Deletion of Six-6 has been reported to be associated with human pituitary anomalies [39]. Id, a member of the helix-loop-helix (HLH) family of transcription factors is also found early in development and in some pituitary tumor cell lines but is decreased or absent in differentiated cells [40]. Its role in pituitary cytodifferentiation remains unclear.

The Corticotroph Cell Lineage

Corticotrophs are the first cells to differentiate in the human fetal pituitary [4, 5]. Although expression of the POMC gene is one of the most promiscuous events in endocrine tumors outside the pituitary, adenohypophysial corticotroph lineage is one of the most stable; expression of POMC is rarely associated with expression of other adenohypophysial hormones. A novel T box factor called Tpit was identified selectively in pituitary POMC-expressing lineages and apparently in no other tissue, including hypothalamic POMC neurons. Tpit was shown to activate POMC gene transcription, in cooperation with Pitx1. In gain-of-function experiments, Tpit induced POMC expression in undifferentiated pituitary cells, and in contrast, TPIT gene mutations were found in patients with isolated deficiency of pituitary POMC-derived ACTH [41]. These data indicate an essential role for Tpit in differentiation of the pituitary POMC lineage.

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A protein that binds the POMC promoter was identified in nuclear extracts of the murine pituitary corticotroph cell line AtT-20 and was named corticotroph upstream transcription element-binding (CUTE) protein. CUTE was identified in cells expressing POMC but not in other pituitary-derived cell lines, and was implicated as an important determinant of cell-specific expression of the POMC gene in the pituitary and other sites [42]. Subsequent studies have shown that CUTE complexes contain the HLH transcription factor NeuroD1/␤2 and this is thought to be important for POMC expression [43].

The Pit-1 Family: Somatotrophs, Mammosomatotrophs, Lactotrophs and Thyrotrophs

Pit-1, also known as GHF-1, is a 33-kD 291-amino acid protein which belongs to the homeobox family of developmental regulatory proteins [44, 45]. The presence of an additional domain, conserved in Pit-1 and the proteins OCT-1, OCT-2 and UNC-86, gave rise to the term ‘POU-domain’ that characterizes this family of homeodomain proteins [46, 47]. This protein, as its name suggests, exhibits pituitary-restricted expression. It activates the structurally related GH and PRL genes in rat and human [47]. Pit-1 expression in the developing rodent pituitary is associated with the onset of GH and PRL production [48, 49] and correlates temporally and spatially with expression of ␤-TSH in the fetal rodent adenohypophysis [49, 50]. This latter finding led to the identification of Pit-1 binding sites in the ␤-TSH promoter [51], however, other factors in addition to Pit-1 are required for [52] ␤-TSH gene expression [53] and thyrotroph differentiation [54]. Isoforms of Pit-1 that result from alternative mRNA splicing, Pit-1␤ [55–57] and Pit-1T [58, 59], have different selective effects on target gene transcription and can act as repressors of gene expression [60]. Early studies using in situ hybridization and immunocytochemistry identified Pit-1 mRNA transcripts in all 5 phenotypically distinct pituitary cell types and Pit-1 protein only in the nuclei of somatotrophs, lactotrophs and thyrotrophs [49], suggesting that translational controls dictate the pattern of rodent Pit-1 expression. However, analysis of human pituitary adenomas that represent relatively homogeneous cell populations have shown that transcriptional control dictates selective expression of the pit-1 gene in human adenohypophysial cells responsible for GH, PRL and ␤-TSH synthesis [61–63]. In the human fetal pituitary, Pit-1 mRNA and protein are identified as early as 6 weeks of gestation; GH immunoreactivity is detectable by 8 weeks [64]. Pit-1 is found only in cells containing GH, PRL and/or TSH throughout human gestation. The temporal association between Pit-1 and GH expression in human

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gestation are consistent with the data obtained in rodents [48, 49] where Pit-1 appears by day 15–16, immediately preceding the onset of PRL and GH mRNA. In contrast, the sequence of cytodifferentiation is different in the human; PRL, ␤-TSH and the ␤-subunits of the gonadotropins only appear 4 weeks later at 12 weeks of gestation [2, 4–6]. These findings support the hypothesis that Pit-1 is insufficient for the cytodifferentiation of lactotrophs and thyrotrophs that occurs much later than the onset of Pit-1 expression. The prolonged time span of human adenohypophysial cytodifferentiation allows careful and accurate dissection of the factors that must be required to act in concert with Pit-1 to promote the subsequent expression of PRL and ␤-TSH. In rodents and humans, differentiation and/or maintenance of somatotroph, lactotroph and thyrotroph phenotypes are dependent on expression of a functional pit-1 gene; mutations in the pit-1 gene result in hypopituitarism [65–68] and hypoplasia of somatotrophs, lactotrophs and thyrotrophs [65]. An interesting observation is that Pit-1 mRNA and protein are highly expressed during human pituitary development at 17–19 weeks [64] when GH levels are extremely high [5] and near term [64] when there is proliferation of lactotrophs [4]. These data suggest that Pit-1 plays an important role not only in the differentiation process, but also in the regulation of hormonal activity and possibly also of cell proliferation. Zn-15 is a zinc finger transcription factor with an unusual DNA-binding domain that binds the proximal GH promoter. In transient transfection studies, it stimulates GH expression and shows synergistic effects with Pit-1 [69]. Little is known of its potential role in pituitary cytogenesis. The factors responsible for PRL gene expression in Pit-1-positive cells are not entirely clear. A number of studies have established that estrogen acting directly through the estrogen receptor regulates PRL gene transcription, synthesis and secretion. Molecular analyses have confirmed that the PRL promoter contains a nonpalindromic estrogen response element (ERE) which functions as weak transcription activator and is enhanced by cooperation with Pit-1 to activate PRL gene transcription [70]. Studies of estradiol binding implied ER expression in 85% of cells in the anterior lobe but not in intermediate or posterior lobe cells [71]; uptake of radiolabeled estrogen was reportedly found in cells containing immunoreactivity for PRL, ␤-FSH, and ␤-LH, as well as in cells containing ␤-TSH, and GH [72]. Immunohistochemical studies initially failed to reliably localize ER␣ in the human pituitary and its adenomas due to the limited sensitivity of the detection method employed [73]. Using antigen retrieval methods, however, ER␣ can now be localized by immunocytochemistry in the nontumorous adenohypophysis [74, 75] in cells containing PRL or gonadotropin ␤-subunits. GH-immunoreactive cells containing nuclear positivity for ER␣ may be mammosomatotrophs that

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are known to exist in the human pituitary [76]. ACTH-containing cells are reported to be negative for ER␣ [74, 75]. Biochemical analyses reliably identify ER in PRL-producing adenomas [74, 75, 77, 78]. However, this detection method requires large amounts of protein and has relatively low sensitivity; comparative studies have shown no correlation between detection of ER␣ mRNA and the presence or amount of protein detected by radioactive ligand binding [74]. The closest correlations between hormone production and ER␣ expression have been documented using either RNase protection assay [75] or RT-PCR [74] which are the most sensitive and specific methods to identify even low levels of expression. These studies have found a correlation between ER␣ expression and the production of PRL or gonadotropins [74, 75]. Splice variants of ER␣ mRNA are also selectively expressed by those types of pituitary adenomas [79]. Corticotroph adenomas do not express ER␣. Somatotroph adenomas that do not produce PRL as well as GH are devoid of ER␣; the lack of ER␣ expression in these cells suggests that the GH-releasing activity of estrogen [80] either is mediated by other pathways or involves a selective effect on mammosomatotrophs. These data suggest that ER␣ may be the factor responsible for the development of PRL expression in somatotrophs that express Pit-1. This factor must have its onset after GH expression during gestation, since two models of disruption by targeting of diphtheria toxin (DT) [81] or by thymidine kinase obliteration (TKO) [82] in GH-expressing cells prevent further development along this pathway. Clearly there must also be a factor responsible for silencing GH expression in the progression from mammosomatotrophs to mature lactotrophs [5, 83, 84]. Regulation of ER␣ expression could also account for the fluctuations in adenohypophysial cell populations during pregnancy, when there is transition from somatotrophs to mammosomatotrophs and lactotrophs [85]. The sequence of differentiation of adenohypophysial cells in the human fetal pituitary, in contrast to the rodent gland, implicates a transcription activator that is distinct from Pit-1, may be common to lactotrophs and gonadotrophs, and has its onset at or just before 12 weeks of gestation in the human fetal pituitary [2, 4–6]. ER␣ may be the common factor implicated in the regulation of hormone production and cytodifferentiation of mammosomatotrophs/ lactotrophs and gonadotrophs in a cell type-specific fashion. Preliminary data suggest that ER␣ expression is initiated in the fetal pituitary around 12 weeks of gestation [86]; if so, it would explain the development of PRL secretion and the differentiation of gonadotrophs at that gestational age. Mice with disrupted ER␣ have decreased circulating PRL levels but PRL is not undetectable [87, 88] and structurally, there is evidence of lactotroph differentiation [89]. A human with an ER␣ mutation has also been described [90]; he too demonstrated similar hormonal profiles. These data would suggest

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that ER␣ is not required for lactotroph differentiation, however, the description of the ER␤ gene and analysis of its distribution in human tissues [91] indicates the redundancy of this system. Further studies involving disruption of both ER genes are required to clearly define the role of ER in pituitary cell differentiation. Members of the Ets transcription factor superfamily play significant roles in the control of growth and development. Co-transfection of Ets-1 and Pit-1 results in synergistic activation of the PRL promoter, suggesting that Ets-1 may mediate ras activation of pituitary-specific gene expression [92, 93]. Again, however, it remains to be seen whether this factor is involved in lactotroph differentiation. Thyrotroph embryonic factor (TEF) is a trans-acting factor that belongs to the leucine zipper gene family of transcription factors. It is thought to be thyrotroph-specific and activates the expression of the human ␤-TSH gene [54]. The proximal ␤-TSH promoter contains three independent TEF binding sites and TEF is able to activate a reporter gene under the control of that promoter. As its name implies, TEF is expressed in a pattern that correlates temporally and spatially with ␤-TSH gene expression in the rodent fetal pituitary [54]; subsequently it is expressed in several other tissues. It remains to be proven, however, that it is expressed in a cell-specific pattern in the adenohypophysis. These data suggest an intriguing possibility that TEF is the factor required for the onset of TSH production in cells that produce Pit-1. The relationship between thyrotrophs and somatotrophs has been recognized previously in rats with prolonged hypothyroidism; the development of thyrotroph hyperplasia is associated with trans-differentiation of somatotrophs into thyroidectomy cells [94]. It is therefore likely that there is a continuum and that the maturation from somatotrophs to differentiated thyrotrophs requires both the onset of TEF expression and the production of a GH repressor that silences GH gene expression. GATA-2, initially identified as a transcription factor required for hematopoietic development, plays a role in thyrotroph and gonadotroph differentiation [95]. There is extensive literature concerning the interactions of Gata-2 and Pit-1 in thyrotrophs that attempt to explain the activation of gonadotrophspecific genes by Gata-2 in gonadotrophs but not thyrotrophs [3] but other mechanisms, such as SF-1 (discussed below) likely also play a role in regulating gene expression in gonadotrophs.

The Gonadotroph Lineage

The nuclear receptor steroidogenic factor-1 (SF-1) is a member of the steroid receptor superfamily [96–98]. This transcription factor regulates expression of

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the steroidogenic enzymes cytochrome P450 CYP11A and CYP11B. It is also known as Ad4BP since it binds to the Ad4 site in the 5⬘ region of the bovine cytochrome P450 CYP11A and CYP11B genes [97–100]. SF-1 is expressed by all zones of the adrenal cortex, granulosa and theca cells of the ovary and Leydig cells of the testis [99, 101]. In situ hybridization has demonstrated that expression of SF-1 in the adrenal anlage immediately precedes expression of the cytochrome P450 enzymes in that tissue [99]. Expression of SF-1 is sexually dimorphic in the developing gonad where it may play a role distinct from the regulation of the steroidogenic enzymes [102]. SF-1 also regulates the müllerian inhibiting substance (MIS) gene to determine müllerian duct regression in the developing embryo [103]. Targeted disruption of this gene shows that it is essential for adrenal and gonadal development and sexual differentiation [104]. The factors determining gonadotroph differentiation were unknown until it was found that SF-1 is necessary for their development [105] as well as for the formation of the ventromedial nucleus of the hypothalamus [106]. SF-1 is expressed in the embryonic mouse forebrain and in the developing mouse pituitary prior to the onset of expression of the gonadotropin ␤-subunits [102, 105]. SF-1 mRNA transcripts are detected in normal mouse gonadotrophs and in an immortalized murine pituitary gonadotroph-derived cell line (␣T3–1), and the protein interacts with a regulatory element in the murine gonadotropin ␣-subunit gene to enhance transcription [105, 107]. Studies of human pituitaries indicate that SF-1 is also expressed in a cellspecific pattern in human adenohypophysial cells, where it correlates with gonadotropin production [108, 109]. In the nontumorous gland, SF-1 is localized in the nuclei of gonadotropin-containing cells but not in other cell types [108]. In the relatively homogeneous populations of tumors, SF-1 expression is characteristic of gonadotropin-producing adenomas, including the classical gonadotroph adenomas and also null cell adenomas and oncocytomas that are known to produce gonadotropins [110–113] and are thought to be related lesions [114]. Interestingly, in the human adenohypophysis, SF-1 expression correlates with gonadotropin ␤-subunits rather than ␣-subunit, since many GH-producing nontumorous cells and adenomas express ␣-subunit but not SF-1[1]. It appears that SF-1 expression is initiated in the fetal pituitary at 12 weeks of gestation [86]; if confirmed, this finding would explain the development of ␤-subunit gonadotropin secretion and the differentiation of gonadotrophs at that gestational age. Many studies also demonstrate a role for estrogen in mediating a positive or negative effect on the expression of the ␤-FSH and ␤-LH hormone genes and on levels of secretion of these gonadotropins [115–117]. There is direct evidence that the classical estrogen receptor (ER␣) binds to the upstream region of the rat ␤-LH gene [118]. Estradiol binding studies have shown uptake of

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radiolabeled estrogen in cells containing immunoreactivity ␤-FSH, and ␤-LH [71, 72], and ER␣ is localized by immunocytochemistry in gonadotrophs [74, 75]. The temporal onset of ER␣ expression in the human fetal pituitary is around 12 weeks of gestation [86], at the time of differentiation of gonadotrophs. However, mice with disrupted ER␣ have differentiated gonadotrophs with elevated circulating gonadotropins due to lack of negative feedback from their maldeveloped gonads, indicating that ER␣ is not required for terminal differentiation of those cells [87, 88]. GATA-2 plays a role in gonadotroph differentiation [95] where it is involved in activation of gonadotroph-specific genes [3].

Regulation of Post-Differentiation Population Expansion and Hormonal Activity

Cyclic AMP response element binding-protein (CREB) binding sites are present in many gene promoters and the factors that bind these sites are implicated in the regulation of numerous hormone genes. In the anterior pituitary, the rodent Pit-1 gene promoter [119, 120] and the human ␣-subunit gene promoter [121] appear to be regulated by cAMP via CREs. Transgenic mice that overexpress a dominant negative CREB exhibit dwarfism with somatotroph hypoplasia [122]. Although the ubiquitous nature of CREB makes it an unlikely candidate to control cell-specific differentiation, it appears that in concert with other factors, this transcription element plays an important and necessary role in rodent somatotroph development. Glucocorticoid receptors, thyroid hormone receptors, and retinoic acid receptors play essential roles in transcriptional regulation of pituitary hormones but these are not expressed in cell-specific fashions and, therefore, are not considered to control terminal cell differentiation. Mutations of factors that regulate target organ differentiation and hormone activity, like Dax-1 and TTF-1, result in abnormal pituitary feedback regulation but are not documented to alter pituitary development directly. A number of factors regulate post-differentiation cell proliferation that is required for normal pituitary development. Hypothalamic hormones and target organ hormones are implicated in proliferation of adenohypophysial cells [123] and the pituitary abnormalities as well as target organ hypoplasia seen in anencephalic fetuses prove the importance of these elements [124]. Growth factors are almost certainly required for expansion of pituitary cell populations in the developing gland, however, in this field, the redundancy of ligands and receptors in each growth factor family complicates our ability to dissect the role of each of these proteins. Signaling through these families of receptors is likely the target

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Corticotroph Rpx/HesX-1 Pax-6 Six-1,3 Isl-1 Ptx 1,2

Somatotroph

Tpit NeuroD1/beta2

?Pit-1␤ ER␣

Oral ectoderm

Rathke’s pouch stem cell

Lhx-3 Lhx-4 Prop-1 GATA-2

Pit-1

Somatotroph stem cell

Lhx-4 GATA-2 SF-1 ER␣

GH-Repressor

ER␣ Mammosomatotroph

TEF GATA-2 GH-Repressor Pit-1␤

Lactotroph

Thyrotroph

Gonadotroph

Fig. 1. Pathways of adenohypophysial cytodifferentiation. Transcription factors implicated in the development of Rathke’s pouch and subsequently of the individual pituitary cell types are shown. The proposed schema involves early determination of pituitary development from the oral ectoderm by a number of factors, including Rpx/Hesx-1, Pax-6, Six-1,3, Isl-1, Ptx-1 and -2, Lhx-3 and -4, Prop-1 and Gata-2. Molecular determinants of corticotroph lineage, the first to occur, involve Tpit and NeuroD1/␤2. This is followed by Pit-1 expression that designates a somatotroph stem cell which, in the absence of other transcription factors, retains somatotroph morphology and function. Expression of estrogen receptor (ER) enhances prolactin gene expression in mammosomatotrophs; a putative GH repressor is implicated in silencing GH transcription to allow the emergence of mature lactotrophs. Other cells in the Pit-1 lineage express TEF (thyrotroph embryonic factor) and GATA-2 to develop into thyrotrophs; again a GH repressor must be implicated in silencing of GH transcription. Prolactin repression may rely on Pit-1␤. The mammosomatotroph is at the center of a 3-way fluctuation indicated by 2-directional arrows; these cells transdifferentiate physiologically. The third pathway of cytodifferentiation is dictated by SF-1 (steroidogenic factor-1) and GATA-2, which, in conjunction with ER and Lhx-4, determine gonadotroph differentiation and gonadotropin hormone gene transcription.

disrupted by mutation of phosphatases, such as the protein tyrosine phosphatase sigma (PTP␴) that results in pituitary dysgenesis [125].

A Model of Adenohypophysial Cytodifferentiation

The various transcription factors discussed above regulate cell differentiation and hormone production in the pituitary. They provide the framework for understanding cell lineages in the adenohypophysis (fig. 1). Advances in determining

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the factors that regulate hormone gene expression have provided a clearer understanding of the hormonal activity of pituitary adenomas. Old concepts of plurihormonality have taken on new significance as these factors are shown to account for the patterns of hormone expression that have long been recognized in human pituitary adenomas. One must ask, however, what determines the next level of regulation. Future studies of the molecular regulation of hormone gene expression will continue to clarify the ability of adenohypophysial cells to maintain cytologic differentiation or to undergo multidirectional differentiation, both functional and morphologic.

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96 Lala DS, Rice DA, Parker KL: Steroidogenic factor I a key regulator of steroidogenic enzyme expression is the mouse homolog of fushi tarazu-factor I. Mol Endocrinol 1992;6:1249–1258. 97 Morohashi KI, Honda SI, Inomata Y, Handa H, Omura T: A common trans-acting factor, Ad4binding protein, to the promoters of steroidogenic P-450s. J Biol Chem 1992;267:17913–17919. 98 Honda SI, Morohashi KI, Nomura M, Takeya H, Kitajima M, Omura T: Ad4BP regulating steroidogenic P-450 gene is a member of steroid hormone receptor superfamily. J Biol Chem 1993;268: 7494–7502. 99 Ikeda Y, Lala DS, Luo X, Kim E, Moisan MP, Parker KL: Characterization of the mouse FTZ-F1 gene which encodes a key regulator of steroid hydroxylase gene expression. Mol Endocrinol 1993;7: 852–860. 100 Morohashi KI, Zanger UM, Honda SI, Hara M, Waterman MR, Omura T: Activation of CYP11A and CYP11B gene promoters by the steroidogenic cell-specific transcription factor, Ad4BP. Mol Endocrinol 1993;7:1196–1204. 101 Morohashi KI, Iida H, Nomura M, Hatano O, Honda SI, Tsukiyama T, Niwa O, Hara T, Takakusu A, Shibata Y, Omura T: Functional difference between Ad4BP and ELP and their distributions in steroidogenic tissues. Mol Endocrinol 1994;8:643–653. 102 Ikeda Y, Shen WH, Ingraham HA, Parker KL: Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Mol Endocrinol 1994;8:654–662. 103 Shen WH, Moore CCD, Ikeda Y, Parker KL, Ingraham HA: Nuclear receptor steroidogenic factor 1 regulates the müllerian inhibiting substance gene: A link to the sex determination cascade. Cell 1994;77:651–661. 104 Luo X, Ikeda Y, Parker KL: A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 1994;77:481–490. 105 Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen WH, Nachtigal MW, Abbud R, Nilson JH, Parker KL: The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 1994;8:2302–2312. 106 Ikeda Y, Luo X, Abbud R, Nilson JH, Parker KL: The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleus. Mol Endocrinol 1995;9: 478–486. 107 Barnhart KM, Mellon PL: The orphan nuclear receptor, steroidogenic factor-1, regulates the glycoprotein hormone ␣-subunit gene in pituitary gonadotropes. Mol Endocrinol 1994;8:878–885. 108 Asa SL, Bamberger AM, Cao B, Wong M, Parker KL, Ezzat S: The transcription activator steroidogenic factor-1 is preferentially expressed in the human pituitary gonadotroph. J Clin Endocrinol Metab 1996;81:2165–2170. 109 Ikuyama S, Ohe K, Sakai Y, Nakagaki H, Fukushima T, Kato Y, Morohashi K, Takayanagi R, Nawata H: Follicle-stimulating hormone-␤ subunit gene is expressed in parallel with a transcription factor Ad4BP/SF-1 in human pituitary adenomas. Clin Endocrinol (Oxf) 1996;45:187–193. 110 Asa SL, Gerrie BM, Singer W, Horvath E, Kovacs K, Smyth HS: Gonadotropin secretion in vitro by human pituitary null cell adenomas and oncocytomas. J Clin Endocrinol Metab 1986;62:1011–1019. 111 Yamada S, Asa SL, Kovacs K, Muller P, Smyth HS: Analysis of hormone secretion by clinically nonfunctioning human pituitary adenomas using the reverse hemolytic plaque assay. J Clin Endocrinol Metab 1989;68:73–80. 112 Yamada S, Asa SL, Kovacs K: Oncocytomas and null cell adenomas of the human pituitary: Morphometric and in vitro functional comparison. Virchows Arch A 1988;413:333–339. 113 Asa SL, Cheng Z, Ramyar L, Singer W, Kovacs K, Smyth HS, Muller P: Human pituitary null cell adenomas and oncocytomas in vitro: Effects of adenohypophysiotropic hormones and gonadal steroids on hormone secretion and tumor cell morphology. J Clin Endocrinol Metab 1992;74: 1128–1134. 114 Kumar TR, Graham KE, Asa SL, Low MJ: Simian virus 40 T antigen-induced gonadotroph adenomas: A model of human null cell adenomas. Endocrinology 1998;139:3342–3351. 115 Chaidarun SS, Eggo MC, Stewart PM, Barber PC, Sheppard MC: Role of growth factors and estrogen as modulators of growth differentiation and expression of gonadotropin subunit genes in primary cultured sheep pituitary cells. Endocrinology 1994;134:935–944. 116 Gharib SD, Wierman ME, Shupnik MA, Chin WW: Molecular biology of the pituitary gonadotropins. Endocr Rev 1990;11:177–198.

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117 Shupnik MA, Gharib SD, Chin WW: Divergent effects of estradiol on gonadotropin gene transcription in pituitary fragments. Mol Endocrinol 1989;3:474–480. 118 Shupnik MA, Weinmann CM, Notides AC, Chin WW: An upstream region of the rat luteinizing hormone 1 gene binds estrogen receptor and confers estrogen responsiveness. J Biol Chem 1989;264: 80–86. 119 McCormick A, Brady H, Theill LE, Karin M: Regulation of the pituitary-specific homeobox gene GHF-1 by cell-autonomous and environmental cues. Nature 1990;345:829–832. 120 Theill LE, Karin M: Transcriptional control of GH expression and anterior pituitary development. Endocr Rev 1993;14:670–689. 121 Delegeane AM, Ferland LH, Mellon P: Tissue-specific enhancer of the human glycoprotein hormone ␣-subunit gene: Dependence on cyclic AMP-inducible elements. Mol Cell Biol 1987;7: 3994–4002. 122 Struthers RS, Vale WW, Arias C, Sawchenko PE, Montminy MR: Somatotroph hypoplasia and dwarfism in transgenic mice expressing a non-phosphorylatable CREB mutant. Nature 1991;350: 622–624. 123 Asa SL: The role of hypothalamic hormones in the pathogenesis of pituitary adenomas. Pathol Res Pract 1991;187:581–583. 124 Pilavdzic D, Kovacs K, Asa SL: Pituitary morphology in anencephalic human fetuses. Neuroendocrinology 1997;65:164–172. 125 Batt J, Asa S, Fladd C, Rotin D: Pituitary pancreatic and gut neuroendocrine defects in protein tyrosine phosphatase-␴-deficient mice. Mol Endocrinol 2002;16:155–169.

Sylvia L. Asa, MD, PhD Department of Pathology, University Health Network 610 University Avenue, Suite 4-302 Toronto, Ont M5G 2M9 (Canada) Tel. ⫹1 416 9462099, Fax ⫹1 416 9466579, E-Mail [email protected]

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Kontogeorgos G, Kovacs K (eds): Molecular Pathology of the Pituitary. Front Horm Res. Basel, Karger, 2004, vol 32, pp 20–33

Molecular Pathology of the Pituitary Development and Functional Differentiation of Pituitary Adenomas

Robert Yoshiyuki Osamuraa, Noboru Egashiraa, Shunsuke Miyaia, Michio Yamazakib, Susumu Takekoshia, Naoko Sannob, Akira Teramotob a

Department of Pathology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa and bDepartment of Neurosurgery, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo, Japan

Abstract This review article describes functional differentiation of the pituitary cells and pituitary adenomas with special emphasis on transcription factors and co-factors. Human pituitary adenomas generally follow the combination of transcription factors and co-factors, which are similar to those of physiologic anterior pituitary cells. On very rare occasions, the single pituitary adenoma produces two hormones, which belong to different cell lineage ‘trans-cell lineage’. Basic mechanism for this was considered to be ‘aberrant expression’ of transcription factors, i.e. NeuroD1 and Pit-1. This was experimentally supported by the induction of GH (mRNA and protein) in AtT-20 cells by transfecting Pit-1 gene. Various mechanisms have been reported for the experimental pituitary oncogenesis. Among these, GHRH has been emphasized as one of oncogenic factors for both human GHomas as well as in the transgenic animals. Copyright © 2004 S. Karger AG, Basel

Introduction

General Outline of the Pituitary Cells It has been well known that the pituitary gland is composed of the anterior lobe, intermediate lobe (clear in rodents) and posterior lobe. The anterior lobe secretes total six hormones including growth hormone (GH), prolactin (PRL),

thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), and follicle-stimulating hormone (FSH)/luteinizing hormone (LH). It is worth pointing out that ACTH is derived from the large prohormone which is designated as proopiomelanocortin (POMC). POMC undergoes post-translational digestion (which is called processing) by the specific enzymes, prohormone convertase (PC) 1/3 and PC2 [1]. It is also worthwhile noting that TSH, FSH and LH share common subunit (SU), ␣SU. Structural and functional specificity lies in ␤SU. Hormonal function can be achieved by combining ␣SU and ␤SU. It is also known that the anterior pituitary cells are assigned to produce certain hormones by particular transcription mechanism. Morphologically, the anterior pituitary cells are characterized by the presence of secretory granules, which are the structures composed of central dense cores and outer limiting membrane by electron microscopy. It is considered that the post-translational processing and combining ␣SU with ␤SU occur in these secretory granules. The secretory granules also play roles in secretion of the hormones through exocytosis of the secretory granules. This secretory process is designated as regulated pathway contrasting to constitutive pathway, which skips secretory granules (fig. 1). The other interesting cells in the anterior pituitary gland are the folliculostellate cells (FS cells) which lack secretory granules and hormone secretion, but are known to secrete cytokines for the local regulation of the endocrine function. The FS cells are positive for S-100 protein, but are negative for hormones by immunohistochemistry. The intermediate lobe is a well-defined structural unit in rodents which is known to secrete ␣MSH (melanocyte-stimulating hormone) derived from ACTH by the function of PC2. No other hormones are produced in this structure. In humans, no definite structural unit of the intermediate lobe is present. Instead, the ‘intermediate-derived’ cells extend into the posterior neural lobe from the transitional zone between the anterior lobe and posterior lobe. These cells have been called ‘invading anterior cells’ and are predominantly positive for ACTH immunohistochemically [2]. The posterior lobe is derived by the neural tissue and secrete hypothalamic hormones, i.e. vasopressin and oxytocin, which are produced in the hypothalamic neurons in the paraventicular and preoptic neurons. Development of the Pituitary Gland (fig. 2) Structural development of the pituitary starts from the formation of Rathke’s pouch, which is derived from the oral ectoderm. It is known that the anterior limb of the Rathke’s pouch develops into the anterior lobe and the posterior limb into the intermediate lobe [3]. The anterior limb proliferates and differentiates into the above six hormone-producing cells, but the posterior limb

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Nucleus Gene Transcription factor

Transcription RNA RNA splicing

mRNA Ribosome

RNA transport

Translation of mRNA

Protein Sorting and processing PC1/3, PC2 Granin family SNAP25

Fig. 1. The anterior pituitary cells are characterized by the presence of secretory granules. It is considered that the post-translational processing and combining ␣SU and ␤SU occur in these secretory granules. The secretory granules also play roles in secretion of the hormones through exocytosis of the secretory granules. This secretory process is designated as a regulated pathway contrasting to a constitutive pathway which skips secretory granules.

into only one hormone production, ␣MSH. The pituitary cells start possessing secretory granules according to the functional development. In rodent pituitary gland, definite intermediate lobe appears in the early developmental stage and stays as functional cells for ␣MSH production. In human pituitary gland, during fetal development, definite intermediate structure exists and produces ACTH and ␣MSH. But it changes its structures to the ‘invading anterior cells’ after birth. In the human anterior pituitary gland, ACTH-positive cells, which are located peripherally in early development, migrate to the center of the gland according to the development.

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a

b Fig. 2. Human pituitary gland and fetal rat pituitary gland. a Human pituitary gland: In human pituitary, during fetal development, a definite intermediate structure exists and produces ACTH and ␣MSH, but it changes its structures to the ‘invading anterior cells’ after birth. In human pituitary, ACTH-positive cells which are located peripherally in early development migrate to the gland. b Development of the pituitary gland: Structural development of pituitary starts from the formation of Rathke’s pouch which is derived from the oral ectoderm. It is known that the anterior limb of Rathke’s pouch develops into the anterior lobe and the posterior limb into the intermediate lobe.

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Functional Differentiation of the Pituitary Gland

Immunohistochemistry has played an important role to clarify the functional differentiation of the pituitary gland in the rodents and in humans. In the rodent anterior pituitary gland, GH, PRL, TSH and ACTH are generally produced and secreted from the different cells. FSH and LH are mostly secreted from the same cells. In female rats, PRL cells and FSH/LH cells are located in close vicinity [4]. In human anterior pituitary gland, GH, PRL, TSH and ACTH are produced primarily in the different cells, but GH and PRL are co-localized in the same cells. The unique functional differentiation in human pituitary gland lies in that GH cells also contain ␣SU. In the adult anterior pituitary gland in both rodents and humans, many S-100 protein-positive FS cells show slender cytoplasmic processes. In rodent pituitary gland, recent molecular studies have disclosed particular transcription factors and their combination with co-factors (as depicted in figure 3). The molecular studies have been stimulated by the cloning of the pituitaryspecific transcription factor-1(Pit-1) by Rosenfeld [5] and Karin [6] in different laboratories. It is known that Pit-1 regulates the functional differentiation of the pituitary cells to GH, PRL and TSH. GHRH-R, estrogen receptor (ER) and GATA-2 are the co-factors to further regulate the cells into GH, PRL and TSH respectively with the synergistic function with Pit-1 [7–10] (fig. 4).

Functional Differentiation of the Human Pituitary Adenomas

In humans, pituitary adenomas account for about 5% of the intracranial tumors [11]. Now the adenomas are classified according to the function (hormone production) into the categories as follows: GH-producing adenomas (GHoms); PRL-producing adenomas (PRLomas); TSH-producing adenomas (TSHomas); ACTH-producing adenomas (ACTHomas); FSH-producing adenomas (FSHomas); gonadotropin subunit-positive adenomas (GnSUomas), and null cell adenomas. Among these, the human pituitary adenomas are subdivided into (1) functioning adenomas and (2) non-functioning adenomas. The former group is the tumor with clinical symptoms and elevated serum hormone levels, and the latter is defined as the one without apparent symptoms or elevated hormone levels. The functioning adenomas include GHomas, PRLomas, TSHomas, ACTHomas and FSHomas. Immunohistochemical studies and other molecular techniques have disclosed that the tumor cells of the ‘non-functioning adenomas’ are frequently positive for gonadotropin subunits. And further, some non-functioning

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Ptx1 NeuroD1 Tpit

ACTH

NeuroD1 Ptx1 Tpit

Ptx1 Tpit

␣MSH

Tpit

Ptx1

FSH

LH GATA-2

Ptx1 Rpx Lhx3

Ptx1 Rpx

SF-1

Ptx1 Lhx3 SF-1 GATA-2 Dax-1

Dax-1

Ptx1 Lhx3 Pit-1 GATA-2

Prop-1 Ptx1 Prop-1 Lhx3

Ptx1 Pit-1 Lhx3

GH

Zn-15

ER

Ptx1 Lhx3 Pit-1 ER

PRL

Fig. 3. Pituitary cell lineage. Several transcription factors have been reported to be active in all or a subset of cell lineages, in many cases during a specific period of development. The anterior pituitary cells are well known to be classified into three cells lineages, i.e. GH-PRL-TSH cells (Pit-1-dependent), POMC (ACTH-␣MSH) (NeuroD1, Tpit-dependent) and FSH/LH (SF-1, Dax-1, GATA-2-dependent).

adenomas are positive for GH, ACTH by immunohistochemistry and have been designated as ‘silent adenomas’ in which electron microscopy has also contributed. Our investigative interests have been focused on the issue why the tumor cells differentiated toward the particular hormone production [11]. According to the previous studies on the functional development of the pituitary adenomas, it has been clarified that the functioning pituitary adenomas generally follow the combination of transcription factors and co-factors, which have been known, to function in the physiologic condition. For example, GHomas are regulated by Pit-1 and GHRH-R [7, 8], PRLomas by Pit-1 and ER [9] and TSHomas by Pit-1 and GATA-2 [10] (fig. 4). It is of particular interest for pathology that the distinction between TSHomas and FSHomas/LHomas

Molecular Pathology of the Pituitary

Ptx1 Lhx3 SF-1 GATA-2 Dax-1?

TSH Ptx1 Lhx3 Pit-1 Zn-15

GATA-2

Pit-1

?

25

GHoma

GHRH-R signal Pit-1

GH

GHRH-R TSHoma Pit-1 GATA-2

GATA-2

TSH

Po s co itive ntr ol

Pit-1 PRL ⫹

Pit-1

ER

PRL

ER

Fig. 4. Pit-1 and co-factors. It is known that Pit-1 regulates the functional differentiation of the pituitary cells to GH, PRL and TSH. GHRH-R, estrogen receptor (ER) and GATA-2 are the co-factors to further regulate the cells into GH, PRL and TSH respectively with the synergistic function with Pit-1.

lies on the expression of Pit-1 as they share GATA-2 as common transcription factors. ACTHomas are the tumors of unique transcription mechanisms as they are regulated by Tpit and NeuroD1 [12, 13]. All pituitary tumors share the early transcription factors such as Ptx1 as an essential basic factor for the additional transcription factors toward specific hormone production. These early transcription factors include Lhx3, Rpx and Prop-1. Prop-1 is known as an enhancer of Pit-1 for later functional differentiation and as a Pit-1 non-dependent transcription factor for early appearance of TSH, FSH/LH and ACTH. The ‘non-functioning’ adenomas are defined as the tumors without apparent clinical symptoms or elevated serum hormone levels (table 1). However, at the cellular levels, the tumor cells are not infrequently detected to express gonadotropin subunits such as ␣SU, FSH␤ and/or LH␤. Also, it has been speculated that a significant proportion of this group of adenomas is derived from the FSH/LH cell lineage (table 2, fig. 5). The silent adenomas are often depicted to be produce (or immuno- or mRNA-positive for) GH or ACTH. In case of silent ACTH-producing adenomas (silent corticotroph adenomas), one possibility lies in inappropriate processing of POMC by lack of PC1/3 and PC2 [1]. Null cell adenomas are the ones without apparent hormone production at the cellular levels and count for approximately 40.4% of non-functioning

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LH␤

␣SU

FSH␤

Fig. 5. Expression of ␣SU FSH␤ and LH␣ in non-functioning pituitary adenoma. The tumor cells are not infrequently detected to express gonadotropin subunits such as ␣SU, FSH␤, and/or LH␤. Also, it has been speculated that a significant proportion of this group of adenomas is derived from the FSH/LH cell lineage.

Table 1. Frequency of pituitary hormones GHoma

PRLoma

ACTHoma

FSHoma

TSHoma

Non-functioning adenoma

Others

26.8%

17.8%

7.7%

4.4%

1.1%

31.5%

10.6%

Table 2. Expression of hormones in non-functioning pituitary adenomas detected by immunohistochemistry GH

PRL

ACTH

FSH␤

LH␤

TSH␤

␣SU

Null

4.6%

10.2%

5.1%

42.6%

19.8%

2.0%

43.2%

37.6%

adenomas. This category has been also claimed from the ultrastructural studies of the pituitary adenomas [14].

Human Pituitary Adenomas with ‘Trans-Cell Lineage’ Differentiation, GH and ACTH

As mentioned earlier in this chapter, most of the functioning human pituitary adenomas follow the combination of transcription factors and co-factors similar to those in the physiological conditions. On rare occasions, the functioning adenomas do express the production of hormones, which belong different developmental cell lineages (designated as trans-cell lineage). Good examples for this include GHomas with Cushing’s syndrome (ACTH production) and ACTHomas

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a

b

c

d

e

f

ACTH NeuroD1 Tpit FSH␤ⲐLH␤ Pit-1 ACTH GH

SF-1 GH Pit-1 PRL TSH␤

g

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(Cushing syndrome) with GH production. Further, some FSHomas have been reported to be immuno-positive for GH or ACTH [15]. With some unexplained mechanisms, the combination of GH and ACTH has been most frequent [16, 17]. In ACTHoma, which disclosed GH production, immunohistochemical detection disclosed that, in some tumor cells, ACTH and GH are co-localized. Regarding transcription factors, the tumor cells in this case showed expression of Pit-1 in addition to NeuroD1. Usually, ACTHomas express NeuroD1 and Tpit, but not Pit-1, therefore, the appearance of Pit-1 is considered ‘aberrant’. And this aberrant expression of transcription factors is considered to be one of the mechanisms of trans-cell lineage hormone production in the functioning pituitary adenomas (fig. 6). Kurotani et al. [18] demonstrated the induction of new GH mRNA and GH protein in AtT-20 cells, which are the mouse pituitary ACTHoma-derived cell line, by transfection of Pit-1 gene. The transformed AtT-20 cells produce GH in addition to ACTH and transcription factors of Pit-1 as well as NeuroD1. This phenomenon was very similar to that in the human pituitary ACTHomas with GH production. This is considered to be as a good experimental model for ‘aberrant expression’ of transcription factors and ‘trans-cell lineage production’ of pituitary hormones (fig. 7). Development of Pituitary Adenomas in Rodents as an Animal Model for Human Adenomas

In human pituitary adenomas, from the tumor tissues we examine as pathological specimens, no transition from normal to hyperplasia and from hyperplasia to adenoma can be appreciated. The pituitary adenomas in the rodents can be induced by various procedures, which are listed as follows: estrogeninduced PRLomas [9]; GHRH and GHomas [8]; GHRH transgenic mice and rats [19]; PTTF [20]; P27 knockout mice [21], and D2R knockout mice [22]. Among these, here, GHRH and its role in pituitary oncogenesis are discussed. In human pituitary GH cells, Pit-1 and GHRH-R are known to function synergistically for its proper function. In human GHomas, GHRH-R has been detected by RT-PCR and is considered to function with Pit-1.[8] Fig. 6. ACTHoma with GH production. Histochemical studies. The adenoma showed basophilic cells (a). ACTH (brown) (b, arrow) and GH (blue) were detected in the adenoma by immunohistochemical double staining, and both were expressed in same adenoma cells (b, arrowhead). GH mRNA was also detected in the adenoma by in situ hybridization (c, antisense probe; d, sense probe). NeuroD1 (e) and Pit-1 (f) were expressed in the nuclei of the adenoma. (g) Schematic drawing of the mechanisms for ‘trans-cell lineage’ expression of ACTH and GH in a pituitary adenoma.

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b

4 AtT-20

5 AtT/Pit-1

a

6 Mouse pituitary

c

Fig. 7. Induction of aberrant expression of transcription factors in vitro. a Visualization of GFP fluorescence in transfected AtT-20 cell line: The transfected gene was constructed by fusing Pit-1 and Green Fluorescent Protein (GFP). Thus it was shown that Pit-1 protein was bound to the nuclei in the living cells as GFP could be traced that the fusion protein moved from the cytoplasm and into the cell. b Immunohistochemistry for GH: AtT-20 cells which transfected GFP-Pit-1 fusion gene were expressed GH in cytosol near the nuclei. c Immunoblot analysis for GH protein: The 22-kDa immunoreactive band was detected for AtT-20 transfected GFP-Pit-1 fusion gene.

have reported a very rare case of GHoma which also secrete GHRH. This case was suggested to serve as a model for the oncogenic role of GHRH by paracrine and autocrine mechanism through GHRH-R. They also investigated the expression of GHRH mRNA by RT-PCR in GHomas and found frequent expression of GHRH in GHomas suggesting that one of oncogenic mechanisms for GHomas is via GHRH production [8]. Experimentally, human GHRH transgenic mice and rats produced pituitary hyperplasia and subsequent adenoma formation at about 10 months of age. The induced pituitary adenomas showed the immunohistochemical localization of GH, PRL and focal TSH. Pit-1 was diffusely present in their nuclei. ACTH, FSH and LH are negative. The non-adenomatous pituitary gland shows hyperplastic GH cells admixed with other hormone-producing cells. Chronological studies on the pituitaries in GHRH Tg mice exhibited hyperplastic GH cells without forming discrete nodular lesion (adenomas) resulting in adenoma formation (fig. 8). These findings suggest that hyperplasia-adenoma sequence exists in the GHRH Tg mice. GHRH stimulates GHRH-R-mediated signal transduction and subsequent cell proliferation and further promoting GH production by synergistic action with Pit-1 [19].

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MT-1 promoter

GHRH gene

713 bp

220bp

a

SV40 poly(A)

b

GHRH

PRL

Pit-1

GH

TSH␤

c

GHRH GHRH-R

d

GHRH

Tumorigenesis

Fig. 8. GHRH transgenic mice (GHRH-Tg) develop pituitary adenomas. a Construct of GHRH transgene. The construct consisted of a 713bp fragment of the mouse MT-1 promoter, containing elements responsible for metal induction and transcription initiation fused to the hGHRH gene. b GHRH-Tg had markedly enlarged and congested pituitary gland. c Pathological analysis: The induced pituitary adenomas showed the immunohistochemical localization of GH, PRL and focal TSH. Pit-1 was diffusely present in the nuclei. d Scheme of mechanism for tumorigenesis in GHRH-Tg mouse.

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of human GRF transgenic mice: Its relationship with hormonal expressions. Endocr J 1993;40: 133–139. Heaney AP, Melmed S: Pituitary tumour-transforming gene: A novel factor in pituitary tumour formation. Baillières Best Pract Res Clin Endocrinol Metab 1999;13:367–380. Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N, Horii I, Loh DY, Nakayama K: Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia and pituitary tumors. Cell 1996;85:707–720. Hentges ST, Low MJ: Ovarian dependence for pituitary tumorigenesis in D2 dopamine receptordeficient mice. Endocrinology 2002;143:4536–4543.

Robert Y. Osamura, MD Department of Pathology, Tokai University School of Medicine Bohseidai, Isehara, Kanagawa 259-1193 (Japan) Tel. ⫹81 463 931121, Fax ⫹81 463 911370, E-Mail [email protected]

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Kontogeorgos G, Kovacs K (eds): Molecular Pathology of the Pituitary. Front Horm Res. Basel, Karger, 2004, vol 32, pp 34–62

Cell Cycle Dysregulation in Pituitary Oncogenesis ˆ ˆ

Madalina Mus¸ata,b, Vladimir V. Vaxb, Ninetta Borbolib, Maria Gueorguievb, Sarah Bonnerb, Márta Korbonitsb, Ashley B. Grossmanb a

Carol Davila University of Medicine and Pharmacy, Bucharest, Romania; bDepartment of Endocrinology, St. Bartholomew’s Hospital, London, UK

Abstract The cell cycle is the process by which cells grow, replicate their genome and divide. The cell cycle control system is a cyclically-operating biochemical device constructed from a set of interacting proteins that induce and coordinate proper progression through the cycle, and includes cyclins, cyclin-dependent kinases (CDK) and their inhibitors (CDKI). There are mainly two families of CDKI, the INK family (INK4a/p16; INK4b/p15; INK4c/p18 and INK4d/p19) and the WAF/KIP family (WAF1/p21; KIP1/p27; KIP2/p57). Progression through the cell cycle is mainly dependent on fluctuations in the concentration of cyclins and CDKI achieved through the programmed degradation of these proteins by proteolysis within the ubiquitin-proteasome system. There is also a transcriptional regulation of cyclin expression, probably dependent on CDK phosphorylation. The p53 family – p53, p63 and p73 – function as transcription factors that play a major role in regulating the response of mammalian cells to stressors and damage, in part through the transcriptional activation of genes involved in cell cycle control (e.g. p21), DNA repair, senescence, angiogenesis and apoptosis. Essential for the maintenance of euploidy during mitosis is human securin, identical to the product of the pituitary tumour-transforming gene (PTTG). Loss of regulation at the G1/S transition appears to be a common event among virtually all types of human tumours. Aberrations of one or more components of the pRb/p16/cyclin D1/CDK4 pathway seem to be a frequent event (80%) in pituitary tumours. The role of p27 is rather that of a haploinsufficient gene. p27/ mice show an increased growth rate, due to increased cellularity, testicular and ovarian cell hyperplasia and infertility, and hyperplasia of the pituitary intermediate lobe with nearly 100% mortality caused by such a benign pituitary tumour. Although the p27 gene was not found to be mutated in human pituitary tumours and its mRNA expression was similar in tumour samples in comparison with normal pituitaries, the load of p27 protein expression in corticotroph adenomas and pituitary carcinomas was shown

to be much lower than those in normal pituitary tissue or other types of pituitary adenoma, suggesting that post-translational processing of p27 accelerates its removal from the nucleus. In respect to p27 degradation and its cellular compartmentalization, several pathways have been explored. Malignant tumours are associated with increased nuclear immunostaining for Jun-activation binding protein-1 (Jab1) which is responsible for phosphorylated p27 export from the nucleus. Corticotrophinomas are characterized by massively increased phosphorylation of p27 on Thr187, but are not associated with changes in Jab1. Macrophage inhibitory factor (MIF), which binds and inactivates Jab1, was noted to be over-expressed in tumours with abundant Jab1, suggesting that it may be part of a compensatory mechanism to moderate Jab1 activity. Proteasomal degradation of p27 requires its ubiquitylation by the SCF ubiquitin ligase, with specific addressing by the F-box protein Skp2 and its co-factor Cks1. Pituitary tumours with high p27 protein expression showed significantly less Skp2 expression than samples with low p27 immunostaining, suggesting that increased Skp2 could play at least a part in this process. No difference was observed in Cks1 mRNA levels between normal pituitaries and pituitary adenomas. The present data suggest that inhibition of growth and tumour development is sensitive not only to the absolute levels of p27 protein, but also to its cellular compartmentalization. Very recent findings from our group have established up-regulation of the serine-threonine kinase Akt in pituitary tumours compared to normal pituitary, which may cause phosphorylation of p27 on Thr157 and cytoplasmic retention of p27. PTTG protein is highly expressed in various human tumours, including pituitary tumours. While its mRNA levels are low in normal pituitary, increases in PTTG transcripts from more than 50% to more than 10-fold were recorded in the majority of a series of pituitary adenomas. Control of the cell cycle is a vital part of the cell’s replication machinery. Disruption of this process is commonly seen in pituitary tumours and we are now beginning to identify regulatory elements which are likely to play a major role in pituitary oncogenesis. Copyright © 2004 S. Karger AG, Basel

Cell Cycle Machinery

The cell cycle is the process by which cells grow, replicate their genome and divide. It is traditionally divided in four phases: S (synthesis), generation of a faithful copy of the genetic material of the cell; M (mitosis), the phase in which the partitioning of all the cellular components between two identical daughter cells occurs; between these two phases there are two gap periods that provide additional time for cell growth, the G1 phase – the interval between end of mitosis and the beginning of DNA synthesis, and the G2 phase – the interval between the end of DNA synthesis and the beginning of mitosis. When cells in G1 cease proliferation, in the absence of mitogenic signals or in the presence of anti-mitogens, they exit the cell cycle and enter a quiescent state, G0, where they can remain for days, weeks or even years before resuming proliferation (fig. 1). Thus, one of the most crucial decisions that every proliferating cell must make is whether to continue another round of cell division or to exit the cell cycle. To ensure proper progression through the cell cycle, cells have developed a series of

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Mitogenic signals (e.g. bFGF) G0 P Rb

G1 CDK inhibitors: p16, p15, p18, p19

Cyclin DCDK4/6 P Rb

PTTG

p21, p27, p57

Cyclin ECDK2

Restriction point P P P P Rb P P

M

Cyclin B/ CDK1 MPC

Cyclin A/ CDK2 S

G2

Fig. 1. Cell cycle progression. In early G1, in response to mitogenic stimuli, cyclin D activates CDK4/6 which partially phosphorylates Rb protein. After progression beyond the restriction point, CDK2 in complex with cyclin E further phosphorylates pRb rendering it inactive and thus allowing transcription of cyclin A. The accumulating cyclin A captures CDK2 from cyclin E and promotes progression through S phase. At the transition between G2-M phase, cyclin B complexes with and activates CDK1 to further activate metaphasepromoting complex (MPC) required for entering M phase. CDK inhibitors oppose CDK activation: in early G1, the INK4 family bind to CDK4/6 and prevent cyclin D activation of this kinase. At G1-S transition, WAF/KIP family members sequester CDK2 away from cyclin E/A when they are in stoichiometric amounts with CDK2. When CDK2 exceeds p27 levels, the former phosphorylates the latter and triggers its degradation. Resetting of pRb occurs in M-phase by dephosphorylation, probably by protein-phosphatase type 1 and this correlates with the time of cyclin A degradation. PTTG is a securin which blocks the separin protein that contributes to chromatid separation in M phase, until activation of the anaphasepromoting complex.

‘checkpoints’ where feedback signals conveying information regarding downstream processes can delay progress into a new phase until they have successfully completed the previous one, and this also allows regulation by signals from the environment, such as the presence of mitogens, growth factors, etc. The major checkpoint in mammalian cells is in G1, known as the restriction point (R) (fig. 1). As mammalian cells undergo a period of mitogen dependence before entering the cell cycle, the transition beyond the restriction point represents a commitment to a new round of division regardless of the presence of mitogens. The cell cycle control system is a cyclically-operating biochemical device constructed from a set of interacting proteins that induce and coordinate proper progression through the cycle.

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There are three main families of proteins involved in this process, namely cyclins, cyclin-dependent kinases (CDK) and their inhibitors (CDKI), whose fluctuation in concentration drive cells through G1 into S phase and further to G2 and M phase. CDK are a group of serine/threonine kinases that form active heterodimeric complexes following binding to cyclins, their regulatory subunits. Regulation of CDK activity is effected at multiple levels: the accumulation of the specific cyclin, the effective assembly in the CDK-cyclin complex, the association with cyclin-dependent kinase inhibitors, and by subsequent phosphorylation and dephosphorylation events [1, 2]. Cyclins are so-called because they undergo cyclic changes in synthesis and degradation in each division cycle of the cell. There are four main classes of cyclins: G1 cyclins (i.e. cyclin D), which bind to CDK molecules during G1 and are required for by-passing the restriction point (R); G1/S cyclins (i.e. cyclin E) necessary for entry into S phase; S cyclins (i.e. cyclin A), which bind CDK2 in S-phase in order to initiate DNA replication, and mitotic cyclins, which bind to CDK1 molecules at G2/M transition and are required for entry into mitosis [3]. Current knowledge indicates that the D-type cyclins stimulate progression through the early G1 phase which is dependent on mitogenic stimuli (e.g. FGF, EGF), prior to the restriction point where commitment to proliferation has been made and after which mitogens are no longer necessary for progression through the cell cycle. The various forms of cyclin D (D1, D2, D3) bind and activate CDK4 and CDK6, which are involved in early G1 transition, being activated by mitogenic signals. As cells enter S phase, cyclin D1 vanishes from the nucleus [4]. Subsequently, activity of the cyclin E-CDK2 complex peaks at the G1-S transition, after which CDK2 is captured by cyclin A. Free cyclin E is now the target for proteolysis via the ubiquitin pathway. Cyclin A (A1 and A2) activates CDK2 when the cell enters the S phase by binding to one side of CDK2’s catalytic cleft, inducing large conformational changes that realign the active site residues and relieve the blockade at the entrance of the catalytic cleft [5]. Cyclin A shows a steady accumulation throughout interphase until G2/M transition, followed by rapid disappearance at the onset of anaphase. Cyclin A and B (B1 and B2) also complex with CDK1/CDC2/p34 to form the mitosis-promoting factor whose activity peaks at G2/M transition, being required for the cell to enter M phase [6]. For full catalytic activity, CDKs also need phosphorylation on a threonine (Thr) residue (T172 in CDK4 and T160 in CDK2). This is carried out by the CDK7-cyclin H complex (known as CDK-activating kinase/CAK) [7], which in turn can be regulated by the CDK8-cyclin C complex (repressing transcription of the CDK7-cyclin H subunits) [8]. The role of CDK3 remains obscure at the present time.

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The primary substrates of CDKs in G1 progression are the members of the retinoblastoma protein family: pRb/p105 [9, 10], p107 [11] and Rb2/p130 [12, 13]. The three retinoblastoma family members negatively control cell cycle progression between G1 and S phases. These molecules are very similar in sequence and structure, mostly in their highly conserved ‘pocket domain’ that gives rise to a particular steric conformation allowing them to function as docking sites for a series of proteins that must be tightly regulated throughout the cell cycle [10, 11, 13]. Retinoblastoma proteins potentially interact with more than 100 different cellular proteins [14]. With respect to cell cycle regulation, pRb binds to the E2F/DP family of transcription factors to change their role from active transcriptional activators to inhibitors of transcription [15–17]. E2Fbinding sites are located on the promoters of several growth-promoting genes, such as c-myc, c-myb, dehydrofolate reductase, thymidine kinase, thymidine synthetase, DNA polymerase , cyclin A, cyclin E, cyclin D1, CDK1/cdc2 and E2F1 whose products are necessary for DNA synthesis, but E2F-binding sites are also found on the promoters of cell cycle inhibitors such as the retinoblastoma gene (RB1) [18–20]. Blocking the active form of E2F by sequestrating it in an Rb-E2F heterodimer prevents cell cycle progression. Retinoblastoma family members act in a phase-specific manner, as p130-E2F complexes are generally found in growth-arrested cells, while p107-E2F complexes are predominant in S-phase [21]. Rb activity as a tumour suppressor is dependent on its phosphorylation status [22]. Before G1 phase progression is initiated, Rb is underphosphorylated and is thus able to repress cell cycle progression. Cyclin/CDK-mediated phosphorylation of Rb is the most likely mechanism that turns off the anti-proliferative actions of Rb at G1/S transition. In early G1, in response to mitogenic signals, CDK4/6-cyclin D complexes partially phosphorylate Rb, resulting in partial activation of E2F/DP transcription factors which participate in the generation of molecules required for G1/S transition, such as cyclin E. CDK2 is sequentially activated by E-type cyclins (E1 and E2). The cyclin E-CDK2 complex completes Rb phosphorylation that now releases the transcription factors, allowing them to carry out specific tasks in cell cycle progression, such as the synthesis of cyclin A. It has recently been reported that Rb family members suppress cell proliferation by regulating not only E2F-dependent mRNA transcription, but also rRNA and tRNA transcription, and also by stimulation of histone deacetylase 1 and DNA packaging [23]. In addition to these, pRb has been implicated in the regulation of differentiation [24, 25] and apoptosis [26]. Cyclin-dependent kinase inhibitors (CDKI) counteract CDK actions, either by blocking their activation, or by impairing substrate/ATP access [27]. There are two types of CDKIs: the INK family and the WAF/KIP family. The INK family (INK4a/p16; INK4b/p15; INK4c/p18 and INK4d/p19) exert inhibitory activity by binding to CDK4 and CDK6 [28, 29]. These proteins exert

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their actions by competing with D-type cyclins for the CDK subunit and thus preventing phosphorylation of pRb thereby inhibiting progress through G1/S. Members of the WAF/KIP family (WAF1/p21; KIP1/p27; KIP2/p57) form heterodimeric complexes with G1/S CDKs and inhibit kinase activity of CDK2-cyclin E complexes [30, 31]. The identification of new targets for p21 and p27, as well as evidence of p27 cytoplasmic relocalization, have revealed unexpected functions for these proteins in the control of CDK activation, the regulation of apoptosis and transcriptional activation [32]. Progression through the cell cycle is mainly dependent on fluctuations in the concentration of cyclins and CDKI achieved through the programmed degradation of these proteins by proteolysis within the ubiquitin-proteasome system (described below: CDK2-p27 Pathway). In more complex cells, there is also a transcriptional regulation of cyclin expression, probably dependent on CDK phosphorylation, though this process remains obscure at the present time [3]. Negative regulation of the cell cycle is not restricted to CDK. One critical target of negative regulation that acts in M phase is a protease named separin/ separase/ESP1 [33]. The critical target of separin is cohesin Scc1p, which tethers sister chromatids together at metaphase. The cleavage of cohesin by separin triggers anaphase. Negative regulation of separin is achieved by another protein known as securin [34]. Securin blocks the separin protein until activation of the anaphase-promoting complex (APC). APC is a ubiquitin ligase that targets securin for proteolysis, thus releasing separin that contributes to chromatid separation in M phase: the securin-separin interaction is thus essential for the maintenance of euploidy. Human securin is identical to the product of the gene called pituitary tumour-transforming gene (PTTG), which is over-expressed in some tumours and exhibits transforming activity in NIH 3T3 cells [35]. The oncogenic nature of increased expression of securin may result from chromosome gain or loss, produced by errors in chromatid separation [34]. The p53 family consists of three proteins – p53, p63 and p73 – which function as transcription factors that play a major role in regulating the response of mammalian cells to stressors and damage, in part through the transcriptional activation of genes involved in cell cycle control (e.g. p21), DNA repair, senescence, angiogenesis and apoptosis [reviewed in 36, 37]. The three members of the family share a significant degree of sequence homology, particularly in the DNA-binding domain, the amino-terminal activation domain and the carboxyterminal oligomerization domain [38–40]. Although structurally and functionally related, p63 and p73 have clearly distinct roles in normal development [39], while p53 is activated in response to oncogenes, DNA damage or other stress signals, resulting in the inhibition of tumour cell growth most decisively by activating apoptosis [41]. The responses to p53 activation can vary between cell cycle arrest, senescence, differentiation or apoptosis, depending on intrinsic and

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extrinsic factors. Under some circumstances, p53 also contributes to the repair of genotoxic damage in G2, potentially allowing for the release of the rehabilitated cell back into the proliferating pool [36]. Malignant progression is dependent on loss of p53 function due to mutations in its gene (TP53) or defects in the signalling pathway up- or downstream of p53.

Cell Cycle Dysregulation in Pituitary Tumorigenesis

Loss of regulation at the G1/S restriction point appears to be a common event among virtually all types of human tumours, including pituitary tumours. CDK4-Rb Protein-INK4 Pathway The importance of the CDK4-p16-Rb protein pathway has been established in a range of different tumour types. The cyclin D1 gene (CCND1), located on 11q13, is one of the most frequently amplified genes observed in human tumours, amplification leading to cyclin D1 protein over-expression [42]. Allelic imbalance of CCND1 gene in invasive pituitary tumours has been described [43]; nevertheless, no correlation was found with immunohistochemical expression of the protein. The results would suggest that over-expression of cyclin D1 occurs early in pituitary tumorigenesis and more frequently in non-functional tumours than in somatotrophinomas [44]. CDK4 gene amplification and over-expression have been found in a number of human tumours including gliomas, sarcomas, breast tumours and colorectal carcinomas. CDK4 mutations involving codon 24 of CDK4 (Arg to Cys or Arg to His), which prevents binding of p16 to the CDK4-binding domain rendering CDK4 constitutively active, have been found in familiar melanoma kindreds [45–47]. CDK4 mutant (R24C) knock-in mice [48] develop pituitary adenomas as well as insulinomas and Leydig cell tumours. Human pituitary adenomas have been studied for mutations at codon 24, but no mutation was found [49, 50]. Simpson et al. [49] examined 45 pituitary tumours and looked at pRb, p16, cyclin D1 and CDK4 protein expression, and analysed the CDK4 gene for the characterized activating mutations within codon 24. Whilst a significant percentage of tumours had abnormal expression of pRb, p16 and cyclin D1, not a single pituitary sampled harboured a R24C mutation in the CDK4 gene. More recently, we have studied 61 pituitary tumours, 14 insulinomas and 6 Leydig cell tumours for R24C or R24H mutations, but no mutation was found in any of these neoplasms at codon 24, or in the surrounding area from codon 1 to codon 41 [50]. However, a role for CDK4 cannot be totally ruled out as several non-contiguous amino-acid sequences on CDK4 are require for binding to p16 (codons 22, 24, 25, 97, 281) [51].

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Retinoblastoma gene (RB1) is a prototype of a tumour suppressor gene, as its loss of heterozygosity (LOH) both in familial and sporadic forms of retinoblastoma is oncogenic when accompanied by concomitant mutations in the remaining allele. Heterozygote retinoblastoma knockout mice develop pituitary adenocarcinomas of intermediate lobe origin [52, 53], but studies on human pRb loss in pituitary tumours have been controversial. While some of these found sustained LOH of RB1 gene in highly invasive or malignant pituitary tumours [54], this finding was not sustained by loss of pRb on immunohistochemical analysis. On the contrary, other studies [55, 56] have reported lack of expression pRb in a small proportion of pituitary tumours, but this did not associate with loss of an RB1 intragenic marker. Loss of pRb protein expression in these tumours was more recently suggested to be due to methylation in the gene-promoter region [57]. LOH at 13q, the locus of the RB1 gene, has been identified in human pituitary adenomas, but there is also evidence that an independent putative tumour suppressor gene at that locus is linked with RB1 and may play a role in pituitary tumorigenesis [58]. p16 preferentially binds and sequestrates CDK4. Loss of p16 results in hyperphosphorylated pRb unable to inhibit G1/S transition. p16 is recognized as a classic tumour suppressor gene and is inactivated in numerous different human cancers, either by homozygous deletion or gene silencing through methylation of its associated CpG island [59]. Hypermethylation of p16 gene and loss of protein expression was shown to be associated with non-functional pituitary adenomas, but no mutation was revealed in the coding region of p16 [60, 61]. Among the non-functional tumours, it was usually null cell adenomas that were hypermethylated [62]. The cell cycle inhibitor p16 is inactivated in many human tumours, this occurring in 70% of head and neck cancers [63]. Disruption of p16 and the colocalized sequence of p19/ARF in mice results in the development of spontaneous tumours at an early age in numerous cell types, but does not cause any pituitary abnormality [64]. Nevertheless, mutually exclusive loss of pRb or p16 was found in a series of pituitary tumours and aberrations of one or more components of the pRb/p16/cyclin D1/CDK4 pathway seem to be a frequent event (80%) in such tumours [49]. Moreover, induced expression of p16 caused hypophosphorylation of pRb and was shown to inhibit AtT-20 pituitary tumour-cell growth and induce cell cycle arrest in G1 [65]. p18 has been shown to play a role in growth control, lymphocyte mitogenic activation and pituitary tumorigenesis in mice [66]. p18 knockout mice develop organomegaly, increased body size, expansion of T lymphocytes (which are hyper-responsive to mitogenic stimulation) and pituitary hyperplasia with progression to pituitary adenomas. This last feature occurs without impairment of p27 levels or its complexes with CDK2, CDK4 or CDK6, indicating that p18 and p27 mediate two partially-independent pathways in controlling pituitary

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tumorigenesis in mice [66]. Moreover, pituitary tumorigenesis in double knockouts (p18/ and p27/) is greatly accelerated compared to the separate knockouts, and can lead to death of these mice by 3–4 months of age [67]. The specificity of this function is further underscored by the fact that mice deficient in other CDKI genes, p16 [64], p21 [68] or p57 [69], do not develop pituitary abnormalities. CDK2-p27 Pathway p27 is a 197-amino-acid protein which was initially identified in complexes with cyclin E-CDK2 in cells that had been growth arrested [70, 71]. Its gene is located on 12p13, a region that is frequently rearranged or deleted in haematological malignancies [72, 73]. p27 Function p27 protein expression is higher in quiescent cells, but falls as cells begin to proliferate. To its main function as ‘gatekeeper’ of the quiescent state in mammalian cells [74, 75] has recently been added new roles in apoptosis, regulation of transcription, cell-cell adhesion, drug sensitivity, differentiation, and tumour virus biology [32, 76]. The effects of p27 on cell cycle progression are mainly due to binding and inactivation of cyclin E-CDK2 complexes at G1/S transition. The crystal structure of bound p27 to cyclin A-CDK2 revealed that p27 inserts itself within the CDK catalytic site, blocking ATP access [77], while important CDK2 conformational changes further lock the catalytic cleft in an inactive form. Since the activated form of CDK2 is located in the nucleus, only the nuclear form of p27 should probably be considered as a catalytic inhibitor of CDKs. Besides its nuclear inhibitory function, cytoplasmic p27 also activates cyclin D1-CDK4 complexes [78]. If cells are persistently activated by mitogens, cyclin D-dependent kinase (CDK4 and CDK6) activity remains high in subsequent cycles and sequestrates virtually all the p27, which seems to promote activation of cyclin D-CDK complexes via directing them to the nucleus and increasing the stability of cyclin D [75]. When mitogens are withdrawn, cyclin D is rapidly degraded and p27 released to inhibit cyclin E-CDK2, thereby arresting progression, usually within a single cell cycle. p27 has a less well understood effect on apoptosis. Over-expression of p27 induces not only cell cycle arrest and loss of cyclin-CDK activity, but also triggers apoptosis in human breast cancer cells and several other cancer cell lines [79, 80]. Induced p27 deficiency disables the trigger for an apoptotic response in Rb / mouse pituitary tumour cells [81]. On the contrary, in carcinoma cells and in leukaemic cell lines, p27 has been shown to prevent drug-induced apoptosis [82]. Whether p27 promotes or inhibits apoptosis depends on the integrity of p27, cell type and the status of the cell, transformed versus non-transformed.

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It has been suggested that certain tumour cells have a mechanism whereby they induce truncation of p27 and p21 through a caspase-like mechanism, thus impairing their apoptotic activity [83]. The role of p27 in cell-to-cell adhesion is even less clarified. A rise in p27 level was shown to be induced by contact inhibition in non-transformed fibroblast and epithelial cells [84, 85]. This suggested a direct role of p27 in cellular adhesion. Furthermore, it was claimed that cell shape and cytoskeletal tension control p27 expression and cell cycle progression as a response of integrin receptors binding to the extracellular matrix [86]. Another link of p27 with intercellular adhesion came from evidence that E-cadherin (the major calciumdependent cell adhesion molecule on normal epithelial cells) can elevate p27 through inhibition of mitogenic signalling pathways initiated by receptor tyrosine kinases such as the epidermal growth factor receptor [87]. More recently, N-terminal phosphorylation of p27 and its cytoplasmic translocation have been reported to induce cell motility [126], but forced expression of p27 also inhibits endothelial cell migration and participates in the down-regulation of motility in vascular smooth muscle cells [88]. Evidence on the yeast homologue of CDKIs, Far1p [89], indicates that cytoplasmic transfer of p27 by a nucleocytoplasmic transporter may ensure proper cellular polarization and morphology of the cell [76]. However, this function remains to be demonstrated in mammals. Several viral gene products have been shown to bind and/or inactivate p27: adenovirus oncoprotein E1A [90], E7 oncogene of HPV-16 [91] polyoma virus small T antigen [92], v-ras and v-src [93], thus allowing the virus to override G0/G1 arrest. Regulation of p27 Expression Regulation of p27 is achieved mainly at a post-transcriptional level. The programmed degradation of many proteins that regulate the cell cycle is carried out by the ubiquitin-proteasome system (fig. 2). Ubiquitin (Ub) is a highly conserved 76-amino-acid protein which functions as a label tag when bound in polymers to a target protein, allowing the latter’s destruction by the proteasome. The proteasome is a 26S multiprotein complex that catalyses the breakdown of polyubiquitylated proteins. It consists of a 20S catalytic and a 19S regulatory subunit. Although the ubiquitin-proteasome system came to light in the context of protein destruction [94], it is now clear that both ubiquitin and the proteasome can also carry out various non-proteolytic tasks, controlling receptor internalization [95], ribosome function [96] and nucleotide excision repair [97]. Recent evidence supports a role for the ubiquitin-proteasome system in controlling gene transcription in several diverse ways, from the regulation of chromatin, RNA polymerase II, through destruction of transcriptional activators [98]. Ubiquitylation is a specific process that is signalled by a degradation signal – degron – in the

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Ub

Ub

E1

Ub

Ub

Ub Ub

Ub

Ub Ub

Ub

Ub Ub

Rbx 1 Cul 1 E2 SCF complex (E3)

Ub

Skp 1 F box

26S Proteasome Substrate P

P

Substrate recognition domain

F-box protein

Fig. 2. The ubiquitin-proteasome system. Many proteins that regulate the cell cycle are degraded via the ubiquitin-proteasome system. Ubiquitylation is the process by which a polyubiquitin (Ub) chain is attached to the substrate protein to be degraded. A cascade of enzymes catalyses the addition of ubiquitin polymers to the protein substrates. E3 (ubiquitin ligase) recognizes the degradation signal, binds to the substrate protein and recruits E2 (ubiquitin-conjugating enzyme) to the substrate. E1 (ubiquitin-activating enzyme) activates Ub and transfer it to E2; E2 forms a thioster bound with activated ubiquitin and subsequently transfers it to E3. The E3 then catalyses the transfer of Ub groups to a lysine residue that is somewhere in the target protein. E3 enzymes, known as ubiquitin ligases/SCF complexes, control specificity in the ubiquitin system. The SCF complex consist of three core subunits (Cul, Skp1, Roc/Rbx1) that couple to one of several F-box proteins. While the F-box is the SCF-binding domain, the F-box protein has also a substrate-binding domain to ensure specificity. The specific F-box protein is Skp2 in the case of p27 and archipelago in the case of cyclin E ubiquitylation [103–105]. If a poly-Ub chain – linked by lysine 48 in ubiquitin itself – is formed, the substrate is targeted for destruction by the 26S proteasome. The 19S subunit of the proteasome recognizes the polyubiquitylated substrate, removes the Ub groups, unfolds the substrate and feeds it into the core of the 20S subunit where it is destroyed (adapted from Nakayama et al. [169]).

substrate protein. In response, a cascade of enzymes generically termed E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) catalyse the addition of ubiquitin polymers to the protein substrates. E3 recognizes the degron, binds to the substrate protein and recruits E2 to the substrate. E1 activates Ub and transfers it to E2; E2 forms a thioster bound with activated ubiquitin and subsequently transfers it to E3. The E3 then catalyses the transfer of Ub groups to a lysine residue that is somewhere in the target protein. If a poly-Ub chain forms – linked by lysine 48 in ubiquitin itself – the substrate is targeted for destruction by the 26S proteasome. The 19S subcomplex of the proteasome recognizes the polyubiquitylated substrate, removes the Ub groups, unfolds the substrate and feeds it into the core of the

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20S subcomplex where it is destroyed. If the poly-Ub chain is linked by lysine 63 in the ubiquitin molecule, or if it has less than four Ub-tags, proteolysis does not occur [98]. Generally, E3 enzymes control the specificity in the ubiquitin system (fig. 2). There are different types of E3 enzymes: the ring finger type E3 that contains a SCF complex consists of three core subunits (Cul, Skp1, Roc/Rbx1) that couple to one of several F-box proteins, so-called because they contain the F-box motif – a highly-conserved sequence of amino acids, first identified in cyclin F1. While the F-box is the SCF-binding domain, the F-box protein has also a substrate-binding domain to ensure specificity. In the case of p27, the specific F-box protein is Skp2 (S-phase kinase interacting protein 2, named as it was discovered through its interaction with the cyclin A-CDK2 complex in S phase). Skp2 cooperates with Cks1 to undergo allosteric alterations allowing it to bind phosphorylated p27. The highest level of p27 ubiquitylation occurs at the G1/S transition targeting lysine residues 134, 135 and 165 on the p27 molecule [99]. A number of other members of the cell cycle are also degraded via the ubiquitin-proteasome system: cyclin E, cyclin D, cyclin A, p58, p21 and E2F-1 [100–102]. In the case of cyclin E, the specific F-box protein is the recently-identified hCDC4/Fbw/Archipelago/Ago [103–105]. Mutations of this F-box protein have been described in various human cancers including breast, ovarian and endometrial tumours. Our preliminary studies indicate that Ago mRNA, contrary to expectation, is over-expressed in all subtypes of pituitary adenomas compared to normal pituitary, and we currently await the results of sequence analysis of these tumours [D.G. Morris et al., unpubl. observations]. In order to be recognized by the F-box protein and thus to lead to degradation, the ubiquitylation substrate (e.g. p27 or cyclin E) needs a prior phosphorylation step. In the case of p27 this process is operational in S and G2 phase as a result of increasing cyclin E/CDK2 activity which is responsible for nuclear phosphorylation of p27 on Thr187 [106]. p27 therefore can bind to cyclin E/CDK2 in two conformations: in a tight state, in the presence of high ATP concentrations, under which the kinase activity is inhibited [107], and secondly in a loose state, at low concentrations of ATP, under which, conversely, CDK2 phosphorylates p27. Thus, once cyclin E/CDK2 is activated, it can trigger p27 degradation accounting for the irreversibility of the subsequent entry into S phase [108]. p27 mutants (Thr187Ala) which are resistant to phosphorylation by cyclin E-CDK2 have been considered to be resistant to ubiquitylation [107]. p27 mutants that can be phosphorylated, but cannot bind the cyclin E-CDK2 complex, were also claimed to be refractory to ubiquitylation. In order to bind the cyclin E-CDK2 complex, p27 needs to be imported into the nucleus. Nuclear import of p27 occurs either via a nuclear localization signal-dependent mechanism (which requires a p27-CDK2 or p27-CDK4 complex) or via a mechanism that requires

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Mitogens Cytoplasm

Nucleus PI3K pathway

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Cyclin D p27 CDK P Thr157 assembly functions CDK4

Fig. 3. p27 degradation and cellular compartmentalization. Nuclear phosphorylation of p27 at Ser10 by an unknown kinase (?pK) allows the export of p27 to the cytoplasm, thus lessening p27 nuclear concentration. At G1/S transition, when the concentration of cyclin E exceeds that of p27, the CDK2/cyclinE complex triggers p27 phosphorylation at Thr187 within the nucleus. Phosphorylated p27 is then bound by Jab1 and exported to the cytoplasm, where the F-box protein Skp2 recognizes phosphorylated p27 and mediates its ubiquitylation. Cks1 allows Skp2 substrate recognition. Ubiquitinated p27 is then targetted to proteolysis by the 26S proteasome. Within the nucleus, MIF sequesters Jab1 away from p27, opposing its nuclear export. A parallel process takes place in the cytoplasm, where protein kinase B (AKT), activated via the PI3K pathway in response to mitogens, phosphorylates p27 at Thr157 and thus sequesters it in the cytoplasm away from its nuclear targets. It is believed that Thr157 phosphorylation of p27 promotes the interaction between cyclin D and CDK4 [adapted from 124].

a nuclear pore-associated protein (mNPAP60) that interacts with Arg90 of p27 and supports its transport across the nuclear membrane [109]. Until recently, it was thought that after nuclear phosphorylation on Thr187, p27 needed to be exported back into the cytoplasm in order to enter the ubiquitylation-degradation machinery. Nuclear p27 interacts with Jab1 (Jun activation domain-binding protein 1 [110]), a component of the signalosome which has been implicated in the nuclear export of phosphorylated p27 to the cytoplasm (fig. 3) [111, 112]. Mutated p27 that cannot bind to Jab1 is neither exported nor destroyed [112].

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However, other data suggest that cyclin E/CDK2 forms a trimeric complex with p27 and in a concentration-dependent manner facilitates its ubiquitylation and degradation by the proteasome within the nucleus without the need to be transported out [113, 114]. Although Jab1 translocates to the cytoplasm to increase the degradation of p27 (fig. 2), it also functions also as a transcriptional cofactor for AP-1 [115]. Therefore, the cytoplasmic translocation of the p27-Jab1 complex should also prevent the association between Jab1 and AP-1 transcription factor [115]. This would down-regulate the activity of the transcription factor in early G1 and prevent the activation of cell cycle regulatory genes [32]. Therefore, Coqueret [32] has suggested that p27 targets transcription factors involved in the activation of growth-promoting genes. While the importance of Thr187 phosphorylation in ubiquitin-mediated degradation of p27 is undisputed, two recent studies have shown that p27 can be degraded by the proteasome in early-mid G1, in a manner independent of Thr187 phosphorylation. Nakayama and colleagues [116] reported a novel cytoplasmic ubiquitin-ligase in Skp2 / mice that operates at G0/G1 boundary, and is responsible for an earlier phosphorylation-independent degradation of p27, while Malek et al. [117] showed that p27 mutant (Thr187Ala) animals have an operational proteolytic pathway that degrades p27 in early-mid G1 phase in a Skp2-dependent manner, implying phosphorylation at an unidentified site. Other sites of p27 subject to phosphorylation are of equal importance in the regulation of the CDKI activity. Phosphorylation on Ser10, an event occurring primarily during G0 and G1, results in p27 export from the nucleus to the cytoplasm. It is likely that phosphorylation on Ser10 and translocation to the cytoplasm allows the threshold levels of nuclear p27 to drop such that cyclin D/CDK2 complexes become active for efficient phosphorylation of p27 on Thr187 and its consequent ubiquitylation and degradation in the nucleus [118]. The kinase that phosphorylates p27 on Ser10 has not yet been identified, nor has the additional factor(s) necessary for the translocation of p27 from the nucleus to the cytoplasm. Some recent evidence points to Kis as the kinase responsible for Ser10 phosphorylation in G1 [119], and to protein kinase B (PKB/Akt) as the additional kinase necessary for p27 nuclear export [120]. Cytoplasmic phosphorylation of p27 due to Akt takes place on Thr157 of p27 [121–123], and leads to cytoplasmic displacement of p27. Because the Akt phosphorylation site in p27 is within its nuclear localization sequence (amino acids 151–166), this process affects cellular localization of p27. Phosphorylation at Thr157 by Akt does not alter total levels of p27, but rather modulates its cellular localization and hence its function as a CDKI. Subcellular compartmentalization and mislocalization of p27 in the cytoplasm sequesters it away from its nuclear target – CDK2 – and in addition cytoplasmic phosphorylation of p27 specifically

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enables cyclin D-CDK4 assembly function [124]. Recent findings suggest that cytoplasmic mislocalization of p27 induced by Akt phosphorylation, regardless of total p27 content, correlates with aggressive phenotype and poor survival prognosis in human breast cancer [122]. This finding provides a novel link between activation of the Akt pathway and cell cycle progression. Cytoplasmic mislocalization of p27 has been previously reported in human tumours and cell lines, including Barrett’s oesophageal adenocarcinoma, and is secondary to the loss of tuberin protein in tuberous sclerosis complex [125, 126]. The HER-2/Grb2/MAPK pathway is also involved in decreasing p27 stability leading to nuclear export of p27 and Jab1 and subsequent enhanced ubiquitin-mediated degradation of p27 in breast cancer cell lines [127]. Translational regulation can also occur via a U-rich element in the 5-UTR of p27 mRNA [128]. Hengst and colleagues [129] reported that p27 is translated primarily by an internal ribosome entry site (IRES) in the 5-UTR of the p27 mRNA that allows efficient cap-independent translation to occur. A class of RNA-binding proteins, known as embryonic lethal abnormal vision (ELAV/Hu), bind to 5-UTR of the p27, inactivate the IRES and inhibit p27 translation (fig. 3). Changes in active p27 levels can also occur in the absence of increased or decreased expression of p27 protein, by its sequestration in cyclin D/CDK4-p27 complexes. This mechanism occurs mainly in early G1, when mitogens activate the MEK/ERK pathway resulting in over-expression of cyclin D/CDK4. Binding of p27 to cyclin D/CDK4 in the cytoplasm seems to be needed for the stability of the complex and its nuclear import. In contrast, the arrest of the cell cycle initiated by TGF- stimulates another CDKI, p15, that binds to cyclin D/CDK4, rendering p27 free to inhibit cyclin E/CDK2. Regulation of p27 Transcription Transcriptional regulation of p27 has also been observed [130]. The promoter region of p27 gene contains binding sites for several transcription factors including Sp1, CRE, Myb, NFB and AFX. Forkhead transcription factors (FKHRL/AFX) which interact with the p27 promoter increase p27 mRNA synthesis and cause cell cycle arrest [131]. Tyrosine kinase receptor-mediated activation of phosphatidylinositol 3-kinase (PI(3)K) targets phosphorylation of PKB/Akt, which is then activated and causes phosphorylation of p27 and hence its sequestration in the cytoplasm, and also direct inhibition of AFX-mediated transcription of p27 and increased cyclin D [132]. A tumour suppressor gene encoding the dual specificity phosphatase PTEN inhibits phosphorylation of Akt by PI3K, leading eventually to transcriptional activation of p27. A striking correlation between PTEN expression and the level of p27 protein has been reported in primary thyroid carcinomas [133]. It has recently been shown that Skp2 can reverse the effect of PTEN on p27 accumulation, suggesting that Skp2

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is a rate-limiting factor in p27 ubiquitylation and degradation and that PTEN regulates this process [134]. Silencing of the p27 gene in the promoter region by increased methylation has also been described in rat pituitary GH3 cells [135]. p27 and Pituitary Tumorigenesis The role of p27 as a tumour suppressor gene does not fit well with Knudson’s ‘two-hit’ model, but is rather that of a haploinsufficient gene. p27/ mice show an increased growth rate, due to increased cellularity, testicular and ovarian cell hyperplasia and infertility, and hyperplasia of the pituitary intermediate lobe with nearly 100% mortality caused by these benign pituitary tumours [136]. p27/ mice express roughly 50% of the normal level of p27 protein and show an intermediate growth rate and also develop pituitary tumours. The wild-type p27 allele is not mutated and protein expression is not silenced in p27/ tumours as it is the case for tumours from p53/, Rb/, p19/, PTEN/ mice, which all show frequent mutation or loss of the remaining wild-type allele [76] consistent with Kundson’s ‘two-hit’ model [137]. This genetic behaviour is also observed in human tumours, where bi-allelic mutations in these tumour suppressor genes are frequently seen. LOH within the p27 region is a common feature of some human cancers such as acute myeloid leukaemia, acute lymphoblastic leukaemia, ovarian cancer and prostate cancer [73, 138–140]. In contrast, bi-allelic mutations in p27 or somatic mutation of p27, either by insertion/deletion/point mutation or by a large rearrangement, occur very infrequently in all human cancers examined to date [76]. However, a reduction in p27 appears to cooperate with mutations of other cell cycle-related genes towards tumour growth. Pituitary and thyroid C cell tumorigenesis are accelerated in Rb/ and p27/ mutant mice [141], leading to pituitary adenomas, while pituitary tumorigenesis is more aggressive in p18/ and p27/ doublemutant mice than in the separate knockouts [66]. Although the p27 gene was not found to be mutated in human pituitary tumours [142, 143], and its mRNA expression was similar in tumour samples in comparison with normal pituitaries, the load of p27 protein expression in corticotroph adenomas and pituitary carcinomas was shown to be much lower than those in normal pituitary tissue or other types of pituitary adenoma [144–146]. In respect of p27 degradation, in most pituitary adenomas phosphorylation at Thr 187 occurred in a similar manner to that seen in the normal pituitary (fig. 4) [147]. However, this appeared to be greatly increased in corticotroph adenomas. Jab1, which as noted above enables p27 to be exported from the nucleus, was not obviously over-expressed in adenomas sufficient to account for diminished nuclear p27 (fig. 4) [147]. Macrophage inhibitory factor (MIF), which has been reported to bind and hence inactivate Jab1, was also not changed in a direction

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*

* * *

* l

ma

r No

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*

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H ic ve TS essi stat a r t g Me Ag

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* *

*

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L

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H ve atic t TS essi tas gr g Me A

*p 0.05 Fig. 4. p27, P-p27 (Thr187), Jab1 and Ki67 immunostaining in pituitary tumours. Jab1, which enables p27 to be exported from the nucleus, was not obviously over-expressed in adenomas sufficient to account for diminished nuclear p27. However, it was noted that the very low levels of p27, both native and Thr-phosphorylated, seen in pituitary carcinoma appeared to correlate with increased Jab1. Data shown as mean SEM. *p 0.05, p 0.05 compared to normal [147].

which would explain the loss of nuclear p27 (fig. 5) [148]. However, it was noted that the very low levels of p27, both native and Thr-phosphorylated, seen in pituitary carcinoma appeared to correlate with increased Jab1 [147]. Since nuclear p27 is under-expressed in pituitary adenomas, we speculated that increased Skp2 could target it for increased degradation if its level was enhanced in adenomas in general. This proved not to be the case, with similar levels of mRNA and protein expression compared to normal pituitary. However, samples with high p27 protein expression showed significantly less Skp2 expression than samples with low p27 immunostaining, suggesting that increased Skp2 could play at least a part in this process (fig. 6) [149]. We further hypothesized that the Skp2 co-factor, Cks1, may be altered to increase degradation of p27, but no difference was observed in mRNA levels between

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Mean (SEM) nuclear MIF immunostaining (% of positive cells)

70

60

50

40

30

20

10

0 Normal pituitaries n 10

Pituitary adenomas n 44

Fig. 5. MIF immunostaining in pituitary tumours. Higher nuclear staining in the adenomas group has been observed, but whether this is a cause or a consequence of the tumoural process remains to be clarified [148].

normal pituitaries and pituitary adenomas (fig. 7) [M. Musat et al., unpubl. observations]. The present data suggest that inhibition of growth and tumour development is sensitive not only to the absolute levels of p27 protein, but also to its cellular compartmentalization. At a biochemical level this could be explained by the stoichiometric inhibition of CDK2 by p27 present in the nucleus. Very recent findings from our group have established up-regulation of Akt mRNA and protein in pituitary tumours compared to the normal pituitary [150], which may cause phosphorylation of p27 on Thr157 and cytoplasmic retention of p27. This remains to be confirmed. We also found Akt activity to be increased in GH3 cells kept in full serum media or stimulated with EGF compared to serum-starved cells, while this activity was blocked by the PI(3)K antagonist, wortmannin, implicating PI(3)K in the activation of Akt in these tumour cells. Studies of Akt activity in human pituitary tumour primary culture are awaited. Thus, p27 seems to play a role in inhibiting tumour growth in response to multiple oncogenic signals rather than that of a classic tumour suppressor gene, and this may be secondary to activation of Akt.

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Mean (SEM) nuclear Skp2 immunostaining (% strong moderate)

18 16 14 12 10

p 0.008

8 p 0.5

6 4 2 0

Normal pituitaries

Pituitary adenomas

Samples Samples with high p27 with low p27

Fig. 6. Skp2 immunostaining in pituitary tumours. No significant difference was seen between normal and tumorous pituitary tissue; individual tumour types had similar mRNA expression and variable protein expression. However, samples with high p27 protein expression showed significantly less Skp2 expression than samples with low p27 immunostaining [149].

Mean (SEM) Cks1 log input amount/ g RNA

1,000

100

10

L

ca Pit rc ui in tar om y a

PR

FS H

H G

FP A N

TH AC

N

or

m

al

0

Fig. 7. Cks1 mRNA expression in normal and tumorous pituitary. No significant difference was observed in Cks1 mRNA expression between normal pituitary and any of the pituitary adenomas [M. Musat, unpubl. data].

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Pituitary Tumour-Transforming Gene PTTG shows low expression normal human pituitary. By contrast, PTTG protein is highly expressed in various human tumours, including the pituitary, adrenal, kidney, liver and ovarian tumours [151]. While its mRNA levels are low in normal pituitary, increases in PTTG transcripts from more than 50% to more than 10-fold were recorded in the majority of a series of pituitary adenomas [152], with highest expression observed in hormone-secreting pituitary tumours which had invaded the sphenoid bone (stages III and IV) and concordant bFGF expression in all the tumours. The expression of PTTG in most pituitary tumours studied suggests that it represents an early change in pituitary tumorigenesis and may also be a novel marker of invasiveness in secreting pituitary tumours. This is discussed in more detail in a related chapter. p53 Family The p53 gene has been considered the most frequently mutated gene in human malignancies, being abnormal in approximately 50% of all human malignant tumours [153]. Several studies investigating p53 status in corticotroph tumours are available. While more than 50% of both invasive and non-invasive corticotroph adenomas from one series showed abnormal p53 immunostaining [154], no mutations were found in an independent series examined at the nucleotide level [155, 156]. On the basis of the existing data, the potential role of p53 as a major contributor to pituitary tumorigenesis, in particular with regard to the corticotroph lineage, cannot be entirely excluded but appears unlikely. At present there are no studies to assess the relationship between p53 and its ubiquitin ligase MDM2, and with the tumour suppressor genes p14/p19ARF and PTEN – both preventing p53 degradation by MDM2 – in pituitary tumours. Studies on the status of the new members of p53 family – p63 and p73 – in pituitary tumours and in corticotroph adenomas in particular are also awaited. Miscellaneous Peroxisome proliferator-activated receptor- (PPAR-) is an orphan receptor, a member of the nuclear receptor superfamily [157, 158] that functions as a transcription factor [159]. A recent report from Heaney et al. [160] showed that PPAR- expression in normal pituitary is restricted and colocalizes with ACTHsecreting cells in the pituitary intermediate region, and that treatment of corticotroph pituitary tumour cells in vitro induced G0/G1 arrest and apoptosis, indicating the functional significance of PPAR- expression in corticotroph tumours. PPAR- synthetic ligands such as thiazolidindione compounds induce cell cycle arrest and the proposed mechanism includes blockade of pRb phosphorylation by inhibiting cyclin D1-CDK4/6 activity [161], increasing p21 [162] and p27 expression [163]. Our own preliminary data show that PPAR- is

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expressed in all subtypes of pituitary tumours [C. Merulli et al., unpubl. observations]. More recent work from Heaney et al. [164] shows abundant expression of PPAR- in all types of human pituitary tumours, and demonstrated that high doses of the PPAR- ligand rosiglitazone is effective in limiting tumour growth in vitro. These authors have proposed the use of thiazolidinediones for managing pituitary tumours which are unresponsive to more conventional therapies. Krüppel-like factor 6 (KLF6) is a zinc finger transcription factor of unknown function which was recently shown to be a candidate tumour suppressor gene in prostate and colon cancer, showing LOH and being mutated in the remaining allele in more than 70% of tumours [165]. Functional studies have shown that wild-type KLF6 up-regulates p21 in a p53-independent manner and significantly reduces cell proliferation, while tumour-derived cell mutants do not [165, 166]. In order to investigate whether mutations of KLF6 are involved in the pathogenesis of sporadic pituitary tumours, we performed a sequence analysis of the coding region for KLF6 in 52 and genomic DNA in 11 pituitary tumours, finding three polymorphisms but no somatic mutation [167]. Conclusions

The cell cycle is the engine of cell proliferation, executing a well-oiled machinery according to a finely regulated set of instructions which are fundamental to tissue growth, and relatively invariant over several hundred, if not thousands, of millions of years of evolution. A series of perturbations of the process have been recorded in pituitary tumours, while transgenic disruption of the machinery frequently leads to pituitary adenomas in animal models. Very recent studies have also demonstrated how relatively minor changes to the control pathways can lead to deregulated DNA multiplication, which could in turn lead to major chromosomal duplications and deletions [168]. However, whether the cell cycle changes reported in pituitary tumours are truly causal remains unclear; at present, it is at least as probable that cytoplasmic changes in signalling pathways, such as activation of growth factor receptors and/or protein kinase B (Akt), feed into the cell cycle which then executes an aberrant set of instructions. It seems highly likely that the details of these mutant input pathways will become much more clear over the next few years. References 1 2

Morgan DO: Cyclin-dependent kinases: Engines, clocks and microprocessors. Annu Rev Cell Dev Biol 1997;13:261–291. Vidal A, Koff A: Cell-cycle inhibitors: Three families united by a common cause. Gene 2000; 247:1–15.

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Morgan D, Murray A, Hunt T, Nurse P: The cell cycle and programmed cell death; in Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (eds): Molecular Biology of the Cell. New York, Garland Science, 2002, pp 983–1026. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G: Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev 1993;7:812–821. Jeffrey PD, Russo AA, Polyak K, Gibbs E, Hurwitz J, Massague J, et al: Mechanism of CDK activation revealed by the structure of a cyclin A-CDK2 complex. Nature 1995;376:313–320. Pines J, Hunter T: Isolation of a human cyclin cDNA: Evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2. Cell 1989;58:833–846. Fisher RP, Morgan DO: A novel cyclin associates with MO15/CDK7 to form the CDK-activating kinase. Cell 1994;78:713–724. Akoulitchev S, Chuikov S, Reinberg D: TFIIH is negatively regulated by cdk8-containing mediator complexes. Nature 2000;407:102–106. Lee WH, Bookstein R, Hong F, Young LJ, Shew JY, Lee EY: Human retinoblastoma susceptibility gene: Cloning, identification and sequence. Science 1987;235:1394–1399. Lee WH, Shew JY, Hong FD, Sery TW, Donoso LA, Young LJ, et al: The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity. Nature 1987; 329:642–645. Zhu L, van den HS, Helin K, Fattaey A, Ewen M, Livingston D, et al: Inhibition of cell proliferation by p107, a relative of the retinoblastoma protein. Genes Dev 1993;7:1111–1125. Yeung RS, Bell DW, Testa JR, Mayol X, Baldi A, Grana X, et al: The retinoblastoma-related gene, RB2, maps to human chromosome 16q12 and rat chromosome 19. Oncogene 1993;8:3465–3468. Mayol X, Grana X, Baldi A, Sang N, Hu Q, Giordano A: Cloning of a new member of the retinoblastoma gene family (pRb2) which binds to the E1A transforming domain. Oncogene 1993;8: 2561–2566. Morris EJ, Dyson NJ: Retinoblastoma protein partners. Adv Cancer Res 2001;82:1–54. Weintraub SJ, Prater CA, Dean DC: Retinoblastoma protein switches the E2F site from positive to negative element. Nature 1992;358:259–261. Bremner R, Cohen BL, Sopta M, Hamel PA, Ingles CJ, Gallie BL, et al: Direct transcriptional repression by pRB and its reversal by specific cyclins. Mol Cell Biol 1995;15:3256–3265. Bosco G, Du W, Orr-Weaver TL: DNA replication control through interaction of E2F-RB and the origin recognition complex. Nat Cell Biol 2001;3:289–295. Paggi MG, Baldi A, Bonetto F, Giordano A: Retinoblastoma protein family in cell cycle and cancer: A review. J Cell Biochem 1996;62:418–430. Sala A, Nicolaides NC, Engelhard A, Bellon T, Lawe DC, Arnold A, et al: Correlation between E2F-1 requirement in the S phase and E2F-1 transactivation of cell cycle-related genes in human cells. Cancer Res 1994;54:1402–1406. Zhu L, Zhu L, Xie E, Chang LS: Differential roles of two tandem E2F sites in repression of the human p107 promoter by retinoblastoma and p107 proteins. Mol Cell Biol 1995;15:3552–3562. Moberg K, Starz MA, Lees JA: E2F-4 switches from p130 to p107 and pRB in response to cell cycle re-entry. Mol Cell Biol 1996;16:1436–1449. Weinberg RA: The retinoblastoma protein and cell cycle control. Cell 1995;81:323–330. Cress WD, Seto E: Histone deacetylases, transcriptional control and cancer. J Cell Physiol 2000; 184:1–16. Lipinski MM, Jacks T: The retinoblastoma gene family in differentiation and development. Oncogene 1999;18:7873–7882. Lipinski MM, Macleod KF, Williams BO, Mullaney TL, Crowley D, Jacks T: Cell-autonomous and non-cell-autonomous functions of the Rb tumor suppressor in developing central nervous system. EMBO J 2001;20:3402–3413. Hickman ES, Moroni MC, Helin K: The role of p53 and pRB in apoptosis and cancer. Curr Opin Genet Dev 2002;12:60–66. Pavletich NP: Mechanisms of cyclin-dependent kinase regulation: Structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors. J Mol Biol 1999;287:821–828. Serrano M, Hannon GJ, Beach D: A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 1993;366:704–707.

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Prof. Ashley B. Grossman Department of Endocrinology St. Bartholomew’s Hospital, London EC1A 7BE (UK) Tel. 44 20 7601 8343, Fax 44 20 7601 8505, E-Mail [email protected]

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Kontogeorgos G, Kovacs K (eds): Molecular Pathology of the Pituitary. Front Horm Res. Basel, Karger, 2004, vol 32, pp 63–95

Role of Regulatory Factors in Pituitary Tumour Formation Márta Korbonits, Damian G. Morris, Alexandra Nanzer, Blerina Kola, Ashley B. Grossman Department of Endocrinology, St. Bartholomew’s Hospital, London, UK

Abstract The molecular basis of pituitary tumorigenesis remains controversial, but there are two major theories which have been subject to most investigation: hormonal (usually hypothalamic factors) and/or growth factor overstimulation, or a molecular defect within the pituitary itself. It has been shown, for example, that excessive regulatory hormone stimulation can lead to an increased number of cells in the pituitary in various physiological or pathological states such as pregnancy (lactotrophs), untreated primary hypothyroidism (thyrotrophs and lactotrophs), primary hypoadrenalism (corticotrophs) and ectopic GHRH-secreting tumours (somatotrophs). Animal models also provide data that in the presence of excessive hypothalamic hormone stimulation, adenoma formation can occur. However, evidence in favour of the monoclonal nature of pituitary tumours argues for an intrinsic molecular defect as the primary initiating event in tumour formation. We review the various hormonal factors and their receptors effecting the different types of pituitary cells, such as CRH, AVP and cortisol feedback on corticotrophs; GHRH, G, PKA, somatostatin and GH and IGF feedback on somatotrophs; GnRH, LH/FSH, activin and oestrogen feedback on gonadotrophs; dopamine, oestrogen and prolactin feedback on lactotrophs; and TRH, TSH and thyroid hormone feedback on thyrotrophs. The monoclonal origin of adenomas makes it unlikely that hypothalamic factors could initiate pituitary transformation, but they could still create an environment where there is a higher chance that a possible causative tumorigenic mutation may occur in one (or several) of the overstimulated pituitary cells, or enhance the proliferation of an already-mutated cell. Copyright © 2004 S. Karger AG, Basel

The molecular basis of pituitary tumorigenesis remains controversial, but there are two major theories which have been subject to investigation: hormonal (usually hypothalamic factors) and/or growth factor overstimulation, or a molecular defect within the pituitary itself. Support for extrinsic hormonal

stimulation is suggested by certain clinical situations. It has been shown, for example, that excessive regulatory hormone stimulation can lead to an increased number of cells in the pituitary in various physiological or pathological states such as pregnancy (lactotrophs), untreated primary hypothyroidism (thyrotrophs and lactotrophs), primary hypoadrenalism (corticotrophs) and ectopic GHRH-secreting tumours (somatotrophs). Animal models also provide data that in the presence of excessive hypothalamic hormone stimulation adenoma formation can occur [1]. However, evidence in favour of the monoclonal nature of pituitary tumours argues for an intrinsic molecular defect as the primary initiating event in tumour formation. Using X-chromosomal inactivation analysis, the monoclonal origin of pituitary tumours was confirmed in female patients heterozygous for variant alleles of the X-linked genes [2, 3]. Recently, a modifying concept has been suggested regarding the clonality of pituitary adenomas: Farrell and colleagues [4] found that in 58% of recurrent pituitary adenomas the recurrent tumour was not from the same clone as the original tumour. This suggests that while there may be an intrinsic clonal defect, this may in some situations be oligoclonal rather than purely monoclonal. The monoclonal origin of adenomas makes it unlikely that hypothalamic factors could initiate pituitary transformation, but they could still create an environment where there is a higher chance that a possible causative tumorigenic mutation may occur in one (or several) of the overstimulated pituitary cells. It is likely that both theories have merit and that pituitary tumours fit into the multistep model of tumour formation dependent on the accumulation of mutational events coupled with hormonal and growth factor stimulation.

Corticotroph Adenomas

The hypercortisolaemia seen in Cushing’s disease is due to excessive secretion of ACTH by the pituitary. Pathologically, this is usually caused by a small pituitary adenoma only a few millimetres in diameter. Abnormal regulation of a corticotroph cell could, however, create an environment which would favour a possible tumorigenic process. Possible mechanisms of excess ACTH secretion from the pituitary gland may include (fig. 1): (1) increased amounts and/or overactive CRH and CRH receptors (CRH-R); (2) increased amounts and/or overactive AVP receptors; (3) a decreased level of ACTH receptors; (4) a decreased level of cortisol in the circulation; (5) decreased amounts or abnormal glucocorticoid receptors (GR) or increased amounts of the inactive GR isoform; (6) changes in pre-receptor regulatory mechanisms, such as 11-HSD, and (7) dysregulation of the steroid feedback factor, annexin-1 (lipocortin-1).

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Cortisol 4 Corticotroph cell Annexin-1 7 (Lipocortin-1)

6 Cortisol

Cortisol 11 -HSD2 Cortisone

11 -HSD1 Cortisone

1 CRH-R CRH

GR

Nucleus

5

3

2 V1b

ACTH-R

Fig. 1. Possible mechanisms of excess ACTH secretion from corticotroph cells (the numbers correspond to the list of possible mechanisms in the text).

1. Increased Amounts and/or Overactive CRH and CRH Receptors Excess CRH induces hyperplasia but cannot induce tumour formation in corticotrophs in animal models [5] or in humans with CRH-secreting tumours [6–10], but it may be a factor sustaining tumour development. In a recent study of 47 corticotroph tumours including 22 microadenomas, 15 macroadenomas and 6 locally-invasive tumours, CRH mRNA transcripts were detected in the adenoma samples using in situ hybridisation. The CRH mRNA signal intensity was linearly correlated with Ki-67 staining. The macroadenomas and the locally-invasive tumours showed significantly higher expression than the microadenomas, while CRH mRNA was expressed at much lower levels in 10 normal pituitaries [11]. This suggests that CRH can act as a local paracrine/autocrine factor and could also be an important factor associated with proliferative potential. Increased amounts of CRH-R were found in corticotroph adenomas in a study of 15 corticotroph tumours together with increased amounts of the V1b receptor [12]. Activating mutations in the CRH1-R was sought in 15 corticotroph adenomas but no abnormalities were found [13]. A two-fold increase was detected in the relative expression of the CRH1-R compared to normal pituitaries, but this could have been caused by the pure corticotroph cell population of the adenomas, since only 10–15% of the cells in normal pituitaries are corticotrophs. On the other hand, while corticotrophs in the normal pituitary show desensitisation after prolonged CRH stimulation, corticotroph adenoma cells lack any CRH1-R desensitisation [14]. CRH up-regulates CRH1-R mRNA levels in human corticotroph adenoma cells and in AtT-20 cells in vitro [14–16] but not in normal rat pituitary. Abnormal processing of CRH1-R may also be present in corticotroph adenomas [17]. Urocortin is a CRH-like molecule

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with strong expression in the normal pituitary, but it is absent from pituitary tumours [18]. 2. Increased Amounts and/or Overactive AVP Receptors Arginine-vasopressin is a potent ACTH-releasing hormone which acts synergistically with CRH. Although the pituitary-specific V1b receptor seemed to be overexpressed in some corticotroph tumours, no mutations have been reported [12, 19]. The overexpression may be a consequence rather than a cause of the excess levels of circulating cortisol. Mice overexpressing the V1b receptor in the pituitary have increased circulating corticosterone concentrations, but no tumour development was observed [20]. There are no data available regarding the expression of AVP itself in pituitary adenomas. 3. Decreased Amount of ACTH Receptors It has been postulated that ACTH may regulate its own secretion through an ultra-short loop feedback within the pituitary; as ACTH-secreting adenomas are characterised by relative resistance to glucocorticoid feedback, this may occur via dysregulated ACTH feedback. Early studies in animals suggested that an elevated plasma ACTH concentration could in itself inhibit the secretion of ACTH, independent of steroid feedback, and this was subsequently confirmed in murine corticotroph tumour cells [21], although not in normal rat pituitary tissue [22]. ACTH implants into the rat median eminence have also been shown to reduce circulating corticosteroid levels [23]. In the human, there is also evidence that ACTH may inhibit its own secretion via an ultra-short loop feedback on the pituitary. Infusions of a shorter [1–24 amino-acid] biologically-active ACTH were performed in patients with Addison’s disease treated with conventional replacement therapy [24]: endogenous ACTH secretion was measured using a highly specific two-site immunometric assay which did not cross-react with the injected short ACTH[1–24]. Endogenous ACTH was shown to be significantly reduced 15 min into the infusion compared to placebo; in addition, the endogenous ACTH response to CRH was inhibited by ACTH[1–24] infusion, suggesting that the effect was likely to be at the level of the pituitary rather than the hypothalamus. These results were contrary to an earlier paper in Addison’s patients that had apparently demonstrated no evidence of inhibition of endogenous ACTH by the synthetic ACTH analogue alsactide (-Ala1, Lys17-ACTH1–17-4-amino-N-butylamide) [25]. The reason for the discrepancy in findings between these two studies is not clear, although it has been postulated to be due to a possible difference in specificity between ACTH[1–17] and ACTH[1–24] at the receptor level [24]. We have studied the expression of the ACTH receptor (ACTH-R or melanocortin-2 receptor) in 13 normal pituitary specimens and 40 pituitary

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adenomas using real-time quantitative PCR [26]. ACTH-R mRNA was detected in normal human pituitary, but levels were undetectable in 11 out of 14 ACTHsecreting tumours. The corticotroph tumours which still contained ACTH-R transcripts did not show any mutations. Diagnostic pre-operative plasma ACTH levels were significantly lower in the ACTH-R-positive ACTH-secreting tumours compared with those which were ACTH-R-negative. These results provide further evidence compatible with an ultra-short ACTH feedback loop in the pituitary, and suggest that loss of expression of the ACTH-R in corticotroph adenomas of patients with Cushing’s disease may play a role in the resistance to feedback of the pituitary-adrenal axis seen in these patients. However, we were unable to demonstrate the expression of ACTH-R mRNA in normal mouse pituitary or in the mouse corticotroph adenoma cell line AtT20, and no effect of ACTH[1–24] was observed in normal rat pituitary culture or in AtT20 cells on either ACTH release or cAMP formation [27]. This may indicate a species difference between humans and rodents, as it has been described previously that the HPA feedback regulation is different in rats [28]. Interestingly, an activating mutation in the ACTH receptor has been identified in a patient with ACTH-independent Cushing’s syndrome, where a single nucleotide mutation caused abnormal desensitisation of the receptor which led to uncontrolled cortisol secretion from the adrenals [29]. 4. Decreased Amount of Cortisol in the Circulation One of the most characteristic biochemical features of Cushing’s disease is the relative resistance to corticosteroid feedback where the level of ACTH is reset to a new higher level of circulating cortisol; this relative resistance can also be used diagnostically, as demonstrated by dexamethasone suppression studies [30]. Lack of cortisol feedback occurs in untreated Addison’s disease and congenital adrenal hyperplasia, and the lack of negative feedback and unopposed hypothalamic stimulation could theoretically lead to pituitary tumour formation. ACTH-secreting adenomas have been described in untreated Addison’s disease and congenital adrenal hyperplasia [31–35]. The rarity of such cases, however, seems to indicate that diminished feedback suppression of ACTH-producing cells in Addison’s disease does not by itself induce the development of a pituitary adenoma, but might promote the growth of an independently and coincidentally occurring microadenoma. The lack of corticotroph hyperplasia in the non-tumorous adenohypophysis neighbouring the ACTH-secreting adenoma in a patient with Addison’s disease favours the interpretation that hypothalamic stimulation played no major role in adenoma formation in these patients [34], or at least it is not present by the time the tumour has formed. In an already abnormal corticotroph cell the complete lack of feedback can lead to further rapid tumour growth, such as after bilateral

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adrenalectomy with rapid enlargement of ACTH-secreting tumours (Nelson’s syndrome). 5. Decreased Amounts or Abnormal Active GRa or Increased Amounts of GRb Isoform The glucocorticoid receptor (GR) is present in the normal corticotroph and folliculo-stellate cells and all types of pituitary adenomas [36]. It has been speculated that a change in the number or function of the corticotroph GRs might decrease the activity of corticosteroid feedback, and thus allow selective clonal expansion of a mutated phenotype, but in general there is little evidence that the GR is abnormal in structure or function in the majority of corticotrophinomas [37, 38]. The GR has two splice variants, GR and GR. The GR is the active form of the receptor while the GR inhibits the function of the isoform. No significant abnormality was found in the relative expression of GR and GR, and no mutations were found in the GR gene, in 17 sporadic corticotroph adenomas [37]. An abnormal GR causing glucocorticoid-resistance syndrome, due to a dominant negative heterozygous mutation in one patient, caused a corticotroph adenoma, probably due to chronic corticotroph hyperstimulation and decreased glucocorticoid-negative feedback [39]. A somatic GR mutation has been described in the corticotroph adenoma from a patient with Nelson’s syndrome [40]. 6. Pre-Receptor Regulatory Mechanisms – 11b-HSD In the past, it had been thought that the major determinants of steroid hormone action were solely the amount of circulating hormone, the amount of binding proteins in the circulation and the amount of receptors in a particular tissue. However, there is an important additional level of regulatory control – enzyme-mediated pre-receptor metabolism (activation or inactivation) of a ligand. This control has been described for thyroid hormones (T4 to T3 conversion) and for androgens (testosterone-dihydrotestosterone conversion) as well. It is now recognised that 11-hydroxysteroid dehydrogenase (11-HSD), either by activating cortisol from cortisone (type 1 isoenzyme), or conversely inactivating cortisol to cortisone (type 2 isoenzyme), may play an important prereceptor role in regulating corticosteroid hormone action at some sites [41–44]. If 11-HSD1 and/or -2 were to be expressed within the anterior pituitary gland itself, they could, in theory, modulate glucocorticoid feedback: one could speculate that this might be deranged in Cushing’s disease. Furthermore, corticosteroids are also known to modify the secretion of other pituitary hormones such as growth hormone, thyroid-stimulating hormone, gonadotrophins and prolactin [45–48]. The expression and localisation of 11-HSD types 1 and 2 in normal and tumorous pituitary tissue were therefore investigated in order to

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11 -HSD1

11 -HSD2

Strong HSD1 staining

No HSD2 staining

Human kidney 11-HSD2

Normal human pituitary

11-HSD1

11-HSD2

No HSD1 staining

Strong HSD2 staining

Positive control

Corticotroph tumour

a

Cortisol

Cortisol

Reduced cortisol feedback

11 -HSD2

11 -HSD1

b

Cortisone

Cortisone

Fig. 2. a 11-HSD1 and 11-HSD2 staining in normal pituitary and corticotroph adenomas. Strong HSD1 staining was observed in normal pituitary, with no HSD2 staining, while in corticotroph adenomas strong HSD2 staining was seen with no HSD1 staining. Magn. 100 [modified from 49]. b Suggested mechanism of abnormal cortisol regulation by 11-HSD enzymes in corticotroph adenomas.

explore their possible role(s) in pituitary tumorigenesis, and specifically in Cushing’s disease [49]. In the normal pituitary, both 11-HSD type 1 mRNA and type 1 immunoreactivity was detected in each of the samples studied. Double-antigen labelling of the pituitary sections revealed co-localisation of 11-HSD type 1 enzyme with GH- and prolactin-secreting cells, while no co-localisation was observed with ACTH, TSH or LH/FSH [49]. The presence of 11-HSD1 in folliculo-stellate cells, identified by the specific non-hormonal marker S-100, was also detected. The majority of the normal pituitaries studied showed a detectable level of expression of 11-HSD type 2 mRNA. However, no 11-HSD type 2 immunoreactivity was detected by immunohistochemistry, in spite of positive immunofluorescence seen in the kidney control tissue (fig. 2). The majority of the pituitary adenomas showed the presence of both mRNA and immunoreactivity for 11-HSD1 and 2. Corticotroph tumours showed very little 11-HSD1 staining but strong 11-HSD2 staining, suggesting an induction of 11-HSD type 2 in these adenomas resulting in lack

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GHRH IGF rec

10

GH-R

GHRH 1 R

2

AC

ATP cAMP 9

Somatotroph cell

Pit1 Nucleus 5

8 SS-R

3 R R PKA 4 C C

7 SS

GHS-R TRH-R

C

C

CREB

P

6

Fig. 3. Possible mechanisms of excess GH secretion from somatotroph cells (the numbers correspond to the list of possible mechanisms in the text).

of cortisol ‘seen’ by the tumour which might play a role in the deranged glucocorticoid feedback in this tissue (fig. 2) [49]. 7. Annexin-1 (Lipocortin-1) Glucocorticoid inhibition of ACTH secretion also involves annexin-1 (lipocortin-1). Annexin-1 is produced by folliculo-stellate cells; these express GRs, and glucocorticoids induce annexin-1 synthesis. Annexin-1 is exported by folliculo-stellate cells and depresses peptide release from other pituitary cells (ACTH, GH, prolactin) by binding to cell surface annexin-1 receptors on endocrine cells and thereby serving as an autocrine and/or paracrine agent [50]. Pituitary adenomas express annexin-1 in non-endocrine cells and annexin-5 in both endocrine and non-endocrine cells in moderate amounts, but a pituitary carcinoma showed higher level of expression of both proteins [51]. Somatotroph Adenomas

Regulation of the somatotroph cell involves hypothalamic stimulating (GHRH) and inhibiting factors (somatostatin, SS), as well as feedback regulation by growth hormone and IGF-I. Possible derangements in the regulation of the somatotroph cell are shown in figure 3: (1) increased amounts and/or overactive GHRH and/or GHRH receptors; (2) constitutionally active G protein -subunit; (3) inactive PKA regulatory subunit; (4) constitutively-active PKA catalytic subunit; (5) constitutively-active CREB or Pit1 transcription factor; (6) abnormal TRH receptors; (7) increased amounts and/or overactive GHS-R receptors or ghrelin; (8) decreased amount of somatostatin or somatostatin

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receptors; (9) decreased amount or mutation of the GH receptor, and (10) decreased amount or mutation of the IGF-I receptor. 1. Increased Amounts and/or Overactive GHRH and GHRH Receptors GHRH is crucial in the control of pituitary somatotroph cell proliferation. GHRH-R inactivating mutations cause isolated GH deficiency and a hypoplastic pituitary gland [52–54]. GHRH-secreting ectopic tumours cause somatotroph hyperplasia in humans [55, 56] but not necessarily adenoma formation [57], while GHRH overexpression in transgenic animals resulted in true pituitary tumour formation in a large proportion, but not all, mice [1]. To study whether this effect occurs directly via GHRH or via the increased GH or IGF-I levels, transgenic mice with GHRH overexpression but lacking the GH receptor were created, with very low GH and IGF-I levels. Somatotroph hyperplasia and adenoma formation still occurred suggesting a direct role for GHRH [58]. Mice lacking the GH receptor also show hyperplasia with a distorted reticulin pattern [59]. However, the increased GHRH stimulation leading to high GH levels in patients with GH receptor abnormalities (GH insensitivity, Laron syndrome), or in the single patient described with an IGF-I mutation, does not cause pituitary enlargement [60, 61], possibly due to a lack of the trophic influence of IGF-I. These data suggest species-specific differences in the feedback regulation of the growth axis. GHRH mRNA and protein expression is present in the pituitary gland and was found to be higher in acromegalic tumours compared to the normal pituitary using in situ hybridisation, RT-PCR, Northern blotting and Western blotting [62, 63]. The GHRH mRNA signal was linearly correlated with invasiveness, pre-operative GH levels and Ki-67 staining, and was highest among tumours with post-operative recurrence [64, 65]. Interestingly, a lack of desensitisation of GH release in the presence of continuous GHRH was observed in somatotroph adenomas, unlike the normal pituitary, and it is therefore tempting to speculate that this might play a role in the development of acromegaly [66]. Similar lack of desensitisation was observed for CRH in human corticotroph adenoma cells [14], as mentioned above. As activating mutations of other G protein-coupled receptors cause the development of endocrine tumours, it has been speculated that somatic activating mutations of the GHRH-R might provide the molecular basis for somatotroph cell proliferation in a subset of human GH-secreting pituitary adenomas. The search for activating mutations in somatotroph adenomas of the GHRH-R gene led to the discovery of a relatively common single nucleotide polymorphism (SNP), A57T, which causes an increased cAMP response to GHRH in vitro, as observed by three different studies [67–70]. However, this change had been identified in the germ-line DNA of these acromegalic patients, suggesting that this is a SNP. The reported heterozygote frequency is 7–20%

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[67, 69]. It is very unlikely that this SNP plays a part in pituitary tumorigenesis, but could theoretically cause enhanced GH response to GHRH challenge [71, 72]. Another substitution (V225I) was also identified in the GHRH-R, which caused a similarly enhanced response. However, this was not confirmed in another study [73]. An alternatively spliced mRNA of the GHRH-R gene is also expressed in some somatotroph adenomas. The splice variant encodes a truncated, non-functional GHRH-R protein, although the pathophysiological importance of this variant has not been established [74]. 2. Constitutively-Active G Protein a-Subunit The first mutation ever identified in human pituitary tumours was the activating mutation in the -subunit of the G protein (gsp [Gs protein] mutation) linked to the GHRH receptor. It is present in about 40% of human somatotroph adenomas (less in Far-Eastern populations) causing constitutively-activated cAMP synthesis [75, 76]. These mutations change residue Arg201 to Cys or His and Glu227 to Arg or Leu. These residues are critically involved in ADP ribosylation and GTPase activity, and mutations constitutively activate the -subunit of the G protein by inhibiting its GTPase activity. According to the original hypothesis, the resulting elevated cAMP levels activate protein kinase A, which phosphorylates the cAMP response element binding protein (CREB) and leads to sustained GH hypersecretion and cell proliferation. However, there are several lines of evidence to challenge this hypothesis. While the gsp mutation would, in principle, confer growth advantage, the low growth rate of tumours with such mutations probably reflects the existence of mechanisms able to counteract the activation of the cAMP pathway. In gsp adenomas the activity of the cAMPdegrading enzyme phosphodiesterase is elevated compared to wild-type tissues. Moreover, gsp tumours highly express two nuclear transcription factors, CREB and the inducible cAMP early repressor (ICER), that are the final targets of the cAMP-dependent pathway and are positively regulated by cAMP signalling, i.e. the CREB and the ICER. The increased expression of the repressor transcription factor ICER, which competes with the binding of CREB to CREs, may inhibit the transcription of several cAMP responsive genes, including CREB itself. Finally, it has been shown that gsp adenomas contain very little gsp protein as detected by immunoblotting [77]. This is probably due to the increased degradation of the constantly activated Gs protein. The gene coding for Gs (GNAS1) gene is a tissue-specifically imprinted gene and is only expressed from the maternal allele in the normal pituitary [78]. Tumours with gsp mutations have the mutation on the maternal allele. During the later phase of tumorigenesis the imprinting is relaxed and a small amount of paternal expression also occurs both in gsp and gsp tumours [78]. Therefore, in gsp tumours, where the mutation is on the maternal allele and

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only this allele is expressed, the resulting constantly activated protein product is rapidly degraded: this could explain some of the phenotypic characteristics of patients with gsp somatotroph adenomas, since somatotroph adenomas with gsp mutations are smaller, have lower GH levels, do not respond briskly to GHRH, and are extremely sensitive to the effects of somatostatin analogues [76, 79–81]. Somatic, usually mosaic, gsp mutations in the GNAS1 gene cause the McCune-Albright syndrome. The McCune-Albright syndrome is characterised by polyostotic fibrous dysplasia, pigment patches of the skin and endocrinological abnormalities, including precocious puberty, thyrotoxicosis, pituitary gigantism, and Cushing’s syndrome. Gsp mutations have been (rarely) described in nonfunctioning pituitary adenomas (NFPAs) and corticotroph adenomas as well, and their occurrence together with a Gi2 mutation has also been described [82, 83]. Gsp mutations also occur in other endocrine tissues, either related or unrelated to pituitary disorders [84]. 3. Inactive PKA Regulatory Subunit The protein kinase A regulatory subunit 1 (PRKAR1A) has been identified as the gene responsible for Carney complex type I [85, 86]. Carney complex is a multiple neoplasia syndrome characterised by spotty skin pigmentation, cardiac and other (skin, mucous membrane) myxomas and endocrine tumours: Cushing’s syndrome from nodular adrenocortical dysplasia, acromegaly (or prolactinoma) caused by a pituitary adenoma, large-cell calcifying Sertoli cell tumours, Leydig cell tumours and psammomatous melanotic schwannomas [85, 87, 88]. The mechanism of tumorigenesis in patients with Carney complex involves constitutive activation of cAMP-dependent protein kinase A. In the absence of cAMP, PKA is an inactive tetrameric complex consisting of two regulatory (R) and two catalytic subunits (C) (fig. 3). Upon binding of two molecules of cAMP to each R subunit, activation proceeds by the dissociation of the PKA holoenzyme into a two R subunit dimer with four molecules of cAMP bound, and two free active C subunits [89]. The catalytic subunits promote downstream signalling by phosphorylation of serine and threonine residues on specific substrates [90]. In Carney complex type I, mutations of the PRKAR1A gene lead to defective binding of the regulatory subunit, and hence constitutive activation even in the absence of cAMP [85]. The Carney complex has been thought to exist in at least two genetically distinct forms, one of which maps to chromosome 17 and the other to chromosome 2. The chromosome 17 form is designated Carney complex type I. Is there a similar mechanism operating in sporadic somatotroph adenomas? We studied the PRKAR1A gene in 17 sporadic somatotroph as well as 17 other types of pituitary tumours, but found no mutations in the coding region of the gene [91]. The lack of mutations in sporadic somatotroph tumours has now been confirmed in another series [92]. We also

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analysed the relative expression of the gene in these adenomas and compared it to gene expression in cells from a patient with Carney complex type I. We found that all the sporadic pituitary adenomas expressed an equal amount of the gene to normal tissue, while the Carney complex positive controls had an undetectable level of expression due to nonsense-mediated decay. 4. Constitutively-Active PKA Regulatory Subunit The catalytic subunit of PKA has also been studied in 59 pituitary tumours and 22 normal pituitaries for activating mutations: none was found [93]. Protein kinase C is a calcium and phospholipase C-dependent protein participating in signal transduction. Its activity is increased in pituitary adenomas; a mutation was found in 4 invasive tumours, but this does not appear to be a common phenomenon [94]. 5. Constitutively-Active CREB or Pit-1 Transcription Factor The role of CREB has been studied in pituitary tumours: elevated levels of the phosphorylated form were found in almost all somatotroph tumours compared to NFPAs, but only 25% contained a gsp mutation [95]. This implies that there is upstream overactivity in this pathway in addition to, or in place of, gsp mutations. As yet, the nature of such factor or factors remains elusive. Pit-1 is a somato-, lacto- and thyrotroph cell-specific transcription factor; it is expressed in GH-, PRL- and TSH-secreting adenomas but occasionally in NFPAs as well [96]. Fifteen pituitary adenomas were studied but no mutations were found except a splice variant of 26 bp insertion which was identified in both tumour and germ-line DNA [97, 98]. 6. Abnormal TRH Receptors In patients with acromegaly, TRH can cause a paradoxical increase in GH and -subunit release, which is not observed in normal patients. The TRH receptor is expressed in somatotroph adenomas but no abnormality has been found in the TRH receptor gene [99, 100]. It seems unlikely that the paradoxical response to TRH, which is also seen in other non-tumorous situations, is an important causal factor in tumorigenesis. 7. Increased Amounts and/or Overactive GHS-R Receptors or Ghrelin Growth hormone secretagogues (GHSs) are synthetic compounds with GH-releasing ability. Their functional receptor, the GHS-R type 1a, was first identified from pituitary and hypothalamic tissue [101]. Functional GHS-R was shown to be present in human pituitary adenomas even before the receptor had been cloned, demonstrating GH release in response to GHS stimulation [102, 103]. Using both conventional and real-time RT-PCR we demonstrated that

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GHS-R1a was overexpressed in somatotroph adenomas [104, 105]. The increased expression of the GHS-R gene in the somatotroph tumours was obvious even when taking into account the fact that about 50% of the normal pituitary consists of somatotroph cells, while in a somatotroph adenoma all the cells are somatotrophs. The increased number of GHS-Rs in somatotroph tumours might explain why acromegalic patients show enormous GH rises after GHS injection [106–110]. Several other studies have also demonstrated the presence GHS-R1a mRNA in normal and adenomatous pituitary [111–115]. One of the studies suggested that somatotroph tumours express 200 times more GHS-R mRNA, and thyrotroph tumours 10 times more, than normal pituitaries [115]. The functionally-inactive GHS receptor type 1b mRNA was also detected in normal and neoplastic pituitary samples. Pre-operative GH values showed a significant correlation with GHS-R1a mRNA. The GHS-R1a was also sequenced for activating mutations, but none was found [116]. Somatotroph adenomas show expression of ghrelin mRNA (the endogenous GHS-R1a ligand) [117]. Ghrelin expression was relatively high in somatotroph adenomas compared to normal tissue. Comparing the expression of the mRNA of ghrelin, GHS-R1a and 1b, none showed any significant difference between the gsp and gsp groups. Recently, Kim et al. [118] found a negative correlation between the amount of ghrelin mRNA present in the sample and the size of the adenoma. The presence of ghrelin and its receptor within the pituitary suggest the possibility of a local paracrine effect. This possibility is supported by our recent studies where we have shown a proliferative effect of ghrelin on a rat somatotroph adenoma cell line [119]. We demonstrated that ghrelin stimulates [3H]-thymidine incorporation; the effect is via the MAPK pathway as phosphorylated ERK1/2 was increased on Western blotting, and both effects could be inhibited by the MAPK inhibitor U0126. We also investigated the signalling pathways and found that both the PKC and the tyrosine kinase pathway are involved in this effect [120]. It is therefore possible that up-regulation of ghrelin and the GHS-R1 in somatotroph tumours is involved in the proliferation of somatotroph adenoma cells, but the changes are quantitative rather than qualitative. 8. Decreased Amount of Somatostatin Receptors Somatostatin is produced in the periventricular and to a lesser degree in the arcuate nucleus of the hypothalamus, and has a profound inhibitory effect on GH release directly via the median eminence as well as indirectly via inhibiting GHRH neurons in the arcuate nucleus. It has been suggested that decreased somatostatic drive could be a factor in somatotroph adenomas. There have been conflicting reports regarding the hypothalamic somatostatinergic tone in acromegaly [121, 122], but a recent study using glucose-induced TSH suppression in patients with active and inactive acromegaly suggest an increased

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35

GH-R

IGF-R

% Positive cells on immunostaining

30 25 20 15

** ***

10 5 0

Normal acro

Normal acro

Fig. 4. GH-R and IGF-R expression in somatotroph adenomas compared to normal pituitary using immunohistochemistry. Somatotroph adenomas have significantly lower expression of both GH and IGF receptors. **p 0.01 [adapted from 131].

hypothalamic tone in active acromegalic patients, presumably as a consequence of the elevated GH and IGF-I levels [123]. Somatostatin is also expressed in the pituitary and therefore local paracrine or autocrine effects are possible [62]. In somatotroph adenomas, somatostatin expression is diminished compared to the normal pituitary resulting in a possibly reduced local effect [62, 124]. All the somatostatin receptors (SSTR15) are expressed in normal and adenomatous pituitary tissue with SSTR2 and SSTR5 being the most abundant and probably most important as well [125, 126]. Previously, it was thought that the effect of somatostatin on pituitary GH release is crucial for the maintenance of the pulsatile GH release from the pituitary; however, this theory has recently been challenged [127]. Absent or inactive somatostatin receptors could play a role in the increased GH release from the pituitary. However, no mutations were detected in 15 somatotroph tumours [128]. More recently, a germ-line mutation has been described in the SSTR5 in a patient with an octreotide-resistant somatotroph adenoma, but it is improbable that the somatostatin receptor is an important pathogenetic factor in the majority of somatotroph adenomas [129]. 9. & 10. Decreased Amounts or Mutation of the GH & IGF-I Receptor Feedback regulation of GH release at the pituitary level is exerted by GH and IGF-I via their receptors [130]. We therefore studied the mRNA and protein expression of the GH receptor and the type 1 IGF receptor genes in a range of pituitary tumours, and found decreased expression of both these genes in somatotroph adenomas (fig. 4), suggesting that decreased negative feedback via GH and IGF-I might play a role in the uncontrolled GH release in somatotroph adenomas [131]. We also sequenced the coding region of the GH receptor gene in 18 somatotroph adenomas but, apart from known polymorphisms, no other alteration of the sequence was identified [131]. No mutations were found in the IGF receptor -subunit in 19 somatotroph adenomas [132]. IGF-I is synthesised in both the normal and adenomatous pituitary gland [133] and lower

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GnRH

1 Activin

GnRH-R

Leptin 5

IIII IIII

3 Follistatin

II 2 LH/FSH

Gonadotroph cell

LEPRb 4 ER Nucleus ER

Fig. 5. Possible mechanisms of NFPA and gonadotroph tumorigenesis (the numbers correspond to the list of possible mechanisms in the text).

IGF-I and IGF-I receptor levels have previously been reported in somatotroph adenomas [134].

Non-Functioning and Gonadotroph Adenomas

Clinically, NFPAs represent approximately 25% of all pituitary tumours. Recent studies using a number of in vitro techniques have shown that the majority of such tumours produce gonadotrophins. Possible derangements of hormonal regulation of these tumours are shown in figure 5: (1) increased amounts and/or overactive GnRH and GnRH receptors; (2) altered LH or FSH isoforms; (3) decreased activin activity; (4) abnormal oestrogen feedback, and (5) leptin and leptin receptor. 1. Increased Amounts and/or Overactive GnRH and GnRH Receptors Gonadotrophin-releasing hormone (GnRH or LHRH) is synthesised in the hypothalamus and releases both LH and FSH from the pituitary gland in a highly regulated pulsatile fashion. Increased GnRH drive in patients with untreated primary hypogonadism could lead to the appearance of castration cells, to gonadotroph hyperplasia and possible adenoma formation [135], but it is difficult to rule out coincidence in these cases. In life-long hypogonadism, such as in Klinefelter’s syndrome, gonadotroph hyperplasia can occur. An intriguing, but unresolved, question is why gonadotroph hyperplasia is not apparent in old age when the

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negative feedback of peripheral hormones is lacking. Gonadotroph adenomas usually occur in the elderly but a causal relationship has not been proven [136]. GnRH has been shown to be expressed both in normal pituitary and in pituitary tumours [137, 138]. In the presence of the GnRH receptor, adenomas respond to GnRH with hormone release [137]. The presence of both GnRH and the GnRH receptor suggests that GnRH may be a paracrine/autocrine regulator of cell function in the pituitary and may affect gonadotroph tumour hormone phenotype [138]. 2. Altered LH or FSH Isoforms LH and FSH expression has been studied in gonadotroph and nonfunctioning adenomas. FSH -subunit mRNA was found to be in excess compared to -subunit mRNA in one-third of gonadotroph tumours, including 9 tumours where -subunit mRNA was undetectable. This is in contrast to data in the normal pituitary where -subunit is present in excess of -subunits at both the mRNA and protein levels. The free -subunit hypersecretion identified in pituitary adenomas may be due to biosynthetic abnormalities intrinsic to neoplastic gonadotrophs [139]. 3. Decreased Activin Activity Although activin stimulates FSH release, activin functions as an antiproliferative cytokine in some non-functioning pituitary tumours [140]. Activins interact with a dual receptor system involving TGF--type transmembrane serine/ threonine kinase receptors classed as type I or type II receptors (fig. 5). Although some tumours have been shown to have loss-of-function mutations in TGF--type receptors, a mutational analysis of the intracellular kinase domains of the type I and II activin receptors in 64 pituitary adenomas found that such somatic mutations are rare [141]. The activity of activins is modulated by follistatin via an extracellular protein-protein interaction. Follistatin has been identified in folliculo-stellate cells and could modulate the response of pituitary cells to activins [142]. Follistatin expression was reduced in gonadotroph adenomas compared with the normal pituitary [143], which might simply be explained by the lack of folliculo-stellate cells in pituitary adenomas. The effectors of downstream signalling events (e.g. Smads) and their role in pituitary tumours remains to be studied in detail. 4. Oestrogen Feedback Oestrogen has a negative feedback effect on gonadotroph cells. This effect was investigated in patients with gonadotroph-secreting adenomas as well as non-functioning adenomas. An oestrogen challenge caused inhibition of LH and FSH levels in control subjects while none of the gonadotroph adenoma

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patients and only 50% of the null cell NFPA patients showed similar inhibition, suggesting that loss of negative feedback and oestrogen resistance is present in these tumours, similar to that of thyroid hormone and glucocorticoid resistance observed in TSH- and ACTH-secreting tumours respectively [144]. The -subtype of the oestrogen receptor (ER) is expressed in the normal pituitary and in pituitary adenomas containing prolactin or gonadotroph-secreting cells [145, 146]. Several ER isoforms generated by alternative mRNA splicing have been identified [147]. Some of these have enhancing effects on wild-type ER while others are inhibitory. Coexpression of variant and wild-type receptors has been observed and is compatible with potential interactions between ER and its variant isoforms in oestrogen-sensitive pituitary cell types [148]. More recently, a form of the ER has also been recognised [149]: this is present in all types of pituitary adenomas [150]. ER was found to be coexpressed with ER and ER splice variants in 47% of oestrogen-responsive adenomas, i.e. prolactinomas and gonadotroph tumours. It seems that ER plays a minor role in oestrogenic effects when ER is also expressed but could have important transactivating roles in ER-negative pituitary tumours. Coexpression and interaction of various ER isoforms in pituitary tumours may be of pathophysiological relevance for the regulation of pituitary neoplastic cell proliferation and hormone biosynthesis in response to oestradiol. 5. Leptin and Leptin Receptor Leptin mRNA expression was detected in human pituitary tissues [151–153] and during in vitro culture pituitary tumours release leptin into the incubation media [153]. Expression of the leptin receptor long isoform (LEPRb) mRNA was also detected in the normal pituitary as well as in the majority of pituitary adenomas assessed [151, 154, 155], while in tissue culture leptin stimulated the specific release of FSH, -subunit or TSH from NFPAs or somatotroph tumours [153]. These data suggest that leptin is contained within and may be released from a variety of different pituitary cell types, and may particularly affect the local secretion of FSH. The observed co-localisation with several pituitary hormones [152] suggests that hypothalamic stimulating and inhibitory factors may influence its release. Since leptin has also been shown to have anti-proliferative effects in pituitary cell lines [151], the current data are supportive of a role for pituitary-derived leptin in the local regulation of pituitary function as well as systemic effects.

Lactotroph Cell

Possible derangements of hormonal regulation of lactotroph cells are shown in figure 6: (1) dopamine receptor; (2) prolactin-releasing peptide;

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Dopamine

3

BMP-4

1

5

PRL-R IIII IIII

D2R

2

noggin Noggin

I II PrRP-R PrRP Lactotroph cell ER 4 Nucleus

ER

PACAP-R

6 PACAP & VIP

Fig. 6. Possible mechanisms of lactotroph tumorigenesis (the numbers correspond to the list of possible mechanisms in the text).

(3) prolactin feedback; (4) oestrogen feedback and oestrogen receptors; (5) bone morphogenetic protein-4 (BMP-4), and (6) PACAP and VIP. 1. Dopamine Receptor Dopamine is the hypothalamic inhibitor hormone for lactotroph cells. Its main receptor in the pituitary is the type 2 dopamine receptor (D2R), and D2R knockout mice show development of lactotroph hyperplasia followed by lactotroph tumour formation [156]. Typically, male animals develop small tumours later in life while females develop large tumours much earlier [157]. No mutations were identified in the D2R in 79 human pituitary adenomas including prolactinomas and somato-mammotroph tumours [158]. Some lactotroph adenomas are unresponsive to dopaminergic therapy and cell lines derived from these types of tumours show more rapid growth. D2R expression is low in these tumours but can be increased with nerve growth factor treatment [159]. It has been recently suggested that D2R expression is regulated by nerve growth factor via a NF-B-dependent pathway [160]. 2. Prolactin-Releasing Peptide Prolactin-releasing peptide (PrRP) is a hypothalamic peptide whose specific receptor is present in the normal pituitary gland [161] as well as in all types

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of pituitary tumours [162]. PrRP receptor (PrRP-R) expression is inhibited by dopamine agonist treatment in human lactotroph adenomas [163], which could be important in the therapeutic effect of dopamine agonists, although the level of the PrRP-R mRNA expression shows no correlation between plasma prolactin levels and PrRP-R expression levels [162]. Interestingly, PrRP may have a much wider role in human biology following the finding that there is an association of polymorphisms in the gene encoding the PrRP-R with blood pressure, but not obesity, in a UK Caucasian population [164]. 3. Prolactin Feedback Prolactin has negative feedback on its own secretion via the prolactin receptor both at the level of the hypothalamus and the pituitary [165]. Prolactin receptor-deficient mice develop large prolactinomas, larger than the ones with D2R deficiency [165]. Normal lactotrophs express high levels of the prolactin receptor and prolactinomas have a higher prolactin receptor expression, while dopamine agonist treatment decreases the level of prolactin receptors [166]. 4. Oestrogen Rising oestrogen levels are responsible for the rising prolactin levels and lactotroph hyperplasia resulting in pituitary enlargement during pregnancy, and this can be especially pronounced in pre-existing prolactinomas where careful monitoring and occasionally medical therapy is necessary [167]. Interestingly, somatotroph cells can ‘switch’ to lactotroph cells during pregnancy [168]. Oral oestrogen-containing contraceptives do not increase the risk of prolactinomas [169] and they are not associated with increased tumour growth, at least in the short term [170]. However, unopposed oestrogen administration has been implicated in the pathogenesis of prolactinoma in a male-female transsexual individual [171]. In animal studies, oestrogen treatment is associated with an increase in many factors which have been shown to promote tumorigenesis, including vascular endothelial growth factor, pituitary tumour transforming gene and galanin, and oestrogens can cause true adenomas in rodents [172]. Somatostatin receptor expression is inhibited by oestrogen in lactotroph cells [173]. Prolactinomas contain the highest concentrations of ERs of all the pituitary tumour types, and especially high concentrations have been found in macroprolactinomas [174]. In a study of ER splice variants, 9 of 11 prolactinomas expressed multiple ER variants apart from the wild-type ER [148]. ER is also expressed in lactotroph cells and the ER mRNA was coexpressed with ER and its splice variants in 60% of prolactinomas and in 100% of mixed GH/PRL adenomas [150]. Oestrogen-mediated effects in normal and neoplastic pituitary appear to be highly dependent on the expression of ER and ER isoforms, which have varying transcriptional activities. Dominant

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negative mutant ER suppresses lactotroph cell growth and the prolactin promoter [175]. 5. Bone Morphogenetic Protein 4 (BMP-4) Bone morphogenetic proteins (BMPs), similar to activins, belong to the TGF- family and are multifunctional growth factors involved in many aspects of tissue development and morphogenesis. It has been shown that BMP-4 is important in pituitary development [176]. The secreted polypeptide, noggin, binds and inactivates BMP-4, with an interaction similar to activin and follistatin. BMP-4 is overexpressed in prolactinomas, including oestradiol-induced rat prolactinomas and human prolactinomas, compared with normal tissue and other pituitary adenoma types. BMP-4 stimulates, and noggin blocks cell proliferation and the expression of c-myc in human prolactinomas, whereas BMP-4 has no action in other human pituitary tumours. Smad 4 is often involved in the signalling pathway of TGF- family members and it has been shown that Smad 4 stimulated by BMP-4 interacts with ER in prolactinoma cells [177]. 6. PACAP and VIP Pituitary adenylate cyclase-activating peptide (PACAP) is present in normal and all types of pituitary adenoma cells and there is a positive response in terms of proliferation and hormone synthesis by most of the pituitary cell lines, as well as primary cultures of human adenomas. PACAP receptors recognise VIP as well and may signal the effect of VIP in lactotroph cells [178]. Their role in pituitary tumorigenesis is unknown.

Thyrotroph Cell

Possible derangements of hormonal regulation of thyrotroph cells are shown in figure 7: (1) increased amounts and/or overactive TRH and TRH receptors; (2) increased TSH, and (3) decreased amounts thyroid hormone receptor. 1. TRH Pituitary hyperplasia and enlargement occurs in patients with untreated hypothyroidism which could also lead to increased prolactin levels due to high TRH stimulation of the pituitary, and might create a differential diagnostic problem for patients with pituitary enlargement and high prolactin levels [179, 180]. TRH is expressed in the normal pituitary and pituitary adenomas and has been shown to be under thyroid hormone control [181, 182]. TRH receptor

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TRH TRH-R 2 TSH

1 q/11

Thyrotroph cell

TSH-R

3 Nucleus

TR T3/T4

Fig. 7. Possible mechanisms of thyrotroph tumorigenesis (the numbers correspond to the list of possible mechanisms in the text).

mRNA is expressed in normal pituitary and all types of pituitary tumours [183]. No activating mutations were detected in the TRH receptor gene in thyrotroph or any other pituitary tumours [99, 100]. The TRH receptor signals through the Gq and G11 G proteins but no dominant somatic mutations have been found in these genes in pituitary adenomas [99]. 2. TSH TSH synthesis and secretion is under the positive control of thyrotrophinreleasing hormone and under the negative control of the thyroid hormones. However, it is hypothesised that TSH has a direct effect on the regulation of its own synthesis through an intrapituitary loop mediated by pituitary TSH receptors [184]. The TSH receptor was identified in thyrotroph and folliculo-stellate cells only [184]. Examination of 58 pituitary adenomas, including two clinically-active and two clinically-inactive thyrotroph adenomas, with double staining for pituitary hormones and the TSH receptor, revealed TSH-R immunopositivity in only the two clinically-inactive thyrotroph adenomas [185]. Mice homozygous for the targeted disruption of the glycoprotein hormone -subunit (part of the TSH molecule) display hypertrophy and hyperplasia of the anterior pituitary thyrotrophs which can be reversed by thyroid hormone treatment [186]. Recently, a patient has been described with thyroid hormone resistance due to a mutation in the -isoform of the thyroid hormone receptor, who also

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Table 1. Factors that are potential messengers in the anterior pituitary gland -MSH -Subunit Activin Adenosine, ATP Angiotensin II Bombesin and gastrinreleasing peptide Bone morphogenic protein and noggin Brain-derived neurotrophic factor Calcitonin gene-related peptide Eicosanoids Endothelins Epidermal growth factor

Follistatin Galanin Kinins, kallikrein GnRH GH Hypoxia-inducible factor IGF-I IL-6, IL-11 Leukaemia-inhibitory factor Nerve growth factor Macrophage inhibitory factor Neuromedin B, neuromedin U

Nitric oxide NPY Opioid peptides Oxytocin POMC fragments PRL Substance P and neurokinin A Transforming growth factors TRH Urocortin VEGF VIP

harboured a pituitary adenoma, raising the question that thyroid hormone resistance predisposes to pituitary hyperplasia and adenoma development [187]. 3. Thyroid Hormone Receptor Thyroid hormone receptor mRNA was detected at different amounts in pituitary adenomas [188]. In the thyroid resistance syndrome, caused by a mutation in the thyroid hormone receptor, pituitary enlargement was observed due to thyrotroph hyperplasia, but this could be reversed with high-dose thyroid hormone treatment [189]. Aberrant alternative splicing of thyroid hormone receptor in a TSH-secreting pituitary tumour is a mechanism which may cause hormone resistance [190]. Normal mRNA levels but undetectable protein levels of the thyroid hormone receptor were observed in two TSH-secreting adenomas, suggesting abnormal post-translational processing and a possible role of the resulting thyroid resistance in the tumour formation [191].

Local Paracrine/Autocrine Effects

In addition to the classical hypothalamic stimulatory and inhibitory factors and feedback signals from the target organs, there is an increasing catalogue of factors known to be produced in the pituitary (table 1). The local regulation of pituitary function by these factors has been described in some excellent reviews [192–195].

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Conclusions

There are a plethora of alterations in the regulatory pathways in many types of pituitary adenoma, including those affecting feedback pathways and cell signalling messengers. However, most of these are quantitatively rather than qualitatively different to the normal pituitary, and it is difficult to avoid the conclusion that these are mostly a consequence of the proliferative process rather than being directly causal. As such, these are likely to be epiphenomena which are of some interest in understanding pituitary pathophysiology but are not directly pathogenetic. Nevertheless, there may be certain changes, such as the decreased expression of some of the feedback receptor pathways in some tumours, which may be additional changes to other, more fundamental mutations, but which aid in the survival and proliferative advantage of certain clones. The tumorigenic process has been divided into two basic steps: initiation and progression. These processes may be supported by different mechanisms acting in parallel as well as in sequence. In our opinion, it seems probable that changes in feedback pathways may occur in pituitary adenomas to enhance the survival of a selected clone, or which engender a proliferative process which acts as an enhancing landscape to mutant selection.

Acknowledgements We are grateful for Prof. K. Kovacs for helpful suggestions on the manuscript. Support was provided by the Medical Research Council, the Joint Research Board and the Cancer Research Committee of St. Bartholomew’s Hospital.

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167 Abboud CF, Ebersold MJ: Prolactinomas; in Thapar K, Kovacs K, Scheithauer BW, Lloyd RV (eds): Diagnosis and Management of Pituitary Tumors. Totawa, Humana Press, 2001, pp 279–294. 168 Vidal S, Horvath E, Kovacs K, Lloyd RV, Smyth HS: Reversible transdifferentiation: Interconversion of somatotrophs and lactotrophs in pituitary hyperplasia. Mod Pathol 2001;14:20–28. 169 Wingrave SJ, Kay CR, Vessey MP: Oral contraceptives and pituitary adenomas. Br Med J 1980; 280:685–686. 170 Corenblum B, Donovan L: The safety of physiological estrogen plus progestin replacement therapy and with oral contraceptive therapy in women with pathological hyperprolactinemia. Fertil Steril 1993;59:671–673. 171 Kovacs K, Stefaneanu L, Ezzat S, Smyth HS: Prolactin-producing pituitary adenoma in a maleto-female transsexual patient with protracted estrogen administration. A morphologic study. Arch Pathol Lab Med 1994;118:562–565. 172 Asa SL, Ezzat S: The pathogenesis of pituitary tumours. Nat Rev Cancer 2002;2:836–849. 173 Visser-Wisselaar HA, Van Uffelen CJ, Van Koetsveld PM, Lichtenauer-Kaligis EG, Waaijers AM, Uitterlinden P, et al: 17-Estradiol-dependent regulation of somatostatin receptor subtype expression in the 7315b prolactin secreting rat pituitary tumor in vitro and in vivo. Endocrinology 1997; 138:1180–1189. 174 Stefaneanu L, Kovacs K, Horvath E, Lloyd RV, Buchfelder M, Fahlbusch R, et al: In situ hybridization study of estrogen receptor messenger ribonucleic acid in human adenohypophysial cells and pituitary adenomas. J Clin Endocrinol Metab 1994;78:83–88. 175 Lee EJ, Duan WR, Jakacka M, Gehm BD, Jameson JL: Dominant negative ER induces apoptosis in GH(4) pituitary lactotrope cells and inhibits tumor growth in nude mice. Endocrinology 2001; 142:3756–3763. 176 Rosenfeld MG, Briata P, Dasen J, Gleiberman AS, Kioussi C, Lin C, et al: Multistep signaling and transcriptional requirements for pituitary organogenesis in vivo. Recent Prog Horm Res 2000;55: 1–13. 177 Paez-Pereda M, Giacomini D, Refojo D, Nagashima AC, Hopfner U, Grubler Y, et al: Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk. Proc Natl Acad Sci USA 2003;100:1034–1039. 178 Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, Vaudry H: Pituitary adenylate cyclase-activating polypeptide and its receptors: From structure to functions. Pharmacol Rev 2000;52: 269–324. 179 Ghannam NN, Hammami MM, Muttair Z, Bakheet SM: Primary hypothyroidism-associated TSHsecreting pituitary adenoma/hyperplasia presenting as a bleeding nasal mass and extremely elevated TSH level. J Endocrinol Invest 1999;22:419–423. 180 Alkhani AM, Cusimano M, Kovacs K, Bilbao JM, Horvath E, Singer W: Cytology of pituitary thyrotroph hyperplasia in protracted primary hypothyroidism. Pituitary 1999;1:291–295. 181 Pagesy P, Croissandeau G, Le Dafniet M, Peillon F, Li JY: Detection of thyrotropin-releasing hormone mRNA by the reverse transcription-polymerase chain reaction in the human normal and tumoral anterior pituitary. Biochem Biophys Res Commun 1992;182:182–187. 182 Le Dafniet M, Brandi AM, Kujas M, Chanson P, Peillon F: Thyrotropin-releasing hormone (TRH)-binding sites and thyrotropin response to TRH are regulated by thyroid hormones in human thyrotropic adenomas. Eur J Endocrinol 1994;130:559–564. 183 Kim K, Arai K, Sanno N, Teramoto A, Shibasaki T: The expression of thyrotrophin-releasing hormone receptor 1 messenger ribonucleic acid in human pituitary adenomas. Clin Endocrinol (Oxf) 2001;54:309–316. 184 Prummel MF, Brokken LJ, Meduri G, Misrahi M, Bakker O, Wiersinga WM: Expression of the thyroid-stimulating hormone receptor in the folliculo-stellate cells of the human anterior pituitary. J Clin Endocrinol Metab 2000;85:4347–4353. 185 Theodoropoulou M, Arzberger T, Gruebler Y, Korali Z, Mortini P, Joba W, et al: Thyrotrophin receptor protein expression in normal and adenomatous human pituitary. J Endocrinol 2000;167:7–13. 186 Brinkmeier ML, Stahl JH, Gordon DF, Ross BD, Sarapura VD, Dowding JM, et al: Thyroid hormone-responsive pituitary hyperplasia independent of somatostatin receptor 2. Mol Endocrinol 2001;15:2129–2136. 187 Safer JD, Colan SD, Fraser LM, Wondisford FE: A pituitary tumor in a patient with thyroid hormone resistance: A diagnostic dilemma. Thyroid 2001;11:281–291.

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188 Wang CJ, Howng SL, Lin KH: Expression of thyroid hormone receptors in human pituitary tumor cells. Cancer Lett 1995;91:79–83. 189 Gurnell M, Rajanayagam O, Barbar I, Jones MK, Chatterjee VK: Reversible pituitary enlargement in the syndrome of resistance to thyroid hormone. Thyroid 1998;8:679–682. 190 Ando S, Sarlis NJ, Krishnan J, Feng X, Refetoff S, Zhang MQ, et al: Aberrant alternative splicing of thyroid hormone receptor in a TSH-secreting pituitary tumor is a mechanism for hormone resistance. Mol Endocrinol 2001;15:1529–1538. 191 Gittoes NJ, McCabe CJ, Verhaeg J, Sheppard MC, Franklyn JA: An abnormality of thyroid hormone receptor expression may explain abnormal thyrotropin production in thyrotropin-secreting pituitary tumors. Thyroid 1998;8:9–14. 192 Denef C: Autocrine/paracrine intermediates in hormonal action and modulation of cellular responses to hormone; in Conn M (ed): Handbook of Physiology – Cellular Endocrinology. New York, Oxford University Press, 1998, pp 461–514. 193 Denef C: Paracrine mechanisms in the pituitary; in Imura H (ed): The Pituitary Gland. New York, Raven Press, 1994, pp 351–378. 194 McNicol AM: Gene expression in pituitary adenomas: New insights. Microsc Res Tech 1997;39:182–193. 195 Jones TH, Brown BL, Dobson PRM: Paracrine control of anterior pituitary hormone secretion. J Endocrinol 1990;127:5–13.

Márta Korbonits MD, PhD MRC Clinician Scientist, Senior Lecturer in Endocrinology Endocrine Oncology, Department of Endocrinology, St. Bartholomew’s Hospital 59 Bartholomew Close, Unit 1.1, London EC1A 7BE (UK) Tel. 44 20 7601 8613/8746, Fax 44 20 7601 7015, E-Mail [email protected]

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Kontogeorgos G, Kovacs K (eds): Molecular Pathology of the Pituitary. Front Horm Res. Basel, Karger, 2004, vol 32, pp 96–109

Growth Factors and Cytokines: Function and Molecular Regulation in Pituitary Adenomas Ulrich Renner, Marcelo Paez-Pereda, Eduardo Arzt, Günter K. Stalla Department of Endocrinology, Max Planck Institute of Psychiatry, Munich, Germany

Abstract The growth and functions of the anterior pituitary cells are regulated by hypothalamic factors, peripheral hormones and growth factors. However, the expression of numerous growth factors and cytokines, as well as their receptors, within the anterior pituitary suggests that these factors could be locally involved in the control of pituitary development, function and proliferation by auto-/paracrine mechanisms. In the normal pituitary, intrapituitary factors probably play a role in modifying endocrine signals in the pituitary cells. However, since alterations in growth factor production and receptor expression have been found in pituitary adenomas, these factors may also contribute to the pathophysiology and progression of pituitary tumours. The potential roles of the most important growth factors and cytokines in pituitary adenoma pathogenesis are reviewed. Copyright © 2004 S. Karger AG, Basel

Auto-/Paracrine Interactions in Pituitary and Pituitary Adenomas

Pituitary cells synthesize and release numerous growth factors and cytokines (table 1), and express their corresponding receptors [1]. This has led to the hypothesis that anterior pituitary cells are not only regulated in an endocrine manner by hypothalamic factors and circulating peripheral hormones, but also by locally produced auto- or paracrine-acting factors [1, 2]. It has been speculated that intrapituitary factors could play a role in pituitary physiology by modulating the response of pituitary cells to extrapituitary stimulation [1, 2]. There is increasing evidence that intrinsic growth factor production could be stimulated or inhibited by extrapituitary factors, which suggests that the effects of the latter could partially be mediated, enhanced or dampened by intrapituitary factors [1, 2].

Table 1. Overview about the expression of the most relevant growth factors and cytokines in normal pituitary and pituitary tumours Growth factors and cytokines

Expression in normal pituitary

Expression in pituitary adenomas

TGF-␤1 TGF-␤3 Activin G

L, FS L, FS

Inhibin

G

Follistatin

FS

BMP-4

n.k.

EGF

C, S, G, T

TGF-␣ FGF-2 NGF

C, S, G, T FS, L L

IL-6

FS

LIF

C, FS

Reduced expression in rat prolactinomasa Increased expression in rat prolactinomasa Expressed in human inactive and gonadotroph adenomas Expressed in human inactive and gonadotroph adenomas Reduced expression in human gonadotroph adenomas Over-expression in mice prolactinomasb and human prolactinomas No over-expression in human pituitary adenomas Over-expression in rat prolactinomasa Over-expression in rat prolactinomasa Reduced expression in dopamine-resistant human prolactinomas Tumor cell-derived production in the majority of human pituitary adenomas Reduced expression in human prolactinomas

C ⫽ Corticotroph cells; FS ⫽ folliculostellate cells; G ⫽ gonadotroph cells; L ⫽ lactotroph cells; S ⫽ somatotroph cells; T ⫽ thyreotroph cells. See text for abbreviations of growth factors. aEstradiol-induced prolactinomas in Fischer 344 rats. bProlactinomas in dopamine D2 receptor knock-out mice.

In pituitary adenomas, an altered expression of cytokines/growth factors and their receptors has been observed [1–3] (tables 1, 2). Although it is unlikely that these alterations play a causative role in pituitary tumour pathogenesis, the intratumoural changes of these factors and their receptors may contribute to the excessive hormone production and the loss of growth control in pituitary adenomas [4, 5].

The Transforming Growth Factor-␤ Protein Family

Among the more than 30 members of the transforming growth factor-␤ (TGF-␤) protein family [6], TGF-␤1, TGF-␤3, activin, inhibin and, more

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Table 2. Overview about growth factor receptors which are probably involved in pituitary tumour development Growth factor receptor

Expression and function in pituitary tumours

Truncated ALK4 activin receptor

Expressed in different types of pituitary adenomas. Truncated ALK4 binds activin without inducing its antiproliferative effects Epidermal growth factor receptor expression correlates with tumour aggressiveness in somatotropinomas and nonfunctioning tumours Constitutively active, oncogenic variant of the EGF receptor. Membrane expression was found in a single corticotroph carcinoma Pituitary tumour-derived, N-terminally truncated isoform of wild-type fibroblast growth factor receptor type 4. ptd-FGFR4 is abundantly expressed in human pituitary adenomas. ptd-FGFR4 transgenic mice develop pituitary adenomas Missing in dopamine agonist-resistant human prolactinomas. Re-expression induces dopamine agonist sensitivity

EGFR

ERBB2

ptd-FGFR4

Nerve growth factor receptor p75NGFR

recently, bone morphogenetic factor 4 (BMP-4) have all been identified as important modulators of normal and/or adenomatous anterior pituitary cell function and growth. The TGF-␤ isoforms, activin and BMP-4, bind to different, specific type 1 and type 2 receptor heterodimers, which then phosphorylate different cytoplasmic, receptor-specific R-Smad proteins (Smad1, Smad2, Smad3). The phosphorylated R-Smads form a complex with a common Co-Smad protein (Smad4). The R-Smad/Co-Smad protein complex is transported to the cell nucleus where it interacts with different DNA-binding co-factors, as well as co-activators, or repressors, which induce or suppress the transcription of numerous target genes [6]. TGF-b1 and TGF-b3 On the whole, TGF-␤ isoforms inhibit normal epithelial cell functions and growth and act in a stimulatory manner in tumours [6]. In the normal pituitary TGF-␤1 was found to inhibit PRL production and lactotroph cell growth. In contrast, TGF-␤3 stimulated lactotroph cell proliferation [7]. Interestingly, estrogen down-regulated TGF-␤1 and up-regulated TGF-␤3 during estrogen-induced prolactinoma formation in Fischer 344 rats [8]. This has led to the hypothesis that an estrogen-induced shift in the intrapituitary TGF-␤1/-␤3 balance towards growth-promoting TGF-␤3 is involved in prolactinoma formation [9]. TGF-␤3 was further shown to up-regulate FGF-2 [10], which is known to stimulate

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both angiogenesis and lactotroph cell proliferation, and both TGF-␤ isoforms enhance the production of the angiogenic factor VEGF (vascular endothelial growth factor; see chapter 11) [11]. Therefore, it has been speculated that TGF-␤ might support prolactinoma development by stimulating both tumour cell proliferation and neovascularization through auto-/paracrine mechanisms [9, 11]. Activin and Inhibin Activin stimulates FSH production in pituitary gonadotropes, whereas inhibin suppresses it [12]. Common protein subunits (␣, ␤A, ␤B) form the variants of both inhibin (␣/␤A and ␣/␤B heterodimers) and activin (␤A/␤A and ␤B/␤B homodimers or ␤A/␤B heterodimer) [6]. Activin signals through ALK4/ActIIRA (or ActIIRB) receptor heterodimers [6]. Inhibin suppresses the action of activin by blocking receptor binding or inhibiting intracellular activin signalling [13]. Moreover, activin action is suppressed by follistatin, an activinbinding protein [14]. Activin normally suppresses the growth of pituitary tumour cells through ALK4 wild-type receptors and the Smad2/3/4 pathway [6, 15]. However, in nonfunctioning adenomas truncated isoforms of ALK4 have been found which compete with wild-type ALK4 for dimerization with ActIIRA or ActIIRB [16]. Receptor heterodimers containing truncated ALK4 isoforms are not able to induce the suppressive effects of activin on cell growth, which may play a role in tumour development in a subset of nonfunctioning adenomas [16]. Moreover, disturbances in the intratumoural expression of activin, inhibin and follistatin in gonadotroph adenomas underline the potential pathological impact of these factors in pituitary tumours [15, 17]. Bone Morphogenetic Factors BMP-2 and BMP-4 are important regulators of embryonic pituitary organogenesis by inducing Rathke’s pouch formation and participating in the control of lactotroph cell population development [18, 19]. The role of BMPs in normal adult pituitary has so far not been studied, but recently, BMP-4 was shown to be over-expressed in lactotroph tumours from D2-receptor knock-out mice and in human prolactinomas [20] (fig. 1a). BMP-4 is a potent stimulator of proliferation in lactosomatotroph GH3 tumour cells (fig. 1b), and studies in GH3 tumours in nude mice indicate that BMP-4 is involved in prolactinoma pathogenesis via a cross-talk between Smad1, Smad4 and estrogen receptors [20].

Transforming Growth Factor-␣ and Epidermal Growth Factor

Both transforming growth factor-␣ (TGF-␣) and epidermal growth factor (EGF) bind to the same tyrosine kinase receptor, EGFR, which in normal

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1.4

* 1.2

OD (WST-1 assay)

BMP-4(OD/control)

4

3

2

1

* 0.8

*

0.6 0.4

1 0.2 0

a

0 n ⫽ 12 n ⫽11 n ⫽12 n ⫽13 n ⫽3

Basal

b

50ng/ml 100ng/ml 200ng/ml BMP-4 BMP-4 BMP-4

Fig. 1. Expression and action of bone morphogenetic protein-4 (BMP-4) in pituitary tumours [20]. a BMP-4 was examined by Western blot in 51 protein homogenates, each obtained from one of 51 individual samples from normal human pituitary or pituitary adenomas. The BMP-4 signals were analysed by densitometry and normalized using actin values as loading control. 䊉 ⫽ Normal human pituitary, 䊐 ⫽ human prolactinomas, 䊊 ⫽ human ACTH-secreting tumours, ◊ ⫽ human GH-secreting tumours, ⌬ ⫽ clinically inactive pituitary tumours. b GH3 cells were treated with different doses of BMP-4 for 72 h. Cell proliferation was measured by WST-1 assay. Results represent the mean ⫾ SE of quadruplicates from four independent experiments. *p ⬍ 0.01 with respect to basal, (ANOVA with Scheffé’s test).

pituitary was found to be expressed in all endocrine cell types, although it was most abundant in the corticotropes and somatotropes [1]. Both factors predominantly stimulate the production of anterior pituitary hormones and moreover, both EGF and TGF-␣ act as mitogens in endocrine pituitary cells [1, 2]. Blockade of EGFR signalling in somatotropes during embryogenesis resulted in dwarf mice and pituitary hypoplasia, indicating a role for EGFR and its ligands in embryonic pituitary organogenesis [21]. EGFR is expressed in all types of pituitary adenomas, however, it is most abundant in corticotropinomas [22]. In the group of somatotropinomas and nonfunctioning adenomas, it has been shown that EGFR expression correlates with the aggressiveness of the pituitary tumours [23]. ERBB2, an oncogenic form of the EGFR, which is expressed in many human neoplasias, is not of relevance in pituitary adenomas [24]. Membrane over-expression of ERBB2 has only been reported in a corticotroph carcinoma with a high proliferation index [25]. TGF-␣ seems to support the development of experimental lactosomatotroph rodent pituitary tumours in an autocrine manner [26–28]. Targeted over-expression

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of TGF-␣ in pituitary lactotropes induces lactotroph hyperplasia and subsequent prolactinoma formation in female, but not male mice [27]. In Fischer 344 rats, estrogen-induced prolactinoma formation involves over-expression of TGF-␣ [28]. The dopamine D2 receptor antagonist bromocriptine suppressed TGF-␣ over-expression and could prevent prolactinoma development [28]. All these observations suggest that TGF-␣ contributes to prolactinoma formation.

The Fibroblast Growth Factor Family

Among the more than 20 members of the fibroblast growth factor (FGF) protein family which act through four tyrosine kinase receptors (FGFR1–4) [29], FGF8 and FGF10 participate in early pituitary determination during embryogenesis [18]. In the adult pituitary, FGF2 (basic FGF) seems to be the most important member of this protein family. It is not only a potent angiogenic factor but also stimulates PRL production and lactotroph cell proliferation [29]. FGF2 expression is up-regulated early in estrogen-induced prolactinoma formation in Fischer 344 rats, and it has been speculated that FGF2 could stimulate both cell proliferation and angiogenesis in these tumours [30]. In patients with either sporadic or MEN1-associated pituitary adenomas, significantly elevated levels of circulating FGF2 have been measured, which suggests that FGF2 maybe an additional diagnostic marker for pituitary tumours [31, 32]. In addition to proliferative-acting FGF2, a growth-suppressing protein, GFG, is produced from FGF2 antisense mRNA [33]. In the normal pituitary, GFG levels were higher than those of FGF2, whereas the opposite was found in pituitary tumours [33]. Heterogeneous expression of FGFR1, 2 and 3 isoforms has been detected in the normal pituitary and pituitary adenomas [34]. Interestingly, ptdFGFR4, a pituitary tumour-derived, N-terminally truncated form of the FGFR4, which lacks the signal peptide and the first two extracellular IG-like domains but containing the two intracellular kinase domains, was only found in pituitary adenomas [34, 35]. ptd-FGFR4 was shown to induce pituitary tumours in transgenic mice which express ptd-FGFR4 in lactotropes, whereas wild-type FGFR4 had no tumorigenic potential [35, 36]. All together, these findings underline the importance of FGF2 and its receptors in pituitary tumour formation.

Nerve Growth Factor

In addition to its multiple effects on neuronal cells, nerve growth factor (NGF) is a regulator of neuroendocrine and endocrine cell functions and growth via its two receptors trkA and p75NGFR. In the pituitary, the most

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impressive effect of NGF is to change dopamine agonist-resistant prolactinomas to dopamine agonist-responsive tumours by inducing re-expression of dopamine D2 receptors (D2R) [37]. Dopamine-resistant prolactinomas not only lack D2R, but are also devoid of NGF and p75NGFR. In contrast, responsive prolactinomas express D2R, trkA, p75NGFR and produce NGF [38]. Transient treatment of dopamine agonist-resistant prolactinomas with NGF leads to p75NGFR expression. NGF could then induce re-expression of D2R, mediated by the activation of nuclear factor-␬B [39]. It seems that an autocrine loop involving NGF and p75NGFR participates in controlling D2R expression in normal lactotropes. Loss of this autocrine mechanism after tumoural transformation leads to the formation of dopamine agonist-resistant prolactinomas [40]. NGF-induced transformation of the latter to dopamine-responsive prolactinomas could be of high clinical relevance.

The gp130 Cytokine Family

The group of gp130 cytokines consists of interleukin (IL)-6, IL-11, leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM) and cardiotropin-1 which bind to different receptors, but all act through the common gp130 signal-transducing protein [41]. Recently, it has been shown that the suppression of gp130 protein production in lactosomatotroph GH3 tumour cells by an antisense approach abolished the ability of these cells to form tumours in nude mice (fig. 2). This indicates that one or more of the gp130 cytokines may play a role in pituitary tumorigenesis [42]. The expression of almost all of the gp130 cytokines and their corresponding receptors has been detected in pituitary or pituitary adenomas [43], but only the roles of IL-6 and LIF in pituitary physiology and pathophysiology have been extensively studied. Interleukin-6 IL-6 is produced in the normal pituitary by folliculostellate (FS) cells [44], whereas in the majority of pituitary adenomas, in which FS cells are rare or absent, tumour cells are the source of IL-6 [45]. It is a potent stimulator of secretion for nearly all the hormones in the normal pituitary and contributes to excessive ACTH production in corticotroph adenomas [1, 3, 46]. Interestingly, IL-6 inhibits the growth of normal pituitary cells [47] but differently regulates c-fos expression in pituitary adenomas [48] and stimulates pituitary tumour cell proliferation [47]. IL-6 is linked through gp130 to different signalling pathways such as the JAK/STAT pathway or the MAP kinase (MAPk) pathway; however, gp130 also induces cytokine-signalling inhibitors like SOCS-3 [41]. Although the underlying mechanism for the opposing growth effects of IL-6 in normal

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GH3 control

gp130-AS

Tumor volume (mm3)

2,000

gp130-AS GH3 control

1,500 1,000 500 0 14

17

20

23

26

Days after injection

Fig. 2. Effect of gp130 suppression on GH3 cell tumour development in nude mice. GH3 cell clones with suppressed gp130 production (gp130-AS) did not develop tumours in nude mice whereas GH3 control cells formed large tumours within 4 weeks after injection [for details, see 42].

and adenomatous pituitary cells has not yet been studied, differences in the induction of activating signal pathways or stimulation of cytokine-signalling inhibitor production by the IL-6/gp130 complex may be responsible for the different mitogenic effects of IL-6 in the normal and adenomatous pituitary. In summary, the intratumoural production of tumour cell growth-stimulating IL-6 in the majority of pituitary adenomas makes this cytokine an attractive candidate as an auto-/paracrine stimulator of adenoma progression. Leukaemia Inhibitory Factor

Similar to IL-6, LIF is a pleiotropic cytokine, which exerts different effects in the pituitary. LIF is an important stimulator of POMC expression and ACTH

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secretion through the JAK/STAT pathway and plays an important role in the activation of the HPA axis during stress and inflammation [49]. Transgenic mice that over-express LIF in the pituitary have corticotroph hyperplasia, which is accompanied by a reduced somatotroph and lactotroph cell development and function [50]. These mice exhibit symptoms of Cushing’s syndrome [50], however, in human corticotropinomas no over-expression of LIF has been found [43, 51]. Recently it has been shown that LIF might play a role in prolactinoma pathophysiology. This type of pituitary tumour seems to be the only one in which LIF is not expressed, and stimulation of prolactinoma cell cultures with LIF inhibited prolactin secretion [52]. Interestingly, sulpiride, a dopamine D2 receptor antagonist, reverted the suppressive effect of LIF on PRL. Therefore, it has been suggested that LIF might inhibit PRL secretion through an interaction between the gp130 pathway and the D2 receptor [52]. Loss of this suppressive autocrine loop may participate in prolactinoma development.

The Intrinsic Growth Factor Network in Normal and Adenomatous Pituitary

The local production of multiple growth factors and cytokines within the anterior pituitary has led to speculations and hypotheses that an intrapituitary network of these factors may exist that could play a role in pituitary physiology [1–3, 41]. This idea is supported by the observation that the expression of intrapituitary factors and their receptors is a dynamic process as they fluctuate during embryogenesis and postnatal development, as well as during the menstrual cycle, pregnancy, stress, infectious processes and so on. The changes in intrapituitary factors/receptors may reflect local adaptive processes during general alterations in endocrine homeostasis. For example, in cycling female rats, alterations in FGF2 have been reported. Intrapituitary FGF2 expression is low during diestrus, increases during proestrus and reaches its maximum at estrus [53]. Estradiol (E2), which rises during proestrus, peaks at estrus and declines during diestrus, was found to be responsible for the fluctuations in FGF2 [53]. E2 or FGF2, or a combination of both, might be responsible for alterations in the intrapituitary proliferative index (PI) in cycling females, which have the highest PI during estrus and lowest PI during diestrus [54, 55]. The observation that lactotroph cells represent the vast majority of proliferating cells during estrus cycle-related changes in PI could be explained by the fact that both E2 and FGF2 are potent lactotroph mitogens [55]. However, in cycling female mammalians, other E2-regulated growth factors such as TGF-␤1, TGF-␤3, TGF-␣, BMP-4 and VEGF might

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change, which probably all contribute to the estrus cycle-dependent alterations in the growth and function of lactotroph anterior pituitary cells. E2-induced alterations in intrapituitary growth factors may also play a role in the development of lactotroph pituitary hyperplasia during pregnancy. Increases in the above-mentioned intrapituitary lactotroph mitogens, which are under the control of E2, could account for the pregnancy-related increase in size of about 50–100% of the anterior pituitary [56]. Since E2-induced FGF2 and VEGF [53] are angiogenic factors, pregnancy-associated hyperplasia of lactotroph pituitary cells might be accompanied by neovascularization through angiogenesis. However, neither continuous cycling nor repetitive pregnancies result in a permanent enlargement of the pituitary. This indicates that there are mechanisms present in the pituitary which counteract the E2-driven increase in the lactotroph cell population. After lactation, an increased apoptotic activity, which is largely confined to the lactotropes has been found in the hyperplastic pituitary [56–58]. In this way, the formerly enhanced numbers of lactotropes are normalized to previous levels. All together, these findings suggest that there is an intrapituitary growth factor network, which controls the lactotroph cell population. This network seems to be under estrogenic control at least in female mammalians. Physiological alterations in E2 during cycling or pregnancy result in enhanced lactotroph proliferation and in lactotroph hyperplasia in the pituitary. After reduction of elevated E2, the activity of the growth factor network declines, and other mechanisms that have yet to be identified induce normalization of lactotroph cell numbers through apoptosis. These observations have led to speculations that estrogens could be a risk factor for prolactinoma development in humans [4, 5]. However, apart from an enhanced incidence of microprolactinomas in women [4], there is little to support this hypothesis. For example, male-to-female transsexuals treated with excessive dosages of E2 rarely develop prolactinomas [59]. Except for a particular rat strain (Fischer 344 rats), animals treated with high concentrations of estrogens develop at best lactotroph hyperplasia but not prolactinomas. However, in Fischer 344 rats, estrogens rapidly induce prolactinomas both in males and in females [30, 53]. The induction of prolactinomas in these animals is associated with E2-induced over-expression of FGF2, TGF-␣, TGF-␤3 [9, 28, 30, 53], which all have growth-stimulating properties on lactotropes, and suppression of TGF-␤1 [9] which inhibits lactotroph cell growth. Moreover, E2-induced VEGF [53] could act in concert with FGF2 to induce tumour neovascularization in addition to tumour expansion. It is evident that E2-regulated mechanisms observed in the pituitaries of cycling and pregnant female mammalians contribute in Fischer rats to prolactinoma formation. However, a still unknown

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causative genetic alteration (e.g. expression of an oncogene or loss of a tumour suppressor) must be present which makes this particular rat strain susceptible for prolactinoma development in response to E2 and E2-induced growth factors.

Conclusion and Perspectives

To date, there is increasing evidence that intrapituitary growth factors and cytokines play distinct roles in pituitary physiology. There are no hints that spontaneous over-expression of any of these factors, or expression of an oncogenic-like, constitutively active growth factor receptor plays a causative role in human pituitary tumour pathogenesis. However, after tumoural transformation, some growth factors, cytokines and their receptors are often differently expressed or differently activated, and in this way may act as factors supporting pituitary tumour expansion. Therefore, some growth factors/cytokines may be considered as pituitary tumour progression factors and blockade of these factors may slow down, or at best, stop further tumour expansion. Treatment of dopamine agonist-resistant prolactinomas with NGF is practically impossible for different reasons (inactivation of NGF through binding to serum proteins, NGF-induced pain, high costs of treatment). Therefore, at present there is no evidence that intratumoural growth factors/cytokines, or their receptors could provide a basis for the development of a therapeutically concept for pituitary tumour treatment.

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Hentges S, Boyadjieva N, Sarkar DK: Transforming growth factor-␤3 stimulates lactotrope cell growth by increasing basic fibroblast growth factor from folliculo-stellate cells. Endocrinology 2000;141:859–867. Renner U, Lohrer P, Schaaf L, Feirer M, Schmitt K, Onofri C, Arzt E, Stalla GK: Transforming growth factor-␤ stimulates vascular endothelial growth factor production by folliculostellate pituitary cells. Endocrinology 2000;143:3759–3765. Weiss J, Harris PE, Halvorson LM, Crowley WF Jr, Jameson JL: Perfusion of rat pituitary cells with gonadotropin-releasing hormone, activin, and inhibin reveals distinct effects on gonadotropin gene expression and secretion. Endocrinology 1993;132:2307–2311. Matzuk MM: Editorial: In search of binding – Identification of inhibin receptors. Endocrinology 2000;141:2281–2284. Hashimoto O, Nakamura T, Shoji H, Shimasaki S, Hayashi Y, Sugino H: A novel role of follistatin, an activin-binding protein, in the inhibition of activin action in rat pituitary cells. Endocytotic degradation of activin and its acceleration by follistatin associated with cell-surface heparan sulfate. J Biol Chem 1997;272:13835–13842. Danila DC, Inder WJ, Zhang X, Alexander JM, Swearingen B, Hedley-Whyte ET, Klibanski A: Activin effects on neoplastic proliferation of human pituitary tumors. J Clin Endocrinol Metab 2000;85:1009–1015. Zhou Y, Sun H, Danila DC, Johnson SR, Sigai DP, Zhang X, Klibanski A: Truncated activin type I receptor Alk4 isoforms are dominant negative receptors inhibiting activin signaling. Mol Endocrinol 2000;14:2066–2075. Penabad JL, Bashey HM, Asa SL, Haddad G, Davis KD, Herbst AB, Gennarelli TA, Kaiser UB, Chin WW, Snyder PJ: Decreased follistatin gene expression in gonadotroph adenomas. J Clin Endocrinol Metab 1996;81:3397–3403. Treier M, Gleiberman AS, O’Connell SM, Szeto DP, McMahon JA, McMahon AP, Rosenfeld MG: Multistep signaling requirements for pituitary organogenesis in vivo. Genes Dev 1998;12: 1691–1704. Scully KM, Rosenfeld MG: Pituitary development: Regulatory codes in mammalian organogenesis. Science 2002;295:2231–2235. Paez-Pereda M, Giacomini D, Refojo D, Carbia Nagashima A, Hopfner U, Grübler Y, Chervin A, Goldberg V, Goya R, Hentges ST, Low MJ, Holsboer F, Stalla GK, Arzt E: Involvement of bone morphogenetic protein 4 in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk. Proc Natl Acad Sci USA 2003;100:1034–1039. Roh M, Paterson AJ, Asa SL, Chin E, Kudlow JE: Stage-sensitive blockade of pituitary somatomammotrope development by targeted expression of a dominant negative epidermal growth factor receptor in transgenic mice. Mol Endocrinol 2001;15:600–613. Kontogeorgos G, Stefaneanu L, Kovacs K, Cheng Z: Localization of epidermal growth factor and epidermal growth factor receptor in human pituitary adenomas and nontumorous pituitaries: An immunocytochemical study. Endocr Pathol 1996;7:63–70. LeRiche VK, Asa SL, Ezzat S: Epidermal growth factor and its receptor (EGF-R) in human pituitary adenomas: EGF-R correlates with tumor aggressiveness. J Clin Endocrinol Metab 1996; 81:656–662. Ezzat S, Zheng L, Smyth HS, Asa SL: The c-erbB-2/neu proto-oncogene in human pituitary tumours. Clin Endocrinol 1997;46:599–606. Nose-Alberti V, Mesquita MI, Martin LC, Kayath MJ: Adrenocorticotropin-producing pituitary carcinoma with expression of c-erbB-2 and high PCNA index: A comparative study with pituitary adenomas and normal pituitary tissues. Endocr Pathol 1998;9:53–62. Finley EL, Ramsdell JS: A transforming growth factor-␣ pathway is expressed in GH4C1 rat pituitary tumors and appears necessary for tumor formation. Endocrinology 1994;135:416–422. McAndrew J, Paterson AJ, Asa SL, McCarthy KJ, Kudlow JE: Targeting of transforming growth factor-␣ expression to pituitary lactotrophs in transgenic mice results in selective lactotroph proliferation and adenomas. Endocrinology 1995;136:4479–4488. Borgundvaag B, Kudlow JE, Mueller SG, George SR: Dopamine receptor activation inhibits estrogen-stimulated transforming growth factor-␣ gene expression and growth in anterior pituitary, but not in uterus. Endocrinology 1992;130:3453–3458.

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Galzie Z, Kinsella AR, Smith JA: Fibroblast growth factors and their receptors. Biochem Cell Biol 1997;75:669–685. Heaney AP, Horwitz GA, Wang Z, Singson R, Melmed S: Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis. Nat Med 1999;5:1317–1321. Zimering MB, Katsumata N, Sato Y, Brandi ML, Aurbach GD, Marx SJ, Friesen HG: Increased basic fibroblast growth factor in plasma from multiple endocrine neoplasia type 1: Relation to pituitary tumor. J Clin Endocrinol Metab 1993;76:1182–1187. Ezzat S, Smyth HS, Ramyar L, Asa SL: Heterogenous in vivo and in vitro expression of basic fibroblast growth factor by human pituitary adenomas. J Clin Endocrinol Metab 1995;80:878–884. Asa SL, Ramyar L, Murphy PR, Li AW, Ezzat S: The endogenous fibroblast growth factor-2 antisense gene product regulates pituitary cell growth and hormone production. Mol Endocrinol 2001;15:589–599. Asghar Abbass SA, Asa SL, Ezzat S: Altered expression of fibroblast growth factor receptors in human pituitary adenomas. J Clin Endocrinol Metab 1997;82:1160–1166. Ezzat S, Zheng L, Zhu XF, Wu GE, Asa SL: Targeted expression of a human pituitary tumor-derived isoform of FGF receptor-4 recapitulates pituitary tumorigenesis. J Clin Invest 2002;109:69–78. Low MJ: Pituitary adenomas in man and mouse: Oncogenic potential of a truncated fibroblast growth factor receptor 4. J Clin Invest 2002;109:15–16. Missale C, Boroni F, Losa M, Giovanelli M, Zanellato A, Dal Toso R, Balsari A, Spano P: Nerve growth factor suppresses the transforming phenotype of human prolactinomas. Proc Natl Acad Sci USA 1993;90:7961–7965. Missale C, Losa M, Sigala S, Balsari A, Giovanelli M, Spano PF: Nerve growth factor controls proliferation and progression of human prolactinoma cell lines through an autocrine mechanism. Mol Endocrinol 1996;10:272–285. Fiorentini C, Guerra N, Facchetti M, Finardi A, Tiberio L, Schiaffonati L, Spano P, Missale C: Nerve growth factor regulates dopamine D2 receptor expression in prolactinoma cell lines via p75NGFR-mediated activation of nuclear factor-␬B. Mol Endocrinol 2002;16:353–366. Missale C, Spano P: Nerve growth factor in pituitary development and pituitary tumors. Front Neuroendocrinol 1998;19:128–150. Arzt E: gp130 cytokine signaling in the pituitary gland: A paradigm for cytokine-neuroendocrine pathways. J Clin Invest 2001;108:1729–1733. Castro CP, Giacomini D, Nagashima AC, Onofri C, Graciarena M, Kobayashi K, Paez-Pereda M, Renner U, Stalla GK, Arzt E: Reduced expression of the cytokine transducer gp130 inhibits hormone secretion, cell growth, and tumor development in pituitary lactosomatotrophic GH3 cells. Endocrinology 2003;144:693–700. Hanisch A, Dieterich KD, Dietzmann K, Lüdecke K, Buchfelder M, Fahlbusch R, Lehnert H: Expression of members of the interleukin-6 family of cytokines and their receptors in human pituitary and pituitary adenomas. J Clin Endocrinol Metab 2000;85:4411–4414. Renner U, Gloddek J, Paez Pereda M, Arzt E, Stalla GK: Regulation and role of intrapituitary IL-6 production by folliculostellate cells. Domest Anim Endocrinol 1998;15:353–362. Jones TH, Daniels M, James RA, Justice SK, McCorkle R, Price A, Kendall-Taylor P, Weetman AP: Production of bioactive and immunoreactive interleukin-6 (IL-6) and expression of IL-6 messenger ribonucleic acid by human pituitary adenomas. J Clin Endoccrinol Metab 1994;78: 180–187. Paez Pereda M, Lohrer P, Kovalovsky D, Perez Castro C, Goldberg V, Losa M, Chervin A, Berner S, Molina H, Stalla GK, Renner U, Arzt E: Interleukin-6 is inhibited by glucocorticoids and stimulates ACTH secretion and POMC expression in human corticotroph pituitary adenomas. Exp Clin Endocrinol Diabetes 2000;108:202–207. Arzt E, Buric R, Stelzer G, Stalla J, Sauer J, Renner U, Stalla GK: Interleukin (IL) involvement in anterior pituitary cell growth regulation: Effects of IL-2 and IL-6. Endocrinology 1993;132: 459–467. Paez Pereda M, Goldberg V, Chervin A, Carrizo G, Molina A, Andrada J, Sauer J, Renner U, Stalla GK, Arzt E: Interleukin (IL)-2 and IL-6 regulate c-fos protooncogene expression in human pituitary adenoma explants. Mol Cell Endocrinol 1996;124:33–42.

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Auernammer CJ, Melmed S: Leukemia inhibitory factor – neuroimmune modulator of endocrine function. Endocr Rev 1999;21:313–345. Yano H, Readhead C, Nakashima M, Ren SG, Melmed S: Pituitary-directed leukemia inhibitory factor transgene causes Cushing’s syndrome: Neuro-immune-endocrine modulation of pituitary development. Mol Endocrinol 1998;12:1708–1720. Kontogeorgos G, Patralexis H, Tran A, Kovacs K, Melmed S: Expression of leukemia inhibitory factor in human pituitary adenomas: A morphologic and immunocytochemical study. Pituitary 2000;2:245–251. Ben-Shlomo A, Miklovsky I, Ren SG, Yong WH, Heaney AP, Culler MD, Melmed S: Leukemia inhibitory factor regulates prolactin secretion in prolactinoma and lactotroph cells. J Clin Endocrinol Metab 2003;88:858–863. Heaney AP, Fernando M, Melmed S: Functional role of estrogen in pituitary tumor pathogenesis. J Clin Invest 2002;109:277–283. Oishi Y, Okuda M, Takahashi H, Fujii T, Morii S: Cellular proliferation in the anterior pituitary gland of normal adult rats: Influence of sex, estrous cycle, and circadian change. Anat Rec 1993;235:111–120. Yin P, Arita J: Differential regulation of prolactin release and lactotrope proliferation during pregnancy, lactation and the estrous cycle. Neuroendocrinology 2000;72:72–79. Orgnero de Gaisan E, Maldonado CA, Aoki A: Fate of degenerating lactotropes in rat pituitary gland after interruption of lactation: A histochemical and immunocytochemical study. Histochem J 1993;25:150–165. Aoki A, de Gaisan EO, Pasolli HA, Torres AI: Disposal of cell debris from surplus lactotrophs of pituitary gland. Exp Clin Endocrinol Diabetes 1996;104:256–262. Drewett N, Jacobi JM, Willgoss DA, Lloyd HM: Apoptosis in the anterior pituitary gland of the rat: Studies with estrogen and bromocriptine. Neuroendocrinology 1993;57:89–95. Kovacs K, Stefaneanu L, Ezzat S, Smyth HS: Prolactin-producing pituitary adenoma in a maleto-female transsexual patient with protracted estrogen administration. A morphological study. Arch Pathol Lab Med 1994;118:562–565.

Dr. Ulrich Renner Department of Endocrinology, Max Planck Institute of Psychiatry Kraepelinstrasse 10, DE–80804 Munich (Germany) Tel. ⫹49 89 30 622349, Fax ⫹49 89 30 622605, E-Mail [email protected]

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Proliferation Markers and Cell Cycle Inhibitors in Pituitary Adenomas Wolfgang Saeger Institute of Pathology of the Marienkrankenhaus Hamburg, Hamburg, Germany

Abstract Proliferation markers are widely used in general surgical pathology and also in pituitary pathology. They should help for differing aggressive or rapidly growing tumors from those with slower growth, as cellular atypia is not helpful for identifying aggressive adenomas of the pituitary. Only the number of mitoses is important for prognosis. A lot of markers can be used: antibodies for cyclins A, B, D and E, for proliferating cell nuclear antigen, Ki-67/Mib-1, antibodies for the inhibitory proteins p16, p27, p53, and for DNA topoisomerase II␣. A marker for apoptosis and its inhibitors may be also important. From our experiences, Mib-1 is the most reliable marker. The recommendation of this marker in the WHO classification of pituitary adenomas is fully justified. Copyright © 2004 S. Karger AG, Basel

Introduction

Mechanisms of tumorigenesis and tumor cell multiplication have been elucidated in many points during the last two decades. The normal cell cycle can be influenced, stimulated or inhibited by many different principles which may also be important for tumor cell cycle. In the late G1 phase, when transcription and translation had occurred, and in the G2 phase, cell cycle checkpoints are installed for stopping the replication process if critical genes are mutated or DNA is damaged. The process is mediated by protein kinases which phosphorylate and activate enzymes and other proteins and controlled by proteins called cyclins. In the G1 phase, cyclins D1, D2 and D3 and the protein kinases 2, 4, 5 and 6 are regulatory. Cyclin-dependent protein kinases (CdK) are inhibited by multiple inhibitory proteins, especially p15, p16, p18,

p19, p21 and p27. For G1-S transition, cyclin E and CdK2 regulated by retinoblastoma gene are necessary. They are inhibited by p53 protein, which enhances the gene transcription of p21. In patients with tumorous diseases the aims of surgical pathology are the identification and classification of the tumors and the discussion of prognosis. Since cell pleomorphism is of minor importance in pituitary adenoma pathology and small foci with invasion of surrounding tissues are frequently found (in 40–50% of adenomas), we have to use other parameters for identifying adenomas with increased risk of recurrences after surgical resection. Recurrences depend on residual adenoma tissue and their proliferation properties. The best proliferation marker would be an antibody that demonstrates proliferating cells and indicates a more rapid growth, a probably higher recurrence rate and the danger of carcinoma development. From all our studies and reviews of the literature we know that such a marker does not exist up to now. For those questions we can use DNA cytometry, counting mitoses, immunostainings for cyclins, growth factors, inhibitory proteins and uncharacterized proliferation markers but also molecular pathology of genes responsible for the cell cycle protein synthesis.

DNA Cytometry

Flow and image cytometry provide information about the fraction of cells in the G0/G1 phase, the S phase, the G2 and M phase of the cell cycle [1]. Additionally, the DNA index, the 2c-deviation index, and the ratio of 5c-exceeding events can be measured and provide information on the stem line, the distribution, and single cells with a DNA content greater than 2c. Using flow cytophotometry, the highest rates of aneuploidy were found in prolactinomas (36% [2] up to 70%, in GH/prolactin cell adenomas 80% [3]) and lowest aneuploid DNA pattern (20%) in inactive adenomas [3]. Correlations existed between aneuploidy and suprasellar extension [2] and especially between aneuploidy and mitotic index [2]. Using image cytophotometry the rate of aneuploidy varied between 31% [4] and 50% [2]. A correlation between tumor size and aneuploidy but not dura invasion was found [4]. The mean S-phase fraction was lower in euploid (mean 1.7%) [4] respectively mostly less than 10% [1] and higher in aneuploid adenomas (mean 3.6% [4], respectively mostly more than 10% [1]). Although DNA cytometry is undoubtedly an essential method in pathology, its value for assessment of prognosis of pituitary adenomas appears to be very limited.

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Fig. 1. Cyclin D3 in a FSH/LH cell adenoma: some strongly positive nuclei. ⫻270.

Cyclins

Cyclins regulate transitions through various phases of cell cycle by activating cyclin-dependent kinases. Cyclins D1 and D3 allow the cell to proceed into the S phase. Cyclin D1 was immunohistologically sparsely demonstrated and more frequently in non-functioning and aggressive adenomas than in other adenoma types [5]. The amplification of cyclin D1 gene was shown by Yu and Melmed [6]. Others [7] found cyclin D expression in pituitary adenomas related to size and tumor regrowth. The differences between regrowing and non-regrowing tumors were related to reduced bcl-2 expression, increased cell proliferation, more cells of the G2/M stage and reduced cell differentiation with more aggressive subsequent behavior. In our material, cyclin D1 was positive in only one invasive null cell adenoma whereas cyclin D3 was overexpressed in the nuclei of 68% of our inactive adenomas [8] (fig. 1). Overexpression correlated to the labeling index (LI) of Ki-67 in our series but also in the studies of others [7]. Cyclins A, B and E were demonstrated in all adenoma types and were significantly higher in macroadenomas compared to microadenomas [7]. For cyclin A, a positive linear correlation with the Mib-1 index [9] and a more than twofold higher LI of recurrent adenomas in comparison with non-recurrent adenomas were found.

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Fig. 2. Proliferating cell nuclear antigen (PCNA) in a sparsely granulated GH cell adenoma: two positive nuclei. ⫻480.

The cyclin proliferating cell nuclear antigen (PCNA) is an auxiliary 36-kD non-histone intranuclear protein serving as a cofactor of DNA polymerase delta during the DNA synthesis phase of the cell cycle [10]. The PCNA index is extremely low in normal pituitary [11] whereas in adenomas different data were published. Some authors measured an index of 1% [12], others an index of up to 19% [12]. It was found to be higher in recurrent than in primary pituitary adenomas (1.88 vs. 1.06%) [12] but the very little although significant difference is without significance for the prognosis in the single case, whereas in another study [11] the greater differences (13 vs. 19%) appear to be useful for predicting the likelihood of recurrence. In our studies, a low index (⬍1%) was found in inactive adenomas that could not be correlated to growth or recurrence [13]. In GH cell adenomas a stronger expression could be demonstrated (fig. 2). In the same way, differences between invasive and non-invasive adenomas (2.9 vs. 1.3% LI) were found by one group [14], whereas others could not confirm these data [15, 16]. Adenoma-type specific differences do also not exist [17].

Ki-67 resp. Mib-1

Ki-67 is a monoclonal antibody recognizing nuclear proteins of 345- and 395-kD molecular weights in proliferating cells during all non-G0 phases of the

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Fig. 3. Mib-1 in an oncocytic adenoma: about 3% positive nuclei. ⫻440.

cell cycle [18], but cannot be used in paraffin-embedded tissue. The monoclonal antibody Mib-1 binds the same antigen as Ki-67 but can be used in formalin-fixed paraffin-embedded specimens [19]. In the WHO Classification 2000 [20], Mib-1 labeling of more than 3% of adenoma nuclei was presented as one main criterion for atypical adenomas. The other main criterion is an elevated mitotic index. These definitions implicate that Mib-1 is the most important marker for proliferation in pituitary pathology. Counting 1,000 nuclei of inactive adenomas the Ki-67 LI was 2.4% (range 0–23%) whereas others found lower values (1%) [21]. ACTH adenomas showed the highest values (5.9%) [22]. Previous studies detected a positive correlation between dural invasion of adenomas and increased Ki-67 LI [22–26]. However, although Thapar et al. [26] found a higher Mib-1 labeling in invasive adenomas than in non-invasive adenomas, they state that proliferative activity alone might be too simplistic to alone be casually associated with invasive potential. These modifications are underlined by the fact that more than 25% of invasive adenomas show relatively low Mib-1 labeling. Others found no significant differences between invasive and non-invasive adenomas [15, 16]. In our studies [13] we measured small differences in Mib-1 LI in invasive null cell adenomas (LI of 0.149) in contrast to non-invasive null cell adenomas (LI of 0.004) and invasive oncocytomas (LI of 0.159) (fig. 3) in contrast to non-invasive oncocytomas (LI of 0.08). Higher LI were present in acidophil stem cell adenomas (fig. 4).

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Fig. 4. Mib-1 in an acidophil stem cell adenoma: about 3% positive nuclei. ⫻220.

Regrowing adenomas after partial resection showed a significantly higher Ki-67 LI than those which remained cured [27]. Such correlations could not be confirmed by others [28]. There is an inverse correlation between age and Mib-1 index: elderly patients have lower values correlating to the fact that the tumor volume doubling time is much longer in elderly patients [29]. Others [30] found a positive association between preoperative prolactin levels and Mib-1 LI, which was lower in young female patients than in older female and male patients. Carcinomas and their metastases showed highest LI [26] (fig. 5).

Inhibitory Proteins

The p16 gene encodes a physiological inhibitor of the cyclin D-CDK4 complex and is considered as an important tumor suppressor gene. Methylation of the CpG island within the p16 gene is associated with loss of expression of p16 protein in pituitary tumors [31]. By PCR analysis it was shown [32] that 71% of null cell adenomas, 29% of gonadotroph adenomas but no GH or prolactin adenoma were hypermethylated. Immunostainings for nuclear expression of p16 protein supported these data: hypermethylated tumors were negative. Significant differences between invasive and non-invasive adenoma did not exist [33].

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Fig. 5. Mib-1 in a metastasis of an ACTH-secreting pituitary carcinoma: about 5% positive nuclei. ⫻320.

The p27 kip1 (p27) gene encodes an inhibitor of cyclin-dependent kinase activity. In endocrine and other tumors, p27 nuclear expression is inversely related to the Mib-1 immunostaining [34]. As parathyroid hyperplasias have threefold more p27-positive nuclei than parathyroid adenomas [34], it may be used to classify and differentiate hyperplastic and neoplastic tissues. By immunostaining in our material, normal pituitaries showed a strong nuclear expression of p27 [35]. 40% of pituitary adenomas were completely negative [35], whereas in 33% less than 10% of nuclei were stained and in 27% a strong expression was found. Lowest staining indices were present in the sparsely granulated GH cell adenomas, the densely granulated prolactin cell adenomas and the ACTH cell adenomas. In another study, the most marked decrease of p27 protein expression was noted in non-functioning adenomas (80%) and GH adenomas (76%) [36]. Comparing corticotroph adenomas and carcinomas, the expression of p27 protein is lower in carcinomas than in adenomas [37–39]. In recurrent adenomas a lower mean LI for p27 (47%) was found than in non-recurrent adenomas (67%) [9]. In general, p27 expression was inversely related to the proliferation marker Mib-1 [34, 39]. Dopamine agonist treatment of prolactin adenomas did not significantly influence the p27 expression [40]. Vitamin D3 hypophosphorylates p27 and can accumulate p27 protein in pituitary adenomas and was found to arrest the growth of ACTH cells [41].

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p53 belongs to the family of tumor suppressor genes which encode a 53-kD nuclear phosphoprotein. Point mutations in highly conserved regions of the p53 gene induce conformational alterations that stabilize the protein in transformed cells. In normal cells, p53 protein is undetectable by immunocytochemistry. On the other hand, immunostaining is able to demonstrate a mutant p53 protein [42], since specific monoclonal antibodies exist for the mutant form. Loss of p53 function can develop from mutations of one or both alleles or from deletions of both alleles. In the latter situation, p53 is useless. Allele loss in the region on chromosome 17p where the p53 gene is located is frequent in malignant tumors. It was shown in many tumors that one allele is deleted and the other is mutated. If normal p53 allele is retained, mutant p53 can bind wild-type protein and inactivate it or prevent transcription of the normal gene. First immunohistological studies [43] of p53 protein immunostaining in pituitary adenomas were without positive results whereas all pituitary tumors in AVP/SV40 transgenic mice showed p53 immunoreactive nuclei. Later studies demonstrated p53 protein in 16% of invasive adenomas [44], especially in ACTH adenomas, whereas in non-invasive ACTH adenomas p53 was not expressed [45]. Others found a lower LI in invasive adenomas [46]. In recurrent adenomas it was more often demonstrated than in non-recurrent ones [47]. p53 protein expression correlated with a higher LI of Ki-67 [48]. Comparing invasive ACTH adenomas with ACTH carcinomas, the LI of p53 was higher in carcinomas (37.3 vs. 49.9%) [37], and there was a correlation between duration of survival and p53 expression [49], but carcinomas may be p53-negative [14, 50, 51]. In our own series, we found p53 positivity restricted to invasive adenomas [13]. 20% of all invasive clinically inactive adenomas (null cell adenomas and oncocytomas) harbored p53-positive nuclei (fig. 6). All invasive gonadotroph adenomas were p53-negative. In sparsely granulated prolactin cell adenomas [52], bi- and plurihormonal adenomas [53] with acromegaly p53 were not demonstrated. Even in our 7 pituitary carcinomas, p53 could not be immunostained [51]. From our experiences we conclude that positive p53 protein in pituitary adenomas can be interpreted as a sign of increased or invasive growth but that on the other hand a negative p53 protein does not exclude a more rapid or even malignant tumor growth.

Topoisomerase

DNA topoisomerase II␣ is a molecular and immunohistochemical marker for proliferation rate and is the target for several anti-neoplastic agents.

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Fig. 6. p53 protein in an oncocytic adenoma: two positive nuclei. ⫻440.

Topoisomerase II␣ immunopositivity was detected only in the nuclei of tumor cells. Gonadotroph adenomas, null cell adenomas, and ACTH-producing adenomas were found [54] to have the lowest topoisomerase II␣ indices, whereas primary pituitary carcinomas and some silent adenomas presented the highest counts. Significant correlations between topoisomerase II␣ expression, patient gender, and vascularity were not found. In contrast, a significant negative correlation was found between topoisomerase II␣ expression and patient age. Its expression was significantly higher in invasive than non-invasive adenomas. A tendency to have higher counts was also observed in microadenomas compared with in macroadenomas. Although the indices of topoisomerase II␣ and the proliferation marker Mib-1 were similar in most tumor types, a significant correlation between both indices was demonstrable only in nonfunctioning adenomas [54]. Our own studies on inactive adenomas [8] showed no differences in topoisomerase II␣ immunostaining (fig. 7) between gonadotroph adenomas, null cell adenomas and oncocytic adenomas, and no significant correlation to the time of development of adenoma symptoms but a correlation of topoisomerase II␣ with cyclin D3 and the proliferation marker Mib-1. In octreotide-treated GH-producing adenomas, topoisomerase II␣ was significantly decreased compared with untreated tumors. No significant changes were observed in bromocriptine-treated prolactin-producing adenomas [54].

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Fig. 7. Topoisomerase II␣ in a FSH/LH cell adenoma: many positive nuclei with different staining intensity. ⫻270.

Urokinase, Urokinase Receptor, Urokinase Inhibitor,Tissue Plasminogen Activator, and Matrix Metalloproteinase

Although not real markers of proliferation, urokinase-type plasminogen activator (UPA), its receptor (UPAR), its inhibitor (PAI-I), matrix metalloproteinase 9 (MMP9) and tissue plasminogen activator (tPA) are considered to be correlated with invasion and malignancy in several carcinoma types. UPA, UPAR and PAI-I are found in many pituitary adenomas [55, 56], but also normal anterior lobe. All three markers are differently overexpressed in adenomas (fig. 8, 9) in contrast to normal pituitary cells which showed weaker and more focal immunoreactions. Comparing invasive and non-invasive adenomas, UPA is expressed in significant higher degrees in inactive invasive adenomas. For all other adenoma types no correlations between tumor type, invasiveness and expression of UPA, IPAR and PAI-I are found. MMP9 (fig. 10) is demonstrable in normal and hyperplastic pituitaries and in adenomas of different types in nearly identical degrees [57]. TPA was overexpressed in 48% of ACTH-secreting adenomas and in 44% of inactive adenomas of FSH/LH cell type or null cell type or oncocytic type and in one TSH cell adenoma. Only slight expression can be noticed in GH and/or prolactin-producing adenomas and in normal or hyperplastic pituitaries [57].

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Fig. 8. Plasminogen activator inhibitor in a sparsely granulated ACTH cell adenoma: most cells with positive cytoplasm. ⫻440.

Fig. 9. Urokinase receptor in a sparsely granulated ACTH cell adenoma: all cells with positive cytoplasm. ⫻440.

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Fig. 10. Matrix metalloproteinase-2 in a sparsely granulated ACTH cell adenoma: all cells with positive cytoplasm. ⫻440.

Apoptosis

Apoptosis represents a spontaneous form of programmed cell death. This phenomenon occurs under several physiological and pathological conditions and shows characteristic morphological and biochemical features. A clear-cut border to necroses does not exist. Probably the injured cells can elect to commit suicide by apoptosis instead of necrosis [58]. bcl-2 is a protein derived from the inner mitochondrial membrane and restricted to cells with long life spans. It is related to programmed cell death: overexpression of bcl-2 gene blocks apoptosis [59] and bcl-2 oncogene induces a prolonged cell survival. Furthermore, a positive relationship between bcl-2 protein expression and markers of angiogenesis was found [60] in prolactin adenomas, GH adenomas and inactive adenomas (fig. 11), showing higher levels of bcl-2 expression in more vascularized adenomas. The lowest levels of bcl-2 and other apoptosis regulatory proteins Bar and bcl-X were detected in pituitary carcinomas [61]. Estrogens and dopamine agonists influence apoptoses in the pituitary as was shown in studies of rat pituitaries [62]. In estrogen-induced prolactin cell hyperplasia, apoptoses are increased if estrogens are withdrawn and they are much more increased if the rats are treated with dopamine agonists. For demonstration of apoptoses, the ISEL/TUNEL technique is established. Its principle is the demonstration of nuclear DNA fragmentation by labeling DNA containing free 3⬘-OH ends (3⬘-OH nick end labeling). The

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Fig. 11. bcl-2 protein in a null cell adenoma: about 10% of cell with positive cytoplasm. ⫻440.

enzyme polymerase is used to generate 5⬘ overhangs from any 3⬘-OH ends of DNA strand beaks and to end-fill the overhangs and incorporate biotinylated deoxyadenosine or deoxyuridine triphosphate. For visualization of the labeled sites, immunoperoxidase detection systems are used [63, 64]. By using this technique, apoptosis was found in 24% of adenomas [65] (in 11% of GH-secreting adenomas and in 33% of non-functioning adenomas) (fig. 12). Others [66] counted a higher apoptopic LI in functioning adenomas (LI 5.6%) than in non-functioning adenomas (LI 1.8%). The LI was higher in TSH adenomas (10.3%) and lower in ACTH adenomas (5.9%), GH adenomas (5.5%), prolactin adenomas (5.2%) and in mixed GH/prolactin-producing adenomas (5.1%) [66]. Apoptosis was not demonstrable in normal pituitaries [65]. In adenomas, there was a positive correlation between Ki-67 LI and the apoptotic index [67]. Octreotide treatment did not influence the number of apoptotic cells [67]. Apoptoses can also be identified by electron microscopy showing a marked shrinkage of the cell volume, nuclear pyknosis with margination of chromatin and crescent formations, a perinuclear halo and lytic changes of cytoplasmic organelles [66, 68]. With monoclonal antibodies to single-stranded DNA a better differentiation than between the TUNEL technique between apoptoses and necrosis was reported [69], but studies of pituitaries are not known to us. The pituitary adenylate cyclase-activating polypeptide (PACAP) was found to inhibit TGF-␤1-induced apoptosis in cultured human pituitary adenoma

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Fig. 12. Apoptotic cells in TUNEL technique: many positive nuclei in a sparsely granulated GH cell adenoma. ⫻440.

cell line HP-75 [70]. This effect can be blocked by PACAP receptor antagonists. The estrogen receptor antagonist tamoxifen induced growth arrest and apoptosis in human primary pituitary tumor cultures [71]. Apoptosis was also increased in pituitary adenoma cultures by hypericin as an inhibitor of protein kinase C [72]. References 1 2 3 4

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Dr. Wolfgang Saeger Institute of Pathology of the Marienkrankenhaus Alfredstrasse 9, DE–22087 Hamburg (Germany) Tel. ⫹49 40 2546 2701, Fax ⫹49 40 2546 2730, E-Mail [email protected]

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Down-Regulation of E-Cadherin and Catenins in Human Pituitary Growth Hormone-Producing Adenomas Toshiaki Sanoa, Qian Zhi Ronga, Noriko Kagawaa, Shozo Yamadab a

Department of Pathology, University of Tokushima School of Medicine, Tokushima, and bDivision of Neurosurgery, Toranomon Hospital, Tokyo, Japan

Abstract Growth hormone (GH)-producing pituitary adenomas can be ultrastructurally divided into two major types: densely granulated and sparsely granulated. The latter type of adenoma characteristically exhibits globular accumulations of cytokeratin filaments known as fibrous bodies, which are immunohistochemically identifiable as juxtanuclear dot-like immunoreactivity. We hypothesize that the formation of fibrous body might be related to dysfunction of adhesion molecules, because of the functional relationship between intermediate filaments and the cadherin-catenin complex and frequent observation of loss of cohesiveness of the adenoma cells. Our recent immunohistochemical study showed that expression of E-cadherin and its undercoat proteins, ␣-, ␤- and ␥-catenin, in GH cell adenomas with prominent fibrous bodies was significantly reduced compared with GH cell adenomas without fibrous bodies and the normal adenohypophysial cells. Although no mutation of exon 3 of the ␤-catenin gene was found in any GH cell adenomas with fibrous bodies, methylation-specific polymerase chain reaction analysis revealed that the E-cadherin promoter region was methylated in 37.5% of these adenomas, two of which displayed total methylation, but not in GH cell adenomas without fibrous bodies. We conclude that the decreased expression of the E-cadherin-catenin complex and methylation of the E-cadherin gene promoter region are events associated with the formation of fibrous bodies in GH cell adenomas. It remains to be clarified to explain the mechanism by which down-regulation of adhesion molecules is involved in the abnormal assembly of intermediate filaments. Copyright © 2004 S. Karger AG, Basel

Two Types of GH-Producing Pituitary Adenomas

It has been known that there are ultrastructurally two different types of growth hormone (GH)-producing pituitary adenomas: densely granulated and

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Fig. 1. Adenoma without fibrous bodies. a A perinuclear staining pattern of CK is seen. b Expression of E-cadherin is preserved. c Expression of ␤-catenin is also preserved.

sparsely granulated somatotroph adenomas [1]. The latter type of adenomas characteristically exhibit a globular structure known as fibrous body which is composed of aggregation of cytokeratin (CK) intermediate filaments [2, 3]. By immunohistochemistry using low molecular weight CK antibodies, fibrous body can be easily identified as a juxtanuclear dot-like immunoreactivity (fig. 1a) [4]. The fact that tumor cells of GH-producing adenomas with prominent fibrous bodies contain less secretory granules than those without fibrous bodies suggests that cellular functions including production of secretory granules, GH synthesis and cell growth regulation may be impaired in this type of tumor. In fact, GH cell adenomas with prominent fibrous bodies have been reported to show decreased levels of GH mRNA [5] and different clinical and biochemical features compared with those without fibrous body [4]. Recently, Mazal et al. [6] reported that GH-producing adenomas with prominent fibrous bodies showed aggressive growth indicated several parameters including MIB1 index, apoptotic activity, and frequency of invasion. An extreme phenotype of GH cell adenomas with prominent fibrous bodies may be silent somatotroph adenoma, that is, clinically nonfunctioning GH cell adenoma in which only a few granules and a very faint GH immunoreactivity can be observed [5]. Histologically, GH cell adenomas with fibrous bodies often display scattered tumor cells, suggesting lack of tumor cell-cell adhesiveness and dysfunction of adhesion molecules.

Abnormal Assembly of Intermediate Filaments and Adhesion Molecules

Abnormal paranuclear aggregation of CK filaments is also seen in some neuroendocrine carcinomas such as small cell carcinomas [7] and Merkel cell carcinomas of the skin [8]. Similar structural alteration of intermediate

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filaments known as rhabdoid feature, which is characterized by a clear globular, inclusion body-like area in the cytoplasm, is mainly composed of vimentin with or without CK. Rhabdoid feature is seen in various tumors, such as malignant rhabdoid tumor of the kidney [9], carcinomas arising in various sites [10–12], and epithelioid sarcoma [13]. All these tumors with spherical accumulation of intermediate filaments are known as aggressive tumors with frequent recurrence and metastasis. Loss of cohesiveness of tumor cells is a common finding of these tumors. Decreased membranous expression of adhesion molecules has been reported in some of these tumors [13]. Intermediate filaments are physiologically linked directly to the adherens junction, in which intracellular adhesion is caused by classic cadherins such as E- or N-cadherin, or indirectly by involving a cross-linking element such as plectin [14, 15]. In order to exhibit their functional adhesion activity, cadherins must form complexes with cytoplasmic plaque proteins, ␤- or ␥-catenin, which, in turn, bind ␣-catenin that is attached to the actin microfilament-based cytoskeleton [16]. Therefore, it is likely that abnormal assemble of intermediate filaments in the cytoplasm may be due to impaired function of these adhesion molecules. We thus hypothesize that the formation of fibrous body in GH cell adenomas might correlate to dysfunction of adhesion molecules. To clarify the role of classic cadherin adhesion molecules in the two types of GH cell adenomas, we studied expression of the E-cadherin and its undercoat proteins, ␣-, ␤- and ␥-catenins, by use of specific monoclonal antibodies [17]. Immunohistochemistry for E-Cadherin and Its Undercoat Proteins in GH Cell Adenomas

In normal adenohypophyseal glands, E-cadherin (1:500, Transduction Laboratories, Lexington, Ky., USA) and ␣- and ␤-catenins (1:500, Transduction Laboratories) were strongly expressed on nearly entire hormone producing cell-cell boundaries, showing that the E-cadherin-catenin complex was one of the most principal intercellular adhesion molecules. In 24 GH cell adenomas with prominent fibrous bodies (fig. 1a), expression of E-cadherin (fig.1b), ␣-catenin and ␤-catenin (fig. 1c) was significantly decreased compared with that in GH cell adenomas without fibrous bodies (fig. 2a–c). In GH cell adenomas, mixed cells with and without fibrous bodies, E-cadherin and its undercoat proteins were exclusively immunopositive in the cells without fibrous bodies. There was no significant difference in E-cadherin expression between invasive and noninvasive GH cell adenomas in keeping with a recent report [18]. Our results suggested that there is a significant down-regulation

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Fig. 2. Adenoma with prominent fibrous bodies. a A juxtanuclear dot-like immunoreactive pattern of CK (CAM5.2) is obvious. b Expression of E-cadherin is decreased. c Expression of ␤-catenin is also decreased.

of the E-cadherin-catenin complex in GH cell adenomas with fibrous bodies, which might be an event associated with formation of fibrous bodies. Although our study does not explain the mechanism by which downregulation of adhesion molecules in some GH-producing adenoma cells in vivo is associated with formation of fibrous bodies, a few findings about the relationship between cytoskeleton components and cell membrane proteins may support this correlation. First, CK filaments form a dense filament network radiating from the nucleus and extending to the plasma membrane, and interact with the plasma membrane, nuclear envelope, mitochondria, microtubules, and actin microfilaments. Actin microfilaments appear to be a key element in linking intermediate filaments to the plasma membrane [19–21]. Second, destabilization of microtubules and microfilaments could induce significant alterations in the cytoskeletal organization of CK filaments in epithelial cells from a uniform distribution to an open lattice of CK filaments stabilized by membrane-associated focal centers, like fibrous bodies [22]. Finally, GH, platelet-derived growth factor, insulin and epidermal growth factor, binding to their specific cell membrane receptors, could induce perturbation of the cytoskeletal elements [23, 24]. Nuclear localization of ␤-catenin has been often observed in a number of tumors including endocrine tumors [25–27]. Semba et al. [26] reported that nuclear localization of ␤-catenin was demonstrated in 21 of 37 (56.8%) pituitary adenomas, but Tziortzioti et al. [27] found nuclear localization of ␤-catenin in only 2 of 154 pituitary tumors (1.3%). In our series, we could not find positive nuclear staining of ␤-catenin in any adenomas. Genetic Alterations in the E-Cadherin-Catenin Complex of GH Cell Adenomas

To see the mechanism of inactivation of the E-cadherin-catenin complex, mutations in exon 3 of ␤-catenin gene were investigated in 16 GH cell Sano/Rong/Kagawa/Yamada

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adenomas with prominent fibrous bodies [17]. Using PCR and sequencing of cloned plasmids, mutations were not detected in these cases. Although mutations in exon 3 of ␤-catenin gene have been reported in a number of tumors [28, 29], our study indicates that mutations in exon 3 of ␤-catenin gene appear to be uncommon in GH cell pituitary adenomas. We also examined the methylation status of E-cadherin promoter region in the same 16 GH cell adenomas with fibrous bodies and found that 6 (37.5%) adenomas were methylated around the transcription start site of E-cadherin [17]. Moreover, two of them displayed total methylation at the E-cadherin promoter, as revealed by the detection of band corresponding to methylated DNA alone, and the remaining 4 tumors displayed partial methylation, as revealed by the detection of bands corresponding to both methylated and unmethylated DNA. On the other hand, the methylation status of the E-cadherin promoter region was not detected in 10 GH cell adenomas without fibrous bodies. In conclusion, the decreased expression of the E-cadherin-catenin complex and methylation of the E-cadherin gene promoter region might be events associated with the formation of fibrous bodies in GH cell adenomas with prominent fibrous bodies. It remains to be clarified to explain the mechanism by which down-regulation of adhesion molecules is involved in the abnormal assemble of intermediate filaments.

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Morphologic Changes and Molecular Regulation of Angiogenesis in Pituitary Adenomas N. Garcia de la Torre, J.A.H. Wass, H.E. Turner Endocrinology Department, The Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Headington, Oxford, UK

Abstract Angiogenesis, the process of development of a new vasculature, plays a crucial role in tumour growth. In the pituitary, unlike other tissues, vascularization is lower in adenomas compared to the normal gland. Despite this finding, a relationship between increased vascularity and some aspects of tumour behaviour such as size, invasiveness, surgical outcome and malignancy, has been demonstrated. The process of angiogenesis is the result of a balance of stimulating and inhibiting factors. It is likely that an interaction between gene expression (such as pituitary tumour transforming gene), hormonal stimuli including oestrogens, corticosteroids, dopamine, 16-kDa fragments of prolactin and growth hormone, somatostatin analogues, and pro- and anti-angiogenic growth factors (e.g. vascular endothelial growth factor and fibroblast growth factor), determine the final angiogenic phenotype of pituitary tumours, and thus subsequent tumour behaviour. Copyright © 2004 S. Karger AG, Basel

Introduction

Angiogenesis describes the process of development of new blood vessels from existing vasculature. Physiological angiogenesis during adult life is mainly restricted to the female reproductive cycle and wound healing. With the exception of these two processes, angiogenesis is usually inhibited in the normal tissues of the adult [1], but may be activated in some pathological diseases, for example psoriasis [2], retinal neovascularization [3], arthritis [4], and malignancy [5]. Angiogenesis plays a crucial role in tumour growth, in that it promotes oxygenation, nutrient perfusion, and the removal of metabolic waste [6]. Additionally, the breakdown of extracellular matrix through the action of matrix metalloproteinases

allows tumour invasion of surrounding structures and the new blood vessels provide a route of entry for metastatic tumour cells to enter the systemic circulation. There are now several experimental [7] and clinical data [8] showing that growth of solid tumours is angiogenesis-dependent [9]. In addition, angiogenesis (measured as tumour microvessel density) has been shown to be related to tumour behaviour. In many human tumours including prostate, breast, stomach, and bladder, increased angiogenesis has been shown to be correlated with development of metastasis [10], poor prognosis [11], and reduced survival [12, 13]. Premalignant lesions have also been shown to be more vascular than normal tissue. Pre-carcinoma of the cervix exhibits high levels of angiogenesis [14]. Histologically normal lobules from breast harbouring cancer have been shown to be significantly more angiogenic than lobules from breast without cancer [15] and transgenic mice with an oncogene in the pancreatic ␤-cells demonstrate increased angiogenesis in hyperplastic islets prior to the development of frank neoplastic change [16]. Angiogenesis is a complex multistep process, involving stimulation by various pro-angiogenic growth factors, and reduction in inhibitors of angiogenesis. It is the net balance of the pro-angiogenic factors and the inhibitors of angiogenesis that determine the final angiogenic phenotype of the tumour [17].

Morphologic Vascular Changes in Pituitary Adenomas

The observation of reduced vascularization in the parenchyma of pituitary tumours compared to autopsy specimens of normal pituitary tissue was first reported by Schechter [18]. Subsequent studies assessed vascularization in a more comprehensive manner using immunostaining for different endothelial markers [19, 20] and confirmed that the microvascular density (MVD) of pituitary adenomas is significantly lower than in the normal gland. Low vascular density and/or inhibition of angiogenesis is unusual for a tumour but may play a role in the usually slow growth of pituitary adenomas and may at least in part explain the common finding of incidental non-progressive pituitary microadenomas in 10% of normal glands [21]. Alternatively, the low growth rate of these tumours may not influence the metabolic demand significantly, so that vascularization does not limit the growth.

Relationship with Tumour Behaviour

Several studies have shown that angiogenesis in pituitary tumours is related to tumour behaviour and outcome (table 1). Despite the fact that the parenchyma of pituitary adenomas is less vascular than normal tissue, it has been shown that

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Table 1. Studies reporting relationship between angiogenesis and/or angiogenic growth factors expression and behaviour in pituitary tumours Relationship with tumour behaviour

Reference

Macroprolactinomas are significantly more vascular than microprolactinomas Invasive prolactinomas are significantly more vascular than non-invasive tumours Macroprolactinomas and ACTH-secreting tumours with lower MVD are more likely to be surgically cured Pituitary carcinomas show higher vascularization than pituitary adenomas VEGF protein expression is higher in pituitary carcinomas compared to adenomas VEGF mRNA expression is higher in pituitary adenomas than in normal glands

20 22 22 19, 25, 26 46 49

microprolactinomas are significantly less vascularized than macroprolactinomas [20], that invasive prolactinomas are significantly more vascular than noninvasive tumours and that surgical cure is more likely in macroprolactinomas and in ACTH-secreting tumours with lower MVD than in those of higher MVD [22]. In addition, different pituitary tumours vary in the relationship between size and vascular density. There is no difference in vascular density between GH-secreting macroadenomas and microadenomas, in contrast to microprolactinomas which are significantly less vascular than macroprolactinomas [20]. This fits with the clinical observation that microprolactinomas are a distinct clinical entity from macroprolactinomas, rarely progress in size [23] and are not part of the same pathological process [24]. In contrast, different size GH-secreting tumours are clinically part of the same spectrum of the disease. Further evidence of a relationship between angiogenesis and aggressiveness has been demonstrated in several studies that showed that vascularization is higher in the rare pituitary carcinomas than in benign adenomas of the pituitary gland [19, 25, 26]. However, there was no relation between angiogenesis and tumour regrowth. Thus, there is evidence of a relationship between increased vascularity and some, but not all, aspects of pituitary tumour behaviour.

Vascular Supply in Pituitary Tumours

Unlike other sites of tumour formation, the anterior pituitary has a dual blood supply. The hypothalamo-pituitary portal supply is the main source,

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carrying blood from the median eminence with hypothalamic releasing and inhibitory factors, but there is an additional direct arterial supply from the loral and capsular arteries [27]. The liver also has a dual blood supply (portal and direct arterial blood supply), and most metastases derive their blood supply directly from the hepatic artery rather than the hepatic portal vein [28]. The source of the blood vessels supplying the pituitary adenomas is unclear, although there are several studies suggesting that a direct arterial supply from the systemic circulation may develop. These reports include radiological data from angiography [29] and dynamic magnetic resonance imaging [30]. Anatomical studies in human pituitary tumours have reported an arterial blood supply [31, 32], that has been further confirmed by an animal model of oestrogeninduced lactotroph hyperplasia and tumorigenesis in rats [33, 34]. The apparently lower MVD of pituitary tumours may perhaps reflect a completely or partially de novo blood supply from the extraportal system. Therefore, although the tumours are less vascular overall, they may have induced new vessel development from the systemic circulation, escaping hypothalamic influences on hormone production and the lower MVD may be the end result of an increase in angiogenesis after all.

Regulation of Angiogenesis in Pituitary Adenomas

The sinusoid-capillary network of the anterior lobe of the pituitary gland has a fenestrated layer of endothelial cells, as in all endocrine organs, which allows soluble factors (growth factors or hormones) to diffuse into the surrounding tissue and vice versa. In that way, pro- and anti-angiogenic growth factors of the pituitary can bind to endothelial cells, and hormones produced in peripheral endocrine glands (e.g. ovary, adrenal gland) or their synthetic analogues can influence hormone and growth factor production by tumour cells. In addition to this interaction between hormones and growth factors, genetic events involved in pituitary tumour pathogenesis may play an important role in the regulation of angiogenesis in pituitary adenomas.

Regulation of Angiogenesis by Growth Factors Folliculostellate cells (FSC) were first described in the pituitary gland by Rinehart and Farquhar [35] and are characterized by their shape and long slender cytoplasm processes. Ferrara et al. [36] were able to study follicular cell function following enzymatic dispersal of bovine anterior pituitary gland, and in vitro culture. They demonstrated the production of fibroblast growth factor

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(FGF-2), a then new growth factor named vascular endothelial growth factor (VEGF) [37], and leukaemia inhibitory factor (LIF) [38] from these cultures. VEGF plays a key role in both physiological and pathological angiogenesis through the increase of proliferation and migration of endothelial cells [39] and also increasing endothelial permeability by inducing pores or fenestrations in the endothelium [40]. In addition, VEGF functions as an anti-apoptotic factor promoting the survival of endothelial cells in newly formed vessels [41]. Its presence has been demonstrated in the pituitary gland of several species and in the GH3 cell line [37, 42, 43]. VEGF has been demonstrated to be expressed in the normal pituitary only in FSC [37]. However, approximately 90% of human pituitary tumours showed measurable VEGF secretion in vitro [44], and VEGF expression could be detected by immunohistochemistry and in situ hybridization in pituitary adenomas [45, 46]; but it is not clear by which cell types VEGF is produced inside the tumours. There are two main VEGF receptors involved in angiogenesis: the VEGF receptor 1 or Flt1 and VEGF receptor 2 or KDR/Flk1. Compelling evidence indicates that VEGFR-2 is the major mediator of the mitogenic, angiogenic, and permeability-enhancing effects of VEGF [47]. In normal rat pituitaries, VEGFR-2 immunoreactivity was evident in both endothelial cells and adenohypophyseal cells, supporting the concept that VEGF functions as an autocrine/paracrine factor in the pituitary [48]. The data are conflicting regarding VEGF expression and pituitary tumour behaviour. Lloyd et al. [46] analyzed VEGF protein expression by immunohistochemistry in 148 pituitary adenomas and showed that, in agreement with the microvascular data, VEGF expression is decreased in pituitary adenomas compared to the normal pituitary gland, and that carcinomas had higher VEGF expression than adenomas. However, a further study analyzing VEGF mRNA and VEGFR-2 mRNA by PCR from 121 pituitary tumours reported a significant 3.2-fold increase (p ⬍ 0.05) of VEGF mRNA in non-functioning tumours, which represented 77% of the pituitary tumours studied, compared with normal pituitaries [49]. TSH-omas (3 cases) were unique in demonstrating significantly reduced expression of VEGF mRNA, compared with the normal gland (92% reduction, p ⫽ 0.002). In addition, VEGFR-2 mRNA expression was significantly increased 13.9-fold (p ⬍ 0.0001) in pituitary tumours of all types. Western blot analysis from 24 randomly selected pituitary specimens revealed that protein data were in agreement with the mRNA results in most, but not all, the non-functioning adenomas showing increased VEGF expression. A different distribution of histological types in both studies and lack of close correlation between mRNA and protein expression could be the reasons for these discrepancies. FGF-2 is a potent angiogenic factor produced by endothelial, stromal and tumoural cells as well as released from the extracellular matrix, and stimulates proliferation of endothelial cells [50]. Although elevated FGF-2, in addition to

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VEGF, plasma concentrations have been demonstrated in patients harbouring pituitary tumours [51], there were no data directly relating this factor to angiogenesis in pituitary adenomas until the recent discovery of the human pituitary tumour transforming gene (PTTG) and its involvement in pituitary tumour pathogenesis (see below). Although up-regulation of angiogenic factors is necessary to stimulate angiogenesis, simultaneous down-regulation of angiogenesis inhibitors is also required to sufficiently turn on angiogenesis. It is likely that negative regulators may be particularly important to maintain a quiescent endothelium in the very vascular endocrine glands. Rather than increased pro-angiogenic factors, downregulation of angiogenesis inhibitors may be required to activate the angiogenic switch [17]. LIF was also detected in conditioned medium from bovine pituitary FSC [52]. Although LIF inhibited aortic endothelial cell proliferation, it had no effect on smaller vessels (adrenal capillaries). LIF is present in human pituitary adenomas [53], but because of its lack of effect on small vessels, it is unlikely to play a significant role in the angiogenic process in pituitary tumours. Recently, Basu et al. [54] have provided evidence that dopamine (DA) can selectively inhibit VEGF-induced angiogenesis of mouse ovarian tumour in vivo, as well as VEGF-induced endothelial cell proliferation and migration of cultured human umbilical vein endothelial cells (HUVEC) in vitro. Moreover, DA can inhibit VEGFR-2 phosphorylation in HUVEC. This effect of DA is likely to be mediated through DA2-receptors detected on endothelial cells, because other DA2 agonists like bromocriptine and quinagolide had the same effect, and DA1, DA3, and DA4 antagonists could not reverse the effect of DA. The inhibitory effect of DA was selective to VEGF and did not affect the effect of other factors as FGF-2. These findings might provide a novel approach to angiogenic therapy.

Hormonal Regulation of Angiogenesis There is increasing evidence that hormones play an important role in the control of endothelial cell function and growth. Oestrogens have been shown to increase expression of VEGF. Protein expression of VEGF as well as its receptor type 2 increased during the development of oestrogen-induced prolactinomas in the pituitary of rats and this was associated with the growth and enlargement of blood vessels [55]. These findings suggest a role in the modulation of pituitary tumour angiogenesis that could be a part of the mechanism by which oestrogens cause pituitary hyperplasia and possibly prolactinoma formation. Further studies showed a strong inhibition of oestrogen-induced lactotroph

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tumour angiogenesis by methoxyestradiol; furthermore, VEGF expression was down-regulated, concomitant with suppression of tumour angiogenesis [56]. The fact that glucocorticoids inhibit the secretion of VEGF by FSC was demonstrated recently [44, 57] in an analysis of VEGF secretion in different rodent pituitary cell lines as well as in several types of human pituitary adenoma cell cultures. Secretion of VEGF from AtT20, GH3, and TtT/GF rodent cell lines as well as in 84% of the human adenomas tested was inhibited by dexamethasone. These observations could be highly relevant regarding corticotrope adenoma development in vivo. One explanation of the microadenomatous phenotype of most corticotrope adenomas could be that the elevated cortisol levels inhibit VEGF production and subsequent angiogenesis. Many endogenous inhibitors of angiogenesis have been shown to be cleaved products of other larger proteins; for example, angiostatin is a cleaved product of plasminogen, which inhibits angiogenesis and metastasis growth in vivo, although intact plasminogen lacks this activity [58]. Endostatin, is a proteolytic fragment of collagen XVIII and causes inhibition of endothelial proliferation as well as increased tumour cell apoptosis leading to regression of metastasis and dormancy of tumours [59]. In the pituitary gland, there are several potential inhibitors of angiogenesis. The 16-kDa N-terminal fragment of human prolactin (16K PRL) is a potent anti-angiogenic factor in vivo in the chicken chorioallantoic membrane (CAM) assay [60]. 16K PRL inhibits FGF-2 and VEGF-induced cell proliferation of cultured bovine and human capillary endothelial cells [61]. Further studies have shown that 16K PRL inhibits FGF-2-induced angiogenesis in vivo in the rat cornea model [62]. Although 16K PRL has been demonstrated in the pituitary gland of the rat [63], there are no data on its potential role in the human pituitary gland and pituitary tumours. In vivo studies using the CAM assay and in vitro studies of endothelial cell proliferation show the 16-kDa fragment of GH to be angioinhibitory, while GH is stimulatory to angiogenesis [64]. The peptides of the PRL/GH family are novel in that, the N-terminal fragments being anti-angiogenic, the intact molecules are angiogenic. However, the hypothesis that proteolytic cleavage of the PRL/GH members by enzymes as cathepsin D and thrombin, constitute a mechanism for modulating vascularization in human pituitary tumours remains to be proved. The presence of somatostatin receptors on tumour cells and on the proliferating vascular endothelium has led to several in vitro and in vivo studies to investigate the anti-proliferative and anti-angiogenic effects of somatostatin analogues. The anti-angiogenic activity of octreotide was first demonstrated by Woltering et al. [65] using direct visualization of blood vessels on the CAM assay. Several studies have investigated the mechanism for this anti-angiogenic effect. Proposed mechanisms include: (i) direct inhibition of endothelial cell proliferation, (ii) inhibition of VEGF and IGF-I secretion, and (iii) inhibition of

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monocyte chemotaxis [66]. Few studies have focused on the effect of somatostatin analogues in pituitary adenomas. Vidal et al. [26] evaluated microvessel densities in 157 various pituitary adenoma types and showed that GH-producing adenomas treated with octreotide tended to have lower MVD than untreated tumours, although the differences did not reach statistical significance. Inhibition of angiogenesis was proportional to the binding affinity and the GH-inhibiting ability of the somatostatin analogue [67], suggesting a potential inhibition of angiogenesis mediated by a lowering of GH levels resulting in a subsequent decrease in IGF-I levels. Pawlikowski et al. [68] demonstrated that treatment of rats with octreotide inhibited the diethylstilboestrol-induced elevation of prolactin levels and pituitary cell proliferation. It also suppressed some but not all diethylstilboestrol-induced changes in anterior pituitary vascularization. It may be that the effect of octreotide is related rather to reduction of blood supply than to structural changes in the vascular network. The anti-angiogenic effect of somatostatin analogues might be of clinical relevance mediating the tumoural shrinkage observed in some tumours treated with these compounds.

Genes and Angiogenesis The recently isolated pituitary tumour-derived transforming gene (PTTG) has been shown to be highly expressed in malignant human cell lines and pituitary tumours [69]. Pituitary PTTG is regulated in vivo and in vitro by oestrogens. In the rat prolactinoma model, oestradiol was shown to induce PTTG expression early in pituitary transformation (normal cell to hypertrophic/hyperplastic cell), followed within 24 h by increased FGF-2 and VEGF expression associated with pituitary angiogenesis [70]. Using the same animal model, selective anti-oestrogen treatment blocked oestrogen-induced pituitary PTTG expression and inhibited lactotroph tumour growth [71]. PTTG induces an angiogenic phenotype in both in vitro and in vivo angiogenesis models, and increased PTTG mRNA is associated with angiogenic phenotype in human tumours [72]. It has been suggested that FGF-2 may be the effector for PTTGdriven angiogenesis, since rat pituitary tumours with higher vascularity showed increased PTTG expression, and anti-FGF-2 antibodies inhibited PTTG stimulation of new blood vessel formation in vitro [73]. Further studies reported that PTTG also stimulates VEGF expression in vitro independent from FGF-2 up-regulation and that VEGF and PTTG mRNA expression were highly correlated in human pituitary tumours [49]. Thus, PTTG overexpression seems to be an important mechanism of pituitary tumorigenesis and angiogenesis. Until now, no other genes have been involved in the regulation of angiogenesis in pituitary adenomas.

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Estrogens (17-␤-estradiol)

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VEGF

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Fig. 1. Proposed model of hormonal regulation of angiogenesis in pituitary tumours. PTTG: pituitary tumour-transforming gene, FGF-2: fibroblast growth factor-2, VEGF: vascular endothelial growth factor, GH: growth hormone, IGF-1: insulin growth factor-1, EC: endothelial cells, 16K GH: 16-kDa fragment of growth hormone, 16K PRL: 16-kDa fragment of prolactin, DEX: dexamethasone, DA: dopamine.

Conclusions

Pro- and anti-angiogenic growth factors, hormonal stimuli and genes constitute an integrated regulatory system that determines the angiogenic phenotype of pituitary tumours and possibly some aspects of tumour behaviour (fig. 1). However, the regulation of angiogenesis in pituitary tumours is not fully elucidated and the potential efficacy of inhibition of this process is unclear.

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Nevertheless, the regulation of angiogenesis by some drugs as somatostatin analogues and DA agonists, and the likely future release of hybrids of these compounds might open new possibilities in the treatment of pituitary tumours.

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Ferrara N: Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int 1999;56:794–814. Vidal S, Lloyd RV, Moya L, Scheithauer BW, Kovacs K: Expression and distribution of vascular endothelial growth factor receptor Flk-1 in the rat pituitary. J Histochem Cytochem 2002;50:533–540. McCabe CJ, Boelaert K, Tannahill LA, Heaney AP, Stratford AL, Khaira JS, Hussain S, Sheppard MC, Franklyn JA, Gittoes NJ: Vascular endothelial growth factor, its receptor KDR/ Flk-1, and pituitary tumor transforming gene in pituitary tumors. J Clin Endocrinol Metab 2002; 87:4238–4244. Vlodavsky I, Folkman J, Sullivan R, Fridman R, Ishai-Michaaeli R, Sasse J, Klagsbrun M: Endothelial cell-derived basic fibroblast growth factor: Synthesis and deposition into subendothelial extracellular matrix. Proc Natl Acad Sci USA 1987;84:2292–2296. Komorowski J, Jankewicz J, Stepien H: Vascular endothelial growth factor, basic fibroblast growth factor and soluble interleukin-2 receptor concentrations in peripheral blood as markers of pituitary tumours. Cytobios 2000;101:151–159. Ferrara N, Winer J, Henzel WJ: Pituitary follicular cells secrete an inhibitor of aortic endothelial cell growth: Identification as leukemia inhibitory factor. Proc Natl Acad Sci USA 1992;89:698–702. Akita S, Webster J, Ren SG, Takino H, Said J, Zand O, Melmed S: Human and murine pituitary expression of leukemia inhibitory factor. Novel intrapituitary regulation of adrenocorticotropin hormone synthesis and secretion. J Clin Invest 1995;95:1288–1298. Basu S, Nagy JA, Pal S, Vasile E, Eckelhoefer IA, Bliss VS, Manseau EJ, Dasgupta PS, Dvorak HF, Mukhopadhyay D: The neurotransmitter dopamine inhibits angiogenesis induced by vascular permeability factor/vascular endothelial growth factor. Nat Med 2001;7:569–574. Banerjee SK, Sarkar DK, Weston AP, De A, Campbell DR: Over-expression of vascular endothelial growth factor and its receptor during the development of estrogen-induced rat pituitary tumors may mediate estrogen-initiated tumor angiogenesis. Carcinogenesis 1997;18:1155–1161. Banerjee SK, Zoubine MN, Sarkar DK, Weston AP, Shah JH, Campbell DR: 2-Methoxyestradiol blocks estrogen-induced rat pituitary tumor growth and tumor angiogenesis: Possible role of vascular endothelial growth factor. Anticancer Res 2000;20:2641–2645. Gloddek J, Pagotto U, Paez Pereda M, Arzt E, Stalla GK, Renner U: Pituitary adenylate cyclaseactivating polypeptide, interleukin-6 and glucocorticoids regulate the release of vascular endothelial growth factor in pituitary folliculostellate cells. J Endocrinol 1999;160:483–490. O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Sage EH, Folkman J: Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994;79:315–328. O’Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J: Endostatin: An endogenous inhibitor of angiogenesis and tumour growth. Cell 1997; 88:277–285. Clapp C, Martial JA, Guzman RC, Rentier-Delure F, Weiner RI: The 16-kDa N-terminal fragment of human prolactin is a potent inhibitor of angiogenesis. Endocrinology 1993;133:1292–1299. D’Angelo G, Martini JF, Iiri T, Fantl WJ, Martial J, Weiner RI: 16K human prolactin inhibits vascular endothelial growth factor-induced activation of Ras in capillary endothelial cells. Mol Endocrinol 1999;13:692–704. Duenas Z, Torner L, Corbacho AM, Ochoa A, Gutierrez-Ospina G, Lopez-Barrera F, Barrios FA, Berger P, Martinez de la Escalera G, Clapp C: Inhibition of rat corneal angiogenesis by 16-kDa prolactin and by endogenous prolactin-like molecules. Invest Ophthalmol Vis Sci 1999;40:2498–2505. Clapp C, Sears PS, Russell DH, Richards J, Levay-Young BK, Nicoll CS: Biological and immunological characterization of cleaved and 16K forms of rat prolactin. Endocrinology 1988;122: 2892–2898. Struman I, Bentzien F, Lee H, Mainfroid V, D’Angelo G, Goffin V, Weiner RI, Martial JA: Opposing actions of intact and N-terminal fragments of the human prolactin/growth hormone family members on angiogenesis: An efficient mechanism for the regulation of angiogenesis. Proc Natl Acad Sci USA 1999;96:1246–1251. Woltering EA, Barrie R, O’Dorisio TM, Arce D, Ure T, Cramer A, Holmes D, Robertson J, Fassler J: Somatostatin analogues inhibit angiogenesis in the chick chorioallantoic membrane. J Surg Res 1991;50:245–251.

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Garcia de la Torre N, Wass JA, Turner HE: Anti-angiogenic effects of somatostatin analogues. Clin Endocrinol (Oxf) 2002;57:425–441. Woltering EA, Watson JC, Alperin-Lea RC, Sharma C, Keenan E, Kurozawa D, Barrie R: Somatostatin analogs: Angiogenesis inhibitors with novel mechanisms of action. Invest New Drugs 1997;15:77–86. Pawlikowski M, Kunert-Radek J, Grochal M, Zielinski K, Kulig A: The effect of somatostatin analog octreotide on diethylstilbestrol-induced prolactin secretion, cell proliferation and vascular changes in the rat anterior pituitary gland. Histol Histopathol 1997;12:991–994. Zhang X, Horwitz GA, Heaney AP, Nakashima M, Prezant TR, Bronstein MD, Melmed S: Pituitary tumor transforming gene expression in pituitary adenomas. J Clin Endocrinol Metab 1999;84:761–767. Heaney AP, Horwitz GA, Wang Z, Singson R, Melmed S: Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis. Nat Med 1999;5:1317–1321. Heaney AP, Fernando M, Melmed S: Functional role of estrogen in pituitary tumor pathogenesis. J Clin Invest 2002;109:277–283. Heaney AP, Singson R, McCabe CJ, Nelson V, Nakashima M, Melmed S: Expression of pituitarytumour transforming gene in colorectal tumours. Lancet 2000;355:716–719. Ishikawa H, Heaney AP, Yu R, Horwitz GA, Melmed S: Human pituitary tumor-transforming gene induces angiogenesis. J Clin Endocrinol Metab 2001;86:867–874.

Dr. Helen Turner Department of Endocrinology The Oxford Centre for Diabetes, Endocrinology and Metabolism Churchill Hospital, Headington, Oxford OX3 7LJ (UK) Tel. ⫹44 01865 857300, Fax ⫹44 01865 742348, E-Mail [email protected]

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Advances in Pituitary Pathology: Use of Novel Techniques Ricardo V. Lloyd Mayo Clinic and Mayo Foundation, Rochester, Minn., USA

Abstract Many new techniques are rapidly being developed and applied to the study of normal and neoplastic pituitary tissues. These range from techniques used for the past few decades such as in situ hybridization to more recent developments such as comparative genomic hybridization, laser capture microdissection, DNA and tissue arrays, proteomics, and RNA interference technology. These approaches have been applied to answer many challenging questions about pituitary function and pathophysiology. This chapter reviews many of these recent developments and shows their applications to pituitary biology in order to demonstrate how these new techniques are providing insights about basic aspects, clinical, and pharmacologic knowledge of the pituitary gland and of pituitary tumors. Copyright © 2004 S. Karger AG, Basel

In situ Hybridization

Hybridization Methods Hybridization methods which involve paring of complimentary strands of nucleic acid such as DNA-DNA, DNA-RNA, or RNA-RNA hybrids are one of the keystones of molecular studies. Various forms of hybridization including solution hybridization, Northern and Southern hybridization, and in situ hybridization (ISH) have provided major insights into molecular mechanisms and disease development including studies in the pituitary. ISH is a powerful technique used in molecular pathology. The relationship of expression of specific gene products to other cells in the tissue sections or in cell preparations can be readily visualized with this approach by using radioactive or chromogenic reporter products. A combination of ISH analysis and immunohistochemistry can also be used to localize gene transcripts and the translated protein products within the same cell or in adjacent cells.

a

b Fig. 1. a ISH showing localization of PRL mRNA in normal anterior pituitary cells. An oligonucleotide probe was used for hybridization and a chromogenic reporter system with alkaline phosphatase and nitroblue tetrazolium and bromo-chloro-indole phosphate were used. b ISH to detect PRL mRNA in a pituitary adenoma using an 35S-labeled oligonucleotide probe. The use of radioactive probes is more sensitive than chromogenic probes, but the resolution is better with chromogenic probes.

In situ Hybridization Various approaches have been used by investigators for ISH. Preservation of nucleic acid is one of the critical steps in the procedure [1–3]. Messenger RNA is better preserved in frozen tissue sections than in paraffin sections, but highly reproducible results have been obtained in many studies with paraffinembedded tissue sections. Fixatives such as paraformaldehyde, ethanol, and neutral buffered formalin are excellent for messenger RNA preservation. After fixation, tissues can be sectioned and stored for weeks or months without loss of messenger RNA in the tissues. Many different types of probes can be used for ISH including cDNA and cRNA probes and synthetic oligonucleotide probes. The signal can be detected with radioactive reporters or nonradioactive reporters, which is more commonly used [4] (fig. 1a, b). Nonradioactive detection is more rapid, but this is usually not as sensitive as radioactive probe. Studies in the Pituitary Studies using ISH studies with human pituitaries have been done for more than 15 years. Formalin-fixed paraffin-embedded tissue sections have been used extensively in these studies. One of the earliest studies showed a growth hormone secreting pituitary tumors in which the messenger RNA for growth hormone was present, but the protein was not detected in the cells [5]. Many studies have shown that prolactin-producing cells in tumors commonly express only prolactin but not growth hormone message, supporting the concept that prolactin-producing cells in tumors are terminally differentiated. Other studies have examined growth hormone-releasing hormone and somatostatin on growth

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hormone gene expression in human pituitary tumors [6, 7]. Studies by Kovacs et al. [8] and Trouillas et al. [9] showed that some silent growth hormone tumors without clinical evidence of acromegaly usually express the growth hormone messenger RNA within these cells. The functional status of the growth hormone messenger proteins in patients with silent growth hormone tumor is unknown. Other studies [10] examining a series of pituitary tumors from patients with acromegaly compared immunohistochemistry and ISH; there was 100% correlation for growth hormone and 60% for prolactin in the hybridization signal for the hormone content. This finding was in agreement with earlier studies [5]. In other studies of growth hormone and prolactin tumors, investigators [11] found that messenger RNA for prolactin increased during pregnancy [11]. This finding suggests that a transformation of cell types with the development of mammosomatotroph cells expressing both prolactin and growth hormone in the altered physiological state of pregnancy. In another study using ISH, investigators reported that prolactin messenger RNA was decreased by bromocriptine and a population of small cells indicated in these cells responded to the drug with decreased cytoplasmic volume. A subpopulation of the larger prolactin cells did not show a decrease in prolactin messenger RNA [12]. ISH analysis of ACTH tumors has been reported by various investigators [13]. Some studies have shown pro-opiomelanocortin (POMC) messenger RNA expression in most functional tumors as well as in some of the silent pituitary tumors. This has been confirmed by other investigators [14, 15]. Other studies have found that the silent ACTH tumors may have an abnormal messenger RNA [16]. Some investigators have used nonradioactive probes to detect POMC messenger RNA within pituitary tumors [17, 18]. Others have shown that in Nelson’s syndrome, there was a greater detection of POMC messenger compared to Cushing’s disease, suggesting differences in the transcript expression in these two conditions. Other investigators have examined non-neoplastic pituitary for POMC gene expression and have shown that POMC messenger can be detected in postmortem pituitaries up to 66 h after death [19]. Some investigators using quantitative ISH show that there was an increase in POMC messenger in suicide victims compared to patients who had cardiac death [19, 20]. ISH analyses of gonadotroph tumors have been reported. The FSH- and LH- genes have been detected in gonadotroph, null cell, and oncocytic tumors [21–24]. This specific finding suggested a close relationship between gonadotroph and null cell tumors. Other studies of gonadotroph, nonfunctional pituitary tumors [25] show that one-third of the tumors expressed a -subunit of human chorionic gonadotrophin. A small number of these tumors also express growth hormone messenger RNA. Other investigators have found prolactin and POMC messenger RNA in nonfunctional tumors [22].

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A wide variety of other messenger RNA transcripts have been identified in pituitary tumors by ISH. These have ranged from transcripts for secretory granule protein messenger RNAs such as chromogranin/secretogranin family to hypothalamic hormones and hormone receptors as well as transcription factors and galectin-3 expression in normal and neoplastic pituitaries [23–26]. These studies have shown the utility of ISH in analysis of gene expression. Fluorescent in situ hybridization (FISH) has been used to analyze pituitary chromosomal abnormalities [27]. These studies have provided further insight into the pathogenesis of pituitary tumors [27]. In situ Polymerase Chain Reaction Analysis of Gene Products in Pituitary Tumors Many studies have used reverse transcriptase-polymerase chain reaction (RT-PCR) for the analysis of gene expression in pituitary tumors [28–32]. In addition to the use of conventional RT-PCR for studies of pituitary tumors, investigators have used ISH combined with PCR (in situ PCR) to visualize lowabundant messages in cells [33–37]. In situ PCR studies have usually been done with cultured cells or frozen tissue sections and are more difficult to do with paraffin-embedded tissues for messenger RNA localization. These studies have localized low copy numbers of genes in specific cellular compartments. Comparative Genomic Hybridization (CGH) CGH uses the total genome DNA from tumors and reference samples. These DNAs are labeled with different fluorochromes and then hybridized to a normal chromosomal preparation using excess unlabeled Cot 1 DNA to inhibit nonspecific hybridization of labeled repeated sequences (fig. 2). After hybridization and washings, the ratio of the two genomes hybridized to each location on the target chromosomes indicates the relative copy number of the two DNA samples at the specific loci in the genome. One can then determine the level of genomic abnormality which may represent amplification or loss at specific loci [38–41]. The advantages of using CGH include (a) mapping changes in copy numbers in a complex genome so that the abnormalities can be related to the normal reference gene and to physical maps of genes and genomic sequences, and (b) a cell culture is not needed as with cytogenetic analyses. Disadvantages of CGH include (a) limitation to a resolution of 10–20 Mb and (b) there is no quantitative information about gene dosage. Studies in the Pituitary CGH has been applied to studies of pituitary adenomas by various investigators [42–50]. A variety of tumor types have been used and abnormalities of

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Normal DNA

Tumor DNA

Cot I DNA

Competitive hybridization Washings

Fluorescent microscopy Image recording

Fig. 2. Diagrammatic illustration of comparative genomic hybridization. DNA was extracted from a tumor and normal cells and mixed with an excess of Cot I DNA to decrease background hybridization. The various DNA undergo competitive hybridization using blood cells or other normal cells in metaphase. After washing, the chromosomes are analyzed by fluorescent microscopy, the data images are collected and subjected to computer analysis. The gains and losses in the tumor sample chromosome are collected and analyzed.

various chromosomes have been found. In some studies, different investigators using the same tumor types have detected different types of chromosomal gains or losses (table 1). In one CGH study of pituitary carcinoma metastases using two ACTH- and two PRL-producing tumors, the investigators found chromosomal gains in all samples, but losses were detected only in the PRL carcinomas [50]. The most frequent gains were noted on chromosomes 5, 7p, and 14q [50].

Laser Capture Microdissection

The acquisition of homogeneous or pure cell populations for cell biologic and molecular analyses has been a difficult challenge for many decades. A variety of approaches have been tried, including macroscopic dissection of tissues from frozen tissue blocks to increase the population of specific cell types [51]. Some investigators have used irradiation of manually ink-stained sections to destroy cells that were not of interest [52, 53]. Microdissection with the aid of a microscope and needles has been used by many other investigators [54, 55]. This latter approach can provide a great deal of precision in collecting homogenous cells, but it is slow and labor-intensive and requires a high degree of manual dexterity. Most of these approaches have not provided the speed, precision, and efficiency needed for research or routine clinical molecular diagnostic use.

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Table 1. Comparative genomic hybridization analyses of human pituitary adenomas Reference

Tumor types

Cases studied

Abnormality %

Chromosomal abnormalities

42

Nonfunctioning

23

74

Sex chromosome and 18 (34.7%); amplifications of 4q, 5q, 9p, 13q and 17q (10–30%)

43

PRL, GH, TSH, ACTH, nonfunctioning

52

48

11, 7, X, 1, 8, 13, 5, 14, 2, 6, 9, 10, 12, 3, 18 (decreasing frequency); functional tumors nonfunctioning tumors

44

GH

10

80

Gains 5, 9, 22q, 17p12 (20–50%); losses 13q, 18 (20–30%)

45

PRL, GH, nonfunctioning

12

–

Loss 13q most common (5 cases)

46

All types

75

45.3

Gains 4.9 times more frequent than losses Gains X (32%), 19 (16%), 12 (6.7%), 7 and 9 (6.7%) Loss 11 (5.3%), 13 and 10 (4%)

47

Nonfunctional (26) Functional (12)

38

–

Gains 3, 7, 14, 6p and 20q Loss 13q

48

GH adenomas associated with Carney’s complex

4

25

Gains 1p, 2q, 9q, 12q, 16, 17, 19p, 20p, 22p, 22q Loss 6q, 7q, 11p and 11q

49

3 PRL, 1 null cell, 4 gonadotroph adenomas, primary, and recurrences

8

88

Gains 4q, 5q, 13q Loss 2, 1p, 8q, 16, 12q

50

Metastatic pituitary carcinoma (PRL 2) (ACTH 2)

4

100

Gains 1q, 3p, 7, 8, 9, 13q, 14q, 21q Loss 1p, 10, 15, 11q

The recent development of laser capture microdissection (LCM) by the National Cancer Institute (NCI) group led by Liotta and his colleagues [56, 57] has provided a rapid and efficient method to capture pure cell populations for molecular and other studies. Principles of LCM The NCI group first reported on the development of a rapid and reliable method of obtaining homogenous population of cells from complex tissues [56, 57]. The availability of commercial instruments resulted from a joint venture by the NCI and Arcturus Engineering (Mountain View, Calif., USA).

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Laser Transport arm Plastic cap holder

Joystick Plastic cap

Glass slide with specimen

Fig. 3. Laser capture microdissection with the Pix Cell instrument. The laser beam is focused on the cut section of cells on the glass slide. The transport arm carries the plastic cap over to align it with the specimen and laser beam. The focused laser beam captures the cell of interest which adhere to the ethylene vinyl acetate film which is bound to the captured target cells. The captured cells of interest is carried back to the side and deposited in the tube with extraction buffer. These cells can then be used for molecular analyses such as RNA or DNA protein analysis.

With LMC, a complex section of tissues or heterogeneous cell populations on a glass slide is placed on the stage of a specially designed microscope. After the areas or cells of interest are selected, an ethylene vinyl acetate (EVA) transparent film is apposed to the section, and an infrared laser beam is directed at the cell of interest. When the focused laser beam coaxial with the microscope optics is activated, the EVA film above the targeted area melts, surrounds, and holds the cells of interest, which are kept in the film after removal from the glass slide (fig. 3). The film can be placed directly into DNA or RNA extraction buffers for nucleic acid extraction and analysis or into other buffers for protein analysis. While in the buffer, the cellular material becomes detached from the film and can be used for cellular or molecular analyses after extraction. Based on numerous reported studies in the literature and our own observations, the LCM procedure does not lead to significant alterations in the morphologic or apparent molecular features of the cells of interest. Operating Procedure The prototypic instrument used for LCM is the Pix CellTM developed by Arcturus Engineering and the NCI group. The instrument consists of a laser

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optics deck and illumination tower with a halogen bulb housing, a microscope, and a slide stage with a vacuum chuck. A joystick with XY control and video camera attachment are attached to the microscope. The XY control is for fine positioning of the sample. The laser can be controlled by amplitude and pulse width adjustments using digital controls on the front panel of the electronic box. When the laser fires, an emission indicator is lit, and the power supply emits a beeping sound. Typical operating parameters for the laser for a 30-m spot is an amplitude of 30 mW and a pulse width of 5 ms; for a 60-m spot, an amplitude of 50 mW and a pulse width of 5 ms is used. When the operator is ready to capture the cells of interest, a plastic cap is transported with the film carrier and placed on the desired position of the tissue or cells. The laser is then activated, and the EVA film binds to the captured target on the tissue. The laser can be activated as often as is needed to capture the desired numbers of cells. Multiple clusters of homogenous cells can be accumulated into the same polymer EVA film, and individual single laser shots can be used to procure specific cell clusters or individual cells (fig. 3). In addition, multiple shots can be combined to procure complex but homogenous tissue structures. Up to 3,000 shots can be captured on one transfer film cap, which may include up to 6,000 cells. Since each shot takes less than 1 s to perform, a large number of cells can be captured in a relatively short period. Variables in the Procedure Technical variables with LCM include the types of analyses that will be done with the tissues relative to fixation and processing, use of frozen versus paraffin tissue sections, and combination of LCM with other techniques such as immunohistochemistry (immuno-LCM). Tissues fixed in buffered formalin and embedded in paraffin can be used for routine analysis of DNA and in some cases mRNA as well. Tissues are routinely stained with hematoxylin and eosin for optimum visualization of cellular details during LCM. These stains do not affect the integrity of the DNA or RNA. For optimum RNA analysis, frozen tissue sections are preferred. Surprisingly, the nucleic acids are not adversely affected by exposing the tissues to xylene for a short period. RNAse-free conditions are important for obtaining high-quality RNA samples. The section for frozen tissue should be cut at 10 m or less for optimum transfer, because with thicker sections, it is more difficult to visualize single cells. Sections have to be completely dry and not coverslipped for effective LCM transfer so the final xylene rinse facilitates efficient LCM transfer. After the frozen sections are cut, the slides can be used immediately or stained at 80C until use. We have observed that starting with fresh alcohols and xylene for each set of experiments avoids many of the technical problems associated with difficult transfers during LCM.

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The procedure of combining LCM with immunophenotyping for RNA analysis (immuno-LCM) was first reported from the NCI [58] and subsequently by others for analyzing single cells [59] and tissues [60–62]. The critical requirements include using RNAse-free condition and using RNAse inhibitors during the immunostaining procedures. A rapid immunostaining procedure minimizes the time of tissue exposure to RNAse. Various studies have shown that precipitating fixatives such as ethanol and acetone produced better quality RT-PCR product amplification compared with cross-linking fixatives such as formaldehyde and glutaraldehyde [58, 59, 61]. However, precipitating fixatives are less effective in some immunostaining procedures compared with cross-linking fixatives [59]. Other Laser-Based Microdissection Systems Although LCM is the principal system used, other systems have been developed, mainly in Europe, that are somewhat similar to LCM [62–68]. Schutze and Lahr [63] used a low-power laser to create a gap between the cells of interest and adjacent cells; with an increase in the laser power, they were able to ‘catapult’ the microdissected cells into the cap of a microfuge tube for further analysis without requiring any direct contact with the cells of interest. Using this approach, they could amplify simple cells from archival tissues in the analysis of Ki-ras mutations [63]. Fink et al. [64] used an ultraviolet light laser to ablate cells that were not of interest and subsequently collected the cells of interest with a stent needle under the control of a micromanipulator [64], which is a modification of the original method of Shibata et al. [52, 53]. Other investigators have utilized this approach to study microsatellite instability in breast cancer at the single cell level [65]. A combination of ultraviolet microbeam microdissection with laser pressure catapulting for the isolation of single chromosomes for development of chromosome-specific paint probes has been reported [66]. Becker et al. [67] used a combination of ultraviolet laser microbeams with microdissection and molecular characterization of individual cells to detect a novel mutation in the E cadherin gene in single tumor cells [67]. A recent study used the PALM Laser-Microbeam System, which allows the contact-free isolation of single cells or groups of cells using the laser pressure catapulting technique and realtime PCR to study the Her-2/Neu gene and topoisomerase II gene in breast cancer specimens [68]. Disadvantages of LCM and Related Techniques Although LCM is faster and easier to perform than manual microdissection, there are a few disadvantages. The principal one is that the tissue is not coverslipped during LCM, so the refractive indices of the dry sections have a

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refractile quality that obscures cellular details at higher magnifications. This can be partially overcome with a diffusion filter on the instrument or by using a drop of xylene on the tissues, which provides wetting and refractive-index matching; the xylene usually evaporates rapidly before microdissection [69]. Another disadvantage for investigators with a tight budget is the costs of the instrument. Finally, although LCM is faster than manual methods, a great deal of time is still required to collect cells for an experiment compared with biochemical analysis, especially when thousands of cells are collected [70]. Studies in the Pituitary Our laboratory has used LCM from dissociated cells to study gene expression in individual pituitary cells [59]. We have also analyzed normal and tumorous breast tissue by LCM and RT-PCR to show that PRL-R was expressed not only in the epithelial component of normal and tumorous breast tissues but also by stromal cells [60], suggesting modulation of mammary stromal cells by PRL. In more recent studies, our laboratory has, for the first time, obtained pure populations of pituitary folliculostellate cells and used these for molecular and cell biologic studies that have provided new insights into the role of these cells in pituitary function [71]. Analysis of folliculostellate cells in the anterior pituitary has shown that these cells express a variety of mRNAs such as TGF-, TGF--R, leptin, and leptin receptor [72].

DNA Microarrays

Microarray analysis to explore gene expression on a global level is producing a lot of new information. Significant advances have already been made in this new field [73–76]. In the field of tumor biology, new discoveries have been reported in studies of leukemia [77], lymphoma [78], breast cancer [79, 80] and malignant melanomas [81]. Distinction between normal and colonic tumors have been reported by the use of microarrays [82]. The principle of microarray analysis is that mRNA from a specific tissue or cell line is used to generate a labeled sample or target. This is then hybridized in parallel to a large number of DNA sequence immobilized on solid support with a specific order or array (fig. 4). With this approach, many thousand transcripts can be detected and quantified at the same time. Array platforms include cDNA and oligonucleotide arrays. The arrayed material or probe can be produced by PCR generalized from cDNA libraries for DNA arrays which are printed onto glass slides or nylon membrane as spots which are 100–300 m in size. Up to 36,000 or more cDNAs can be fitted onto the surface of a conventional microscope slide.

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Reference RNA

Cy3

First-stand cDNA synthesis Label cDNA

Tumor RNA

Cy5

Hybridization

Ratio of Cy5/Cy3 Computer-assisted analysis bioinformatics

Fig. 4. Schematic overview of cDNA microarray analysis. DNA from two different samples is used to synthesize single-stranded cDNA with nucleotides labeled with two different fluorescent dyes. Both samples are hybridized to the array surface on glass slide or nylon resulting in competitive binding of differentially labeled cDNAs to the specific array elements. High-resolution confocal fluorescence scanning of the array using two different wavelengths (depending on the two dyes used) to produce relative signal intensities and ratios of mRNA abundance for the genes represented on the array. The large amount of data generated about over- and underexpressed genes are then analyzed by computer-assisted bioinformatics.

Oligonucleotide arrays are usually made from short 20–25 mers which are synthesized onto silicon woven or by ink-jet technology. Presynthesized oligonucleotides can also be printed onto glass slides. One advantage of oligonucleotide arrays is that because the sequence information is known as sufficient to generate the DNA to be arrayed, it is relatively easy to prepare the array since thousands of DNAs do not have to be generated, and the most highly specific sequences of a gene can be used for the oligonucleotide arrays avoiding homologous sequences with other genes as well as specific variants. The disadvantage of short oligonucleotides include decreased hybridization specificity and reduced sensitivity. However, longer oligonucleotides of 50–100 base pair size are being used to avoid these potential problems [83]. The sample source is used to extract mRNA for microarray analysis. It is then converted to a cDNA by reverse transcriptase, hybridized to the DNA species on the surface of the array, then detected by fluorescence scanning or phosphoimaging. The oligonucleotide chips are highly reproducible, so

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accurate comparisons can be made by comparing signals generated by samples from separate arrays, but this cannot be done with cDNA arrays. The alternative approach of using fluorescent types of two different wavelengths such as Cy5 and Cy3, which can be scanned using two different wavelengths, can be used to compare the intensity of the two spots (fig. 4). A reference RNA must be used when comparing a large number of samples. After generation of microarray data from an experiment, the most demanding challenge is the subsequent analysis. Since a large number of spot intensities and intensity ratios are generated, the data must be carefully analyzed, then validated. Replication of the experiment is one sure way of reducing potential false positive signals. Many investigators use independent duplicate samples with reciprocal labeling with four microarrays for each experimental point. For genes with low expression levels, the variability of microarray results can be quite great, so replication to establish a high degree of confidence in the data is needed. The use of data analysis software is essential for the bioinformatics analysis of the data. Public sources such as http://genome-www4. stanford.edu/MicroArray/SMD/restech.html as well as private sources such as Silicon Genetics GeneSpring (http://www.sigenetics.com/) are available. Validation of the results by Northern hybridization, quantitative, or real-time RT-PCR, RNAse protection assays and RT-PCR as well as tissue microarrays [83] should be done in the complete analysis of microarray data to improve the reliability of the data. Studies in the Pituitary The pathogenesis of pituitary adenomas is largely unknown. Recent studies have identified a few molecular genetic changes in pituitary adenomas and have shown that they are monoclonal in origin [84–90]. Potentially major advances in the field should be possible with microarray technology on a genomic scale since simultaneous analysis of the expression levels of thousands of genes can be analyzed to allow for detection of genes important in pituitary tumorigenesis. A small number of studies are now available to allow us to take a look at the potential of this approach to study not only pituitary tumorigenesis, but also the characterization of genes and proteins that are potentially useful for the prognosis and medical therapy of pituitary tumors [91–95]. cDNA expression array in pituitary tumors was first performed in a rat model to analyze and identify gene expression patterns linked to aging and associated spontaneous pituitary adenomas in rats [91]. The investigators used RNA from 3-month-old rats and tumor-bearing 20- to 28-month-old rats with cDNAs using 588 known genes. They detected 79 genes, and the GH gene was present in the highest level of both groups. In the old rats, there were 28 genes that were expressed at higher levels while in the younger rats, there were 15 genes expressed

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at higher levels. Galanin, which has PRL-promoting activity, and glutathione 5-transferase were the genes with the largest difference in the older and younger groups, respectively. The results were further validated by relative RT-PCR which was consistent with the microarray data for 14 of the 15 genes tested. Of interest in this study, other genes that were overexpressed in the pituitary induced plateletderived growth factor-associated proteins, CDKS, p27 kip1, and thyroid hormone receptors that were overexpressed in the pituitary of rats with spontaneous tumors [91]. Several of these overexpressed genes including p27 kip1 have been observed to have a role in pituitary tumorigenesis in rodent pituitary [96, 97]. Evans et al. [92] used cDNA arrays to identify gene expression profiles in secretory and nonfunctional human pituitary tumors and compared them to normal pituitary with 7075 genes. They observed differential expression of 128 genes. They analyzed three genes by RT real-time qualitative PCR in 37 pituitaries. In the NF groups, folate receptor (FR)- was overexpressed 2.5-fold compared to the normal pituitary. FR is a glycosyl-phosphatidylinositol-linked protein and is thought to confer growth advances to cells with limited concentrations of cells with a limited concentration of 5-methyltetrahydrofolic acid with increased uptake of folate [98], so the potential therapeutic approach of using FR by targeted drug delivery has been suggested for recurrent NF tumors [99] since such studies have been successful in some patients with ovarian tumor and FR- is overexpressed in ovarian as well as other cancers [100]. Ornithine decarboxylase, a rate-limiting enzyme in the biosynthesis of polyamine such as putrescine, spermidine, and spermine, was overexpressed only in GH tumors. This enzyme correlates with malignancy in pituitary tumors as well as gliomas [101]. Thus, polyamine metabolism is a potential target for antineoplastic therapy [102]. ACTH tumor overexpressed CMP-tk, a transmembrane receptor with tyrosine kinase activity. Tyrosine kinase receptors are involved in control of cellular growth and differentiations [103], so this gene may have an important role in ACTH tumor initiation and progression. PRL adenomas, the most common tumor type in humans, overexpressed Trichohyalin, and TGF- receptor III genes and protease inhibitor 12, gene but overexpression of these genes was not validated by reverse transcriptasequantitative polymerase chain reaction [92]. These results highlight the potential discovery of genes in pituitary tumors that could be used as targets for radiopharmaceutical therapy of pituitary tumors. Some disadvantages of this general approach from this study that will be discussed below. In a more recent study to determine early gene expression changes preceding thyroid hormone-induced involution of a TSH-producing tumor, Wood et al. [93] used a 1,176 DNA microarray to detect 7 up-regulated and 40 down-regulated genes by thyroid hormones. The results were validated by

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Northern hybridization. They reported that several cell cycle regularity proteins were changed including up-regulated of p15 and down-regulation of p57 and CDK2, which is known to activate the expression of p27 and c-myc levels to growth arrest in neuroblastoma cell lines [104]. Many genes expressed in the pituitary are targeted by thyroid hormones were also identified including cell adhesion proteins such as cadherin 4 and -catenin, tyrosine kinase ryk, inhibin, chromogranin B and C, proconvertase 1, and the apoptosis protein DAD1, most of which were down-regulated by thyroid hormone treatment [93]. In a related study of expression, profile of active genes in human pituitary gland but not in pituitary tumors, Tanaka et al. [94] constructed a gene expression profile of the normal human pituitary gland. These investigators used a total of 1,015 randomly collected 31 expressed sequenced tags which were grouped into a 527 gene signature species [94]. They reported on the relative activities of genes that were unique to the pituitary. Not surprisingly, pituitary hormone-related genes such as PRL, GH, LH, FSH, and -subunits were highly expressed. Interestingly, other genes such as chromogranin B, a secretory granule protein present in all anterior pituitary gland cells, CPE, and 7B2 were also highly expressed. Normal pituitary-specific transcripts including pituitary gland specific factors 1a and 1b of 128 and 91 amino acids respectively were identified. However, the identity and functions of these and other related normal genes are not known. The early studies with microarrays have been provided new insights into genes unequally expressed by pituitary tumors. However, there are several disadvantages that emerge in the technical methods used in these studies. The starting materials have usually been contaminated with additional cell types such as fibroblast, endothelial cells, and in the normal pituitary with several other cell types. The use of more sophisticated approaches to identify only the cells of interest such as LCM [56] and immunophenotyping and LCM [59] should help to increase the specificity of the array and increase the discovery of low expressed genes. Levels of mRNA regulation is only one aspect to understand the pathogenesis of dysregulation of various cellular pathways. Other regulatory pathways such as protein regulation and post-translational modifications are also important. Proteomics approaches should also complement DNA microarray analysis in understanding normal and abnormal cell functions [105–107]. New approaches using genomics and proteomics techniques should lead to the discovery of many more important regulatory pathways in pituitary cells and tumors. Summary The use of microarrays to explore gene expression on a global level as well as examination of gene modulation of a given phenotype such as during transformation or protection from apoptosis is in its early stage. The data being generated from microarray analysis are rapidly accumulating and should be

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facilitated by improved advances in bioinformatics with the use of specialized arrays, these approaches should become more hypothesis-driven. A combination of approaches such as LCM and immunophenotyping LCM and microarrays should improve the specificity of these approaches. The studies on human pituitary tumors reviewed here indicate the potential utility of these newly discovered genes in understanding the pathogenesis, and improvements in the medical treatment of human pituitary tumors.

Tissue Microarrays

Recent developments in high-throughput analyses such as DNA arrays and proteomic analyses have generated a tremendous amount of data acquisition. The significance of many of these newly discovered genes and proteins are unknown, so validation and functional studies including high-throughput tissue analysis should facilitate these studies. Techniques such as immunohistochemistry and ISH have been used by morphologists to correlate gene or protein expression with specific tissue and cell types. The development of high-throughput techniques has been applied to tissue analysis with the use of high-density arrays made up of a hundred or more tissue samples which provides an efficient method to validate and analyze molecular studies. Array-based high-throughput analysis to facilitate gene expression and copy number surveys of a large number of tumors was first reported by Kononen et al. [108]. Since then, there have been many reports of applications of tissue arrays for the study of gene and protein expression [108–120]. Most tissue microarrays (TMA) with formalin-fixed paraffin-embedded tissues use multiple ‘donor’ blocks in which tissue cores, usually 0.6 mm in diameter, are extracted with the aid of a micrometer. Manual and automated tissue arrays are available from commercial sources, and it usually takes a few days to weeks to construct the TMA. One of the limiting factors is collecting the blocks and slides and deciding the area of each block to be used in construction of the TMA. Usually at least 109 or 1010 cells are needed to create a TMA cell block of adequate thickness to make a core. The individual cases are placed on the block in duplicate or triplicate and are usually 0.2 mm apart. Once made, the tissues can be cut with a microtome 4–6 m in thickness. Once the TMA are completed, it should be possible to cut 100–200 sections from the block depending on the thickness of the cores. During construction of the block, the orientation of each specimen including the normal tissues or other tissues used as known spacers that are placed in a specific location in the TMA to use as a reference.

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Frozen TMA offers some advantages over paraffin TMA such as better preservation of RNA and some proteins [109, 117]. FISH studies used for TMA produce more consistent results with ethanol fixation [108]. Difficulties with frozen TMA include technical problems working with frozen tissues and poorer preservation of morphology compared to paraffin sections. Validation of the TMA has been reported by various investigators. In their original studies, Kononen et al. [108] compared their results by FISH for gene amplification and by immunohistochemistry for estrogen receptor and p53 in breast cancer to previously published results [108]. Other approaches have compared Her2/neu and hormone receptors in breast cancer tissues using TMA and whole tissue sections and found similar results when using two tissue cores in the TMA [111]. In another study using Ki-67, Rb, and p53 expression, Hoos et al. [118] reported that three tissue cores per case were more accurate than two cores with a concordance rate of up to 98%. Studies with proteins that are expressed only focally such as chromogranin and synaptophysin and prostate carcinoma may lead to a greater discrepancy in the analysis of TMA compared to larger tissue blocks [119]. The quantitation of immunohistochemistry and ISH data from TMA is still in its infancy [121]. Automated instruments such as CAS 200 by Bacus Labs (Lombard, Ill., USA) and the use of automated slide scanners to digitize entire sections such as the BLISS system by Bacus Labs and Interscope Technologies (Pittsburgh, Pa., USA), should facilitate data analysis in the future. There is a great deal of promise for the future of data analysis with TMA because of the advances being made in generating and storing high-quality images by digital imaging technology [122]. TMA Studies in the Pituitary Studies combining TMA with or without DNA arrays in the pituitary have been very limited to date. In a study of chromogranin A processing using chromogranin A region-specific antibodies, Jin et al. [123] showed that different tumor types expressed different chromogranin A proteolytic products (fig. 5a–c). They found that the PRL adenomas and carcinomas had low levels of expression of vasostatin I and II compared to other tumor types. The TMA were validated by comparing the results with the PRL tumors to whole tissue blocks and good agreement was found with these two approaches [123].

Proteomics

Proteomics is the large-scale study of proteins, usually by biochemical methods. Historically, proteomic analyses were developed in the late 1970s with

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c Fig. 5. a Tissue microarray (TMA) of pituitary tumors. The TMA consists of triplicate samples of each specimen. Liver tissues were used as spacers with a vertical and horizontal row in the center of the specimen. These TMAs have been used for ISH and immunohistochemical analyses. b Closer view of pituitary adenoma TMA after immunostaining with a chromogranin A antibody. Most of the tumor cells are positive. c Individual specimen of a gonadotroph adenoma immunostained for chromogranin A showing diffuse staining in all of the tumor cells.

the use of two-dimensional (2-D) gel electrophoresis. In the 1990s, biological mass spectrometry accelerated the proteomic field with more sensitive and rapid analytical methods for protein characterization [124–129]. Proteomics includes analysis of the functional aspects of gene products (functional genomics) such as large-scale identification and/or localization of proteins and study of protein interactions. Functional genomics must be distinguished from ‘structural genomics’ in which studies are focused on the large-scale analysis of protein structure [129]. Verification of a gene product by proteomic methods is a first step in supporting the data from genomic analysis. In addition, the post-translational modifications and existence of isoforms of proteins can only be analyzed with proteomic methods. In some cases, there may not be a direct correlation between mRNA and protein expression, so analysis of protein expression becomes more critical [130, 131]. Other advantages of proteomic analysis

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include studies of gene product localization, mechanisms of regulation of protein function, studies of protein-protein interactions and molecular analysis of the composition of cellular organelles can be done only at the protein level [129]. The quantity of proteins within cells is much greater than genes. A cell line may have 10,000 genes expressed, while the number of proteins may be 10-fold or more greater. At the start of each analysis, one must obtain purified proteins for subsequent analyses. One- or 2-D electrophoreses are the most common methods used. With 2-D, gel electrophoresis, most proteins are solubilized in SDS. Mass spectrophotometric analysis of proteins has replaced the more classical techniques of protein analysis because of its greater sensitivity. It offers advantages of analyzing protein mixtures and can handle high-throughput samples. After digestion of sequences with specific proteases such as trypsin, peptides are separated by gel electrophoresis and then analyzed by mass spectrometry using matrix-assisted laser desorption/ionization (MALDI). MALDI identification has been automated such that hundreds of protein spots can be excised, digested enzymatically followed by mass spectra analysis and automatic searches against databases [132, 133]. Modifications of proteins which are usually not apparent from genomic sequences or mRNA expression data such as glycosylation, phosphorylation, and sulfation, which affects protein function can be readily addressed in proteomic analyses. Analysis of up- or down-regulated proteins by differential-display proteomics is a growing area of proteomics in tumor biology. Use of arrays for proteomic analysis or ‘protein chips’ is still in its infancy, but various approaches are being developed such as the use of antibodies bound to the chip to identify proteins of interest [134–136] (fig. 6). One can use fluorescentlabeled proteins from different cell lines or different treatment using two different fluorophores for labeling. The specificity of the antibodies is very critical. Protein-protein interactions is another important area of proteomic analysis, since it can provide clues to biological function and has potential therapeutic applications. This may involve purification of the entire multi-protein complex by affinity based methods or by using epitope tags and immunoprecipitation techniques [129]. Many studies showing proteomic applications for the early detection of cancer are emerging [137, 138]. In addition, various strategies for protein identification [139] and procedures for the use of ethanol fixation and paraffin embedding of tissues for subsequent analyses such as by mass spectrometry are being developed [140].

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Sample 1

Sample 2

Protein extraction

Protein labels with dye

Cy3

Remove unbound dye by gel filtration

Cy5

Combine samples incubate with antibody arrray

Bioinformatic analysis

Fig. 6. Diagrammatic outline of antibody microarray. Two different samples are used. Proteins are extracted and labeling buffers are added using fluorescent tags. The unbound dye is removed after gel filtration and the samples are combined and incubated with the antibody microarrays. The two ratios of fluorescent dyes such as Cy3 and Cy5 for each protein target is used to calculate a ratio for each spot on the array. The reliability of the approach depends on the specificity and sensitivity of the antibodies.

Studies in the Pituitary Applications of proteomic studies in normal and neoplastic pituitaries have recently been reported [141–143]. In one study, nine proteins from the human pituitary proteome using a protein post-mortem pituitary from a 48-year-old woman who died accidentally was reported. 2-D electrophoresis with silver staining followed by in-gel digestion with trypsin and MALDI time-of-flight mass spectrometry followed by characterization from a protein sequence database led to the identification of various hormones such as GH and PRL as well as enzymes including ubiquitin thiolesterase L1 and glutathione S-transferase P [141].

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In another study by some of the same authors using the same sample, these investigators identified 62 prominent protein spots corresponding to 38 different proteins [142]. Proteins such as galectin-1 and annexin V in addition to hormones and enzymes were identified. In a proteomic study of a pituitary adenoma from a 54-year-old man, Zhan and Desiderio [143] obtained around a thousand protein spots which were separated by 2-D electrophoresis to obtain 135 protein spots representing 111 proteins which were characterized by mass spectrometry. They obtained 96 spots by MALDI-TOF and characterized pituitary hormones, cellular signals, enzymes, cell structure and transport proteins. However, the authors did not indicate what type of adenoma they were studying, and there was no attempt to quantify the proteins that were characterized.

RNA Interference

RNA interference (RNAi) was first discovered in Caenorhabditis elegans as a response to double-stranded DNA (dsRNA) which led to gene-specific gene silencing [144]. The mechanisms of dsRNA-induced silencing of homologous sequences from exogenous sources such as virus-induced gene silencing or endogenous sources exist in many eukaryotic organisms [145–149]. The mechanism of RNAi includes an effector nuclease or RNA-induced silencing complex (RISC) (fig. 7). Initiation of silencing occurs after recognition of dsRNA in the cell that converts the silencing trigger to 21–25 nucleotide RNA fragments. These small interfering RNAs (siRNAs) join an effector complex, the RISC, and guide the complex to homologous substrates. Endogenously expressed small hairpin RNAs regulate gene expression through the RNAi pathway. Short hairpin RNAs (shRNAs) with 19–29 nucleotide dsRNA stems are processed by the RNA polymerase III enzyme Dicer and are incorporated into the RISC which results in the targeting and degradation of cognate mRNAs. Alternatively, siRNAs with 19–21 nucleotides of dsRNA can bypass the need for Dicer and are directly incorporated into RISC. Factors that influence the efficacy of siRNA include (1) the targeted region of the gene, (2) the base composition of the siRNA sequence, and (3) secondary structure of the mRNA target and the types of RNA-binding proteins [147]. Because there is probably no siRNA replication in mammals, siRNAdirected silencing by transfection is limited by its transient nature. DNA vectormedicated mechanisms to express substrates that can be converted into siRNA in vivo have been developed [149]. Limitations of gene silencing by transfected siRNA include (1) the transient nature of the knockdown of the gene of interest

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dsRNA

ATP Dicer ADP Pi P P siRNA duplex P RISC P siRNA–protein complex ATP

RISC activation

ADP Pi siRNA-mediated target recognition

mRNA cleavage

Fig. 7. RNA interference pathway. The long double-stranded RNA (dsRNA) is cleaved by the RNase III polymerase member Dicer enzyme into small interference RNAs (siRNAs) of 19–23 bp. These siRNAs are then incorporated into the RNA-inducing silencing complex (RISC). The single-stranded antisense strand guides the RISC to mRNA that has a complimentary sequence and leads to the cleavage of the target mRNA.

which is dependent on cell growth and dilution of the siRNA below a critical threshold, and (2) the half-life of the protein is also critical since proteins with long half-lives are less effectively silenced [149]. The specificity of gene silencing by siRNA has been addressed by various investigators. Two recent studies have demonstrated that siRNA-induced gene silencing of transient or stably expressed mRNA is highly gene specific and does not provide secondary effects detectable by genome wide expression profiling and that it is a reliable approach for large-scale screening of gene function and drug validation [148, 149].

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Studies in the Pituitary Very few studies have applied this new technology to the pituitary. In one study from our laboratory, Riss et al. [26] used an siRNA for galectin-3 to show that this protein was important for pituitary cell proliferation and apoptosis. Future Directions

The various techniques discussed in this chapter should continue to provide new information and directions to understand the basic biology and pathophysiology of pituitary tumor development. These approaches should also help in the clinical and pathologic diagnosis of pituitary tumors as well as in the development of new pharmacologic agents to treat pituitary tumors and other pituitary disorders. As we move into the new era of individualized disease treatment for patients heralded by advances in genomics and proteomics, there will be continuous challenges in learning more about gene and protein function and the interaction of myriads of molecules within an individual cell such as those in the pituitary which leads to orderly or disorderly growth, hormone secretion, and differentiation. Continued success will come from a conscious integration in our efforts to understand the complex molecular interplay that makes each pituitary cell unique, yet part of a harmoniously functioning gland. References 1

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R.V. Lloyd, MD Mayo Clinic, Laboratory Medicine and Pathology 200 First Street, SW, Rochester, MN 55905 (USA) Tel. 1 507 2845022, Fax 1 507 2841599, E-Mail [email protected]

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Pituitary Tumor Transforming Gene: An Update Run Yu, Shlomo Melmed Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, Calif., USA

Abstract Herein we summarize the recent rapid advances in understanding the pituitary tumor transforming gene (PTTG) oncogene. Clinical studies reveal that PTTG-binding factor, fibroblast growth factor 2, and vascular endothelial growth factor are elevated in pituitary tumors, and mostly correlate with PTTG levels, also confirming the PTTG role in angiogenesis. PTTG overexpression disrupts mitosis and causes aneuploidy in single live cells and PTTG modulates p53 activity and p53 also mediates DNA damage-induced inhibition of PTTG transcription. Physiological functions of PTTG are revealed by PTTG-null mice who exhibit a variety of cell growth abnormalities including diabetes mellitus secondary to defective ␤-cell proliferation. PTTG is therefore an oncogene for pituitary tumors and other neoplasia, and also involved in critical metabolic functions. Further studies are required to address mechanisms for these oncogenic and physiological functions, and more importantly, to understand conditions which determine the switch of PTTG from functioning physiologically to behaving as an oncogene. Copyright © 2004 S. Karger AG, Basel

Introduction

Pituitary tumor-transforming gene (PTTG), first isolated from pituitary tumor cells by our group, transforms NIH 3T3 cell in vitro and in vivo, and is expressed in most pituitary tumors [1]. The human PTTG protein has 202 amino acids and has an N-terminal regulatory domain and a C-terminal functional domain. PTTG expression is cell cycle-dependent and peaks at G2/M. PTTG is localized to both the nucleus and cytoplasm. PTTG is phosphorylated at G2/M phase and is degraded in a proteasomal pathway. PTTG induce p53-dependent and -independent apoptosis. There may be several possible mechanisms for

Normal mitosis

PTTG overexpression Parental cell (interphase)

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Fig. 1. Schematic diagram of securin function and aneuploidy. Left: Normal mitosis. PTTG is a mammalian securin which maintains binding of sister chromatids during mitosis. During mitosis, sister chromatids are bound with cohesins. PTTG inhibits separin, an enzyme that regulates cohesin degradation. At the end of metaphase, securin is degraded by an anaphase-promoting complex, releasing tonic separin inhibition, which in turn mediates degradation of cohesins that hold sister chromatids together. In this manner, sister chromatids are separated equally into daughter cells. Right: Mitosis in cells overexpressing PTTG. PTTG overexpression may render sister chromatid separation difficult and result in aneuploidy [from 16, with permission].

PTTG tumorigenesis. PTTG and FGF together form a positive feedback loop and stimulate tumor angiogenesis. PTTG may transactivate c-myc or other proliferation-promoting genes. PTTG behaves as a mammalian securin and PTTG overexpression is associated with aneuploidy (fig. 1). Here we summarize new results and discuss future research directions, including: (1) PTTG’s expanding role in tumorigenesis in pituitary and other endocrine tumors, as well as in non-endocrine tumors; (2) new insights on mechanisms for PTTG tumorigenesis, and (3) implications of PTTG physiological roles derived from observations in PTTG-null mice and cells. PTTG Role in Tumorigenesis

PTTG overexpression in pituitary tumors has now been further confirmed in several studies [2–4]. A strong correlation between PTTG induction or

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expression levels and pituitary tumor growth has been demonstrated in experimental models [5–7]. Nevertheless, controversies on correlation between PTTG levels and tumor behaviors exist. A small study with 40 patients suggests higher PTTG levels in somatotroph tumors and a correlation between PTTG expression and GH secretion [3]. However, a larger study with 111 patients did not find a correlation between PTTG expression and hormone secretion, consistent with several previous reports [4]. These two studies do not find correlation of PTTG levels and clinical features such age, sex, recurrence, tumor size, or imaged tumor invasiveness [3, 4]. PTTG-binding factor (PBF) was reported in 2000 [8]; it helps translocate PTTG from the cytoplasm to the nucleus. PBF mRNA is dramatically higher (nearly 6-fold) in pituitary tumors than in normal pituitaries, and PBF levels are highest in non-functioning tumors [4]. PBF and PTTG levels correlate significantly, but PTTG and PBF levels do not correlate with tumor clinical features. PTTG promotes angiogenesis by stimulating fibroblast growth factor 2 (FGF-2) in experimental models [9]. Recent papers have examined PTTG expression and angiogenesis growth factors such as FGF-2 and vascular endothelial growth factor (VEGF) [2–4]. FGF-2 mRNA levels are not elevated in pituitary tumors, but FGF-2 protein levels are higher [4]. This observation suggests a mechanism of possible PTTG modulation of FGF-2 function. VEGF mRNA levels and protein concentrations are elevated in 81 non-functioning tumors [2]. VEGF receptor KDR levels are even more elevated in non-functioning tumors and in pituitary tumors in general. VEGF and KDR levels correlate with PTTG levels, and PTTG stimulates VEGF but not KDR in vitro. In another study, all 16 pituitary tumors secreted VEGF in cell culture but there was no clear quantitative correlation between VEGF secretion and PTTG expression levels or tumors sizes [3]. Despite of conflicting results, it appears that PTTG does play an intimate role in pituitary tumor angiogenesis. PTTG tumorigenesis has been explored in other endocrine tumors and the results are largely consistent with findings in pituitary tumors. PTTG is shown to play a critical early role in follicular thyroid cancer [10] and PTTG and FGF-2 are both prognostic markers for recurrence of thyroid cancers [11]. PTTG is also involved in lymphoid tumorigenesis [12], T-cell activation [13], and in gastric and esophageal cancers [14, 15].

New Insights on Mechanisms of PTTG Tumorigenesis

In a review in 2001, we had hypothesized that mitotic disruption by PTTG overexpression is probably a major mechanism for PTTG tumorigenesis [1]. PTTG is a mammalian securin that regulates sister chromatid separation during

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Meta Meta Chromosome tear Chromosome polar movement

Asymmetrical cytokinesis Asymmetrical cytokinesis

Inter

b

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c Fig. 2. Aneuploidy in live cells. Chromosome non-segregation and aneuploidy resulting from failure of PTTG-EGFP degradation. Single live cells expressing PTTG-EGFP were continuously observed and representative images shown. Both phase contrast (bright field)

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mitosis [1], and we have confirmed the hypothesis by single cell imaging [16]. Single cell imaging has the unique advantage of following a particular cell over several days and defects in mitosis can be attributed with confidence to the experimental manipulation. In our imaging system, we observed and recorded digital images of the same single live cells transfected with EGFP-tagged PTTG or other proteins over a period of up to 7 days. The first major observation is that mitosis progression is severely inhibited by PTTG overexpression [16]. Metaphase is prolonged 7 times compared with controls. Mitotic check point protein MAD2 or 3F3/2 does not appear to be important in mediating the mitotic block. Secondly, PTTG overexpression inhibits chromosome separation and results in unsymmetrical cytokinesis (fig. 2). Non-segregated chromosomes move to one pole of the cell en bloc and the cell then divides into a daughter cell containing all the chromosomes and another daughter cell devoid of chromosomes. The former cell also contains two centrosomes and survives into a cell with a macronucleus. The latter dies not unexpectedly. Due to technical difficulties, the macronuclear cell cannot be further observed, but it can be inferred that the next round of mitosis will be even more chaotic due to the multiple copies of chromosomes and centrosomes. Mitotic disruptions caused by PTTG depend on PTTG expression levels [16]. Cells with low PTTG expression undergo complete PTTG degradation at metaphase-to-anaphase transition and essentially normal mitosis whereas cells with high PTTG expression invariably have incomplete PTTG degradation and result in macronuclear daughter cells. The importance of PTTG degradation is further indicated by the fact that a non-degradable PTTG mutant results in macronuclei even at very low expression levels. These lines of evidence point to the conclusion that incomplete PTTG degradation secondary to PTTG overexpression results in doubling of chromosome numbers, a form of aneuploidy. Unsymmetrical cytokinesis induced by PTTG overexpression has been observed by others [17]. Several caveats should be recognized. The results are largely derived from cancer cell lines which are highly resistant to apoptosis. Cells already are aneuploid before PTTG overexpression, and the high PTTG expression levels achieved by transfection probably do not occur under physiological conditions. Nonetheless, our conclusion likely reflects tumorigenesis in vivo to a certain and PTTG-EGFP images (dark field) are shown. a Complete absence of chromosome segregation with completed cytokinesis. b Incomplete chromosome segregation with aborted cytokinesis. c A cell that doubles nuclear size as a result of chromosome non-segregation. Phases in mitosis are shown on the left of images. Meta, metaphase; and inter, interphase. Thick arrow, non-segregated chromosomes; thin arrow, torn chromosomes; asterisk, a micronucleus; D2, second day of observation. Bar: 10 ␮m [from 16, with permission].

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extent. Tumorigenesis is a very rare event and is only successful under ‘atypical’ conditions, where mechanisms for cell senescence and apoptosis fail and pre-tumorous changes already exist. Mechanisms for PTTG expression are being actively studied. In one study, the PTTG promoter in pituitary tumors from 25 patients was amplified by PCR and sequenced in 16 tumors [18]. No sequence insertion, deletion, or point mutations were detected. It appears therefore that epigenetic factors may contribute to PTTG overexpression. As we previously considered in 2001, a highly conserved region in the PTTG promoter that contains SP1, CCAAT, CDE (cell cycledependent element) and CHR (cell cycle homology region) motifs may be important for regulating PTTG expression [1]. This region turns out to be important for p53-mediated downregulation of PTTG transcription by DNA damage [19]. In an attempt to search for proteins that interact with PTTG protein, various screening methods such as yeast two-hybrid system or phage display have been employed. One such protein is Ku-70 [20]. Ku-70 heterodimerizes with Ku-80 to form a regulatory subunit of DNA-dependent protein kinase (DNA-PK), the catalytic subunit being DNA-PKcs. DNA-PK is responsible for DNA repairing double strand breaks. It is not known whether PTTG binding to Ku-70 affects DNA-PK enzymatic activity and which cell compartment this interaction is located. Another protein that interacts directly with PTTG is p53 [21]. In this case, the functional consequence of PTTG-p53 interaction is studied in depth. Generally speaking, PTTG inhibits p53 activity. p53 DNA binding, transactivation, and apoptosis are all inhibited by PTTG. Although p53 inhibition may be an important mechanism of PTTG tumorigenesis, these results differ somewhat from our earlier observation that PTTG induces p53-dependent and -independent apoptosis [1]. The PTTG-p53 relationship becomes very unstable if PTTG inhibits p53 activity because p53 also inhibits PTTG transcription. When a cell sustains an insult, p53 is activated and cells are blocked at G2/M. p53 would inhibit PTTG expression, which in turn decreases PTTG inhibition of p53 activity and more inhibition of PTTG expression. However, at G2/M, PTTG levels are highest, thus not supporting the thesis that PTTG inhibits p53 activity. These concerns should be addressed in future studies.

Implications from PTTG-Null Mice and Cells

To study the physiological role of PTTG, we generated PTTG-null mice and examined the phenotypes [22, 23]. Based on known PTTG functions and tissue distribution at that time, we predicted that PTTG deletion may be lethal because securin function is important for cell division. Surviving mice have defects in testis and platelets based on PTTG localization in testis and megakaryocytes.

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Fig. 3. Premature centromere division. Metaphase spread from MEFs of PTTG-null mice showed premature centromere division. Arrow, premature centromere division [from 22, with permission].

PTTG-null mice also have fewer pituitary tumors since all pituitary tumors have elevated PTTG. Surprisingly, the phenotypes demonstrate once again that in vivo physiology can hardly be directly predicted from in vitro studies. The PTTG-null mice (PTTG⫺/⫺) have a normal life span compared with littermates with normal PTTG (PTTG⫹/⫹), which demonstrates that the mice have a redundant backup securin function. The nature of the backup securin function is not clear but PTTG isoforms [1] and a phosphorylation-dependent mechanism for chromatid separation [24] are possible mechanisms. Whatever the backup mechanisms are, they are not perfect substitutes for PTTG, as mouse embryo fibroblasts (MEFs) derived from PTTG⫺/⫺ have an abnormal cell cycle with partial G2/M block [22]. Metaphase spreads of PTTG⫺/⫺ MEFs showed abnormal chromosomal formations such as quadriradial and triradial chromosomes. As further evidence that PTTG is the index mammalian, premature centromere division can be observed in PTTG⫺/⫺ MEFs (fig. 3). The phenotypes of PTTG-null mice confirm a PTTG securin function but also demonstrate that chromatid separation is governed by redundant mechanisms. PTTG⫺/⫺ mice indeed have small testes and mild thrombocytopenia [22]. It is not clear if the small testes have physiological significance because although the male mice are fertile, litter sizes are smaller. We do not have clear explanations for splenic hypoplasia and thymic hyperplasia in PTTG⫺/⫺ mice. The most interesting and completely unexpected phenotype is a unique form of diabetes arising in the PTTG⫺/⫺ mice [23].

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3 months

8 months

KO

WT

50␮m

a

3 months Insulin

8 months Nuclei

Insulin

Nuclei

KO

WT

b Fig. 4. Small islets and nuclear morphology. a Smaller islet size in PTTGI⫺/⫺ mice. Islets derived from PTTG⫺/⫺ and WT mice at 3 and 8 months were stained with a guinea pig antibody to insulin and goat anti-guinea pig rhodamine. b Pleiotropic ␤ cell nuclei in PTTG⫺/⫺ islets. Islets of PTTG⫺/⫺ and WT mice aged 3 months were stained with a guinea pig antibody to insulin and goat anti-guinea pig rhodamine (left) and nuclei counterstained with Hoechst 33342 (right). Macronucleus is indicated by arrowhead [from 23, with permission].

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The diabetes is only obvious in male mice after approximately 6 months [23]. These male mice exhibit typical hyperglycemia and insulinopenia. The diabetes is not type I, because no signs of islet inflammation such as lymphocyte infiltration or islet destruction such as apoptosis can be found. The diabetes is not type 2 either, because the mice do not have insulin resistance and they are thin and depleted of fat. Rather, PTTG⫺/⫺ ␤ cells have intrinsic defects in proliferation, as shown by the inability of the ␤ cells to expand appropriately with age (fig. 4). The PTTG⫺/⫺ ␤ cells also have macronuclei, consistent with abnormal cell cycle regulation as a result of PTTG deletion (fig. 4). Intriguing questions are why ␤ cells are selectively affected by PTTG deletion and how PTTG deletion causes the ␤-cell proliferation defects. The securin function is critical in mitotic checkpoint and PTTG deletion may also result in mitotic disruption. This hypothesis has actually been confirmed by several studies in cells devoid of PTTG [22, 24]. MEFs derived from PTTG-null mice have abnormal nuclear morphology and a high percentage of double nuclei and macronuclei [22]. PTTG-null colon cancer cells lose chromosomes frequently and exhibit abnormal anaphase [24]. Recently, the Tax oncogene from human T-lymphotropic virus type 1 has been shown to degrade PTTG through the anaphase-promoting complex [25]. Cells expressing Tax have diminished PTTG and develop convoluted nuclei and macronuclei. These results strongly suggest that PTTG is critical in maintaining chromosome stability.

Conclusions and Future Directions

It is rather clear that although PTTG must play an important role in pituitary tumorigenesis, PTTG also is involved in tumorigenesis of other tumors, is not a selective pituitary oncogene. PTTG-induced aneuploidy and PTTG inhibition of p53 may contribute to tumorigenesis. Furthermore, PTTG-null mice models demonstrate that PTTG also has physiological roles in metabolism and other systems. We suggest two major directions for future PTTG research: (1) mechanisms for PTTG tumorigenesis in pituitary and other tumors, and PTTG physiological functions, and (2) conditions required to switch PTTG from its physiological role to a tumorigenic one.

Acknowledgements Supported by NIH grant CA75979, the Doris Factor Molecular Endocrinology Laboratory, and the Annenberg Foundation.

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6 7 8 9 10 11

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13 14

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Yu R, Melmed S: Oncogene activation in pituitary tumors. Brain Pathol 2001;11:328–341. McCabe CJ, Boelaert K, Tannahill LA, Heaney AP, Stratford AL, Khaira JS, Hussain S, Sheppard MC, Franklyn JA, Gittoes NJ: Vascular endothelial growth factor, its receptor KDR/Flk-1, and pituitary tumor-transforming gene in pituitary tumors. J Clin Endocrinol Metab 2002;87: 4238–4244. Hunter JA, Skelly RH, Aylwin SJ, Geddes JF, Evanson J, Besser GM, Monson JP, Burrin JM: The relationship between pituitary tumour-transforming gene expression and in vitro hormone and vascular endothelial growth factor secretion from human pituitary adenomas. Eur J Endocrinol 2003;148:203–211. McCabe CJ, Khaira JS, Boelaert K, Heaney AP, Tannahill LA, Hussain S, Mitchell R, Olliff J, Sheppard MC, Franklyn JA, Gittoes NJ: Expression of pituitary tumour transforming gene and fibroblast growth factor-2 in human pituitary adenomas: Relationships to clinical tumour behaviour. Clin Endocrinol 2003;58:141–150. Yin H, Fujimoto N, Maruyama S, Asano K: Strain difference in regulation of pituitary tumortransforming gene in estrogen-induced pituitary tumorigenesis in rats. Jpn J Cancer Res 2001;92: 1034–1040. Heaney AP, Fernando M, Melmed S: Functional role of estrogen in pituitary tumor pathogenesis. J Clin Invest 2002;109:277–283. Heaney AP, Fernando M, Yong WH, Melmed S: Functional PPAR-␥ receptor is a novel therapeutic target for ACTH-secreting pituitary adenomas. Nat Med 2002;8:1281–1287. Chien W, Pei L: A novel binding factor facilitates nuclear translocation and transcriptional activation function of the pituitary tumor-transforming gene product. J Biol Chem 2000;275:19422–19427. Ishikawa H, Heaney AP, Yu R, Horwitz GA, Melmed S: Human pituitary tumor-transforming gene induces angiogenesis. J Clin Endocrinol Metab 2001;86:867–874. Heaney AP, Nelson V, Fernando M, Horwitz G: Transforming events in thyroid tumorigenesis and their association with follicular lesions. J Clin Endocrinol Metab 2001;86:5025–5032. Boelaert K, McCabe CJ, Tannahill LA, Gittoes NJ, Holder RL, Watkinson JC, Bradwell AR, Sheppard MC, Franklyn JA: Pituitary tumor transforming gene and fibroblast growth factor-2 expression: Potential prognostic indicators in differentiated thyroid cancer. J Clin Endocrinol Metab 2003;88:2341–2347. Saez C, Pereda T, Borrero JJ, Espina A, Romero F, Tortolero M, Pintor-Toro JA, Segura DI, Japon MA: Expression of hpttg proto-oncogene in lymphoid neoplasias. Oncogene 2002;21: 8173–8177. Stoika R, Yu R, Melmed S: Expression and function of pituitary tumour transforming gene for T-lymphocyte activation. Br J Haematol 2002;119:1070–1074. Yamada M, Asanuma K, Yagihashi A, Nakamura M, Kameshima H, Kobayashi D, Watanabe N: Human pituitary tumor transforming gene 1 (hPTTG1) in gastric mucosal tissues. Am J Gastroenterol 2001;96:1313–1314. Shibata Y, Haruki N, Kuwabara Y, Nishiwaki T, Kato J, Shinoda N, Sato A, Kimura M, Koyama H, Toyama T, Ishiguro H, Kudo J, Terashita Y, Konishi S, Fujii Y: Expression of PTTG (pituitary tumor transforming gene) in esophageal cancer. Jpn J Clin Oncol 2002;32:233–237. Yu R, Lu W, Chen J, McCabe CJ, Melmed S: Overexpressed pituitary tumor transforming gene causes aneuploidy in live human cells. Endocrinology 2003;144:4991–4998. Hagting A, Den Elzen N, Vodermaier HC, Waizenegger IC, Peters JM, Pines J: Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1. J Cell Biol 2002;157:1125–1137. Kanakis D, Kirches E, Mawrin C, Dietzmann K: Promoter mutations are no major cause of PTTG overexpression in pituitary adenomas. Clin Endocrinol 2003;58:151–155. Zhou Y, Mehta KR, Choi AP, Scolavino S, Zhang X: DNA damage-induced inhibition of securin expression is mediated by p53. J Biol Chem 2003;278:462–470. Romero F, Multon MC, Ramos-Morales F, Dominguez A, Bernal JA, Pintor-Toro JA, Tortolero M: Human securin, hPTTG, is associated with Ku heterodimer, the regulatory subunit of the DNAdependent protein kinase. Nucleic Acids Res 2001;29:1300–1307.

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Bernal JA, Luna R, Espina A, Lazaro I, Ramos-Morales F, Romero F, Arias C, Silva A, Tortolero M, Pintor-Toro JA: Human securin interacts with p53 and modulates p53-mediated transcriptional activity and apoptosis. Nat Genet 2002;32:306–311. Wang Z, Yu R, Melmed S: Mice lacking pituitary tumor transforming gene show testicular and splenic hypoplasia, thymic hyperplasia, thrombocytopenia, aberrant cell cycle progression, and premature centromere division. Mol Endocrinol 2001;15:1870–1879. Wang Z, Moro E, Kovacs K, Yu R, Melmed S: Pituitary tumor transforming gene-null male mice exhibit impaired pancreatic beta cell proliferation and diabetes. Proc Natl Acad Sci USA 2003; 100:3428–3432. Jallepalli PV, Waizenegger IC, Bunz F, Langer S, Speicher MR, Peters JM, Kinzler KW, Vogelstein B, Lengauer C: Securin is required for chromosomal stability in human cells. Cell 2001;105:445–457. Liu B, Liang MH, Kuo YL, Liao W, Boros I, Kleinberger T, Blancato J, Giam CZ: Human T-lymphotropic virus type 1 oncoprotein tax promotes unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1 and causes chromosomal instability. Mol Cell Biol 2003;23:5269–5281.

Dr. Shlomo Melmed Academic Affairs, 2015, Cedars-Sinai Medical Center UCLA School of Medicine 8700 Beverly Blvd, Los Angeles, CA 90048 (USA) Tel. ⫹1 310 4234691, Fax ⫹1 310 4230119, E-Mail [email protected]

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Kontogeorgos G, Kovacs K (eds): Molecular Pathology of the Pituitary. Front Horm Res. Basel, Karger, 2004, vol 32, pp 186–204

Pituitary Tumour Clonality Revisited R.N. Clayton, W.E. Farrell School of Medicine, Keele University, Stoke on Trent, Staffordshire, UK

Abstract Allelotype analysis and X chromosome inactivation analysis in women enables the assessment of tissue clonality, and has demonstrated that the majority of sporadic human pituitary adenomas are monoclonal. This implies that these tumours arise from de novo somatic genetic change(s) in a single pituitary cell. However, clonality within any given tumour may be multiple or single, multiple tumours arising on the background of hyperplasia may be of identical or different clonality, multiple ‘sporadic’ tumours in a tissue may be of differing clonal origin, and finally morphology cannot predict genetic makeup. These general principles may also apply to the pituitary so it is simplistic to assume that monoclonality is inevitable and that pituitary tumours cannot be multiclonal in origin. Indeed, these observations would be entirely compatible with the initiating stimulus resulting in hyperplasia of specific cell subtypes in the pituitary giving rise to a number of different clones each with variable potential to develop into a discrete tumour depending on their rate of cell division/rate of apotosis. Stimuli might include pituitary-specific oncogenes, intrapituitary growth factors, or extrapituitary trophic factors (e.g. hypothalamic releasing hormones). Copyright © 2004 S. Karger AG, Basel

Background

Because anterior pituitary function is tightly controlled by releasing/ inhibiting hormones elaborated by the hypothalamus it was, for a long time, believed that hypothalamic dysregulation might be the initial stimulus to tumorigenesis in the anterior pituitary. The pros and cons for a hypothalamic vs. pituitary site for initiation of the process are listed in table 1. Some points are worth emphasizing. Even if so-called longstanding lack of negative target gland feedback were to induce chronic hypothalamic stimulation of an anterior pituitary cell subtype with hyperplasia and tumour formation [1–7], it is difficult to see

Table 1. Primary site of initiation for pituitary tumours Hypothalamus In favour • ‘Feedback’ tumours in longstanding untreated end-organ failure • GHRH transgenic mice develop multiple adenomas on background of somatotroph hyperplasia • Dopamine D2 receptor knockout mice develop lactotroph hyperplasia and multiple discrete adenomas • GHRH can induce GH cell proliferation and c-fos gene in vitro • Cushing’s disease with corticotroph ‘hyperplasia’ may be ‘cured’ by surgery Against • No multiple tumours • Normal tissue surrounds tumour • Low recurrence rate • How to account for non-functional tumours

Pituitary

• Anatomically and functionally discrete not surrounded by hyperplasia

• Histologically homogeneous • ‘Mixed’ GH/Prl tumours derived from pluripotential progenitor cell

• Surgery for small adenomas followed by long-term ‘cure’

• Genetically monoclonal

• In MEN-1 hyperplasia occurs in parathyroids and islets (? pituitary)

• Hyperpalsia and multiple adenomas • •

co-exist in Carney complex somatotrophinomas May be multi/oligoclonal from outset Occasionally polyclonal

how this explanation would account for non-functioning tumours for which there is no known hypothalamic trophic hormone. Transgenic mice overproducing GHRH [8] do so in amounts which are probably much higher than could ever be produced by the hypothalamus. Moreover, in those rare patients whose acromegaly [9, 10] or Cushing’s syndrome [11] is caused by ectopic GHRH/CRH production, the pituitary, if removed, shows somatotroph or corticotroph hyperplasia, not a discrete adenoma. In a dopamine D2 receptor knockout mouse, discrete prolactinomas develop on the background of antecedent lactotroph hyperplasia [12]. Thus it seems that all functional subtypes of adenoma can, in unusual circumstances, arise on a background of hyperplasia making it quite conceivable that longstanding hypothalamic stimulation could contribute significantly to initiation of adenoma development. The most persuasive argument that tumours arise ab initio in the pituitary is the genetic one of monoclonality. This argument assumes that a single cell of a given lineage sustains a genetic change(s) conferring on it enhanced proliferation ability. Even if this were the case, it is far from clear that the genetic change(s) is ‘intrinsic’ to the cell rather

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than being driven from outside (i.e. extrinsic), be that from other cells within the pituitary, which may produce hypothalamic releasing factors/growth factors [13], or the hypothalamus. However, not all histologically discrete adenomas are monoclonal, a few Cushing’s adenomas are polyclonal [14] further challenging the monoclonality argument. Perhaps the most persuasive arguments for pituitary origins are the clinical and anatomical ones, especially the long-term ‘cure’ of somatotrophinomas [15] and prolactinomas [16] by initial surgery for microadenomas. If hypothalamic dysfunction was the proximate cause this would not be relieved by surgery and recurrence rates for microadenomas would be high, which they are not being (⬍10% at 7 years [15]). We are afforded more clues to the genetic abnormalities in the rare genetic syndromes in which pituitary adenomas are a component, albeit uncommon, of the phenotype. For example in multiple endocrine neoplasia type 1 (MEN-1) a germ-line deletion or mutation of one allele is accompanied by a mutation in the retained allele in the tumour [17]. Since the MEN-1 gene product is a transcriptional regulator it is likely that total loss of the protein product somehow leads to unrestrained growth. In MEN-1 the second ‘hit’ has clearly occurred in the organ that develops the neoplasia, although on an already abnormal genetic background. However, MEN-1 gene mutations do not appear to play a role in the pathogenesis of sporadic pituitary adenomas [reviewed in 18], nor indeed in familial acromegaly [19, 20]. Carney complex [21] is another autosomal dominant condition which includes somatotrophinomas in the phenotype. Here the genetic abnormality appears to be in the regulatory subunit of protein kinase A (PRKAR1A). Inactivating mutations in PRKAR1A lead to haploinsufficiency and disruption of the normal regulation of PKA activity in somatotrophs [22]. Activation of the cAMP-PKA-CREB pathway seems particularly important in somatotrophs, and indeed is recognised as stimulating cellular proliferation in this cell lineage [23]. Interestingly, similar mutations in PRKAR1A do not occur in sporadic somatotrophinomas [24]. The interesting histological appearance of hyperplasia of somatotrophs and multiple adenomas in pituitaries of Carney complex patients also demonstrates most emphatically that hyperplasia at least accompanies and may even precede adenoma formation, at least in human somatotroph [25]. This is very reminiscent of the situation in GHRH overexpressing transgenic mice [8]. What will be very interesting to discover is whether the multiple discrete adenomas from Carney complex pituitaries arise from the same clone or have individually distinct clonal origins. Notwithstanding the weight of argument in favour of a proximate pituitary site for initiation, it is quite possible that hypothalamic hormones, as well as local pituitary growth factors [26], act as progression factors for already transformed cells.

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Methods for Establishing Clonality and Technical Issues

X Chromosome Inactivation Analysis This is the ‘gold standard’ method for assessment of clonality. The Lyonisation [27] model states that, in females, either the paternal or maternal X chromosome is inactivated during mitosis when a cell divides to produce its progeny. Thus, in any tissue fragment which contains several cell generations there will be an approximately equal complement of cells with paternal and maternal X chromosomes inactivated. The mechanism for X chromosome inactivation is by methylation of cytosine residues in CpG islands at the 5⬘ end of active genes to prevent their transcription thereby resulting in protein production from only one X chromosome gene. Fortuitously, there are several genes on the X chromosome that are ‘polymorphic’, that is each allele can be distinguished by the presence or absence of a restriction enzyme site or the number of tandem (trinucleotide) repeats. Examples of the former are the genes PGK-1, HPRT, M27␤, and of the latter the human androgen receptor (HUMARA). Because HUMARA is more informative (80% polymorphic) it is the preferred method of analysis and is possible by PCR-based techniques, although one must be aware of PCR bias [28]. This method can only be applied to samples from women, and primer extension preamplification (PEP) cannot be used to increase DNA quantity as this procedure destroys the methylation signature required to resolve the two alleles. Autosomal Allelic Deletion (Loss of Heterozygosity) The demonstration that a tissue or tumour contains only one of two heterozygous alleles is also evidence that the sample is monoclonal, and this method of analysis does not depend on the methylation status of DNA. However, as with the X chromosome, it does depend upon the degree of constitutive heterozygosity of DNA markers or microsatellite polymorphisms at a given locus. As with X chromosome inactivation, the microsatellite polymorphism analysis has superseded RFLP analysis since it could be used in PCR-type studies. The key advantage is that these polymorphisms are frequent and more evenly spaced across the genome, so this is generally more informative than either RFLP or X-inactivation analysis. Where there have been direct comparisons in women, autosomal LOH-determined clonality is confirmed by X-chromosomal clonality either by Southern blotting or PCR (see figure 1 for comparison). The other major advantage of autosomal LOH analysis is that it is applicable to samples from men. In general therefore, PCR-based techniques for clonality analysis are preferred when the source of DNA is limited. It has been argued that because

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T2 6. 3

T2 6. 2b

T2 6. 2a

T2 6. 1

B2 6

D13S1246

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Fig. 1. Clonality determined by LOH and X chromosome inactivation analysis. Analysis of LOH at the microsatellite locus D13S1246 in a non-functioning tumour reveals uniform loss of either the upper or lower allele indicating monoclonal tumours (upper panels). A similar interpretation is revealed by the loss of the lower allele at HUMARA locus in T26.1 and the upper allele in the recurrences T26.2 and T26.3. T26.2a and T26.2b are separate fragments from the same tumour sample (the first recurrence).

of the extreme sensitivity of PCR, any contamination of tumour DNA with ‘normal’ constitutive DNA (such as that derived from entrapped leukocytes, blood vessels, or fibrous tissue) may obscure the ability to detect LOH or loss of one X chromosome allele. In practice there is not an issue as we have shown that, for LOH, 30% normal DNA contamination of a tumour DNA sample is required to produce a ‘polymorphic’ signal from a monoclonal sample [29]. Comparison of Southern vs. PCR Microsatellite Analysis for LOH/X Inactivation Early studies to explore clonality employed restriction enzyme digestion of extracted tumour DNA followed by Southern blotting and hybridization with radiolabelled cDNA probes required a relatively large amount of DNA, so was only feasible if the tumour was of reasonable size. With the advent of PCR it is now possible to obtain sufficient DNA from paraffin-embedded slides for X-inactivation/LOH analysis. Where comparison of clonality/LOH has been compared in the same sample by Southern blotting and microsatellite/PCR analysis, identical conclusions regarding clonality are obtained [34]. Although microdissection has considerable advantage, particularly with regard to provision of a homogeneous tumour sample, the DNA retrieved is often limited and detailed molecular studies in a single tumour are not possible. This can be overcome using PEP which has the advantage of producing an almost inexhaustible supply of DNA for future studies. The PEP technique is designed to generate

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sufficient template to allow multiple subsequent DNA analysis using the PCR reaction. PEP, first described by Zhang et al. [30], is reported to result in a minimum of a 30-fold amplification with a probability of amplifying any sequence in the genome being not less than 78%. In those cases where these types of preamplification are employed, the primary tumour extraction is the ‘gold standard’. Thus, in experiments requiring careful and detailed confirmation, the primary stock DNA should be re-examined. Southern analysis from solid whole tumour is more likely to detect gene amplification and rearrangements than PCR-based methods. However, PCR may detect allelic imbalance indicative of gene amplification. In pituitary tumours, amplification of individual genes is rarely detected, so this advantage is more theoretical than real. Extraction of DNA from slide material has the distinct advantage of being almost certain of avoiding ‘contamination’ with DNA from surrounding normal pituitary, if present in the biopsy. This cannot be guaranteed using fresh/frozen solid tissue, without prior microscopy. However, because slide-extracted DNA is inevitably fragmented it is, in practice, not possible to amplify across large stretches of DNA. The maximum is about 500 base pairs but this is not usually a problem because we design amplicons containing the microsatellite to be ⬍300 base pairs in size. This must be borne in mind when choosing PCR primers for any given locus. The theoretical drawback of PCR-based methods is that of infidelity and sequence errors introduced by misincorporation in the procedure, which may be a problem if doing DNA sequencing. The outcome of the PCR microsatellite analysis is critically dependent on having good quality DNA to amplify, and if this is too degraded, the analysis fails. It has been suggested that fragmentation may lead to preferential failure of amplification of the larger of two alleles at a locus. We circumvent this problem by co-amplification of a house-keeping gene (GAPDH) amplicon which is larger than the microsatellite amplicon. A further refinement for examination of discrete areas within a single specimen is the use of laser-assisted microdissection followed by PEP [31]. This would be suitable for LOH, but not X chromosome inactivation, studies.

Clonality of Pituitary Tumours

It is now 13 years since the first reports appeared on the clonality of sporadic adenomas assessed by X chromosome inactivation. These analyses using methylation-sensitive restriction enzyme digestion and RFLP-Southern blot analysis at the PGK-1 and HPRT loci were from cryopreserved solid tissue samples [32, 33]. In the report by Herman et al. [33], 3/3 somatotrophinomas, 2/2 non-functional tumours, 4/4 prolactinomas and 3/4 corticotrophinomas

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were monoclonal. Histologically the monoclonal tumours were homogeneously immunopositive for the respective hormone in the functional tumours. There were three tumours removed from the women with hyperprolactinaemia which contained interspersed non-tumourous anterior pituitary cells, and one corticotrophinoma contaminated by normal pituitary tissue and all these were polyclonal. It is probable that had these latter samples been microdissected to remove contaminating normal tissue that these would prove to be monoclonal. Similarly, Alexander et al. [32], using DNA from freshly frozen preserved tissue pieces, demonstrated that 6/6 non-functioning tumours were monoclonal. Subsequently, autosomal allelic deletion analysis by both RFLP-Southern blotting from whole tissue or by PCR microsatellite analysis of archival sections has confirmed monoclonality in much larger numbers of all the major subtypes of adenoma [18, 34–37]. Of particular interest with respect to the potential influence of the hypothalamus in the initiation of pituitary tumours are the corticotrophinomas [38]. As discussed below, Levy [39] uses this subtype to build an argument for physiological multiple monoclonality within anterior pituitary corticotrophs. If his argument is correct it might be expected that a proportion of corticotroph adenomas may be polyclonal by X-inactivation and LOH analysis. Schulte et al. [14] indeed found this to be true for 3/9 corticotrophinomas (1 micro-, 2 macroadenomas) all of which appeared to have no more (5–10%) fibrous tissue contamination than those tumours which were monoclonal. Using the highly polymorphic microsatellite cDNA probe M27␤, Gicquel et al. [40] demonstrated monoclonality in 8/8 macrocorticotrophinomas (4 Nelson’s syndrome, 4 Cushing’s disease) whose tumour fragments were reported as macroscopically homogeneous. As these authors acknowledge, they could not rule out the possibility that microadenomas may start off polyclonal and become monoclonal under the influence of extraneous growth stimuli (e.g. CRF/vasopression) such that when they became macroadenomas one clone is clearly dominant producing a monoclonal pattern. However, the results of the study by Biller et al. [41] showed that 10/10 microcorticotrophinomas were monoclonal, all of which were confirmed adenomas on histological and immunohistochemical examination, and are not compatible with the idea of a polyclonal initiation of corticotrophinomas. However, predictably a pituitary biopsy which showed multifocal hyperplastic corticotroph nodules from a patient with ectopic CRF production was polyclonal. Our own, unpublished, data adds to this debate in that we have found some microcorticotrophinomas that are polyclonal by HUMARA and others that are monoclonal. However, in patients with Cushing’s disease apparently cured by removal of non-adenomatous hyperplastic or normal pituitary tissue, this has, without exception, been polyclonal [Simpson et al., unpubl. data]. Thus, on balance, from the relatively small number (⬍50) of

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corticotrophinomas analysed, the majority are monoclonal, although a significant minority (maybe 30%) can be polyclonal.

Does Monoclonality Equate with ‘Tumour’?

A recent review by Levy [39] challenges the assumption that monoclonality is synonymous with neoplasia in the pituitary. The grounds put forward for this are: (a) The rare cyclic ACTH secretory activity by corticotroph adenomas with return to normality at intervals. (b) The expression of multiple hormones (both pituitary and hypothalamic) by subsets of cells within the adenoma. (c) During normal pituitary development there is non-random dispersion of cell types such that there are clusters of particular cell types in discrete regions of the pituitary [42]. These might have arisen by repetitive and controlled division from a single progenitor cell thus giving rise to ‘physiological’ zonal clonality within a ‘normal’ gland. (d) There are precedents for microscopic monoclonality, in the absence of neoplasia, in other tissues. Smooth muscle cells in atherosclerotic plaques exhibit a monoclonal pattern [43] and the normal aorta appears to be a mosaic of small overlapping monoclonal ‘tiles’ [44]. The same appears to hold true of the bladder epithelium [45] and uterus [46]. Regenerating liver nodules are frequently monoclonal but not neoplastic [47], though presumably have the potential to become so as hepatocellular carcinoma frequently develops in certain types of cirrhotic livers. Levy [39] proposes that an expansion of a ‘normal’ monoclonal population of cells could occur in response to a growth stimulus from outside or within the pituitary to produce a histologically discrete adenoma which is truly neoplastic in respect of unrestrained cell division. If there were several ‘normal’ monoclonal clusters, all with different genetic and functional characteristics, which responded to the same stimulus, this could produce an oligoclonal tumour, which by conventional clonality analysis would appear as polyclonal. This may be the case in a few Cushing’s adenomas [14] but seems unlikely for the other tumour subtypes which are invariably monoclonal. So ‘monoclonality’ of discrete areas might be ‘normal’ but this could only be tested by carefully examining a normal pituitary gland in multiple discrete areas, which remains to be done. Further, if Levy’s idea of oligoclonal tumours were true it may be possible to detect the different clones by examining multiple biopsies from different regions of the same tumour. Moreover, our own unpublished data indicated that in a small number of biopsies removed from

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Cushing’s patients in which the histology fails to reveal a discrete adenoma, we show no allelic deletion (LOH) with a reasonable number of markers, which implies polyclonality, although this could represent multiple overlapping monoclones. Undoubtedly, in the majority of adenomas, monoclonality indicates benign neoplasia. The most persuasive evidence for this is our own data from recurrent tumours [36], a significant proportion of which show additional genetic changes in the recurrent/regrown tumours. This is a characteristic feature of neoplasia in other tissues (e.g. colon) [48]. However, as described below, our data from recurrent/regrown tumour, and also from several examples of pituitary carcinomas with metastases, provides evidence in support of an oligo/multiclonal nature of the initial tumour.

Multiclonality of Pituitary Tumours

Evidence from Recurrent Tumours No study has previously examined initial and recurrent/regrowth pituitary tumours from the same individual for clonality or allelic deletions. The original hypothesis we set out to test with our subset of recurrent/regrown tumours was that the second and subsequent tumours would show additional allelic losses (LOH), indicative of further DNA damage, as they progressed. This was based on the classical Vogelstein hypothesis of increasing DNA damage with increasingly aggressive biological behaviour of a tumour [48]. We had already obtained some evidence for this from a cross-sectional study [34] where we showed increasing prevalence of allelic losses in carcinomas (77%) and radiologically invasive (33%) compared to non-invasive tumours (9%). Radiological classification is, however, a rather crude means of assessing biological aggressiveness and is only applicable to the moment in the natural history of the tumour at which the patient presents. The best in vivo human model of biological aggressiveness appeared to us to be the rate at which a tumour regrows/recurs after initial surgery. Some tumour remnants appear to regrow very slowly over years while others regrow and require further treatment within months. The subsidiary question we wished to address was whether we could identify which tumours were likely to regrow rapidly/recur by identification of specific allelic losses or the number of allelic losses in the first biopsy specimen. Identification of such predictive changes, or lack of them, would be especially valuable in deciding which patients should receive early post-operative external radiotherapy to any residual tumour remaining. This is a particular issue with large non-functioning tumours where there are no hormonal markers of early regrowth/recurrence.

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It is also important since hypopituitarism, a frequent sequel to radiotherapy, is not without its own morbidity and even excess premature mortality [49]. To date, no classical histological or more recent immunohistochemical or electron microscopic features can reliably predict tumour behaviour, with perhaps the exception that so-called ‘silent’ corticotroph adenomas (which clinically are initially without features of Cushing’s syndrome and are therefore nonfunctional) seem to have a marked propensity for widespread local invasion and even metastasis formation. We retrospectively obtained paraffin-embedded samples from 49 patients who had had two or more pituitary operations separated by at least 1 year. The indications for second and subsequent operations were variable, including regrowth of residual tumour requiring debulking before radiotherapy, regrowth causing significant headache, worsening or drug-resistant hormone hypersecretion in prolactinomas, acromegaly, or Cushing’s disease. In the majority of patients the regrowth/recurrent tumour was large (⬎1 cm diameter). The patients ranged from 16 to 78 years at first operation and the interval between first and second operation varied from 1 to 25 years (median 3 years). All cases were sporadic. All clinical subtypes were represented, the majority (n ⫽ 32) being non-functional tumours. Thirty-six patients received radiotherapy at some stage, and in 21 patients at least one sample, occasionally more, was available for analysis after radiotherapy. Most initial and recurrent tumours were invasive, meaning tumour was present in one or both cavernous sinuses or invading into the sphenoid sinus. DNA was extracted from blood and tumour DNA as described earlier and subjected to LOH analysis and X chromosome inactivation analysis in females (11 informative cases). With the nine microsatellite markers chosen on the basis of previously demonstrated frequent LOH [34], tumours from 33 patients showed allelic loss in the first and subsequent tumours at one or more loci. On the basis of LOH pattern in first and subsequent tumours, two common LOH patterns were seen – pattern A: LOH observed in the original tumour was identical (preserved) in the patient’s subsequent tumour(s) and in some cases this was accompanied by additional losses (fig. 2a), and pattern B: LOH was observed in the first sample but both alleles were retained (loss to retention) in a subsequent sample(s) (fig. 2b). In some cases this was accompanied by additional losses. 14/33 patients showed loss pattern A and 19/33 patients showed loss pattern B (loss to retention). From the patterns of LOH observed, we suggest that LOH pattern A is consistent with a single monoclonal origin, since within each individual patient, the loss pattern seen in the first tumour is preserved in subsequent samples. An example is shown in subject 23 where loss at the marker D13S153 is seen in the first (23.1) and subsequent tumour (23.2) with an additional loss at the marker

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a Patient No. 23, loss pattern A B

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Fig. 2. Examples of LOH in recurrent pituitary tumours representing common plus additional deletion in the regrown non-functional tumour from a 30-year-old woman (patient 23) (loss pattern A: upper panel); or ‘loss-to-retention’ pattern (loss pattern B: lower panel) in a 29-year-old man with a non-functional tumour (patient 6). In this example, markers IFN␣ (9 p), D10S217 and D13S155, which are lost in the first sample (T6.1), are retained in the recurrence (T6.2) which is still monoclonal as evidence by LOH at locus D13S1246. Thus, the second sample is of different clonal origin to the first. B is the matched blood DNA sample.

PYGM consistent with a progressive accumulation of losses with time (fig. 2a). This single monoclone interpretation was seen with all the tumour subtypes. In samples showing retention of heterozygosity in the second or subsequent samples (loss pattern B, fig. 2), this could best be explained by the second sample being derived from a distinctly separate clone, although both are monoclonal tumours. For example in patient 6 (fig. 2b) the tumour showed loss at 4 of the microsatellite markers while in the second sample (6.2) retention of heterozygosity at 3 of these markers was observed making it highly unlikely that this sample is clonally related to the first. The possibility that retention of heterozygosity is due to contamination by normal tissue is excluded since LOH is still found in tumour 6.2 at the marker D13S1246 indicating monoclonality. We were able to confirm the above interpretation of clonality by X-inactivation analysis in the female patients with informative HUMARA or PGK-1 alleles. In the example shown in figure 3 (pt 27), only the lower allele is present in the first tumour and only the upper one in the second tumour (a non-functional tumour). This must indicate separate clonality (different X-chromosone inactivated). In other examples [not shown but see 36], the same HUMARA or PGK-1 allele was present in all samples, indicative of likely same clonal origins. In a few other examples the clonality interpretation from LOH analysis and X chromosome inactivation was not consistent with a single clone in a sample, but with the presence of at least two clones. As previously discussed, because of the consistency between clonality interpretation from

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Fig. 3. ‘Loss-to-retention’ pattern at locus D10S217 in this patient with a nonfunctional tumour suggests a different clone for the second, regrown, sample (T27.2). Loss of the microsatellite marker D13S155 in the recurrent adenoma (T27.2) shows this to be a monoclonal expansion. This is confirmed by the X chromosome inactivation at HUMARA locus, where the opposite allele is lost in the second compared to first sample (B ⫽ blood; T27.1 ⫽ first tumour; T27.2 ⫽ second tumour).

LOH and X-inactivation in females it is likely that the LOH for interpretation of clonality in males is accurate. We considered that radiotherapy could potentially contribute to altered clonality of recurrent/regrowth tumours but we believe this is unlikely since approximately equal numbers of patients in whom clonality changed had received DXT (42%) between sampling and the remainder (58%) had not. In summary, up to 60% of recurrent/regrown pituitary tumours appear to be clonally distinct from the first tumour. This figure may be even higher if more loci had been examined. Perhaps even differing clonality of recurrent tumours is the rule rather than the exception. How are we to interpret the aforementioned data with respect to tumour initiation? Given that a significant proportion of recurrent/regrown tumours have different clonal origins, are two or more clones present from the very beginning of the tumour? Or does the second independent clone arise later in the progression of the pathogenic process? We have attempted to represent two possible options diagrammatically (fig. 4) in relationship to hypothetical genetic damage (‘hits’). Scenario 1 is on the left-hand side of the figure and envisages that the first ‘hit’ results in hyperplasia of several cells of a given lineage. This hit may be exogenous or endogenous to the pituitary. We favour an exogenous source, such as hypothalamic stimulation, since this is more likely to be cell subtype specific. If the stimulus to hyperplasia was from within the pituitary, e.g. growth factor overexpression, this might be expected to target cells of several lineages simultaneously. Hyperplasia, with its associated increased rate of cell turnover then predisposes these cells to a second hit which

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occurs in one or more cells (A and B) causing a different genetic changes (as represented by LOH) in each. The changes in cell A produce more rapid cell division than in cell B such that clone A becomes dominant, although clone B grows slowly. At surgery the dominant clone is removed and this is monoclonal by LOH analysis. Depending on the completeness of surgical removal, a recurrence may derive from either the remnants of clone A, with or without additional genetic damage (LOH), or from clone B. It is also conceivable that clone A might produce factors which inhibit the expansion of clone B, since growth inhibiting as well as stimulating factors may be produced from pituitary tumours [26]. Scenario 2 is on the right and is not predicated by pre-existing

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cellular hyperplasia but assumes that the first ‘hit’ targets two individual normal cells of the same subtype contemporaneously. The second ‘hit’ then only affects cell A (not cell B at this time) enabling clone A to develop while cell B does not expand into even a minor clone but retains its predisposition to expand if it sustains its second hit at a time before or after removal of clone A (the initial tumour). We believe this scenario to be less likely than the first in most cases for the following reasons: (1) Statistically the first ‘hit’ would be expected to randomly target any cell of any lineage, not cells of the same lineage, unless this ‘hit’ was cell subtype specific. If this were the case, clone tumour A and clone tumour B would be expected to be of different subtypes. Clinically, this is rare since most recurrences/ regrowths are of the same cell subtype as the original tumour, with very few rare exceptions, e.g. silent corticotrophinomas, which are originally non-functional (though by definition ACTH immunopositive) and then change phenotype to become highly functional). (2) If cell B sustained genetic damage and did not expand until some time (months/years) later there would be more chance that it would be eliminated through apotosis, which in fact may be enhanced because of the genetic change, and which does occur in the pituitary albeit at a slow rate.

Evidence of Differing Clonal Origin from Other Tumour Types

One crucial question to be answered in LOH and clonal analysis studies relates to that of sampling bias. How do we know that the extracted DNA, especially from a small biopsy represented on a slide, is representative of the whole tumour? Intratumoral DNA heterogeneity has been examined in colorectal carcinomas [50, 51] by LOH analysis. In each of four separate quadrants from the same tumour, LOH or non-LOH varied at random between quadrants within several of the tumours, although this was homogeneous when quadrants were further subdivided into smaller areas prior to DNA extraction [45]. Similarly, in various gastrointestinal tumours [52], prostate [31], and breast [53] cancers, microdissected areas exhibit intratumoral heterogeneity. How is this data to be interpreted? One distinct possibility is that these solid tumours are composed of several distinct clones of cells, some of which may have greater malignant potential than others. This might explain why in the aforementioned cancers prognostic significance of DNA aberrations has been inconclusive, as well as the variability in frequency with which allelic losses or p53 mutations have been reported. One of these studies formally assessed clonal composition by X chromosome inactivation [52] and showed that separate areas within a given tumour showed an identical X-inactivation pattern, although LOH analysis was

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variable within a given tumour. We would interpret this as a single clonal origin for the tumour but different subclones sustaining accumulating genetic damage as cells within the tumour progress at different rates. What has not yet been determined is whether similar genetic heterogeneity applies in larger pituitary tumours, although our studies on patients with metastases suggest this is a possibility. Is it a surprise that many pituitary tumours appear multiclonal? In the sense that these are generally benign adenomas rather than carcinomas perhaps it is but there are several examples in other tumours, some benign and some malignant of different clonal derivations. In multinodular goitres, co-existing individual nodules may be either poly- or monoclonal, and if monoclonal the clones may be different as revealed by X-inactivation analysis using M27-␤ RFLP analysis [54]. This clearly indicates that whatever the goitrogen(s) it is capable of targeting several cells of the same lineage, possibly contemporaneously. Another study indicated that solitary follicular thyroid adenomas or carcinomas are monoclonal although nodules from multinodular goitres are largely polyclonal [55]. In another case of two dominant autonomous nodules within a multinodular gland, each contained a different activating mutation in the TSH receptor gene. While not proving separate clonal origins, this is highly suggestive thereof [56]. While solitary parathyroid adenomas are monoclonal [57], monoclonal parathyroid tissue is common in hyperplastic glands from patients with uraemic hyperparathyroidism as well as in patients with primary parathyroid hyperplasia [58]. Histological categories of nodular versus diffuse hyperplasia did not predict clonal status. Thus, the chronic stimulation of uraemia induces hyperplasia followed by monoclonal expansion from this pool of cells in the parathyroid gland. So why couldn’t the same happen in the pituitary? Contrariwise, a polyclonal pattern of X chromosome inactivation was observed in medullary carcinomas of the thyroid from both MEN-2 patients as well as sporadic cases [59, 60]. This is a rather unexpected finding given earlier studies more suggestive of a single clone of origin of MTC with distinctly progressive subclones [61]. In benign pancreatic endocrine tumours, polyclonality is the rule which progresses to monoclonality as a single more aggressive clone develops the potential for invasiveness and metastatic spread [62]. In this example, too, the clonality pattern could not be predicted on the basis of histochemistry, proliferation index, or growth pattern. Somewhat surprisingly, sporadic gastrinomas occurring at multiple sites in the same patient appear to have the same clonal origin [63], whereas in MEN-1 patients multiple gastrinomas may be of independent clonal origin [64]. The presence of precursor populations of cells with different clonal composition appears quite commonly in other organs prior to the development

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of invasive cancer, e.g. cervix [65], bladder cancers [66], although when these latter are multifocal they appear to have the same clonal origin [67]. Likewise, in prostatic intraepithelial neoplasia and multifocal prostate cancer, the distribution of LOH indicates areas of similar and differing clonality [68]. The aforementioned evidence tells us several things about cancer clonality: (1) morphology cannot predict clonality; (2) clonality within a given tumour may be multiple or single; (3) multiple tumours arising on the background of hyperplasia may be of identical or differing clonality, and (4) multiple ‘sporadic’ tumours within an organ may be of differing clonal origin. We should not therefore be surprised that pituitary tumours may consist of an admixture of clones and that their recurrences/regrowths may be from any one of such clones. Thus, while the early available evidence indicated that pituitary tumours appear largely monoclonal, it is simplistic to assume that this is inevitable and that these cannot be multiclonal in origin. These observations would be entirely compatible with an initiating stimulus resulting in cell subtype-specific hyperplasia which itself gives rise to several distinct clones with variable potential to develop into tumours. Such stimuli might include hypothalamic trophic factors, intrapituitary growth factors, or pituitary-specific oncogenes.

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Kopp P, Kimura ET, Aeschimann S, Oestreicher M, Tobler A, Fey MF, Studer H: Polyclonal and monoclonal thyroid nodules coexist within human multinodular goiters. J Clin Endocrinol Metab 1994;79:134–139. Namba H, Matsuo K, Fagin JA: Clonal composition of benign and malignant human thyroid tumours. J Clin Invest 1990;86:120–125. Duprez L, Hermans J, Van Sande J, Dumont JE, Vassart G, Parma J: Two autonomous nodules of a patient with multinodular goiter harbor different activating mutations of the thyrotropin receptor gene. J Clin Endocrinol Metab 1997;82:306–308. Arnold A, Staunton CE, Kim HG, Gaz RD, Kronenberg HM: Monoclonality and abnormal parathyroid hormone genes in parathyroid adenomas. N Engl J Med 1998;318:658–662. Arnold A, Brown MF, Urena P, Gaz RD, Sarfati E, Drueke TB: Monoclonality of parathyroid tumours in chronic renal failure and in primary parathyroid hyperplasia. J Clin Invest 1995;95: 2047–2053. Ferraris AM, Mangerini R, Gaetani GF, Romei C, Pinchera A, Pacini F: Polyclonal origin of medullary carcinoma of the thyroid in multiple endocrine neoplasia type 2. Hum Genet 1997;99: 202–205. Volante M, Papotti M, Roth J, Saremaslani P, Speel EJ, Lloyd RV, Carney JA, Heitz PU, Bussolati G, Komminoth P: Mixed medullary-follicular thyroid carcinoma. Molecular evidence for a dual origin of tumour components. Am J Pathol 1999;55:1499–1509. Eng C, Mulligan LM, Healey CS, Houghton C, Frilling A, Rane F, Thomas GA, Ponder BAJ: Heterogeneous mutation of the RET proto-oncogene in subpopulations of medullary thyroid carcinoma. Cancer Res 1996;56:2167–2170. Perren A, Roth J, Muletta-Feurer S, Saremaslani P, Speel EJ, Heitz PU, Komminoth P: Clonal analysis of sporadic pancreatic endocrine tumours. J Pathol 1998;186:363–371. Goebel SU, Vortmeyer AO, Zhuang Z, Serrano J, Jensen RT, Lubensky IA: Identical clonality of sporadic gastrinomas at multiple sites. Cancer Res 2000;60:60–63. Debelenko LV, Zhuang Z, Emmert-Buck MR, Chandrasetharappa SC, Manickam P, Guru SC, Marx SJ, Skaralis MC, Spiegel AM, Collins FS, Jensen RT, Liotta LA, Lubensky IA: Allelic deletions on chromosome 11q13 in multiple endocrine neoplasia type 1-associated and sporadic gastrinomas and pancreatic endocrine tumours. Cancer Res 1997;57:2238–2243. Guo Z, Ponten F, Wilander F, Ponten J: Clonality of precursors of cervical cancer and their genetical links to invasive cancer. Med Pathol 2000;13:606–613. Hartmann A, Rosner U, Schlake G, Dietmaier W, Zaak D, Hofstaedter F, Knuechel R: Clonality and genetic divergence in multifocal low-grade superficial urothelial carcinoma as determined by chromosome 9 and p53 deletion analysis. Lab Invest 2000;80:709–718. Louhelainen J, Wÿkstrom H, Hemminki K: Allelic losses demonstrate monoclonality of multifocal bladder tumours. Int J Cancer 2000;87:522–527. Ruijter ET, Miller GJ, Van de Kaa CA, Van Bokhoven A, Bussemakers MJ, Debrwyne FM, Ruiter DJ, Schalken JA: Molecular analysis of multifocal prostate cancer lesions. J Pathol 1999; 188:271–277.

Dr. R.N. Clayton School of Medicine, Keele University Stoke on Trent, Staffordshire ST4 7QB (UK) Tel. ⫹44 1782 55 49 95, Fax ⫹44 1782 74 73 19, E-Mail [email protected]

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Kontogeorgos G, Kovacs K (eds): Molecular Pathology of the Pituitary. Front Horm Res. Basel, Karger, 2004, vol 32, pp 205–216

Molecular Cytogenetics of Pituitary Adenomas, Assessed by FISH Technique George Kontogeorgos Department of Pathology, G. Gennimatas Athens General Hospital, Athens, Greece

Abstract Fluorescent in situ hybridization (FISH) represents a modern molecular pathology technique, alternative to conventional cytogenetics (karyotyping). In addition to metaphase spreads, it can be applied directly to interphase nuclei. The latter makes the FISH technique powerful for pathologists for it integrates molecular genetics and classic cytogenetics and brings them together to a single framework for morphologic evaluation. Interphase FISH can be applied to imprints from fresh tissue or to paraffin sections after proteinase K digestion. Centromeric, telomeric and locus DNA-sequence specific probes can be used to identify aneuploidy or gene mutations. Several protocols combine molecular cytogenetics with classic karyotyping. Other sophisticated, FISH-based protocols have been introduced. Among them, comparative genomic hybridization is very important for it can detect non-balanced chromosomal aberrations of uncultured tumor cells and provide overall genomic information in a single experiment. This review presents the principles and applications of FISH technique for the investigation of the cytogenetic background of pituitary adenomas. Copyright © 2004 S. Karger AG, Basel

Introduction

Fluorescence in situ hybridization (FISH) was first introduced in 1988 [1], using a radioactive DNA probe to demonstrate chromosomal aberrations in nonmitotic human nuclei. The same year, an alternative non-isotopic fluorescence protocol [2] was introduced. Since then, various types of DNA probes and combined labeling systems have been developed. Currently, FISH represents a modern molecular pathology technique, referred to as molecular cytogenetics, for it is considered an alternative to classic or conventional cytogenetics (karyotyping).

Conventional cytogenetics is a well-established technique, which dominated for many years and provided enormous information regarding the cytogenetic background of many diseases and clinical syndromes. However, karyotyping is a time-consuming technique that requires fresh material, tissue culture equipment and expert qualification, not available in all diagnostic laboratories. In addition, there are difficulties in obtaining metaphases from solid tumors, particularly from those with low proliferation rate. Analysis of a small number of metaphases leads to underestimation of cytogenetic abnormalities, which obscures the heterogeneity and complexity of cytogenetic changes in solid tumors [3]. Moreover, selective cell growth of non-tumorous cells, such as fibroblasts, endothelial cells that can easily proliferate in tissue culture environment, in conjunction with the lack of morphology, may lead to erroneous results. For these reasons, in vivo cytogenetic techniques are highly unreliable in estimating the cytogenetic profile of solid tumors [4]. In contrast, FISH is an easy, reliable, sensitive, rapid and reproducible technique with many applications either directly to interphase nuclei (non-mitotic cells) or indirectly to metaphase spreads. For pathologists, FISH represents a very important morphologic technique for it integrates molecular genetics and classic cytogenetics and brings them together to a single framework for morphologic evaluation. In addition to these advantages, the expanded utilities and diagnostic applications have made FISH, in a short period of time, a powerful technique. Among the various DNA probes available, the sequence-specific ones can localize genes and detect structural genetic abnormalities, whereas the centromere-specific and paint DNA probes allow demonstration and enumeration of chromosomes and are useful in estimating aneuploidy [5–9]. Touch imprints from fresh material, tissue samples from biopsies, and frozen or paraffin-embedded tissues are suitable for FISH analysis. Touch preparations from fresh tissue material retain the whole intact nuclear mass and thus, they are ideal to estimate aneuploidy with accuracy. In contrast, the use of paraffin sections requires pretreatment with proteinase K to facilitate penetration of the cell matrix by the probe. Also cautious interpretation in counting of fluorescent signals, due to partial loss of nuclear mass, is always required [10, 11]. Pituitary adenomas have a low proliferation rate. They grow poorly in culture media and they can survive for only few days [9]. For this reason, the use of interphase FISH technique is considered ideal to investigate their cytogenetic background. Comparative genomic hybridization (CGH) is a novel technique that combines FISH technique with automatic digital image analysis and provides overall genomic information in a single experiment. The technique is based on comparative analysis of the hybridization products of tumor DNA and reference DNA with normal metaphase chromosomes, each labeled with different dual-color

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fluorochromes [12, 13]. CGH is appropriate for detecting non-balanced structural and numerical chromosomal abnormalities. The main advantage of this technique is that it can analyze the structure of chromosomes of uncultured cells by direct application to tumor DNA. Although CGH is considered as the state of the art of fluorescence technology, it is appropriate only for crude genetic screening. Several other sophisticated FISH-based protocols have been introduced: spectral karyotyping (SKY), multicolor FISH (MFISH) combined binary ratio (COBRA) labeling, and quantitative FISH analysis [14–16]. Other combined protocols of chromosome banding with metaphase FISH techniques, such as R-banding by primed in situ labeling (PRINS) and immunohistochemistry with FISH, such as GOLDFISH, enable further analysis of chromosomal abnormalities, which is inevitable to detect with conventional cytogenetics [17, 18]. Chromogenic in situ hybridization (CISH) represents a recently introduced alternative protocol of FISH technique, which enables a chromogen-based instead of fluorescent labeling system [19]. This method has also been applied to investigate pituitary carcinomas [20].

Pituitary Adenomas

Conventional Cytogenetics The molecular pathogenesis of pituitary adenomas remains largely unknown. Even more, there is very little information of their cytogenetic background. Only few studies, based on observations with conventional cytogenetics, mostly single case reports, have been performed. These studies have analyzed the karyotype of a limited number of metaphases from cultured pituitary adenoma cells [21–25]. According to these observations, cytogenetic abnormalities often involve several chromosomes with trisomies representing a frequent finding. Analysis of a single somatotroph adenoma showed an abnormal karyotype composed of 58 chromosomes with multiple trisomies 3, 5, 7, 11, 12, 13, 17 and 19 [21]. Trisomy 9 was detected in one prolactinoma and one non-functioning adenoma [23]. In a recent cytogenetic study of 53 pituitary adenomas of various morphologic types analyzed by direct and short-term culture methods, only 8 tumors (15%) showed and abnormal karyotype [25]. In addition, cytogenetic analysis of uncultured cells from pituitary adenomas showed an 8% incidence of trisomy 12 [26]. More recently, direct analysis and short-term cultures revealed an abnormal karyotype in 6 prolactinomas with trisomy 12 observed in 3 of them [27]. Molecular Cytogenetics Cytogenetic changes of chromosome 11 seem to be important for the development and progression of pituitary adenomas. The tumor suppressor

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Fig. 1. The nuclei of this aneuploid thyrotroph adenoma 11 contain aberrant fluorescent signals of chromosome 11 (FITC/PI, ⫻20).

gene men-1, which is responsible for the development of MEN-1 inherited disorder, is located on chromosome 11q13 [28]. FISH was first introduced to pituitary adenomas using an ␣-centromere-specific DNA probe to detect aberrations of chromosome 11 in GH-producing tumors. In this study, intact nuclei from fresh tissue imprints were used [29]. All adenomas were found to exhibit aberrant, often 3 copies of chromosome 11, in 8–23% of their cell population (fig. 1). Chromosome 11 aberrations showed a slight preponderance in the sparsely granulated variant as compared to the densely granulated type of somatotroph adenomas [29]. Morphometric studies have shown that approximately 40% of functioning adenomas, particularly 20% of GH-producing adenomas, are aneuploid, whereas non-functioning tumors rarely show abnormal DNA histograms [30, 31]. A subsequent study investigated 24 adenomas by direct FISH and DNA image analysis, in order to compare chromosome 11 aberrations with tumor DNA ploidy. All pituitary adenoma types showed variable chromosome 11 copy number abnormalities. They were noted mostly in functioning and less frequently in non-functioning tumors [10]. Overall, 87.5% of adenomas displayed aberrant chromosome 11 copy numbers. Aberrations were more frequent in functioning adenomas (94%) and less frequent in nonfunctioning adenomas (71%). Most often functioning tumors demonstrated trisomy, whereas few adenomas contained tumor cells with more than 3 fluorescent signals per nucleus. Chromosome 11 abnormalities were slightly higher

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Fig. 2. Monosomy of chromosome 11 in a case of a pituitary tumor from a patient with MEN-1 (FITC/PI, ⫻20).

in sparsely granulated somatotroph adenomas as compared to the densely granulated variant. The percentages of their cell population showing mostly trisomy of chromosome 11 were 23 and 17% respectively. Monosomy 11 with complete loss of one chromosome copy was observed in a mixed GH/PRLproducing tumor of a patient associated with MEN-1 with clinical signs of acromegaly (fig. 2). In addition, 3 corticotroph adenomas showed partial loss of one copy of chromosome 11 in 21–58% of their cell population. Nuclei from apparently normal, non-tumorous adenohypophysial parenchyma showed two fluorescent signals corresponding to the pair of normal chromosome 11 (fig. 3). In most cases, aneuploidy based on estimation of tumor DNA index (DI) was correlated with increased copy number of chromosome 11 and the severity of chromosomal aberrations in adenomas containing extra copies was often correlated with the increased DI. The most severe chromosome abnormalities were detected in a thyrotroph adenoma, which was associated with a remarkably abnormal DNA histogram showing the highest DI. Silent corticotroph adenomas, gonadotroph adenoma and null cell adenomas showed less frequent aberrations with less abnormal copy numbers of chromosome 11 and they exhibited diploid DI. The increase of DI noted in adenomas with monosomy of chromosome 11 indicates complex cytogenetic abnormalities with increased copy numbers of other chromosomes that balance the partial loss of chromosome 11 mass [10].

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Fig. 3. Interphase FISH analysis with an ␣-centromeric-specific probe for chromosome 11. All nuclei from normal adenohypophysial parenchymal contain two fluorescent signals corresponding to a pair of chromosome 11 (FITC/PI, ⫻20).

Genomic instability in tumor cells as a result of intracellular variation in the number of chromosomes produces aneuploidy [32]. Indeed, comparative studies of DNA ploidy with FISH and CGA techniques disclosed that aneuploid pituitary adenomas show more frequent and more severe chromosomal aberrations [10, 33]. It is well known that in MEN-1, the affected individuals inherit a mutated men-1 allele. Subsequent inactivation of the remaining normal allele by mutation or deletion within the endocrine tumor genome results in the development of MEN-1 phenotype [34]. Pituitary adenomas occur in about half of the patients with MEN-1. Among them, GH- and GH/PRL-producing adenomas represent the most frequent type show identical morphology with the sporadic tumors [35]. Recent molecular cytogenetic studies, employing single- or dualcolor FISH technique with centromere-specific and gene-specific DNA probes, have confirmed allelic loss of the men-1 gene and/or entire loss of one copy of chromosome 11 in GH/PRL-producing tumors from patients associated with MEN-1 [10, 36–38]. It should be noted however that inactivation of the men-1 gene is infrequently implicated in tumorigenesis of sporadic pituitary adenomas. Zhuang et al. [36] have shown allelic deletions of men-1 gene in only 10% of sporadic pituitary adenomas. Trisomy 11 was also reported in 2 of 31 sporadic pituitary adenomas (one somatotroph adenoma and one prolactinoma)

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in a FISH study using an ␣-satellite centromeric probe and examining intact nuclei with confocal microscopy. A 65% decreased mean allelic ratio or 66% for 16 or 13 microsatellite markers suggested trisomy 11 [39]. The authors concluded that inactivation of the men-1 gene represents an infrequent event in sporadic pituitary adenomas. Following CGH experiments highlighted the significance of chromosome 11 in the pathogenesis of pituitary adenomas by confirming the frequency and extend of chromosomal aberrations as FISH indicated [10, 29, 39]. A higher frequency of chromosome 11 aberrations with particular losses in functioning tumors was reported [40–42]. A few other FISH studies have investigated other chromosomes in pituitary adenomas. A series of 33 tumors was studied by molecular techniques including FISH analysis using an ␣-satellite centromeric probe specific for chromosome 12 [43]. Trisomy 12 suggested in 8 adenomas by the uniformly decreased allelic ratios of 18 microsatellite markers on the entire chromosome ranging from 54 to 66%. FISH technique confirmed trisomy 12 in 5 of these tumors including 2 somatotroph adenomas and 3 prolactinomas. An increased incidence of trisomy 12 was found by direct FISH analysis as compared to conventional cytogenetics. Trisomies of chromosomes 8 and 12 were identified in one pituitary adenoma by karyotyping and subsequently confirmed by interphase FISH technique [26]. According to these findings, it seems that trisomy 5, 8 and 12 represent a non-random cytogenetic change, particularly in prolactinomas [27]. From a series of 53 pituitary adenomas, 31 tumors were also investigated by the dual-color interphase FISH technique using probes specific for chromosomes 5, 8, 12 and X. Seventeen of these tumors (54.8%) showed aberrant copy numbers in one or more of these four chromosomes. Single or combined trisomies of chromosomes 5, 8 and 12 were found in all 10 prolactinomas and in 4 of 9 non-functioning adenomas. Combined loss of chromosomes 5 and 8 was observed in 1 of 6 corticotroph and 1 of 6 GH-secreting adenomas [25]. The authors suggested that karyotyping using direct and shortterm culture methods leads to underestimation of the cytogenetic abnormalities as compared to the increased resolution of cytogenetic analysis employing interphase direct FISH to cell preparations. Up to date, nine studies have investigated pituitary adenomas by CGH. In the first study based on a series of 23 non-functioning tumors [44], the majority of adenomas (74%) showed at least one chromosomal region abnormality in 22 chromosomes of the tumor genome. With the exception of chromosome 14, gains and/or losses affected all remaining chromosomes. The most frequent aberrations were noted in chromosome 8 (34.7%). No tumor showed deletion of the MEN-1 gene locus 11q, suggesting that the men-1 gene is not involved in the pathogenesis of non-functioning adenomas. The second study investigated 40 primary and 13 recurrent pituitary adenomas from 52 patients [40].

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A substantial majority of chromosomes showed genetic abnormalities. Copy number aberrations were observed in 48% of tumors with a higher frequency and greater number in functioning adenomas. Approximately 70% of adenomas with copy number aberrations showed multiple structural cytogenetic abnormalities involving part or the entire chromosome mass. Interestingly, chromosome 11 showed the most frequent aberrations. Loss of total chromosome 11 mass except for a preserved common segment containing 11q13 was noted in four adenomas. These results stress the importance of 11q13 and indicate that rearrangement of this subregion in a subset of sporadic adenomas may lead to tumor suppression gene inactivation. The third study analyzed 12 sporadic adenomas including 2 GH- and 1 PRL-secreting and 9 non-functioning tumors, and compared the results with DNA ploidy [33]. CGH disclosed cytogenetic abnormalities in at least one region in all 3 functioning adenomas, whereas chromosome changes were detectable in 4 non-functioning tumors. The average number of aberrations was 3-fold higher and the mean number of copy gains was significantly higher in functioning adenomas than in non-functioning tumors. In addition, aneuploid adenomas showed significantly frequent cytogenetic changes than diploid tumors. Loss of 13q was the most frequent detected aberration. This alteration has also been suggested in previous reports using microsatellite [45] and karyotype analysis [26]. Of the tumors with cytogenetic changes in this study, 71% had loss of chromosome 13, including 13q14 region, which corresponds to the part harboring the retinoblastoma gene. These authors suggest that loss of 13q14 may be an early chromosome alteration in the development of pituitary adenomas and that there is a putative tumor suppressor gene on 13q14. Another study analyzed the genetic imbalances of 10 growth hormone-secreting adenomas [46]. Gains of whole or parts of chromosomes were detected in chromosomes 5, 9, 19, 22q and 17p12-q21, whereas DNA loss were found in chromosomes 13q and 18. Due to frequent gains in chromosomes 19 and 22q, the authors suggest candidate genes residing in these chromosomal regions may be involved in the pathogenesis of GH-secreting adenomas. Material from original and recurrent tumors from 8 gonadotroph, 3 lactotroph and 1 null cell pituitary adenomas were studied by CGH [47]. Overall, pituitary adenomas showed an average of 1.6 chromosomal imbalances in the primary and 3.4 in the recurrent tumor. Lactotroph adenomas showed significantly higher chromosomal aberrations in both original and recurrent tumors, which were significantly higher than of null cell adenomas. In yet another large series of 75 sporadic pituitary adenomas, CGH disclosed at least one chromosomal imbalance in approximately 45% of tumors [42]. Gains were 5-fold greater that losses. The latter involved mostly chromosome 11, followed by chromosomes 10 and 13. No significant differences were observed among various types of pituitary adenomas. The authors conclude that a non-random pattern of

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chromosomal alterations may contribute to the development of in pituitary tumors. Carney complex is a familial multiple neoplasia and lentiginosis syndrome with features overlapping those of MEN-1 [37]. The pituitary tumors correspond to GH and PRL adenomas, whereas mammosomatotroph hyperplasia of the non-tumorous adenohypophysial parenchyma is common. CGH carried out in 4 of such cases showed cytogenetic changes in the most aggressive tumor, an invasive macroadenoma; these alterations were multiple involving several chromosomal regions, including losses of chromosome 11 [41]. Conclusions

Genomic instability as a result of altered genetic control during mitotic cell division and proliferation is critical for tumor development and progression. Chromosome gains and losses, mutations of genes regulating chromosome segregation in cell cycle lead to genetic instability. In addition, genomic instability may favor the allelic loss or the reduced expression of tumor suppressor genes and thus, enables neoplastic cells to grow faster. FISH and CGH findings showed a high frequency of genomic abnormalities involving multiple chromosomes in pituitary adenomas. Most of the presented molecular cytogenetic findings show that functioning adenomas as compared to non-functioning tumors have greater genomic imbalance with complex genomic abnormalities, which correlates with DNA ploidy. During tumor progression, several chromosome gains and losses emerge, which may become dominant and change completely the initial cytogenetic profile. Molecular cytogenetics provided broad knowledge about pathogenesis and progression of pituitary adenomas accumulated during the last decade. However, the importance of many of the affected genes remains to be discovered. References 1

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Pei L, Melmed S, Scheithauer B, Kovacs K, Benedict WF, Prager D: Frequent loss of heterozygosity at the retinoblastoma susceptibility gene (RB) locus in aggressive pituitary tumors: Evidence for chromosome 13 tumor suppressor gene other than RB. Cancer Res 1995;55:1613–1616. Hui AB, Pang JC, Ko CW, Ng HK: Detection of chromosomal imbalances in growth hormonesecreting pituitary tumors by comparative genomic hybridization. Hum Pathol 1999;30:1019–1023. Rickert CH, Dockhorn-Dworniczak B, Busch G, Moskopp D, Albert FK, Rama B, Paulus W: Increased chromosomal imbalances in recurrent pituitary adenomas. Acta Neuropathol 2001;102: 615–620.

George Kontogeorgos, MD, PhD Department of Pathology, G. Gennimatas Athens General Hospital KOFKA Building, 1st Floor, 154 Messogion Avenue, GR–115 27 Athens (Greece) Tel. ⫹30 210 7784302, Fax ⫹30 210 7704765, E-Mail [email protected]

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Kontogeorgos G, Kovacs K (eds): Molecular Pathology of the Pituitary. Front Horm Res. Basel, Karger, 2004, vol 32, pp 217–234

Morphology, Molecular Regulation and Significance of Apoptosis in Pituitary Adenomas Nikiforos Kapranosa, George Kontogeorgosb,c, Eva Horvathc, Kalman Kovacsc aDepartment

of Molecular Pathology, ‘Mitera’ Maternity and Surgical Center, of Pathology, G. Gennimatas General Hospital of Athens, Greece, and cDepartment of Pathology, St. Michael’s Hospital, University of Toronto, Toronto, Ont., Canada

bDepartment

Abstract Apoptosis represents energy-requiring spontaneous single cell death, with specific morphologic and biochemical features. It is a rapidly processed sequence of events resulting in elimination of damaged cells. Apoptosis occurs in physiological remodeling and proliferative conditions, and also in neoplastic lesions. Several molecules and molecular systems such as bcl-2/bax, Fas/FasL and caspases regulate the apoptotic process. Apoptosis is characterized by a stereotypic pattern of morphologic features, which can be illustrated mostly by electron microscopy. DNA and biochemical assays, based on the specific pattern of nucleosomal fragmentation can detect apoptosis. The in situ labeling techniques are currently used to demonstrate apoptosis in paraffin sections. Several studies of pituitary animal models, cell lines and human pituitaries have been performed during the last 6 years. By electron microscopy, pituitary adenoma cells undergoing apoptosis exhibit a common prototypical pathway of changes. Although the results by the situ labeling techniques are not uniform, apoptosis occurs with low frequency in a subset of pituitary adenomas, in carcinomas and in pituitary hyperplasia. Alternative techniques based on remodeling of cytoskeleton by caspase activity can identify early apoptotic stages. This review presents the principles of apoptosis and summarizes the morphologic and functional changes of apoptosis in pituitary. Copyright © 2004 S. Karger AG, Basel

Apoptosis

Several active processes regulate the maintenance of adult tissue homeostasis and embryonic tissue development, the most important of which are cell

proliferation, differentiation and apoptosis, also known as ‘programmed cell death’ [1, 2]. The latter is an ATP-dependent process that involves signal transduction and gene expression [3]. Spontaneous cell loss is characterized by a stereotypic pattern of morphological changes termed apoptosis, named after the Greek word ‘apóptvsi␵’ meaning the ‘falling off’ of petals of flowers and leaves of trees [4]. In addition to physiologic conditions, apoptosis occurs in hyperplastic and neoplastic lesions.

Apoptosis versus Necrosis

Apoptosis is essentially a phenomenon for rapid elimination of damaged single cells without causing any disturbance in the environment [4, 5]. Chromatin and cytoplasm condensation, cell fragmentation with formation of apoptotic bodies and elimination by phagocytosis, represent morphologic findings originally described by Kerr et al. [5]. Apoptosis is distinctly different from ordinary necrosis, which is a passive and non-ATP dependent lethal process caused by a general failure of cellular homeostasis due to external factors and typically involves large groups of cells. In necrosis the cell swells and bursts spilling out its contents and often provoking an inflammatory immune response [6].

Mechanisms of Apoptosis

The current understanding of the mechanisms of apoptosis has its origin in the research carried out 30 years ago on the nematode worm Caenorhabditis elegans. This organism during its development generates 1,090 cells (302 of them being neurons) and 131 of these cells subsequently die through apoptosis. Cellular apoptosis in this organism was found to be regulated by three genes designated ced-3, ced-4 and ced-9. Ced-3 and ced-4 promoted apoptosis and ced-9 opposed it. The mammalian homologues of ced-3 was found to be a family of cysteine proteases called caspases because they cleave proteins after aspartic residues whereas those of ced-4 and ced-9 are Apaf (apoptosis protease-inhibiting factor) and bcl-2 respectively [7, 8].

Caspases

The family of caspases play a critical role in the regulation of apoptosis and according to their function can be divided into two groups: initiator (or upstream)

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caspases, which are activated by pro-apoptotic signals, and effector (or downstream) caspases, which are activated by the initiator ones. Effector caspases carry out the proteolytic cell destruction and produce most of the morphological changes of apoptosis. This family of highly conserved proteases in mammals consists of more than 14 members [9]. Caspases normally exist in the form of proenzymes (procaspases) and are converted to the entirely active form by sequential proteolytic cleavage, which is usually performed by caspases themselves. Once a caspase is activated, the net effect can be exponentially multiplied by sequential activation of other procaspases. This form of caspases activation is called the caspase cascade, and it is widely used for activating effector caspases-3, -6 and -7 [8, 9]. One of the effector caspases (caspase-3) is responsible for the activation of the DNase, which performs the fragmentation of DNA at the end stage of apoptosis. This fragmentation is carried out by caspase-activated DNase (CAD) which normally exists in a complex with an inhibitory subunit called inhibitor of CAD (ICAD) and is activated by cleavage of ICAD by the active caspase-3 [10].

Regulation of Apoptosis

A large number of stimuli can induce apoptosis, some of them are common for all cells and some of them are tissue-specific. However, the end result of apoptotic stimuli depends on the counterbalance of anti-apoptotic factors in the target cell. In general, most cells require growth factors and mitogen signals for their survival and deprivation of these factors may lead to apoptosis [11]. The pathways for apoptosis induction can be classified in two major groups. In the first pathway apoptosis is triggered by extracellular factors acting on transmembrane ‘death receptors’ such as Fas ligand (FasL) and TNF [12]. Fas exists normally as a homotrimeric form and upon binding with FasL produces clustering of its intracellular parts which are called ‘death domains’. Subsequently a cytoplasmic protein called FADD (Fas-associated death domain) which also carries the death domain binds to the intracellular portion of Fas. FADD then recruits procaspase-8 molecules, which also carries death domain and they both form a complex. Fully active caspase molecules are finally generated through autoproteolysis leading to downstream activation of other caspases (caspase-3, -6 and -7) and cell destruction [13]. Fas-dependent apoptosis can be modified by glycosylation of Fas receptor [14] or by transcriptional up-regulation of Fas expression by the activated p53 protein [15]. In addition, proteins that are termed FLIPs (Fas-associated deathdomain-like) IL-1␤-converting enzyme-inhibitory proteins and structurally

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resemble with caspase-8 except from lacking proteolytic activity, can modulate and essentially inhibit Fas-induced apoptosis [16]. In the second pathway, apoptosis is induced by factors that gain entry into the cytoplasm such as free radicals, toxins, calcium ions and ultraviolet radiation. This pathway also induces procaspases activation through sequential proteolytic cleavage and oligomerization. Factors causing DNA damage such as radiation, cytostatic and genotoxic compounds are capable of inducing apoptosis via activation of p53. Depending on the severity of DNA damage and the cell type, p53 will either cause cell cycle arrest or activate the apoptotic cell destruction [17]. The pro-apoptotic gene Bax has been shown to be a p53 target and is up-regulated in certain cell types during p53-mediated apoptosis. Cytosolic Bax is unable to induce apoptosis and translocation of Bax protein from cytosol to mitochondria is required for p53-induced apoptosis. Peg3/Pw1, a protein, which is up-regulated in p53-mediated cell death process, induces this translocation. In contrast, Bcl-2 expression blocks cytochrome c release and therefore can inhibit apoptosis but without having any effect on Bax translocation, suggesting that Bax translocation acts upstream of Bcl-2 [18]. However, the finding that Bax is frequently induced to similar levels in p53-dependent growth arrest further supports that Bax contributes only partly to p53-induced cell death response, and other factors may be involved in p53-mediated apoptosis [19, 20]. A number of other genes such as PIGs, Fas/CD95, and Killer/DR5 have also been associated with p53-mediated apoptosis [21, 22].

Bcl-2 Family and the Mitochondrion

Bcl-2 gene encodes a 26-kDa protein, which is localized in the mitochondrial membrane, the endoplasmic reticulum and the nuclear envelope [23]. Bcl-2 was initially discovered in B-cell neoplasms in which chromosomal translocation juxtaposed the bcl-2 gene locus at chromosome segment 18q21 with the Ig heavy chain locus at 14q32, resulting in overexpression of Bcl-2 protein [24]. Bcl-2 is a member of a large multigene family, which includes genes that can inhibit or promote apoptosis. Currently the Bcl-2 family consists more than 24 members [25, 26]. Among them bcl-2, bcl-xL, bcl-w, bfl-1, brag-1, mcl-1 and A1 belong to apoptosis inhibitors, whereas bax, bad, bak, bcl-xS, bid, bik and Hrk are apoptosis promoters. The bcl-2-related proteins share homology in four highly conserved domains referred to as BH1, BH2, BH3 and BH4. Through these domains they can homo- or heterodimerize and thus, influence cell death or cell cycle entry [27, 28]. Most of the Bcl-2 family members possess another domain called the transmembrane domain (TM). The TM region is

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a hydrophobic sequence of 21 amino acids located in its C-terminus that enables the insertion of the protein into membranes. The importance of TM region for apoptosis is supported by the observation that deletion of TM domain renders both bcl-2 and bax incapable of completing their anti- or pro-apoptotic function [29]. Bcl-2 protein functions as a cell death suppressor in a wide range of cell types and under a variety of stimuli [30, 31]. Bcl-2 overexpression can block death induced by growth factor deprivation in hematopoietic and neural cells. Bcl-2 also prevents apoptosis induced by ␥-irradiation, glucocorticoid hormones and chemotherapeutic agents [8]. The mitochondrion, which is the focus of the actions of the Bcl-2 family, is intimately involved in the delicate network of apoptosis pathways being capable of regulating the apoptotic signals. The significance of mitochondrion in apoptotic mechanisms started to be clear when the apoptotic proteaseactivating factor-1 (Apaf-1) was identified. Apaf-1 is the mammalian homologue to C. elegans Ced-4. Apaf-1 is capable of binding to procaspase-9 through a domain called CARD (caspase recruitment domain), which is on both molecules. CARD domain of Apaf-1 is normally inactive and not available for interaction with procaspase-9. Conformation change of CARD, which is required for its activation, is accomplished through binding of Apaf-1 with cytochrome c and procaspase-9, forming the holoenzyme apoptosome [32]. The apoptosome is capable of activating caspase-3 and other effector caspases required for the final stages of apoptotic cell death [33]. Members of Bcl-2 family, normally residing on intracellular membranes and particularly the outer mitochondrial membrane, can regulate cytochrome c release through the following mechanisms. First, they may alter the permeability of the outer mitochondrial membrane through forming channels or holes. Second, they may cause rupture of the mitochondrial membrane and subsequently caspase activation and apoptosis [34].

Detection of Apoptosis

Morphology Apoptosis is characterized by distinct morphologic changes that morphologists can detect at light and electron microscopic level. Nuclear condensation and fragmentation, plasma membrane blebbing and cell shrinkage represent morphologic characteristics of early apoptotic changes. Progressively, the cell breaks into small membrane-enclosed fragments, called apoptotic bodies, which are phagocytosed by adjacent cells or macrophages without inciting any inflammatory reaction [4, 5]. The nuclear apoptotic changes are found in the

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literature with the descriptive terms ‘karyopyknosis’, ‘karyorexis’, ‘karyolysis’, whereas several other descriptions such as ‘kanivalism’, ‘cell-in-cell’, ‘tiger eye’ were used to illustrate the phagocytotic changes. In addition, descriptions such as ‘hyaline bodies’, ‘Camino bodies’, ‘Shivata bodies’ and the recently introduced term ‘thanatosomes’ deriving from the Greek word ‘ua⬘nato␵’, which means death, refer to remnants of apoptotic cellular material [35]. DNA Study Intranucleosomal DNA cleavage in approximately 180-bp segments offers an important target for apoptosis detection [36]. These changes are due to breaks in DNA strand by activation of endogenous Ca2⫹-dependent endonuclease [6, 36]. DNA extracted from human tissues shows by gel electrophoresis a characteristic ladder pattern of multiples 180-bp fragments. The derived DNA strand breaks have many new 3⬘-OH ends on which the enzyme terminal deoxynucleotidyl transferase (TdT) can incorporate labeled deoxyribonucleotide triphosphates. The TUNEL technique is based on this property [37, 38], whereas the in situ end-labeling (ISEL) technique, using in situ tailing of the 3⬘-OH ends by Klenow DNA polymerase, represents an alternative assay [39]. According to the labeling of the incorporated deoxyribonucleotide triphosphates (radioactive, fluorescent, digoxigenin), 3⬘-OH ends can be detected and measured quantitatively [40].

Apoptosis in Pituitary Animal Models and Cell Lines

A limited number of studies exclusively based on rodent pituitaries and pituitary cell lines have initially investigated apoptosis in pituitary [41–52]. The cell counts of apoptosis in rat pituitaries with estrogen-induced PRL cell hyperplasia have shown to increase after estrogen withdrawal [41]. In addition, an approximately 2-fold greater increase of apoptotic cell counts was noticed after bromocriptine administration as compared to the untreated animals. The authors suggest that dopamine agonists could induce apoptosis and affect the phagocytic properties of stellate cells. In another study, it was also found that bromocriptine and terguride are effective in suppressing cell proliferation and inducing apoptosis on estrogen-stimulated anterior pituitary gland of female Wistar rats [47]. Studies investigating the mechanisms implicated in the remodeling of the anterior rat pituitary after termination of lactation have found that this process occurs through apoptosis involving increased bax, decreased Bcl-2 and increased p53 protein expression [49]. Cell line experiments have shown induction of apoptosis by bromocriptine in GH-producing rat adenoma cells [42] and murine ACTH-secreting

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adenoma cells [43]. It was also shown that somatostatin analogues inhibit cell growth by induction of apoptosis in AtT-20 pituitary cell line and allows to progress through the cell cycle, except in those arrested in G1 or G2 phases [44]. However, other authors have found that somatostatin analogs and octreotide exert a cytostatic effect on GH3 rat pituitary cell proliferation via a transient G0/G1 cell cycle block and they did not induce apoptosis [45]. Lack of induction of apoptosis by tamoxifen was also noted in GH3 rat pituitary cell lines [48]. The molecular mechanisms of apoptosis were studied in AtT-20 adenoma cell line. AtT-20 cells exposed to ␥-irradiation exhibited typical morphologic changes beginning at 24 h [46]. Irradiated cells showed G1 and G2 arrest after 24 h. Western blot analysis showed that G1 arrest coincided with the induction of p53 protein. However, a 15-fold higher expression of bcl-2 gene was found in AtT-20 cells compared to normal mouse pituitary cells and they remained unchanged for 2 days after irradiation. These data show that occurs independently of bcl-2 gene expression in AtT-20 cells and that G1 arrest and apoptosis following DNA damage seem to be induced mostly by p53 protein. Kulig et al. [50] observed increased apoptosis and decreased bcl-X protein expression of HP75 cell line after treatment with transforming growth factor-␤1 and protein kinase C inhibitors. It was suggested that malignant transformation in pituitary is associated with increase in apoptotic rate and alteration of the expression of bcl-2 family proteins [50]. In addition, pituitary adenylate cyclase-activating polypeptide was found to inhibit transforming growth factor-␤1-induced apoptosis in HP75 human pituitary adenoma cell line [51]. Tamoxifen is a well-known estrogen receptor antagonist and also a protein kinase inhibitor. Induction of growth arrest and apoptosis was noted in a tamoxifen-treated AtT-20 cell line and human primary pituitary tumor cell cultures. Incubation with staurosporine and prolonged treatment with phorbol myristate acetate inhibited protein kinase C activity in AtT-20 cells, but not by tamoxifen. These findings suggest that tamoxifen induces apoptosis in AtT-20 cells independent of a classical protein kinase C isozyme pathway [52]. The enzyme protein kinase C (PKC) is involved in the regulation of cell growth, proliferation, and differentiation in several tissues. A greater PKC activity and expression was found to in adenomatous pituitary cells than in normal human and rat pituitary cells and higher in invasive pituitary tumor cells than in noninvasive ones. Two established pituitary adenoma cell lines, AtT-20 and GH4C1, were treated with hypericin, a potent inhibitor of PKC activity. Hypericin induced inhibition of growth and apoptosis. The potential use of hypericin in the therapy of pituitary adenomas warrants additional in vitro investigations with the aim of later moving toward therapeutic trials in selected patients in whom surgical or medical therapy has failed [53].

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Apoptosis in Human Pituitaries

Given the limited number of studies in human pituitaries and pituitary adenomas, very little is known regarding the regulation and significance of apoptosis [50, 54–65]. Pituitary adenomas are neoplasms with low proliferation rate, as it is evident by absence of mitotic activity and infrequent mitoses even in aggressive tumors [67, 68], and by low bromodeoxyuridine labeling index [69], S-phase fraction [70] and Ki-67 proliferation index [71]. Apoptosis and mitoses are asynchronous, adverse events representing the two faces of the same coin. They are involved in the maintenance of optimal cell numbers to provide appropriate tissue homeostasis in physiologic conditions, and in cellular proliferations including those of endocrine glands [72]. Conceptually, in neoplastic conditions the balance between mitoses and apoptoses is altered in favor of mitoses. Even though both events are commonly up-regulated, only scarce mitoses can be identified in pituitary adenomas and therefore, the numbers of apoptotic cells are also expected to be very small. The increased mitotic index in pituitary carcinomas compared to adenomas [71] was found to correlate with an increased apoptotic index [50]. Therefore, due to the very low apoptotic activity in pituitary adenomas, it is difficult to detect apoptosis relying on histology along. This task is even more difficult in electron microscopic studies, due to the low number of cells included in the specimen and to the lower possibility to have the whole cut surface of a cell with ultrathin sections. Even experienced morphologists can miss apoptotic cells in routine examination, unless specifically look to identify apoptosis. However, electron microscopy represents the best tool to illustrate apoptosis with accuracy, particularly to study in detail the sequence of the apoptotic process. Morphology of Apoptosis in Human Pituitary The original morphologic changes of apoptosis in human pituitary adenomas have been originally described in detailed recent studies [55, 63]. On routine histologic stains the cells display shrinkage with marked reduction of their volume due to loss of water (fig. 1). The early apoptotic nuclei show gradual pyknosis due to chromatin condensation, often with slightly irregular contour. Subsequently, they show margination of chromatin with typical crescent formation. The cytoplasm appears compact, the cells gradually lose connections with adjacent cells and finally they appear floating. Nuclear fragmentation into small, highly pyknotic particles with formation of apoptotic bodies represent advanced events of this process [55]. By electron microscopy, pituitary adenoma cells undergoing apoptosis exhibit a common prototypical morphologic pathway of changes that can be

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Fig. 1. Two rare events: apoptosis (lower left corner) and mitosis in the same section (upper right corner) from a case of untreated somatotroph adenoma. Several cells contain cytoplasmic inclusions (fibrous bodies), which are characteristic for the sparsely granulated variant of somatotroph adenoma. HE. ⫻80.

divided into three phases [63]. The first phase is characterized by prominent nuclear alterations. The sequence of these changes includes discrete clumping and condensation of chromatin with margination along the nuclear membrane and crescent formation or accumulation within one pole of the nucleus (fig. 2). Subsequently, the nucleus is separated from the cytoplasm by a perinuclear halo. In contrast, to nuclear changes, in the early apoptotic stage the cytoplasmic organelles such as mitochondria and Golgi apparatus remain well preserved, whereas the endoplasmic reticulum may be dilated. The morphology of secretory granules regarding their size, shape, texture, electron density and distribution appears normal. Reorganization of the subplasmalemmal area and the increase in electron opacity of the cytoplasmic matrix represents the most striking cytoplasmic change observed during the early stage of apoptosis. In the second phase, both nuclear and cytoplasmic changes are striking. The condensed nucleus breaks into smaller dense fragments, which contain highly electron-dense chromatin mass. Typically, the cells become round and the cytoplasm is extensively altered with marked accumulation of size different vacuoles (fig. 3). A few intact mitochondria and secretory granules are present. Subsequently, at the ‘blebbing’ stage the apoptotic cells are detached from the

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Fig. 2. Early stage of apoptosis in a corticotroph adenoma. The clumping of chromatin substance is evident, but other ultrastructural changes are not yet apparent. ⫻12,760.

Fig. 3. Portion of an apoptotic cell within an adjacent cell of a prolactin cell adenoma. As compared to the surrounding adenoma cells, the electron density of the ground cytoplasm is moderately increased and vacuolization of the RER is noted ⫻13,200.

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Fig. 4. Apoptotic cell in a prolactin cell adenoma, apparently breaking up. The electron density of ground cytoplasm is markedly increased, but images of unevenly dilated RER cisternae and those of swollen mitochondria still can be recognized ⫻11,440.

adjacent cells. Their outlines become convoluted with extensions and the plasma membrane surrounds the detached solid cellular material forming apoptotic bodies. The latter are crowded, closely packed highly condensed nuclear fragments, which lost their membranes, and are encircled by a narrow cytoplasmic rim. In the third phase, the apoptotic bodies are rapidly eliminated by phagocytosis by neighboring, mostly macrophages and stellate cells which also have phagocytic properties (fig. 4). Remnants of degraded apoptotic bodies can be identified within these cells (fig. 5). Infrequently, apoptotic bodies are not eliminated by phagocytosis, but they undergo degradation in a sequence known as ‘secondary necrosis’ [5, 65]. Among different types of pituitary adenomas, corticotroph adenomas show the most frequent and striking apoptotic changes at electron microscopic level [63, 64]. A seldom cell death process, distinct from apoptosis, commonly occurring in dopamine-treated PRL-producing adenomas and in oncocytomas, can also be observed. The main feature of this process is the progressively increased density of the cytoplasmic matrix, resulting in overall appearance of a ‘dark’ cell. The nuclei show no fragmentation, the chromatin becomes highly electron-dense, retaining its original distribution, even at advanced stages. Subsequently, the affected cells while maintaining the integrity of the nucleus, undergo severe

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Fig. 5. A large and a small apoptotic body, enclosed within a neighboring tumor cell in a corticotroph adenoma, are shown. In this advanced phase only the secretory granules can be conclusively identified. Note that the cytoplasmic membrane is still intact ⫻13,200.

alterations in cytoplasmic organelles and show extensive vacuolization. Dark cells can be observed concurrently with apoptotic cells in the same tissue section [63]. DNA Assays in Human Pituitary The ISEL or the TUNEL technique, based on the visualization of intranucleosomal DNA strand breaks, represents effective methods to detect easily apoptosis in paraffin sections [38]. However, several problems are encountered in detection and estimation of apoptosis using these techniques. Tissue fixation, enzyme concentration, prolonged proteolysis, active RNA synthesis and DNA damage in necrotic cells may give negative results or non-specific staining. Estimation of in situ labeling techniques in combination with the morphologic features of apoptosis or with the pattern of DNA fragmentation can minimize these artifacts [63]. The ISEL assay was applied to human pituitary adenomas in 1997 to identify apoptotic cells and to evaluate their apoptotic rate by counting the percentage of labeled nuclei [55–57]. Since then, a small number of other studies followed [50, 59–62, 64, 66, 73]. In a study of 85 adenomas, 46 tumors (54%) contained apoptotic labeled nuclei. The apoptotic labeling index (ALI) was significantly higher in

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Fig. 6. High apoptotic labeling index accessed by in situ end-labeling assay ISEL ⫻80.

functioning adenomas compared to nonfunctioning tumors (fig. 6). The highest percentage of labeled nuclei was noted in thyrotroph adenomas, followed by corticotroph, somatotroph, lactotroph and mixed GH-PRL-producing adenomas [55]. The apoptotic index was also investigated in a more detailed study of 95 nontumorous and neoplastic human pituitaries including 35 pituitary adenomas and 8 carcinomas using the TUNEL technique [60]. Regarding the ALIs of various types of pituitary adenomas, these results were comparable to those reported by Sambaziotis et al. [66], but much lower than the initial findings reported by the same authors [55]. Discrepancies in estimation of the ALIs in these studies are probably attributed to different methods used in retrieving, detecting and counting the labeled apoptotic nuclei. In addition, Kulig et al. [50] found a 4-fold higher ALI in pituitary carcinomas than in adenomas. In addition, they reported a 5-fold higher ALI in pituitaries from pregnant and postpartum compared to matched controls from nonpregnant females [50]. In the functioning group of tumors investigated by Kontogeorgos et al. [55], a higher ALI was observed in microadenomas compared with macroadenomas, but no statistically significant differences were observed, although untreated microadenomas, particularly lactotroph adenomas showed higher ALI. The latter finding is in agreement with the study of Kulig et al. [50] who did not detect apoptotic cells in lactotroph adenomas treated with dopamine agonists. Other studies reported no significant differences between functioning and nonfunctioning tumors [49, 56, 57], between micro- and macroadenomas [56, 60],

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between treated and untreated cases [56, 57, 61, 62] and between recurrent and non-recurrent adenomas [73]. Bcl2-Bax Proteins in Human Pituitary Initial immunocytochemical studies have found that approximately one third of pituitary adenomas show abnormal expression of bcl-2 and that most of these tumors co-expressed c-myc oncoprotein as well [54]. In another study, a similar distribution and staining intensity of both bcl-2 and bax was shown in nontumorous pituitaries. In addition, bcl-2 has found to occur in all adenoma types, but less frequently as compared to bax. Nonfunctioning tumors were more often positive for bcl-2. All functioning adenomas and the substantial majority of nonfunctioning tumors were immunopositive for bax. In contrast to bcl-2, bax was less frequently positive in nonfunctioning tumors [58]. These findings are in keeping with those of Kulig et al. [50] who investigated the apoptosis regulatory proteins bcl-2, bax, bcl-X and bad protein in human pituitary tumors. The authors found a moderate expression of the bcl-2 family proteins in all types of pituitary adenomas, but much weaker intense in carcinomas. In a more detailed recent study, a 12 grade Histoscore (HSC) was used to further evaluate the significance of bcl-2 and bax expression [65]. The bcl-2/bax HSC was extracted by multiplying the immunohistostaining grade by the staining intensity grade. The HSC of bax/bcl-2 HSC ratio was significantly higher in functioning tumors with inverse correlation with the ALI. In contrast, the bcl-2/bax HSC ratio showed a statistically significant correlation with the ALI in nonfunctioning tumors [65]. The wider expression of bax than bcl-2 supports the suggestion that this molecule has additional functions besides inhibiting bcl-2 [42, 50]. p53 Protein in Human Pituitary The p53 gene plays an important role in the DNA repair control during cell proliferation [74]. Mutations of the p53 gene have anti-apoptotic effect leading to tumor progression. The presence of p53 and apoptosis was investigated in two different studies in human pituitary adenomas. Green et al. [56] detected apoptosis by ISEL assay in 33% of GH-producing adenomas and 19% of nonfunctioning tumors. No apoptosis and p53 protein were noted in normal anterior pituitary. No significant association was found between apoptosis and p53 protein expression, tumor type or tumor size. The relation of Heat Shock Protein 70 (HSP-70) with apoptosis and p53 expression were investigated in corticotroph adenomas using the ISEL technique and immunocytochemistry [59]. The ALI was significantly lower in the corticotroph adenomas exhibiting nuclear p53 immunoreactivity compared to those with negative nuclear p53. No significant differences in the ALI were found between tumors with positive and negative cytoplasmic immunopositivity for p53. Colocalization of HSP-70

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and p53 protein was detected in the cytoplasm of adenoma cells. Although p53/HSP-70 complex formation leads to nuclear exclusion of p53 protein, no significant effect to apoptosis was found [59]. Detection of Caspase Activity in Human Pituitary The reason that apoptosis represents a transient and rapidly processed phenomenon makes the morphologic identification of apoptotic cells difficult. By in vivo methods the visible duration of a mitotic figure in male rat pituitary in routine histologic sections has been estimated to approximately 80 min and the visible duration of apoptotic bodies approximately 44 min [75]. The in situ labeling techniques provide substantial aid in resolving this problem. However, more sensitive procedures are required to detect early apoptotic changes. The intermediate filaments have been shown to undergo dramatic rearrangement during apoptosis and caspase cleavage of keratin 18 is associated with reorganization of cytoskeleton [76]. The recently introduced cytodeath antibody demonstrates keratin 18 reorganization and therefore, it can be used to identify early apoptotic events. In a recent case report of a corticotroph adenoma containing many apoptotic cells with typical morphologic changes, cytodeath antibody disclosed several preapoptotic cells which show no morphologic signs of apoptosis [64]. In conclusion, although electron microscopy remains the principal tool for documenting the morphologic features of apoptotic cells, a combination with other methods offers a more reliable approach for evaluating apoptosis in pituitary adenomas. The intensive molecular research currently being carried out at a genomic and proteomic level will further highlight the complicated pathways involved in the regulation of apoptosis in normal and neoplastic pituitary.

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Somatostatin Receptors in Pituitary Function, Diagnosis and Therapy Leo J. Hofland, Steven W.J. Lamberts Section Endocrinology, Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands

Abstract Recent studies have demonstrated that human pituitary adenomas express multiple somatostatin receptor (sst) subtypes. The expression of sst subtypes in human pituitary adenomas is highly variable. This variability in sst subtype expression may explain the variable responsiveness of patients with pituitary adenomas to medical treatment with the sst2-preferring SS-analogs octreotide and lanreotide. In human GH-secreting pituitary adenomas, both sst2 and sst5 are involved in the regulation of GH secretion. In prolactinomas, sst5 receptors are the key receptors in regulating responsiveness to SS. The low abundance of sst2 in prolactinomas explains the lack of efficacy of octreotide in lowering the elevated PRL levels in prolactinoma patients. Octreotide and lanreotide successfully suppress TSH levels, including normalization of thyroid hormone levels in the large majority of patients with TSH-secreting pituitary adenomas, probably due to the high level of sst2 expression in the adenomas. Although sst2 receptors are expressed by a significant proportion of gonadotroph and clinically non-functioning pituitary adenomas, the overall efficacy of sst2-preferring SS-analogs seems low in this type of patients. Finally, corticotroph adenomas may express multiple sst subtypes as well. Octapeptide SS-analogs do not lower circulating ACTH levels in patients with untreated pituitary-dependent Cushing’s disease, whereas SS and the SS-analog octreotide suppress pathological ACTH release in some patients with Nelson’s syndrome. This review discusses the expression and potential role of sst subtypes in the different types of human pituitary adenomas. Copyright © 2004 S. Karger AG, Basel

Introduction

Among the many target tissues of the small peptide molecule somatostatin (SS), the anterior pituitary gland was one of the first identified [1, 2]. SS was originally described as a factor present in hypothalamic extracts capable of inhibiting growth hormone (GH) release by cultured rat anterior pituitary cells [3].

Dphe

Cys

Phe DTrp

Octreotide Lys

Ala

Phe

Asn

Lys

Phe

Cys

Gly

Thr (ol)

Thr

Trp Lys

Cys

D␤Nal

SS-14

Cys

Ser

Thr

Phe

Thr

Cys

Tyr DTrp

BIM-23014 (lanreotide) Lys Arg Asp

Arg

Met

Pro Lys

CST-17

Asn

Phe

Phe

Cys

Trp

Cys

Lys Ser

Ser

Phe

Thr NH2

Cys

Val

Cys

Phe

Phe DTrp

Thr BIM-23268

Lys Cys NH2

Phe

Thrl

Fig. 1. Structures of somatostatin-14 (SS-14), human cortistatin-17 (CST-17), octreotide, lanreotide and BIM-23268.

This factor was subsequently characterized as a cyclic peptide, consisting of 14 amino acids (SS-14; fig. 1) [4]. Several years later, a second bioactive form, an extended SS molecule consisting of 28 amino acids (SS-28), was isolated and characterized [5]. SS-14 and SS-28 act via high affinity G-protein-coupled membrane receptors. Five SS receptor (sst) subtypes have been cloned and characterized. The genes encoding the five sst subtypes are localized on different chromosomes [6]. Via alternative splicing two forms of the sst2 receptor can be generated, e.g. sst2A and sst2B [7, 8]. sst2A and sst2B only differ in the length of their cytoplasmic tail. The five sst’s couple differently to the second messenger systems known to be activated upon SS binding to its receptor. These systems include inhibition of adenylate cyclase activity and activity of calcium channels, as well as stimulation of phosphotyrosine phosphatase (PTP) or MAP kinase activity. This has been reviewed extensively [6, 9, 10]. While the inhibitory effects on adenylate cyclase activity and on the influx of Ca2⫹ are linked to inhibition of secretion processes, the activation of PTP or MAP-kinase activity may play a role in the regulatory effects of SS on cell proliferation [10]. More recently, another SS-like peptide was discovered. This peptide, named cortistatin (CST; fig. 1), was shown to bear a strong structural resemblance to SS, which has an

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Table 1. Binding potency of different somatostatin analogs to the five human somatostatin receptor subtypes Ligand

SS-14 SS-28 CST-17 Octreotide BIM-23014 BIM-23197 BIM-23268 BIM-23052 BIM-23244 SOM230 BIM-23A387

Binding potency of SS-analogs (IC50 in nM)

Reference

sst1

sst2

sst3

sst4

sst5

0.93–2.0 2.2 7.0 280–1,140 180–2,129 6,016 12.0 100 1,020 9.3 293

0.05–0.25 4.1 0.6 0.38–0.60 0.54–0.7 0.19 28.0 11.9 0.29 1.0 0.1

0.1–1.2 6.1 0.6 7.1–34.5 14.0–98.0 26.8 5.5 5.6 133 1.5 77.4

0.2–1.7 1.1 0.5 ⬎1,000 230–1,826 3,897 36.0 132 ⬎1,000 ⬎100 Not done

0.2–1.4 0.07 0.4 6.3–7.0 12.7–17.0 9.8 0.42 1.2 0.67 0.16 1,000

identical receptor binding domain [11]. This 17-residue peptide was found to be expressed first in the human brain, but in further studies in other tissues like stomach, kidney and leukocytes as well [12]. Since the discovery of SS, a large number of SS-analogs have been synthesized. Among these, the octapeptide SS-analogs octreotide and lanreotide are the most widely clinically used stable SS-analogs. All five sst subtypes bind SS-14 and SS-28 with high affinity, but can be divided into two subclasses on their ability to bind structural octapeptide SS-analogs. sst1 and sst4 receptors do not bind octapeptide SS-analogs, whereas sst2A, sst3 and sst5 receptors display a high, low, and moderate affinity, respectively, towards octapeptide SS-analogs like the clinically used octreotide and lanreotide. Recently, novel sst subtype-selective and SS-analogs with a more universal binding pattern to the five sst subtypes have been introduced. Table 1 summarizes the binding affinities of a number of these SS-analogs. In the following paragraphs, the expression of sst subtypes in human pituitary adenomas, the role of sst subtypes in regulating tumoral pituitary hormone secretion and the role of SS-analogs in the treatment of patients with pituitary adenomas is discussed.

Expression and Functional Significance of sst Subtypes in the Normal Pituitary

In the normal human pituitary gland, sst1, sst2, sst3 and sst5 mRNAs are expressed. sst4 mRNA has not been detected (table 2). In human fetal pituitaries

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31, 39 92 39, 46 31, 39 20, 31 20, 31 13, 36 31 39 46

Table 2. Expression of somatostatin receptor subtypes in normal human pituitary and pituitary adenomas Adenoma

Somatostatin receptor subtype mRNA expression, number/total (%) sst1

sst2

sst3

sst4

sst5

GH-secreting

24/51 (65)

94/108 (96)

23/66 (49)

2/70 (4)

82/109 (83)

NFA Prolactinoma

17/46 (37) 33/37 (89)

33/46 (72) 27/37 (73)

23/45 (51) 11/35 (31)

3/40 (8) 4/33 (12)

14/40 (35) 24/33 (73)

Corticotroph TSH-secreting Normal pituitary

5/10 (50) 3/3 (100) 5/5 (100)

6/10 (60) 3/3 (100) 5/5 (100)

3/9 (33) 1/3 (33) 1/5 (20)

1/8 (13) 2/3 (67) 0/5 (0)

6/8 (75) 2/3 (67) 5/5 (100)

Reference

14, 20, 21, 24–28, 31, 46 14, 21, 24–27, 61 14, 21, 24–27, 47, 61 14, 24–27 14, 21 14, 27

NFA ⫽ Clinically non-functioning pituitary adenoma.

at the age of 18–25 weeks, the exclusive expression of sst2 and sst5 has been demonstrated [13]. In another study, all five sst were shown to be expressed in normal fetal pituitary tissue, although only a single fetal pituitary (age of 14 weeks) was studied [14]. Little is known with respect to the role of the different sst in regulating normal pituitary hormone GH secretion. Using a series of sst subtype-selective SS-analogs it was shown that SS regulates GH and TSH secretion by human fetal pituitary cultures via both sst2 and sst5, and PRL secretion mainly by sst2 [15]. In addition, ACTH and LH release by human fetal pituitary cells are only modestly effected by SS or its analogs [15]. Therefore, sst2 and sst5 appear to be the main sst subtypes involved in the regulation of hormone secretion by cells of the fetal normal anterior pituitary gland.

Growth Hormone Secreting Pituitary Adenomas

Somatostatin Receptor Expression Human GH-secreting pituitary adenomas express a variable, high density of SS binding sites in the majority of adenomas. In five human GH-secreting pituitary adenomas, Reubi et al. [16] showed the presence of [125-Tyr11]SS-14 binding sites with a number varying between 103 and 1,058 fmol/mg protein (for comparison, rat pituitary tissue contains 100 fmol/mg protein) and a dissociation constant (Kd) between 0.2 and 1.5 nM. Comparable observations were made by Moyse et al. [17], who demonstrated [125I-Tyr]SS binding with a variable sst concentration between 44 and 405 fmol/mg protein and Kd values ranging from 0.2 to 1 nM. Subsequent autoradiographic studies on cryostat

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sections showed that a small subgroup of GH-secreting pituitary adenomas expressed a low density of heterogeneously distributed sst binding sites for the sst2-preferring SS-analog [125I-Tyr3]octreotide, while in the same adenomas binding sites for [125I]-LTT-SS-28 were present [18, 19]. These data clearly suggested the existence of different sst subtypes on the basis of the characteristics of binding of different radiolabeled SS-analogs. More recently, after the cloning of the five different sst, it became clear that GH-secreting pituitary adenomas express transcripts for multiple sst subtypes. A large number of studies have now demonstrated that all sst subtype mRNAs, except sst4, can be expressed by human GH-secreting pituitary adenomas. The results of these studies are summarized in table 2. In most of these studies, sst subtype mRNAs were analyzed by RT-PCR, which gives no information on the number of sst subtype transcripts present in the adenoma tissue. Therefore, more recent studies evaluated the quantitative pattern of expression of the five subtype mRNA levels. In a series of 15 GH-secreting pituitary adenomas, sst2 and sst5 mRNA was found in all adenomas. The level of mRNA expression varied considerably, however, between the adenomas. The average sst5 mRNA level was approximately 10-fold higher, compared to the sst2 mRNA level [20]. A comparable large variability in the expression of sst2 mRNAs was found by others [21]. Treatment with SS-Analogs Current treatment options in patients with acromegaly due to a GH-secreting pituitary adenoma are surgery, medical therapy and radiotherapy. As medical therapy, stable SS-analogs, such as octreotide and the sustained release depot formulations Sandostatin-LAR and SR-lanreotide are widely used, both as primary or secondary therapy [22, 23]. Treatment with this generation of octapeptide SS-analogs results in clinical and biochemical control, i.e. normalization of circulating GH and IGF-I levels, in approximately two-thirds of the acromegalic patients [23]. The successful medical treatment of acromegalic patients with octapeptide SS-analogs is due to the expression of a high density of SS receptors on the adenoma cells, mainly sst2, which is one of the five known sst, and the selective high affinity binding of these analogs to this particular sst subtype [15]. The molecular basis for the clinical experience that one-third of patients with acromegaly are not adequately controlled by treatment with octapeptide SS-analogs is probably formed by a variable expression of the five known SS receptor subtypes (sst) in the adenomas of these patients [14, 20, 21, 24–27]. Mutations in sst2 and sst5 genes seem not to be a cause for the differences between acromegalic patients in sensitivity to SS-analog treatment. In a series of 19 human GH-secreting pituitary adenomas with variable sensitivity to SS-analog treatment in vivo, the sst2 and sst5 genes were found to posses intact coding sequences [28]. Moreover, no mutations affecting the sst2 protein

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were detected in a series of 15 GH-secreting pituitary adenomas [29]. These data demonstrate that mutations in these sst subtypes do not form the basis for resistance of tumoral GH secretion to SS-analogs. Ballare et al. [30] recently described a germ-line mutation (Arg240Trp) in the sst5 gene in an acromegalic patient resistant to SS-analog treatment. This mutation results in decreased sensitivity to the inhibitory effect of SS on adenylate cyclase activity, while cells expressing the mutant sst5 displayed increased proliferation and increased MAPK activity, compared with wild-type cells. These data suggest that this mutation in sst5 abrogated the antiproliferative action by SS and activates mitogenic pathways. Nevertheless, such mutations appear to be very rare. Recently, it was demonstrated that sst2 mRNA expression in GH-secreting pituitary adenomas was highly correlated with the in vivo GH suppression induced by an acute test using a single injection of 200 ␮g octreotide [31]. Therefore, the sst2 seems a predominant receptor in determining the inhibitory effect of octreotide or lanreotide on circulating GH release in acromegalic patients. Apart from reducing circulating GH levels in acromegalic patients, substantial tumor shrinkage is observed in a significant number of patients as well [32]. Nevertheless, the level of tumor shrinkage is significantly lower compared with the effects of dopamine agonist treatment on shrinkage of prolactinoma. The mechanism of the effect of SS-analog treatment on tumor volume is not clear at present. Unlike the potent inhibitory effect of dopamine agonists on RNA synthesis in prolactinoma cells resulting in prolactinoma cell shrinkage [33], SS and its analogues seem not to have profound effects on GH synthesis in [34, 35]. This may be the cause why octreotide treatment induces a significant decrease of circulating GH levels and sometimes mild to moderate tumor shrinkage. Novel Somatostatin Analogs: New Therapeutic Options? Using a series of novel sst subtype-specific SS-analogs, it has become clear that apart from sst2, sst5 receptors play an important role in regulating GH secretion by human GH-secreting pituitary adenoma cells as well. In this respect the regulation of fetal human GH secretion [15] is similar to that in human GH-secreting pituitary adenomas, new SS-analogs with enhanced sst2 binding affinities were more effective in inhibiting GH secretion compared to the clinically used octapeptide SS-analogs octreotide and lanreotide. In addition, some adenomas showed a better response to sst2-specific analogs, whereas in others sst5-specific analogs were more potent in suppressing GH release [36]. Finally, the effects of sst2- and sst5-specific SS-analogs were additive [36]. The basis for the observed differences between GH-secreting pituitary adenomas in their sensitivity to sst2- or sst5-specific analogs was further unraveled by Jaquet et al. [20]. By quantitative evaluation of sst subtype mRNAs, in

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combination with the evaluation of the responsiveness of a series of cultured GH-secreting pituitary adenomas to sst subtype-selective analogs, it was shown that sst2 mRNA, but not sst5 mRNA, expression levels correlated with the degree of GH inhibition induced by SS-14, SS-28 and the sst2-specific compound BIM-23197 [20]. The sst5 preferential analog BIM-23268 inhibited GH release in only 7 of 15 cases. Moreover, in octreotide-partially responding cultures, in agreement with the results of Shimon et al. [36], partial additive effects in suppressing GH release were found when the sst2- and sst5-specific compounds were tested in combination. Taking these data together, it may be concluded that sst2 is the predominant receptor in regulating GH release by GH-secreting pituitary adenoma cells, whereas sst5 receptors can mediate an inhibitory effect on GH secretion as well. Interestingly, in the same studies it was demonstrated that in adenomas co-secreting GH and PRL, PRL secretion was preferentially inhibited by sst5-specific SS-analogs [20, 36]. The additive GH-suppressive effect of activating both sst2 and sst5 initiated the development of analogs with selectivity to multiple sst subtypes. One of these analogs, the sst2- and sst5- bi-specific compound BIM-23244, indeed inhibited GH release in a subgroup of partially octreotide-sensitive adenomas more potently compared with octreotide. In this subgroup of adenomas, sst2 mRNA expression was 9-fold lower, and sst5 mRNA expression approximately 7-fold higher than in the octreotide-sensitive adenomas. These data suggest that in tumors expressing a low sst2 level and a high sst5/sst2 ratio, sst5 may become more important in regulating GH release [31]. In this respect, the recent observation that sst subtypes may form homo- and heterodimers, resulting in receptors with enhanced binding affinity and modified functional properties [37, 38], may form one of the explanations for the enhanced efficacy of bi-specific compounds such as BIM-23244. The above observations initiated the further development of novel stable SS-analogs with a more universal sst binding profile. One of these new compounds, named SOM230, was recently shown to reduce circulating IGF-I levels in rats by 75% after 126 days of continuous infusion. This effect was significantly more potent compared to octreotide, which suppressed IGF-I release under the same experimental condition by only 28% [39]. In rats, the terminal elimination life of SOM230 is 23 h, which is approximately 11 times longer compared to octreotide [39]. Finally, both in cynomolgus monkeys and beagle dogs, infusion of SOM230, but not SMS 201-995, lowered IGF-I levels potently [40]. Compared with octreotide, SOM230 has a 25, 5 and 40 times higher binding affinity to sst1, sst3 and sst5 receptors, respectively, and 2.5 times lower affinity to sst2 (table 1) [39]. Our preliminary observations indeed suggest that SOM230 inhibits GH release in a higher number of GHsecreting pituitary adenomas, both in vitro [41] and in vivo [42]. A significant proportion of GH-secreting pituitary adenomas express sst1 mRNA as well

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(table 2). Recently, it was shown that activation of sst1 by the sst1-selective agonist BIM-23296 caused a dose-dependent inhibitory effect in the nanomolar range on GH and PRL secretion by GH-secreting pituitary adenomas. In addition to lowering GH and PRL secretion, this SS-analog induced a decrease in cell viability as well [43]. The concept of targeting multiple sst was further evaluated with respect to potential cross-talk of sst with other members of the G-protein-coupled receptor family. Recently, it was reported that heterodimerization of sst5 and dopamine D2 receptors (D2DR) results in the formation of a novel receptor with enhanced biological activity [44]. On the basis of these observations and the knowledge that dopamine agonists inhibit GH hypersecretion in approximately 20% of the acromegalic patients [45], Saveanu et al. [46] recently studied the effects of BIM-23A387, which selectively binds to the sst2 (Ki ⫽ 0.10 nM) and the D2DR (Ki ⫽ 22.1 nM), on GH and PRL release by 11 cultured GH-secreting pituitary adenomas. In both octreotide-sensitive as well as in cultures showing partial responsiveness to octreotide, the maximal inhibition of GH release induced by the individual sst2 and D2DR analogs and by BIM-23A387 was similar. However, the mean IC50 for GH suppression by BIM-23A387 (0.2 pM) was 50 times lower than that of the individual sst2 and D2DR-specific compounds. This enhanced potency of chimeric molecules, such as BIM-23A387, may therefore open a new area of potential novel medical treatment options in acromegalic patients. In conclusion, due to the heterogeneous expression of sst2 and sst5 receptors in GH-secreting pituitary adenomas, a bi-specific analog activating both sst2 and sst5, or more universal SS-analogs like SOM230, may achieve a better control of GH and PRL hypersecretion in a larger number of acromegalic patients than octreotide [31]. In addition, the enhanced potency of novel chimeric molecules targeting different G-protein-coupled receptors, like the D2DR and sst2-specific compound BIM-23A387, may become a novel tool in the treatment of octapeptide SS-analog partially responsive acromegalic patients as well.

Prolactinomas

Somatostatin Receptor Expression In vitro binding studies using [125I-Tyr]SS showed that 8 out of 9 prolactinomas contained sst binding sites, although at very low density [17], whereas in other study 4 of 5 prolactinomas did not contain binding sites for the sst2-preferring agonist [125I-Tyr3]octreotide [19]. As shown in table 2, human prolactinomas express multiple sst subtype mRNAs. On the basis of RT-PCR

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analysis, sst1, sst2 and sst5 mRNAs are the most frequently expressed sst in human prolactinoma. Only a low proportion of the adenomas express sst3 receptors. By quantitative evaluation of sst subtype mRNAs, Jaquet et al. [47] showed that the dominant receptor in prolactinoma is sst5. In 7 of 10 adenomas, sst5 (mean level 5,648 ⫾ 1,918 pg/pg GAPDH) was expressed at a significant higher level compared with sst1 (mean level 1,296 ⫾ 669 pg/pg GAPDH) and sst2 (mean level 148 ⫾ 83 pg/pg GAPDH) mRNA. In addition, only a minority of the prolactinomas (3 out of 10) expressed sst3 and sst4 at very low levels between 49–52 and 11–35 pg/pg GAPDH, respectively [47]. Interestingly, in this series, two prolactinomas with relatively low sst5 mRNA levels, expressed a relatively high level of sst1 mRNA. These observations highlight the importance of evaluating sst subtype mRNA expression in a quantitative manner. Table 2 shows that sst1, sst2 and sst5 mRNAs are expressed in approximately the same proportion of prolactinomas. Nevertheless, the studies by Jaquet et al. [47] clearly show that expression levels of sst2 are magnitudes lower compared with sst5 and sst1 receptor mRNAs. Modulation of PRL Secretion by SS Due to the high expression levels of D2DR on [48] prolactinomas, D2DRspecific agonists such as bromocriptine, CV 205–502 and cabergoline are highly effective in reducing PRL hypersecretion and in inducing prolactinoma shrinkage [33, 49]. Nevertheless, a small proportion of patients shows resistance to dopamine agonist treatment [33, 50], probably due to decreased D2DR expression levels and/or post-receptor defects [33]. Therefore, medical treatments targeting other G-protein-coupled receptors may be of potential therapeutic interest in a small subgroup of patients with prolactinoma. In humans, SS infusion has only marginal effects on basal and TRHstimulated PRL secretion [51]. In addition, the sst2-preferring analog octreotide had no effect on PRL levels [52] and no PRL-suppressing effects by SS infusion have been found in most patients with hyperprolactinemia [53]. In line with the absence of binding sites for the SS-analog octreotide in most prolactinomas, octreotide was shown to be unable to suppress circulating PRL levels in patients with microprolactinomas [54], while sst scintigraphy did not visualize the tumor tissue in 3 female patients with microprolactinoma [55]. Interestingly, SS infusion to estrogen-treated agonadal subjects, induced a significant suppression of circulating PRL levels, suggesting a role of estrogens in regulating lactotroph sensitivity to SS [56]. Studies in rat anterior pituitary cultures clearly showed that PRL release by normal lactotrophs was only sensitive to SS in the presence of physiological concentrations of estradiol [57]. In addition, the sst2 and sst3 expression and the antihormonal effect of octreotide in the 7315b rat prolactinoma model is primarily dependent upon the presence

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of estrogens in vivo and in vitro and it was hypothesized that 7315b tumor cells in vivo could downregulate their own receptor status via their host, because of the ensuing hyperprolactinemia results in a hypoestrogenic state [58]. New Developments Recent in vitro studies showed that SS and sst subtype-selective analogs inhibit the release of PRL by cultured human prolactinoma cells. SS inhibited PRL release in a subgroup (approximately 20%) of prolactinoma cultures [59, 60]. In another study, PRL release was inhibited in 4 of 4 prolactinoma cultures by SS-28, whereas octreotide and other octapeptide SS-analog like lanreotide and RC-160 were clearly less effective [61]. Using the sst5-preferring analogs BIM-23268 and BIM-23052 (table 1), Shimon et al. [36] showed that the observed inhibition of PRL secretion by human prolactinoma cells by SS, is most likely to be mediated via sst5 receptors. In 4 prolactinomas, sst2-preferring analogs did not inhibit PRL secretion, while BIM-23268 and BIM-23052 at 10 nM suppressed PRL secretion by 30–40% [36]. This preferential effect of sst5-preferring analogs was confirmed by Jaquet et al. [47] in a larger series of 10 prolactinomas. In this study, the degree of PRL inhibition by SS-14 and BIM-23268 was significantly correlated with sst5 mRNA expression in the adenomas, but not with sst2 or sst1 mRNAs. However, the maximal inhibitory effects of D2DR agonists and the sst5-specific analog BIM-23268 were superimposable and not additive, both in D2DR-responding, as well as in D2DRresistant cultures. On the basis of these observations it was concluded that unless future studies show that in some prolactinomas PRL regulation is preserved by sst5 agonists but is resistant to dopaminergic control, the usefulness of sst5-selective analogs in the treatment of prolactinoma seems of modest interest [47]. The role of sst1 in the regulation of PRL secretion by prolactinoma cells remains to be clarified.

ACTH-Secreting Pituitary Adenomas

Somatostatin Receptor Expression There are only few available data with respect to sst expression in corticotroph adenomas. Using the sst2-selective SS-analog [125I]204–090, no sst binding sites could be detected by autoradiography in one corticotroph adenoma [19]. In more recent studies the sst subtype expression pattern has been studied by molecular biological techniques. Combining the results of different studies, the rank order of frequency of expression of sst subtypes in corticotroph adenomas is sst5 ⬎ sst2 ⬎ sst1 ⬎ sst3 ⬎ sst4 (table 2). Until now, no quantitative data of sst subtype expression in corticotroph adenomas are available, however.

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Our preliminary data show that indeed sst5, but not sst2, is the predominantly expressed receptor subtype in human corticotroph adenomas [62]. Modulation of ACTH Release by SS The first choice of treatment of patients with pituitary-dependent Cushing’s disease is surgery. If surgery fails, radiotherapy, alone or in combination with steroidogenic inhibitors, may be used [63, 64]. Normal ACTH secretion, studied in vitro and in vivo, is not affected by SS or octreotide [for review, see 65]. Octapeptide SS-analogs seem not useful in the treatment of patients with ACTH-secreting pituitary adenomas [55]. However, SS and the SS-analog octreotide suppressed pathological ACTH release in some patients with Nelson’s syndrome and ACTH and cortisol secretion in several patients with Cushing’s syndrome caused by ectopic ACTH secretion [for review, see 66, 67]. These data suggest that in untreated patients with Cushing’s disease sst2 levels, one of the five known sst subtypes to which octreotide binds preferentially, are low and that this receptor subtype may be upregulated when circulating cortisol levels are low. Additional in vitro evidence for this hypothesis comes from in vitro studies using primary cultures of human corticotroph adenomas, in which it was shown that glucocorticoids downregulated the response of corticotrophin-releasing hormone (CRH)-induced ACTH secretion to octreotide [68]. Therefore, the current generation of octapeptide SS-analogs seem not the most suitable analogs to modify ACTH release in patients with untreated pituitary-dependent Cushing’s disease. The observation that sst5 receptors are expressed in a relative high proportion of corticotroph adenomas suggests that SS-analogs with high binding affinity to sst5 may be novel candidates to modulate ACTH secretion by human corticotroph adenomas.

Gonadotroph and Clinically Nonfunctioning Pituitary Adenomas

Somatostatin Receptor Expression Sst have been demonstrated on clinically non-functioning and gonadotroph adenomas by autoradiography and binding studies on membrane preparations. Ikuyama et al. [69] showed a low density (17.2 and 48.0 fmol/mg protein) of high affinity (Kd 0.18 and 0.32 nM) of SS binding sites in 2 out of 5 clinically non-functioning pituitary adenomas. In addition, in 6 out of 15 clinically non-functioning pituitary adenomas a variable density of binding sites for [125I]204-090 was demonstrated. One sst-positive adenoma had a low receptor density (47 fmol/mg protein) of high affinity (Kd 0.79 nM) [19]. In another series, 6 out of 7 clinically non-functioning pituitary adenomas expressed binding sites for [125I-Tyr3]octreotide [70]. Among the different sst subtypes, sst2

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and sst3 seem the most frequently expressed sst in human clinically nonfunctioning pituitary adenomas (table 2). Again, the relative expression levels of these sst remain to be determined. Modulation of Gonadotrophin and a-Subunit Release by SS In vitro, SS and SS-analogs such as octreotide, inhibit the secretion of gonadotrophins and/or their ␣-subunits. In 10 out of 15 cultures SS significantly reduced the secretion of one or more intact hormones and/or free subunits [71]. Octreotide (10 nM) significantly decreased gonadotrophin or subunit release in 3 of 5 cultures in another study [70]. Finally, SS and lanreotide have been shown to inhibit the proliferation of non-functioning pituitary adenoma cells in vitro [72]. These observations formed the basis for several clinical studies evaluating the potential role of octapeptide SS-analogs in the medical treatment of clinically non-functioning adenomas. Although octreotide therapy may lead to a certain degree of lowering of circulating gonadotrophin or subunit levels and/or adenoma shrinkage in some patients with gonadotroph or clinically non-functioning pituitary adenomas, the overall efficacy of sst2-preferring SS-analogs seems low in this type of patient [73, 74]. The significance of the relative high frequency of expression of sst3 mRNA in clinically nonfunctioning adenomas should be further evaluated. On the basis of receptor binding studies it was reported that the majority of inactive pituitary adenomas indeed expressed sst3 binding sites [75]. In vitro, we found that gonadotrophin and/or subunit secretion was inhibited by SS in 7 of 11 cultures, and by octreotide or lanreotide in only 3 out of 10 cultures. In three cultures hormone release was sensitive to SS, but not to the two SS-analogs [61], suggesting that sst other than sst2, are involved. Therefore, SS-analogs targeting sst3, or more universal SS-analogs like SOM230, should be further evaluated for their potency to inhibit cell proliferation and/or hormone secretion by clinically nonfunctioning or gonadotroph pituitary adenomas.

TSH-Secreting Pituitary Adenomas

The majority of TSH-secreting pituitary adenomas also express sst [76, 77]. In a few cases the sst subtype expression pattern has been evaluated and showed that sst1 and sst2 are frequently expressed (table 2). Octreotide treatment of patients with TSH-secreting pituitary adenomas results in a lowering of TSH levels and normalization of T4 levels in 73% of the patients. In this series of 52 patients an escape from therapy was observed in 5 patients (10%). This loss of sensitivity to the inhibitory effect of octreotide on TSH levels was observed in 2 patients receiving short-term therapy and in 3 patients receiving long-term

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therapy [76]. Overall, Beck-Peccoz et al. [78] reported tachyphylaxis in 22% of the patients with a response to increasing octreotide doses, whereas subsequent escape from the inhibitory effects was observed in 10% of the cases. A comparable high response rate (⬎50% reduction of TSH levels in 23/26 cases) was recently reported by Socin et al. [79]. Therefore, both lanreotide and octreotide successfully suppress TSH levels, including normalization of thyroid hormone levels in the large majority of patients with TSH-secreting pituitary adenomas, most likely due to a high expression of sst2 in the adenomas [74].

Role of sst in Diagnosis

The high density of sst2 receptors in GH-secreting pituitary adenomas allows their visualization by the use of sst scintigraphy using [123I-Tyr3]octreotide or octreoscan ([111In-DTPA-D-Phe1]octreotide) [55, 80]. Several studies demonstrated a positive correlation of uptake values of octreoscan in GH-secreting pituitary adenomas and the in vivo responsiveness of acromegalic patients to octreotide [81–83], while other groups failed to demonstrate such correlation [84–86]. Octreoscan seems of limited importance in the differential diagnosis in acromegaly, since other pituitary adenomas, parasellar meningiomas, lymphomas, or granulomatous diseases of the pituitary may be positive as well, although octreoscan may detect ectopic GHRH-secreting tumors [55]. Overall, these data, including the high costs of octreoscan, do not warrant the extensive use of this technique in acromegaly. Due to the low number of sst2 receptors in prolactinomas, sst scintigraphy only visualizes prolactinoma in selected cases. In 3 female patients with microprolactinomas, sst scinitgraphy did not visualize the tumor tissue [55], whereas 1/5 prolactinomas was visualized in another series [87]. As discussed, clinically non-functioning pituitary adenomas express sst, including sst2, in a significant proportion of cases as well. Sst scintigraphy visualizes clinically non-functioning adenomas in 50–100% of the patients [70, 82, 84, 88]. A positive uptake at scintigraphy has no value in predicting responsiveness to medical treatment with SS-analogs. Corticotroph adenomas do not express high levels of sst2. Consequently, sst scintigraphy is normal in patients with pituitary-dependent Cushing’s disease [89]. On the other hand, sst scintigraphy successfully visualizes tumor lesions in most patients with ectopic ACTH-producing tumors [89]. A positive sst scintigraphy has been found in most patients with TSHsecreting pituitary adenomas [90, 91], although recently a lower detection rate was reported [79]. A positive sst scintigraphy was not predictive for the response to SS-analog treatment [79, 90].

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Pellegrini I, Rasolonjanahary R, Gunz G, Bertrand P, Delivet S, Jedynak CP, Kordon C, Peillon F, Jaquet P, Enjalbert A: Resistance to bromocriptine in prolactinomas. J Clin Endocrinol Metab 1989;69:500–509. Hofland LJ, De Herder WW, Visser-Wisselaar HA, Van Uffelen C, Waaijers M, Zuyderwijk J, Uitterlinden P, Kros MJ, Van Koetsveld PM, Lamberts SW: Dissociation between the effects of somatostatin (SS) and octapeptide SS-analogs on hormone release in a small subgroup of pituitary and islet cell tumors. J Clin Endocrinol Metab 1997;82:3011–3018. Hofland LJ, van der Hoek J, van Koetsveld PM, Bruns C, Weckbecker G, Sprij-Mooij D, Waaijers M, van Aken MO, Beckers A, de Herder WW, Lamberts SWJ: The novel somatostatin analog SOM230 inhibits ACTH release by cultured human corticotroph tumors (abstract). 8th International Pituitary Congress, 2003, abstr OC3. Orrego JJ, Barkan AL: Pituitary disorders. Drug treatment options. Drugs 2000;59:93–106. Colao A, Di Sarno A, Marzullo P, Di Somma C, Cerbone G, Landi ML, Faggiano A, Merola B, Lombardi G: New medical approaches in pituitary adenomas. Horm Res 2000;53:76–87. Lamberts SW, Krenning EP, Reubi JC: The role of somatostatin and its analogs in the diagnosis and treatment of tumors. Endocr Rev 1991;12:450–482. Lamberts SW: The role of somatostatin in the regulation of anterior pituitary hormone secretion and the use of its analogs in the treatment of human pituitary tumors. Endocr Rev 1988;9:417–436. De Herder WW, van der Lely AJ, Lamberts SW: Somatostatin analogue treatment of neuroendocrine tumours. Postgrad Med J 1996;72:403–408. Stalla GK, Brockmeier SJ, Renner U, Newton C, Buchfelder M, Stalla J, Muller OA: Octreotide exerts different effects in vivo and in vitro in Cushing’s disease. Eur J Endocrinol 1994;130: 125–131. Ikuyama S, Nawata H, Kato K, Karashima T, Ibayashi H, Nakagaki H: Specific somatostatin receptors on human pituitary adenoma cell membranes. J Clin Endocrinol Metab 1985;61:666–671. De Bruin TW, Kwekkeboom DJ, Van’t Verlaat JW, Reubi JC, Krenning EP, Lamberts SW, Croughs RJ: Clinically nonfunctioning pituitary adenoma and octreotide response to long-term high-dose treatment, and studies in vitro. J Clin Endocrinol Metab 1992;75:1310–1317. Klibanski A, Alexander JM, Bikkal HA, Hsu DW, Swearingen B, Zervas NT: Somatostatin regulation of glycoprotein hormone and free subunit secretion in clinically nonfunctioning and somatotroph adenomas in vitro. J Clin Endocrinol Metab 1991;73:1248–1255. Florio T, Thellung S, Arena S, Corsaro A, Spaziante R, Gussoni G, Acuto G, Giusti M, Giordano G, Schettini G: Somatostatin and its analog lanreotide inhibit the proliferation of dispersed human non-functioning pituitary adenoma cells in vitro. Eur J Endocrinol 1999;141:396–408. Shomali ME, Katznelson L: Medical therapy of gonadotropin-producing and nonfunctioning pituitary adenomas. Pituitary 2002;5:89–98. Colao A, Filippella M, Somma C, Manzi S, Rota F, Pivonello R, Gaccione M, Rosa M, Lombardi G: Somatostatin analogs in treatment of non-growth hormone-secreting pituitary adenomas. Endocrine 2003;20:279–284. Reubi JC, Waser B, Schaer JC, Laissue JA: Somatostatin receptor sst1-sst5 expression in normal and neoplastic human tissues using receptor autoradiography with subtype-selective ligands. Eur J Nucl Med 2001;28:836–846. Chanson P, Weintraub BD, Harris AG: Octreotide therapy for thyroid-stimulating hormone-secreting pituitary adenomas. A follow-up of 52 patients. Ann Intern Med 1993;119:236–240. Bertherat J, Brue T, Enjalbert A, Gunz G, Rasolonjanahary R, Warnet A, Jaquet P, Epelbaum J: Somatostatin receptors on thyrotropin-secreting pituitary adenomas: Comparison with the inhibitory effects of octreotide upon in vivo and in vitro hormonal secretions. J Clin Endocrinol Metab 1992;75:540–546. Beck-Peccoz P, Brucker-Davis F, Persani L, Smallridge RC, Weintraub BD: Thyrotropin-secreting pituitary tumors. Endocr Rev 1996;17:610–638. Socin HV, Chanson P, Delemer B, Tabarin A, Rohmer V, Mockel J, Stevenaert A, Beckers A: The changing spectrum of TSH-secreting pituitary adenomas: Diagnosis and management in 43 patients. Eur J Endocrinol 2003;148:433–442. Lamberts SW, de Herder WW, Kwekkeboom DJ, van der Lely AJ, Nobels FR, Krenning EP: Current tools in the diagnosis of pituitary tumours. Acta Endocrinol (Copenh) 1993;129(suppl 1):6–12.

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Colao A, Ferone D, Lastoria S, Marzullo P, Cerbone G, Di Sarno A, Longobardi S, Merola B, Salvatore M, Lombardi G: Prediction of efficacy of octreotide therapy in patients with acromegaly. J Clin Endocrinol Metab 1996;81:2356–2362. Oppizzi G, Cozzi R, Dallabonzana D, Orlandi P, Benini Z, Petroncini M, Attanasio R, Milella M, Banfi G, Possa M: Scintigraphic imaging of pituitary adenomas: An in vivo evaluation of somatostatin receptors. J Endocrinol Invest 1998;21:512–519. Broson-Chazot F, Houzard C, Ajzenberg C, Nocaudie M, Duet M, Mundler O, Marchandise X, Epelbaum J, Gomez De Alzaga M, Schafer J, Meyerhof W, Sassolas G, Warnet A: Somatostatin receptor imaging in somatotroph and non-functioning pituitary adenomas: Correlation with hormonal and visual responses to octreotide. Clin Endocrinol (Oxf) 1997;47:589–598. Plockinger U, Bader M, Hopfenmuller W, Saeger W, Quabbe HJ: Results of somatostatin receptor scintigraphy do not predict pituitary tumor volume- and hormone-response to ocreotide therapy and do not correlate with tumor histology. Eur J Endocrinol 1997;136:369–376. Legovini P, De Menis E, Billeci D, Conti B, Zoli P, Conte N: 111-Indium-pentetreotide pituitary scintigraphy and hormonal responses to octreotide in acromegalic patients. J Endocrinol Invest 1997;20:424–428. Plockinger U, Reichel M, Fett U, Saeger W, Quabbe HJ: Preoperative octreotide treatment of growth hormone-secreting and clinically nonfunctioning pituitary macroadenomas: Effect on tumor volume and lack of correlation with immunohistochemistry and somatostatin receptor scintigraphy. J Clin Endocrinol Metab 1994;79:1416–1423. Tofani A, Cucchi R, Pompili A, Carapella C, Crecco M, Mottolese M, Maini CL: 111In-octreotide scintigraphy in pituitary adenomas. Q J Nucl Med 1995;39:94–97. Van Royen EA, Verhoeff NP, Meylaerts SA, Miedema AR: Indium-111-DTPA-octreotide uptake measured in normal and abnormal pituitary glands. J Nucl Med 1996;37:1449–1451. De Herder WW, Krenning EP, Malchoff CD, Hofland LJ, Reubi JC, Kwekkeboom DJ, Oei HY, Pols HA, Bruining HA, Nobels FR, et al: Somatostatin receptor scintigraphy: Its value in tumor localization in patients with Cushing’s syndrome caused by ectopic corticotropin or corticotropinreleasing hormone secretion. Am J Med 1994;96:305–312. Losa M, Magnani P, Mortini P, Persani L, Acerno S, Giugni E, Songini C, Fazio F, Beck-Peccoz P, Giovanelli M: Indium-111 pentetreotide single-photon emission tomography in patients with TSH-secreting pituitary adenomas: Correlation with the effect of a single administration of octreotide on serum TSH levels. Eur J Nucl Med 1997;24:728–731. Krenning EP, Kwekkeboom DJ, Bakker WH, Breeman WA, Kooij PP, Oei HY, van Hagen M, Postema PT, de Jong M, Reubi JC, et al: Somatostatin receptor scintigraphy with [111In-DTPA-DPhe1]- and [123I-Tyr3]-octreotide: The Rotterdam experience with more than 1,000 patients. Eur J Nucl Med 1993;20:716–731. Patel YC, Srikant CB: Subtype selectivity of peptide analogs for all five cloned human somatostatin receptors (hsstr 1–5). Endocrinology 1994;135:2814–2817.

L.J. Hofland, PhD Department of Internal Medicine, Section Endocrinology, Room EE585c Erasmus MC, Dr. Molewaterplein 40 NL–3015 GD Rotterdam (The Netherlands) Tel. ⫹31 10 4634633, Fax ⫹31 10 4635430, E-Mail [email protected]

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Pathology and Molecular Genetics of the Pituitary Gland in Patients with the ‘Complex of Spotty Skin Pigmentation, Myxomas, Endocrine Overactivity and Schwannomas’ (Carney Complex)1 Constantine A. Stratakisa, Ludmila Matyakhinaa, Nickolas Courkoutsakisa,b, Nickolas Patronasb, Antonios Voutetakisa, Sotirios Stergiopoulosa, Ioannis Bossisa, J. Aidan Carneyc a

Section on Endocrinology and Genetics (SEGEN), Developmental Endocrinology Branch (DEB), National Institute of Child Health and Human Development (NICHD); bDepartment of Diagnostic Radiology, Warren Magnuson Clinical Center, National Institutes of Health (NIH), Bethesda, Md. and cDepartment of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minn., USA

Abstract Carney complex (CNC) is a familial multiple neoplasia and lentiginosis syndrome with features overlapping those of McCune-Albright syndrome (MAS) and other multiple endocrine neoplasia (MEN) syndromes like MEN type 1 (MEN 1). Pituitary tumors have been described in a number of patients with CNC; all have been growth hormone (GH) and prolactin (PRL)-producing. In at least some patients, pituitary gland involvement is manifested by hyperplastic areas; hyperplasia appears to involve somatomammotrophs only and to precede GH-producing tumor formation, in a pathway similar to that seen in MAS-related pituitary tumors (and in oncogenesis in other CNC tissues). One patient with CNC and advanced acromegaly had a GH-producing macroadenoma that showed extensive genetic changes at the chromosomal level. These changes appeared to represent secondary or tertiary genetic ‘hits’ involved in pituitary oncogenesis and were confirmed at the molecular level. So far, almost half of the patients with CNC have germline-inactivating mutations in the PRKAR1A 1

This work was presented in part at the 80th Annual Meeting of the Endocrine Society, New Orleans, La., June 21–27, 1998 (P3-552).

gene; in their pituitary tumors, the normal allele of the PRKAR1A gene is lost. Loss of heterozygosity suggests that PRKAR1A, which codes for the regulatory subunit type 1␣ of the cAMP-dependent protein kinase A (PKA), may act as a tumor-suppressor gene in pituitary tissue. These data provide evidence for a PKA-induced somatomammotroph hyperplasia in the pituitary tissue of CNC patients; hyperplasia leads to additional genetic changes at the somatic level, which in turn cause the formation of adenomas in some, but not all, patients. Copyright © 2004 S. Karger AG, Basel

Introduction

The complex of ‘spotty skin pigmentation, myxomas, endocrine overactivity, and schwannomas’ or Carney complex (CNC) is a multiple endocrine neoplasia (MEN) and lentiginosis syndrome [1–4] that is inherited in an autosomal dominant manner [5], and is genetically heterogeneous [6–8]. Although growth hormone (GH) and prolactin (PRL) secretion are frequently abnormal in affected patients [9, 10], clinical acromegaly or significant hyperprolactinemia and GH- or PRL-producing tumors, respectively, have been detected in less than one fifth of them [11, 12]. This pattern of abnormal GH and PRL secretion without pituitary tumors (detectable by common imaging modalities) and infrequent development of clinically significant acromegaly is reminiscent of McCune-Albright syndrome (MAS) [13–16]. Studies of tumors excised from patients with CNC have indicated that their molecular abnormality may be in the pathway that involves the stimulatory ␣-subunit of the guanine nucleotidebinding protein (Gs␣) [17, 18], the gene responsible for MAS [13]. However, Gs␣ mutations were not found in a series of CNC tumors [18]. Almost half of the patients with CNC have germline-inactivating mutations in the PRKAR1A gene [19, 20], which codes for the regulatory subunit type 1␣ of the cAMP-dependent protein kinase A (PKA) [21]. Loss of heterozygosity (LOH) in some tumors from the patients suggested that PRKAR1A may act as a tumor-suppressor gene in affected tissues [22, 23]. However, PRKAR1A’s role in human oncogenesis is controversial, and studies mainly from cell line-related investigation did not suggest a tumor-suppressor role [24].

Pituitary Findings in Patients with CNC

Clinical and Histopathologic Analysis GH-producing tumors have been identified so far in 8 patients with clinically diagnosed acromegaly at the National Institutes of Health [25]. Their

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Table 1. Identification, age, clinical manifestations and PRKAR1A defects of patients with CNC and acromegaly Patient

Age

Gender

Family

Other manifestations

PRKAR1A mutation

1 2 3 4 5 6 7 8

19 38 44 42 18 21 38 19

M F F F M F M F

CAR01 CAR110 CAR16 CAR102 CAR07 CAR07 CAR20 Sporadic case

L, P L, P, Mx, A, T L, P, Mx, T L, P, Mx, T L L, P, T L, P, Mx L, P, Mx

c.578delTG 769C⬎T Negative Pending Pending Pending c.578delTG Negative

F ⫽ Female, L ⫽ lentigines, M ⫽ male, Mx ⫽ myxoma, P ⫽ primary pigmented nodular adrenocortical disease, T ⫽ thyroid nodules or thyroid cancer.

identification and PRKAR1A mutation status are given in table 1; additional clinical details have been described elsewhere [25]. An example of progression of a GH-producing tumor over several years is given in figure 1 (case 4). All tumors stained positive for PRL and occasionally for other hormones (see below). Three of 4 patients (table 2) who had acromegaly as the primary manifestation of CNC (cases 1, 5, 6) had macroadenomas. Microadenomas were detected in all operated patients in the CNC prospective study (cases 2–4, 8). One of these patients (case 2) had been administered octreotide for approximately 6 months because of high GH levels; octreotide was discontinued 3 years before TSS in our institution. Acromegaly was cured by TSS in all patients with microadenomas. Only 1 of 3 patients with macroadenoma, however, was cured surgically (case 5). Multiple macroscopic and microscopic tumors were seen in the pituitary gland of 5 patients (cases 1, 4–7), including 1 with a microadenoma (case 7) (fig. 2). In these patients, extratumoral pituitary parenchyma showed evidence of GH- and PRL-producing cell hyperplasia. In 3 patients whose microadenoma (cases 2, 3, 8) was excised completely, extratumoral parenchyma was not available for study. Adenohypophysial hyperplasia, characterized by poorly delineated zones with increased cellularity and an expanded, somewhat irregular reticulin pattern, was seen in 5 cases (an example is provided in figure 2a). A zone of probable transition from hyperplasia to adenoma, characterized by the gradual disappearance of the reticulin pattern and increasing cellularity, was also documented in these cases (an example is shown in figure 2b).

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a

b

c Fig. 1. Progressive GH-producing tumor formation in a female patient with CNC (case 4) from 1989 (a), 1991 (b), and 1996 (c).

Both hyperplastic areas and adenomatous tissue stained for GH and PRL in all patients. PRL staining was less intense and more limited than GH staining [25], although in all cases it was the same cellular population that demonstrated immunoreactivity for both hormones in consecutive sections. Staining for ␣-subunit was also present in 3 of the 5 tumors (table 2) in the same pattern as that of PRL. Occasional staining for TSH (␤-subunit) and LH was also present in diffusely and rarely present cells of some adenomas and within foci of normal cells entrapped within the tumors (table 2). ACTH and FSH staining, when performed, showed only in foci of normal anterior pituitary cells entrapped within the tumors or the hyperplastic zones (fig. 1a, b).

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Presented with acromegaly

5 years

1 year

2 years

Presented with acromegaly

Presented with acromegaly

Presented with acromegaly

1 year

1

2

3

4

5

6

7

8

54.4

38.6

211.5

115.9

33

39.2

80.5

135.9

IGF-I* nmol/l

4.7

8.3

41

12.6

4.8

14.2

5.4

27

GH, ␮g/l

13.3

12

126.1

NA

27

14.8

69.2

NA

PR, ␮g/l

Micro

Micro

Macro

Macro

Micro

Micro

Micro

Macro

Tumor size

Yes

Yes

No

NA

Yes

Yes

Yes

No

Cure* PRL ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

GH ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫺

⫹

NA

⫹

⫺

NA

⫹

NA

␣-sub

Adenoma staining

NA

⫺

NA

NA

⫹

NA

⫹

⫹

TSH

NA

⫹

NA

⫹

⫺

NA

⫺

⫹

LH

NA

Yes**

Yes**

Yes**

Yes**

NA

NA

Yes**

Hyperplasia

No

NA

NA

NA

NA

No

No

Yes

CGH changes

CGH ⫽ Comparative genomic hybridization, GH ⫽ growth hormone, IGF-I ⫽ insulin-like growth factor type I, Macro ⫽ macroadenoma (greatest diameter over 1 cm), Micro ⫽ microadenoma (greatest diameter less than 1 cm), NA ⫽ not available, PRL ⫽ prolactin. *Cure was defined as postoperative GH levels ⬍1 ␮g/l, and normalization of the plasma IGF-I levels (IGF-I levels from all patients preoperatively were higher than the upper normal for the assay used and for the respective age group) and GH responses to oral glucose tolerance test (data not shown). Data are presented in SI units (1 nmol/l ⫽ 7.649 ng/ml for IGF-I). **In these cases, more than one tumor was seen, in addition to the adenoma detected originally by magnetic resonance imaging.

Follow-up (pre-op)

Patient

Table 2. Clinical course and biochemical, histologic and genetic analysis of pituitary adenomas from patients with CNC and acromegaly

a

b

c Fig. 2. Immunohistochemistry in pituitary tumors from patients with CNC and acromegaly. Figures 2a–c are from case 1 (table 2): (a) abnormally expanded and irregular reticulin pattern, consistent with adenohypophysial cell hyperplasia (⫻100); (b) expanded reticulin pattern with beginning disruption (left-to-the-center and center) consistent with adenoma development (center) within hyperplasia (right) (⫻100); (c) disrupted reticulin pattern in the adenomatous tissue from the same patient (⫻40).

Electron Microscopy Studies We recently described the electron microscopy findings of two GH-producing tumors from patients with CNC [26]. The tumors consisted of large, closely apposed, slightly irregular cells. The nuclei were ovoid or variably irregular and possessed a well to extensively developed nucleolus as well as small quantities of stippled heterochromatin. Rough endoplasmic reticulum was abundant and organized in parallel arrays or well-developed and randomly disposed short profiles. Golgi complexes were conspicuous, occupied a large portion of the cytoplasm and contained spherical or irregular, fused, immature secretory granules. Outside the Golgi zone, secretory granules were scant and measured up to 350 nm in diameter, most ranging between 200 and 250 nm. Extrusion of secretory granules was occasionally seen. Cell membranes between neighboring cells often showed complex interdigitation. Cytoplasmic

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fibrous bodies were not identified. Very few adenoma cells (1%) were densely granulated, possessed well-developed rough endoplasmic reticulum and very prominent Golgi complexes. Their secretory granules measured up to 500 nm in diameter, most ranging between 350 and 400 nm. Granule extrusion was seen. Based on immunoelectron microscopy in 2 cases, the tumor cells were considered mammosomatotrophs. In the first tumor [26], many of the tumor cells had lactotrophic characteristics, but secretory granules in their Golgi areas labeled for both PRL and GH. The large secretory granules of the densely granulated cell component were either bi-hormonal or mono-hormonal, labeling either for GH and/or PRL. Thus, the immunoreactivities in this case were not necessarily true to the ultrastructural phenotype. In the second tumor, both non-tumoral adenohypophysis and adenoma were seen [26]. The latter featured large irregular cells with eccentric, sometimes markedly irregular nuclei of which containing moderately developed nucleoli and small amounts of stippled heterochromatin. Rough endoplasmic reticulum was poorly to moderately developed and Golgi complexes were inconspicuous. Secretory granules were small (⬍250 nm) and sparse in some adenoma cells, numerous and measured up to 450 nm in others. Several tumor cells contained a prominent fibrous body. The non-tumoral adenohypophysis was normal. Immunogold labeling for only GH was noted in this tumor [26]. Genetic Analysis of CNC Pituitary Tumors Comparative genomic hybridization (CGH) analysis of three tumors (cases 2, 3, 8) showed no significant changes over normal DNA [25]. In contrast, analysis of the most aggressive tumor, an invasive macroadenoma (case 1), showed multiple changes, including losses of chromosomal regions 6q, 7q, 11p, 11q, and gains of 1pter-p32, 2q35-qter, 9q33-qter, 12q24-qter, 16, 17, 19p, 20p, 20q, 22p, 22q [25]. The greatest contiguous changes were losses of the long arm of chromosome 6 and the entire chromosome 11. The CNC tumors studied to date from patients with inactivating PRKAR1A mutations have demonstrated LOH for the 17q22–24 PRKAR1A locus [19]. There have been no studies of pituitary tissue from patients with CNC that appear to have other genetic defects.

Overview of Pituitary Tumors in CNC

Acromegaly is usually characterized by a slow, progressive course. In CNC this course is even slower and acromegaly is often unmasked by adrenalectomy for primary pigmented nodular adrenocortical disease (PPNAD) [27]. In our

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prospective evaluation of patients with CNC, we have had the opportunity to observe the ‘development’ of pituitary adenomas in 4 of the patients presented in this report (cases 2–4, 8) (fig. 1). In these 4 cases, biochemical abnormalities of GH and PRL secretion were present in advance of radiological detection of the tumor [9, 10]. The incidence of GH-producing pituitary tumors in CNC has been estimated at less than 15% [1, 6, 11], whereas GH ‘paradoxical’ responses to various stimuli (such as to thyrotropin-releasing hormone (TRH)) or IGF-I elevation, without a detectable tumor, may be present in up to 80% of affected patients [25]. These clinical observations are likely a reflection of probable hyperplasia found in pituitary tissue from patients with CNC. The GH-secreting adenoma in 5 of 8 of these patients appeared to be surrounded by regions with expanded irregular reticulin structure, and GH-, PRL-, and occasionally ␣-subunit-immunoreactive cells. These areas were shown to be identical by staining consecutive slides. It is noteworthy that in all patients, multiple tumors were seen; the surface of the gland was covered with macroscopic tumors in at least 4 patients (patients 2, 4, 7, 8; table 2). In most of these cases, multiple additional tumors that were identified microscopically, also in addition to hyperplasia. It should be noted that PRL staining was not present in all the GH-stained areas; accordingly, PRL levels in the peripheral blood were not markedly elevated in most patients with CNC (table 2), in contrast to GH or IGF-I levels in the same patients. Electron microscopy studies showed significant differences between the two CNC-related adenomas [26], suggesting that not all GH-producing adenomas are the same and these lesions are potentially polymorphic. The genetic investigation complemented the above findings by suggesting that in its evolution, the largest and most aggressive CNC-associated tumor [25] had accumulated a series of genetic changes; in contrast, the small adenomas had normal CGH results. These findings are in agreement with the hypothesis that pituitary tumors develop from clonal expansion of transformed somatic cells [28, 29]. They are also consistent with observations in patients with MAS [14–16] and some patients with MEN 1 [30]. CNC and MAS are genetic conditions that share skin pigmentation abnormalities, adrenocortical hyperplasia, thyroid tumors and even myxomas. However, in most tissues, the lesions are histologically and clinically different in the two conditions: skin lentigines and blue nevi versus café-au-lait spots, micronodular and pigmented dysplasia versus adrenocortical macronodular hyperplasia, hormonally silent nodules or cancer versus thyroid hormone hypersecretion, and skin versus intramuscular myxomas [1, 6, 14, 31, 32]. Mammosomatotroph hyperplasia may be the only lesion that is in fact common to CNC and MAS [33]. Clinically, too, both share a ‘pre-acromegalic’ state [34], which only rarely leads to the detection of an

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adenoma [10, 14–16, 34]. Similar, long-standing somatotroph hyperplasia, which only occasionally leads to pituitary adenoma, has been seen in several other patients, albeit GHRH-induced [35, 36]. The genetic changes required for the formation of an adenoma in the background of benign hyperplasia are not known but appear to be multiple. As the present report has demonstrated, and other investigators have shown in tumors with Gs␣ mutations or allelic losses of the MEN 1 locus [37, 38], pituitary genetic changes tend to increase in number and significance in parallel with the clinical behavior of the neoplasm [28, 29]. Thus, pituitary tumorigenesis in CNC patients may follow the pattern of mutation accumulation that has been suggested for other neoplasms [39, 40]. The extensive genetic instability of cells cultured from CNC tumors suggests that secondary ‘hits’ underlie tumor formation in CNC, the first ‘hit’ being the germline PRKAR1A mutation, in patients that have a genetic defect. This corresponds to Knudson’s hypothesis [41].

Is PRKAR1A Mutated in Other Pituitary Tumors?

We speculated above that the germline CNC mutation causes a predisposition towards other molecular events that are necessary for pituitary tumor formation in CNC patients. Although the evidence suggests that this may be the case in CNC, it is not known whether PRKAR1A can cause sporadic pituitary tumors when mutated at the somatic level. Three recent studies [42–44] have demonstrated that PRKAR1A is an unlikely molecular etiology of non-familial pituitary tumors, not unlike the case of menin, the MEN 1 gene [45].

Conclusion

There is evidence that PRKAR1A-inactivating mutations lead to PKAinduced somatomammotroph hyperplasia in the pituitary tissue of CNC patients. Hyperplasia leads to additional genetic changes at the somatic level, which in turn cause the formation of GH-producing adenomas in some, but not all, patients. Molecular investigation of PRKAR1A’s effects on cellular signaling and the cell cycle, as well as mouse model studies, are expected to shed light on this gene’s role in pituitary tumorigenesis. Although epigenetic modification of its function is not unlikely, somatic PRKAR1A mutations are not frequent in sporadic pituitary tumors.

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Carney JA, Young WF: Primary pigmented nodular adrenocortical disease and its associated conditions. Endocrinologist 1992;2:6–21. Stratakis CA: The familial lentiginosis syndromes are emerging from the obscurity imposed by rarity: New genes and genetic loci for multiple tumors and developmental defects. Horm Metabol Res 1998;30:285–290. Carney JA: Carney complex: The complex of myxomas, spotty pigmentation, endocrine overactivity and schwannomas. Semin Dermatol 1995;14:90–98. Stratakis CA, Kirschner LS, Carney JA: Carney complex: Diagnosis and management of the complex of spotty skin pigmentation, myxomas, endocrine overactivity and schwannomas. Am J Med Genet 1998;80:183–185. Carney JA, Hruska LS, Beauchamp GD, Gordon H: Dominant inheritance of the complex of myxomas, spotty pigmentation and endocrine overactivity. Mayo Clin Proc 1986;61:165–172. Stratakis CA, Carney JA, Lin JP, Papanicolaou DA, Karl M, Kastner DL, et al: Carney complex, a familial multiple neoplasia and lentiginosis syndrome: Analysis of 11 kindreds and linkage to the short arm of chromosome 2. J Clin Invest 1996;97:699–705. Casey M, Mah C, Merliss AD, Kirschner LS, Taymans SE, Denio AE, et al: Identification of a novel genetic locus for familial cardiac myxomas and Carney complex. Circulation 1998;98:2560–2566. Stratakis CA, Kirschner LS, Taymans SE, Carney JA, Basson CT: Genetic heterogeneity in Carney complex (OMIM 160980): Contributions of loci at chromosomes 2 and 17 in its genetics. Am J Hum Genet 1999;65(suppl):A447. Stratakis CA, Kirschner LS, Papanicolaou DA, Sarlis NJ, Raff S, Veldhuis JD, et al: Familial acromegaly beyond MEN-1: Genetic and clinical studies in non-GHRH dependent somatomammotroph hyperplasia in patients with Carney complex or an inherited chromosome 11 inversion. 80th Annual Meeting of the Endocrine Society, New Orleans, La, June 1998, P3-552. Raff SB, Carney JA, Krugman D, Doppman JL, Stratakis CA: Prolactin abnormalities in patients with the syndrome of spotty skin pigmentation, myxomas, endocrine overactivity, and skin myxomas (Carney complex). J Pediatr Endocrinol Metab 2000;13:373–379. Watson JC, Stratakis CA, Bryant-Greenwood PK, Koch CA, Kirschner LS, et al: The neurosurgical implications of Carney complex. J Neurosurg 2000;92:413–418. Irvine AD, Armstrong DK, Bingham EA, Hadden DR, Nevin NC, Hughes AE: Evidence for a second genetic locus in Carney complex. Br J Dermatol 1998;139:572–576. Weinstein LS, Shenker A, Gejman PV, Merino MJ, Friedman E, Spiegel AM: Activating mutations of the stimulatory G protein in the McCune-Albright syndrome. N Engl J Med 1991;325:1688–1695. Gessl A, Freissmuth M, Czech T, Matula C, Hainfellner JA, Buchfelder M, et al: Growth hormoneprolactin-thyrotropin-secreting pituitary adenoma in atypical McCune-Albright syndrome with functionally normal Gs␣ protein. J Clin Endocrinol Metab 1994;79:1128–1134. Garcia MB, Koppeschaar HIP, Lips CJ, Thijsen JH, Krenning EP: Acromegaly and hyperprolactinemia in a patient with polyostotic fibrous dysplasia: Dynamic endocrine studies and treatment with the somatostatin analogue octreotide. J Endocrinol Invest 1994;17:59–65. Cuttler L, Jackson JA, Uz-Zafar S, Levitsky LL, Mellinger RC, Frohman LA: Hypersecretion of growth hormone and prolactin in McCune-Albright syndrome. J Clin Endocrinol Metab 1989;68: 1148–1154. Stratakis CA, Jenkins RB, Pras E, Mitsiadis CS, Raff SB, Stalboerger PG, et al: Cytogenetic and microsatellite alterations in tumors from patients with the syndrome of myxomas, spotty skin pigmentation and endocrine overactivity (Carney complex). J Clin Endocrinol Metab 1996;81: 3607–3614. DeMarco L, Stratakis CA, Boson WL, Yakbovitz O, Carson E, Adrade LM, et al: Sporadic cardiac myxomas and tumors from patients with Carney complex are not associated with activating mutations of the Gs␣ gene. Hum Genet 1996;98:185–188. Kirschner LS, Carney JA, Pack SD, Taymans SE, Giatzakis C, Cho YS, Cho-Chung YS, Stratakis CA: Mutations of the gene encoding the protein kinase A type I-␣ regulatory subunit in patients with the Carney complex. Nat Genet 2000;26:89–92.

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Kirschner LS, Sandrini F, Monbo J, Lin JP, Carney JA, Stratakis CA: Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in patients with the Carney complex. Hum Mol Genet 2000;9:3037–3046. Stratakis CA, Kirschner LS, Carney JA: Clinical and molecular features of the Carney complex: Diagnostic criteria and recommendations for patient evaluation. J Clin Endocrinol Metab 2001; 86:4041–4046. Stergiopoulos S, Stratakis CA: Human tumors associated with Carney complex and germline PRKAR1A mutations: A protein kinase A disease! FEBS Lett 2003;546:59–64. Groussin L, Kirschner LS, Vincent-Dejean C, Perlemoine K, Jullian E, Delemer B, Zacharieva S, Pignatelli D, Carney JA, Luton JP, Bertagna X, Stratakis CA, Bertherat J: Molecular analysis of the cyclic AMP-dependent protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene in patients with Carney complex and primary pigmented nodular adrenocortical disease (PPNAD) reveals novel mutations and clues for pathophysiology: Augmented PKA signaling is associated with adrenal tumorigenesis in PPNAD. Am J Hum Genet 2002;71:1433–1442. Cho-Chung Y: cAMP signaling in cancer genesis and treatment. Cancer Treat Res 2003;115:123-143. Pack SD, Kirschner LS, Pak E, Zhuang Z, Carney JA, Stratakis CA: Genetic and histologic studies of somatomammotropic pituitary tumors in patients with the ‘complex of spotty skin pigmentation, myxomas, endocrine overactivity and schwannomas’ (Carney complex). J Clin Endocrinol Metab 2000;85:3860–3865. Kurtkaya-Yapicier O, Scheithauer BW, Carney JA, Kovacs K, Horvath E, Stratakis CA, Vidal S, Vella A, Young WF Jr, Atkinson JL, Lloyd RV, Kontogeorgos G: Pituitary adenoma in Carney complex: An immunohistochemical, ultrastructural and immunoelectron microscopic study. Ultrastruct Pathol 2002;26:345–353. Ogo A, Haji M, Natori S, Kanzaki T, Kabayama Y, Osamura RY, et al: Acromegaly with hyperprolactinemia developed after bilateral adrenalectomy in a patient with Cushing’s syndrome due to adrenocortical nodular hyperplasia. Endocr J 1993;40:17–25. Asa SL, Ezzat S: The cytogenesis and pathogenesis of pituitary adenomas. Endocr Rev 1998;19: 798–827. Farrell WE, Clayton RN: Epigenetic change in pituitary tumorigenesis. Endocr Relat Cancer 2003;10:323–330. Shintani Y, Yoshimoto K, Horie H, Sano T, Kanesaki Y, Hosoi E, et al: Two different pituitary adenomas in a patient with multiple endocrine neoplasia type 1 associated with growth hormonereleasing hormone-producing pancreatic tumor: Clinical and genetic features. Endocr J 1995;42: 331–340. Stratakis CA, Courcoutsakis NA, Abati A, Filie A, Doppman JL, Carney JA, Shawker T: Thyroid gland abnormalities in patients with the ‘syndrome of spotty skin pigmentation, myxomas, and endocrine overactivity’. J Clin Endocrinol Metab 1997;82:2037–2043. Stratakis CA, Sarlis N, Kirschner LS, Carney JA, Doppman JL, Nieman LK, et al: Paradoxical response to dexamethasone in the diagnosis of primary pigmented nodular adrenocortical disease. Ann Intern Med 1999;131:585–591. Kovacs K, Horvath E, Thomer MO, Rogol AD: Mammosomatotroph hyperplasia associated with acromegaly and hyperprolactinemia in a patient with the McCune-Albright syndrome. Virchows Arch Pathol Anat 1984;403:77–86. Feuillan PP, Jones J, Ross JL: Growth hormone hypersecretion in a girl with McCune-Albright syndrome: Comparison with controls and response to a dose of long-acting somatostatin analog. J Clin Endocrinol Metab 1995;80:1357–1360. Sano T, Asa SL, Kovacs K: Growth hormone-releasing hormone-producing tumors: Clinical, biochemical and morphological manifestations. Endocr Rev 1988;9:357–373. Ezzat S, Asa SL, Stefaneanu L, Whittom R, Smyth HS, Horvath E, et al: Somatotroph hyperplasia without pituitary adenoma associated with a long-standing growth hormone-releasing hormone-producing bronchial carcinoid. J Clin Endocrinol Metab 1994;78:555–560. Thakker RV, Pook MA, Wooding C, Boscaro M, Scanarini M, Clayton RN: Association of somatotrophinomas with loss of alleles on chromosome 11 and with gsp mutations. J Clin Invest 1993; 91:2815–2821.

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Kytola S, Makinen MJ, Kahkonen M, Teh BT, Leisti J, Salmela P: Comparative genomic hybridization studies in tumours from a patient with multiple endocrine neoplasia type 1. Eur J Endocrinol 1998;139:202–206. Cho KR, Vogelstein B: Genetic alterations in the adenoma-carcinoma sequence. Cancer 1992;70: 1727–1731. Vogelstein B, Kinzler KW: The multistep nature of cancer. Trends Genet 1993;4:138–141. Knudson AG: Hereditary cancer: Two hits revisited. J Cancer Res Clin Oncol 1996;122:135–140. Sandrini F, Kirschner LS, Bei T, Farmakidis C, Yasufuku-Takano J, Takano K, Prezant TR, Marx SJ, Farrell WE, Clayton RN, Groussin L, Bertherat J, Stratakis CA: PRKAR1A, one of the Carney complex genes, and its locus (17q22–24) are rarely altered in pituitary tumours outside the Carney complex. J Med Genet 2002;39:e78. Kaltsas GA, Kola B, Borboli N, Morris DG, Gueorguiev M, Swords FM, Czirjak S, Kirschner LS, Stratakis CA, Korbonits M, Grossman AB: Sequence analysis of the PRKAR1A gene in sporadic somatotroph and other pituitary tumours. Clin Endocrinol (Oxf) 2002;57:443–448. Yamasaki H, Mizusawa N, Nagahiro S, Yamada S, Sano T, Itakura M, Yoshimoto K: GH-secreting pituitary adenomas infrequently contain inactivating mutations of PRKAR1A and LOH of 17q23–24. Clin Endocrinol (Oxf) 2003;58:464–470. Asa SL, Somers K, Ezzat S: The MEN-1 gene is rarely down-regulated in pituitary adenomas. J Clin Endocrinol Metab 1998;83:3210–3212.

Constantine A. Stratakis, MD, DSc Section on Endocrinology and Genetics, DEB, NICHD, NIH Building 10, Room 10N262, 10 Center Drive, MSC 1862 Bethesda, MD 20892-1862 (USA) Tel. ⫹1 301 4964686/4021998, Fax ⫹1 301 4020574, E-Mail [email protected]

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Recent Advances in MEN1 Gene Study for Pituitary Tumor Pathogenesis Toru Kameyaa, Toshihiko Tsukadab, Ken Yamaguchia a

Pathology Division, Shizuoka Cancer Center Hospital and Research Institute, Shizuoka, and bTumor Endocrinology Project, National Cancer Center Research Institute, Tokyo, Japan

Abstract Evidence of multiple endocrine neoplasia type 1 (MEN1) is found in approximately 2.7% of patients with pituitary adenomas. The multicentricity of pituitary adenomas has not yet been proved. Prolactinomas are most frequent in MEN1 pituitary tumors. Pituitary tumors with MEN1 are larger in size and more aggressive than without MEN1. Heterozygous germline mutations of MEN1 gene are responsible for MEN1 disorders. Various types of mutations likely causing loss of the gene function have been identified throughout the entire region in patients with MEN1 and related disorders. However, the function of menin, the product of MEN1 gene, remains to be established. Neither mutation hot spot nor phenotype-genotype correlation has been established in classical MEN1. A number of recent studies suggest that somatic mutations in the MEN1 gene do not play prominent role in the pathogenesis of sporadic forms of pituitary adenoma. Copyright © 2004 S. Karger AG, Basel

Introduction

The knowledge on pituitary adenomas of hereditary MEN1 (multiple endocrine neoplasia type 1) cases and in sporadic cases is accumulating in relation to MEN1 gene [1–13]. For the past 20 years or so, the multiple endocrine neoplasia (MEN) syndromes have attracted the interest of internists, endocrine surgeons and pathologists, reflecting both the uniqueness of their clinical and pathologic presentations and the new discovery of important cellular mechanism responsible for growth, development, and function of several lineages of hormone-secreting cells.

Reports dealing with these syndromes date back to the beginning of the 20th century, Erdheim [14], in 1903, was the first to bring multiple endocrine hypersecretory states together into a single syndrome. He recognized a patient with a pituitary eosinophilic adenoma and parathyroid gland hyperplasia. In 1954, Wermer [15] identified a group on individuals who presented with adenoma of different endocrine glands in a distribution that would be at present defined as MEN1. Importantly, he recognized that the syndrome was heritable and appeared to be transmitted in an autosomal-dominant pattern. Recent developments, in particular those related to the molecular genetics of the syndromes, have given numerous important insights into their underlying pathogenesis. The present chapter will give an overview of pathologic findings of pituitary lesions associated with the syndrome and of difference from those unassociated with the syndrome, and then focus on more recent advances made in identification and analyses of the gene (MEN1) or putative genes that are responsible for their phenotypic manifestations and pituitary tumorigenesis.

Overview of Pituitary Lesions in the MEN1 [16, 17]

The term multiple endocrine neoplasia – MEN – encompasses genetically determined disorders with predisposition to hyperplastic and neoplastic lesions in two or more endocrine organs of the same patients. MEN1 is a disease with an estimated prevalence between 0.02 and 0.2 per 1,000. It affects both sexes equally and has no geographic, racial or ethnic preferences, although there is a common ‘founder’ (MEN1) effect in some districts of the world. The disorder may either be inherited as an autosomal-dominant trait or may occur sporadically as a result of new mutations. It has a high degree of penetrance. Forty-three percent of patients who are carriers of the disease gene have clinical evidence of MEN1 by the age of 20 years, 85% by the age of 36 years, and 94% by the age of 50 years [19]. The endocrine tumors characterizing MEN1 develop synchronously or metachronously. They involve the parathyroid, pancreas, duodenum and anterior pituitary gland. Less commonly, they involve the thymus, lung, and other gastrointestinal tracts. Furthermore, adrenocortical, lipomatous, skin lesions (angiofibroma, collagenoma, etc.), and spinal ependymomas may also occur. Clinical symptoms are most commonly caused by the lesions of the parathyroid (80–98%), the pancreas and duodenum (40–85%), and the anterior pituitary (9–40%) [20]. In some patients, the clinical presentation is dominated by one manifestation. Evidence of MEN1 is found in approximately 18% of unselected patients with primary hyperparathyroidism, 15–40% of patients with Zollinger-Ellison syndromes, 4% of patients with insulinomas, 1.5% of

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patients with gastrointestinal neuroendocrine (carcinoid) tumors, and 2.7% of patients with pituitary adenomas. Therefore, the distinction between patients with familial and sporadically occurring tumors may be difficult. Approximately 50% of patients with MEN1 die of their disease at a mean age of 51 years and neoplasia rather than peptic ulcer disease is the main cause of death [21].

Pathology of Pituitary Lesions in MEN1 Patients

The presentation of pituitary adenomas, which are commonly said multicentric, but not yet proved from a large series of collected data, has been reported in up to two thirds of patients with MEN1 in combined surgical and autopsy studies [16, 22–24]. Immunocytochemically, most tumors produce prolactin (PRL) and/or growth hormone (GH) and only occasionally stain for adrenocorticotrophic hormone (ACTH) [25, 26]. Diffuse hyperplasia of one of the cell types has not yet been reported. Amenorrhea is the most frequent manifestation of pituitary lesions in women who are carriers of MEN1 disease gene. Local symptoms, for example visual disturbance, hypopituitarism, Cushing disease, acromegaly, or hyperprolactinemia with impotence in men, are less frequently encountered [25, 27]. Trump et al. [28] reported a 30% frequency of pituitary lesions in a study of 709 individuals from 62 MEN1 families. The considerably lower rate of null-cell adenomas among hereditary patients as compared to sporadic series (7% versus up to nearly 30%) can probably be explained by the lower average age of MEN1 patients [22]. A recent paper by Vergés et al. [12] reported the incidence of each type of pituitary adenomas in a total of 136 MEN1 cases from a registry of the Groupe d’Étude des Neoplasies Endocriennes Multiples (GENEM): PRL, 85 (62%); GH, 12 (9%); ACTH, 6 (4%); cosecretor, 13 (10%), and non-secretor, 20 (15%). Prolactinomas are most likely prevalent in MEN1 cases. An interesting finding of pituitary adenomas of MEN1 patients at Mayo Clinic is the high frequency of multihormonality among MEN1 GH-producing tumors (9%) [22]. Details of the pituitary lesions of MEN1 patients can be obtained only by autopsy cases in which pituitary glands could intensively be investigated. Such situations are rare [24] and pituitary tumors usually do not allow a meaningful comparison for multiplicity and are especially rare in MEN1. Figure 1 is a whole profile of pituitary lesions of our rare autopsy MEN1 case (contributed by Dr. N. Kimura, Pathology Division, Tohoku Rosai Hospital, Sendai, Japan). This MEN1 patient, a male of 15 years of age, presented a clinical symptom of hypoglycemic attacks with psychosis. The blood glucose levels frequently fell

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G

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Fig. 1. Pituitary lesions in a case of sporadic MEN1. A, PRL⫹⫹⫹; TSH⫹, FSH⫹; B, PRL⫹⫹⫹, FSH⫹, LH⫹; C–F, PRL cell proliferation in normal background; G, ACTH cell aggregate.

down to 27 mg/dl, in spite of normo- or mild hyperglycemia states, with plasma insulin levels of 30–50 ␮U/ml. An insulinoma was suspected. Intravenous hyperalimention was required and the patient was deceased after several seizures and unconsciousness. In addition to pituitary lesions to be described later, autopsy revealed an insulinoma measuring 2 cm and several microadenomas in the pancreas and parathyroid hyperplasia of all 4 glands. The family history was not recorded for MEN1. In this case, the lesions are multiple, showing 7 nodular lesions, the largest measuring 5 mm in maximal dimensions. Hormone production by each nodule is shown in figure 1. Six nodules were PRL-producing, and the other one was a small aggregate of ACTH cells. Dr. K. Kovacs [pers. commun.] agreed with us that the largest nodule represented an adenoma immunoreactive for PRL and that other 5 nodules were multiple focal hyperplasia of PRL cells. Multifocal adenomatous or hyperplastic nodules theoretically arise in MEN1 anterior pituitaries, although this was not confirmed in most MEN1 cases, while multiplicity and bilaterality of proliferating nodular lesions have been proved in parathyroids and pancreases of numerous MEN1 patients. Capella et al. [24] reviewed the knowledge on pathology of proliferate lesions of the pituitary gland in MEN1. When compared with sporadic tumors occurring in general population, adenomas with MEN1 were more often functioning, they were more often PRL-producing and they were more frequently plurihormonal. The study of their personal 2 autopsy cases disclosed that pituitary PRL or mixed GH-PRL cell adenomas in MEN1 were multiple and were associated with both diffuse and nodular hyperplasia of PRL and GH cells. In these cases, the nodular hyperplastic foci may represent the starting points of inherited tumorigenesis.

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To date, the largest systematic study correlating endocrinologic, surgical and pathologic data of 40 pituitary adenomas of MEN1 patients were reported by Scheithauer et al. [22]. These cases represented 2.9% of an unselected series of 1,500 pituitary tumors surgically resected at Mayo Clinic. Multiple adenomas were reported in only 1 patient. Recently, a prospective clinical database of 660 pituitary surgeries at NIH was analyzed to assess the incidence of multiple pituitary adenomas in Cushing’s disease [26]. Thirteen patients with at least two separate histopathologically confirmed pituitary adenomas were identified. Among the 13 cases, 2 were from MEN1 patients; one pituitary gland had 5 mm ACTHoma and 3 mm PRL and GH cosecretors, and another one 20 mm TSHoma and 4 mm PRLomas. Kontogeorgos et al. [29] reviewed materials from about 9,470 autopsy cases in which they obtained 20 pituitary glands containing multiple adenomas. However, they did not mention any MEN1 case in their material. The most recent report on the largest study of data of pituitary lesions in MEN1 patients has been made by GENEM [12]. Their work included patients from 22 different multidisciplinary centers from the GENEM in France and Belgium. Prevalence of pituitary disease among their MEN1 patients was 42% and equivalent or higher than frequencies mostly reported by other authors [28, 30–32]. Most interesting, convincing but unexpected data of this paper were that pituitary adenomas were characterized by a larger size and a more aggressive presentation than without MEN1. The frequency of macroadenomas was significantly higher in MEN1 patients than in non-MEN1 subjects (85 vs. 42%, p ⬍ 0.001). Clinical manifestations related to the size of the pituitary adenomas were significantly more frequent in MEN1 patients than in non-MEN1 subjects (29 vs. 14%, p ⬍ 0.01). As far as the secreting adenomas were concerned, normalization of pituitary hypersecretion was much less frequent in MEN1 patients than in non-MEN1 subjects (42 vs. 90%, p ⬍ 0.01), and normalization of plasma PRL level was significantly less frequent in MEN1 patients than in non-MEN1 subjects (44 vs. 90%, p ⬍ 0.001). However, the age at onset of prolactinoma is indistinguishable between MEN1 and non-MEN1 pituitary adenomas [12, 21], while the peak age at onset of expression of endocrine tumor in patients with MEN1 was about 20 years earlier than the age in most of non-MEN1 tumors in patients with hyperparathyroidism, gastrinoma, or insulinoma [33–35].

Genetics and Molecular Pathology of MEN1

The responsible gene for MEN1, termed MEN1, had been localized to the long arm of chromosome 11 by linkage analysis and deletion mapping [36–39]

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and in 1997 identified by positional cloning [40, 41]. The gene encodes 610 amino acid protein, referred to menin, which has no apparent structural homology with previously identified proteins. Since loss of heterozygosity of the MEN1 locus has frequently been observed in tumors associated with MEN1 as well as in sporadic parathyroid and pancreatic tumors [36–39, 42, 43], the MEN1 gene has been considered to be a tumor suppressor gene. Numerous heterozygous germline mutations of the MEN1 gene, which likely cause loss of the gene function, have been identified in the majority of MEN1 patients [40, 44–80], as recently reviewed in several articles [2, 11, 64, 65]. In addition to germline mutations, somatic loss of the normal MEN1 allele has almost constantly occurred in the tumors associated with MEN1, in agreement with the Knudson’s two-hit model for tumor development [11, 81]. Somatic inactivation of both MEN1 alleles is also observed in a considerable fraction of sporadic MEN1-related tumors [53, 56, 60, 73], confirming the involvement of the gene in this development of a subset of sporadic tumors. The Knudson’s model was originally based on epidemiologic observations about retinoblastoma, both its early age at onset and its bilaterality in hereditary cases. The two-hit model has been developed to propose that a tumor can arise after two separate mutations of a critical gene have occurred in one cell [11]. For tumorigenesis by a tumor suppressor like the MEN1 gene, the two hits should be inactivating mutations of both MEN1 alleles, thereby removing all inhibitions of cell accumulation. In a hereditary disease like MEN1 syndrome, the first hit is inherited in each cell of the body through germline transmission of an inactivating mutation in one copy of the MEN1 gene. Thereafter, the second hit must occur at the remaining normal copy of the MEN1 gene in one of many pituitary cells in MEN1 syndrome; then the cell can begin expansion into a clonal tumor. After a first mutation, a second mutation occur relatively early, causing tumor onset relatively early and occur, by chance, in more than susceptible cell, causing tumor multiplicity. The nonhereditary (sporadic) tumor of the same pituitary gland may begin after one susceptible cell has had postnatally two separate mutations, inactivating both copies of the same gene. This situation may explain why the sporadic tumor usually begins later and solitarily.

Structure of the MEN1 Gene

The human MEN1 gene is comprised of ten exons distributed over 7 kilobases (kb) in the chromosome region 11q13 and encodes mRNA of approximately 2.8 kb, which is transcribed in the direction from telomere to centromere (fig. 2) [40, 41, 82]. Exons 2 through 10 encode a 610-amino-acid protein termed menin. Surrounding the gene, there are several DNA segments

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⫺COOH

Fig. 2. Chromosomal localization and structural organization of the human MEN1 gene. a Chromosome 11. b An approximately 300-kb genomic region containing the MEN1 gene (closed box) and microsatellites (open bar) polymorphic DNA markers. Cen, centromere; Tel, telomere. c MEN1 gene. Closed and open boxes indicate protein-coding and non-coding regions of exons, respectively. d Menin mRNA. Closed and open boxes indicate translated and untranslated regions of the mRNA, respectively. e Menin. Shaded boxes indicate nuclear localization signals [82].

containing short repeat sequences, called microsatellites, which are highly polymorphic and have been utilized as DNA markers for genetic mapping of the MEN1 locus [36–39] and DNA tests for MEN1 predisposition [83]. Menin mRNA is expressed in all tissues and cell lines examined, but the expression levels are not uniform among different tissues [84–89]. The expression

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is high throughout the early embryo and in several adult tissues including central nervous system, thymus, testis and placenta. Menin homologs have been identified in other species including the mouse [84, 86–88], rat [86, 87], zebrafish [90, 91], and even fruit fly, Drosophila melanogaster [92], and snail [95] but not in the fission yeast Saccharomyces cerevisiae [93] and nematode Caenorhabditis elegans [93]. Ubiquitous expression in diverse tissues and the presence in a wide range of species suggests that menin is involved in fundamental biological processes. Alternatively spliced menin mRNA in its 5⬘-untranslated region, which also encodes the authentic menin, has recently been identified in humans [95] and other species [84, 86–88].

Function of the MEN1 Gene and Its Putative Relation to Tumorigenesis

Function of the MEN1 Gene The MEN1 gene [1, 2] is a tumor suppressor gene and at least one copy of the normal gene is required to prevent tumor development. Various heterozygous germline mutations of the MEN1 gene, which cause loss of the gene function, have been identified in MEN1 patients. Menin has amino acid sequences necessary for nuclear localization at its carboxy-terminal region, and is mainly localized to the nucleus [82, 96], implying a possible role as a nuclear protein including those in transcriptional regulation, DNA replication, cell division and DNA repair. However, several studies also demonstrated menin in cytoplasmic and membrane fractions [13, 82, 85, 96–98]. Thus, an extranuclear role for menin cannot be excluded. Menin has been shown to interact with proteins including JunD [99–101], NF-␬B [102], Smad3 [103], Nm23-H1 [104, 105], Pem [97], glial fibrillary acidic protein (GFAP) [98], vimentin [98], and the 32-kDa subunit of replication protein A [106]. JunD transcription factor is the first that has been demonstrated to interact directly with menin [99]. Menin has been shown to repress JunD-activated transcription through a histone deacetylasedependent mechanism [100]. However, the pathway from this repression to tumor suppression by menin seems to be complex because JunD-mediated transactivation antagonizes cell growth and transformation under certain conditions. Recently, menin has been shown to uncouple Elk-1, JunD and c-Jun phosphorylation from MAP kinase activation and repress c-fos promoter activity [107]. These findings suggest a potential mechanism of tumor suppression by menin, in which menin suppresses the signal output of MAP kinase cascades.

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Menin has also been shown to interact directly with three NF-␬B family members, p65, p52 and p50, and repress NF-␬B-mediated transcription [102]. NF-␬B complexes are regulators of cellular response to various types of stimulation including mitogens, cytokines, growth factors and ionizing radiation, and considered to be a positive mediator of cell growth. Thus, repression of NF-␬B-mediated transcription by menin seems to be relevant to tumor suppressor activity of menin. It is noteworthy that NF-␬B and JunD interact directly and activate transcription cooperatively in certain cells [108, 109]. In contrast to the repression of JunD- and NF-␬B-mediated transcription, menin seems to enhance Smad3-induced transcription [103]. Antisense menin RNA has been shown to antagonize Smad3-induced and TGF-␤-induced transactivation and TGF-␤-mediated inhibition of cell proliferation. Because disruption of TGF-␤ signaling by inactivating mutations of Smad2 and Smad4 has been observed in cancers [110], it is plausible that menin plays a role in the TGF-␤ signaling pathways and regulates cell growth. TGF-␤ has been shown to induce AP-1 complex formation in certain cells, and the primary components of this AP-1 complex are JunD and Fra2 [110]. Menin may be involved in both Smad3- and JunD-dependent TGF-␤ signaling pathways. Recently, menin has been implicated in osteoblastic differentiation of multipotential mesenchymal cells induced by bone morphogenetic protein 2 (BMP-2) [111]. Inactivation of menin by the treatment with menin antisense oligonucleotides suppressed BMP-2-induced alkaline phosphatase activity and the expression of type I collagen, Runx2/cbfa1 and osteocalcin. Menin has also been shown to interact physically and functionally with Smad1 and Smad5. This interaction may be important for the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage. Since menin antisense oligonucleotide also antagonized TGF-␤-induced transcription in the same stem cells, it appears to be important for Smad3-mediated TGF-␤ signaling in this cell line. However, because TGF-␤ does not induce alkaline phosphatase activity in these cells, the interaction of menin and Smad3 may not be essential for the commitment of the cells to osteoblastic lineage. Menin has been shown to interact directly with the homeobox-containing protein Pem [97], which is implicated in the regulation of androgen-dependent genes in the male reproductive system, specifically in regulating the transition between undifferentiated and differentiated cells of the early mouse embryo and in sperm development. Menin has also been implicated in synapse formation between neurons [94]. Thus, the physiological roles of menin in specific cell types and developmental stages appear to be different. Rat nm23␤, the counterpart of the human Nm23-H1, interacts directly with menin [104]. nm23 is an enzyme with nucleoside diphosphate kinase activity mainly localized in the cytoplasm. It has also been implicated as a transcription

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factor and long been known as a tumor metastasis suppressor although its role in transcription regulation and tumor metastasis has not been well established. The finding that menin exhibits GTP-hydrolyzing activity in the presence of nm23␤ [105], the sole biochemical activity so far demonstrated for menin in vitro, suggests that menin is a member of atypical GTPases. Because there are several members of the GTPase superfamily whose loss is implicated in tumor development, it is possible that the GTPase activity of menin is essential for tumor suppression. Although nm23␤ is principally a cytoplasmic protein in contrast to menin, which is localized mainly in the nucleus, the recent findings that the type III intermediate filament proteins GFAP and vimentin interact directly with menin in the cytoplasm [98] and that nm23 is associated with GFAP in rat glioma cells [112] raise the possibility that menin may interact with nm23 associated with GFAP/vimentin. Menin has been shown to be colocalized with GFAP/vimentin in the cytoplasm of glioma cells during S phase and early G2 phase of the cell cycle, implying a possible role of menin in cell cycle regulation [98]. Menin may have an inhibitory role before the start of S phase and might be transferred to the cytoplasm to permit S phase to proceed. Very recently, menin has been demonstrated to interact directly with the 32-kDa subunit of replication protein A [106], a heterotrimeric protein essential for DNA replication, recombination, and repair, and also implicated in the regulation of apoptosis and gene expression. It has long been suspected that menin may have a role in DNA repair or maintenance of genome stability. Elevated frequencies of spontaneous or induced chromosomal alterations have been noticed repeatedly in normal blood leukocytes from patients affected with MEN1 [113–115], which have only one normal allele of the MEN1 gene. These observations might reflect genome instability caused by slightly less menin in the normal cells of the patients although it has been demonstrated that the amount of menin in the non-neoplastic cells is almost the same among MEN1 patients and normal subjects [89]. The evidence for involvement of menin in DNA repair and stability is thus still fragmentary, and biological significance of the interaction between replication protein A and menin remains to be determined. The molecular function of menin has been investigated mainly by exploring interacting proteins, which might provide clues for the menin function [116]. The identified proteins are, however, mostly common factors expressed in many different cell types and do not explain by themselves the development of specific endocrine tumors frequently occurring in MEN1. Several different roles including cell cycle control, cell lineage determination, maintenance of genome stability, have been postulated based on a few clues so far obtained, but none has been established. Further studies are required to elucidate the physiological function of menin and how menin and interacting proteins are involved in the endocrine and pituitary tumorigenesis and its suppression.

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MEN1 and Related Disorders

MEN1 and Its Variants A subset of MEN1 with a usually high frequency of prolactinoma and a low frequency of enteropancreatic tumor is called the prolactinoma variant of MEN1 [44, 118]. This implies as described earlier that there is a common ‘founder’ effect in some districts of the world [18]. Familial MEN1 is usually defined as the state in which there is an individual fulfilling the criteria for MEN1 and at least one first-degree relative with one feature of MEN1. MEN1 without a family history of MEN1-related tumor is defined as sporadic MEN1. The clinical diagnosis of definitive sporadic MEN1 requires exclusion of familial MEN1 by thorough examination of family members including both patterns. The true sporadic MEN1 should be associated with de novo MEN1 mutation if it is considered to have occurred during the germ cell formation in the patient’s parent [45, 47].

Familial Isolated Somatotropinomas

Familial isolated pituitary tumor is a distinct disease entity characterized by hereditary occurrence of pituitary tumors, mostly GH-producing adenomas, without other endocrine and constitutional disorders [119–122]. All pituitary GH tumors of the familial disease exhibited a loss of heterozygosity at polymorphic microsatellite markers closely linked to the MEN1 tumor suppressor gene. Sequencing of the MEN1 gene revealed no germline mutations, nor was a somatic mutation found in tumor DNA from one subject in one of two families. These data indicated that loss of heterozygosity in the affected family members appeared independent of MEN1 gene change and suggested that a novel tumor suppressor gene(s) linked to the marker and expressed in the pituitary is essential for regulation of somatotrope proliferation [123]. A newer report established linkage of the tumor suppressor gene involved in the pathogenesis of the familial disease to chromosome 11q13.1–13.3 and identified a potential second locus at chromosome 2p16–12 [123].

Germline Mutations of the MEN1 Gene

Detection Rate of Mutation Heterozygous germline mutations of the MEN1 gene have been identified in 50–100% of familial MEN1 and in a smaller fraction of sporadic MEN1

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Table 1. Detection rate of germline MEN1 gene mutations in familial and sporadic MEN1 Detection ratea (%)

Method of mutation screening

familial

sporadic

47/50 (94) 9/10 (90)

8/11 (72)

4/5 (80) 43/57 (75) 47/54b (87) 8/9 (89) 9/12 (75) 4/4 (100) 27/55 (49) 11/14 (79) 4/5 (80) 8/8b (100) 9/10 (90) 25/33 (76) 16/16 (100) 11/12 (92) 15/27 (56) 165/170 (97)

4/6 (67) 9/11b (82)

4/4 (100) 9/13 (69) 1/2 (50) 0/7 (0) 6/15 (40)

8/20 (40) 1/8 (13) 4/5 (80)

Dideoxy fingerprinting SEQ SEQ SSCP Heteroduplex analysis/SEQc SEQ SSCP/SEQc SSCP/SEQc SSCP SEQ SEQ SEQ SEQ SSCP SEQ SEQ SEQ DGGE/SEQc Heteroduplex analysis/SEQc

Reference (first author)

Agarwal, 1997 [45] European Consortium for MEN1, 1997 [41] Shimizu, 1997 [74] Bassett, 1998 [47] Giraud, 1998 [54] Mayer, 1998 [67] Bartsch, 1998 [46] Tanaka, 1998 [76] Teh, 1998 [77] Sakurai, 1998 [71] Sato, 1998 [72] Dackiw, 1999 [51] Poncin, 1999 [70] Mutch, 1999 [69] Hai, 1999 [57] Hai, 2000 [58] Bergman, 2000 [48] Morelli, 2000 [68] Wautot, 2002 [13]

DGGE ⫽ Denaturing gradient gel electrophoresis; SEQ ⫽ nucleotide sequencing; SSCP ⫽ singlestrand conformation polymorphism analysis. aNumber of mutation-positive families (familial MEN1) or patients (sporadic MEN1)/number of families or patients examined. bAtypical MEN1 was excluded. cMutation-negative cases in the rapid screening were examined by nucleotide sequencing.

patients (table 1). These incomplete detections obviously result from unsatisfactory methods of mutation analysis as well as heterogeneity of diseases diagnosed as MEN1. Mutation screening by rapid methods may miss some mutations that could be identified by direct sequencing. Direct sequencing should be conducted in the cases that exhibit no mutation in the screening by the rapid methods. More importantly, only limited regions of the gene, usually protein coding exons and its short flanking sequences, have usually been analyzed. Therefore, mutations in other regions that may affect the gene transcription or mRNA splicing would be missed. Furthermore, even if a nucleotide

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change is identified in these regions, it is difficult to distinguish a causative mutation from a benign polymorphism because biological significance of the nucleotide alteration is not obvious. Also, large deletions would escape detection by the standard analysis methods [48, 62]. A kindred with atypical MEN1 demonstrating frequent expression of pituitary tumors and a low penetrance of primary hyperparathyroidism in contrast to classical MEN1 has been shown not to linked to the MEN1 locus, based on haplotype analysis, although the predisposing genetic defect in typical MEN1 families has previously been mapped to chromosome location 11q13 without evidence of heterogeneity among the 87 families analyzed by 1997 [124]. This indicates that one variant of MEN1 is a disease entity distinct from classical MEN1. The detection rates in sporadic MEN1 are generally lower than those in familial MEN1 (table 1) [45, 47, 48, 51, 54, 58, 68, 70, 77], probably reflecting higher incidence of phenocopy in the cases diagnosed as sporadic MEN1. A large portion of mutation-negative sporadic MEN1 cases were diagnosed on the concurrence of pituitary tumor and hyperparathyroidism and devoid of enteropancreatic lesions [45, 48, 51, 58, 70]. In contrast, most of the mutationpositive sporadic MEN1 cases had endocrine-pancreatic endocrine tumors [45, 48, 54, 58, 68, 70, 76, 77]. Thus, pituitary and parathyroid tumor appears to be less specifically associated with germline mutation of the MEN1 gene than enteropancreatic endocrine tumor. As mentioned before, familial isolated pituitary tumor has not been associated with MEN1 gene mutations [48, 67, 70, 76, 77, 120–123, 126]. Distribution of Mutation Germline mutations of the MEN1 gene so far identified in MEN1 and related disorders are summarized in figure 3. Mutations are scattered throughout the entire protein coding exons and their splicing junctions. No clear hot spot of mutation has been identified, but some mutations have been repeatedly encountered in apparently unrelated pedigrees. Some of them appear to be founder mutations originating from the common ancestors as evidenced by common chromosome haplotype [45, 54, 69, 77, 127, 128]. However, patients of different ethnic groups often share identical mutations at these ‘warm spots’. Furthermore, several recurrent mutations have been shown to be linked to different haplotypes, indicating that not all of them are founder mutations [45, 47, 54, 69, 77, 125, 126]. Type of Mutation Various mutations including single nucleotide change, small insertions and deletions, and large deletions have been identified. Approximately 50% and

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3

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P12L L22R E26K R29X 177delT 210ins15 L39W G42D E45G E45K E45X 249del4* 275T⫺⬎GG 298ins5 298ins10 307G⫺⬎AA 310insG 310ins5 311insG 311ins5 313delC 315del11 317ins5 320del2 321ins4 337delC 341ins2 345delC 357del4 359del4

360ins2 361ins2 362del4 366ins18* 377delC 378del2 382delC Q96X R98L R98X 416delC 416del32 426del2 R108X 437ins2 441ins2 K119del K120X 483del2 W126X 500delC Y133X 512delC 523delC H139D H139R H139Y F144V 555GG⫺⬎C

exon 1

2

W183R W183X V184E* E191X T197del W198X 711delA 711ins2* 713delG 734delC 735del4 738del4 739del4 750delG V125M 757del26 764G⫺⬎T 764 ⫹ 1G⫺⬎T 764 ⫹ 3A ⫺⬎G

S145S(AGC/AGT)

556-3C⫺⬎G 560insA 569delC G156D A160P A160T V162F 593ins5 597delG 599insA A164D C165R Q166X L168P D172Y 632delC A176P E179D W183S

76T/A/C 88-16C/G

765-4delT 765-1G⫺⬎T 765-1G⫺⬎C W220X 776delC 778delT L223P G225R Y227X R229L§ 816insT 817del9 C241R C241Y C241F* A242V 842delC§ S253P S253W§ S253X E255K* 879ins7 Q258X Q260P* Q260X Q261X 893⫹1 G⫺⬎T 893⫹1G⫺⬎A 893⫹1G⫺⬎C

3

556-58C/T

5

935-1G⫺⬎C G281R A284E A284Q L286P 1021delA 1022⫹1G⫺⬎A

894-9G⫺⬎A 894-4CTAGA ⫺⬎TCAGCC 894-1G⫺⬎C 896del9 K262X L264P W265X L267P* Y268X 924insC E274A§

4

L256L (CTT/CTC)

R171Q (CGG/CAG)

6

1023-1G⫺⬎C G305D* S308X A309P T311P Y312X Y313X R314P 1054insA H317Y H317R 1059delC P320L 1071delT Y323X 1089delT A337D 1132delG W341R* W341X T344R 1142delG Q349X 1159⫹1G⫺⬎A

7

1160-2A⫺⬎G Y353D Y353X 1170delT D357H* E358del E363del* 1202del2 A368D I372M 1226delC P373S 1264delC 1267delG E388X 1280delG Q393X

8

1298del4* 1300delC 1325delG 1325insA 1328del5 L414del* R415P R415X D418N D418del 1362del12 1363delA 1363del2 1364delC 1374delA 1377insG W423S W423X 1381insG E424del S427R

9

1408del11 1412delG W436R W436X 1419delg 1422insA 1424del4 1429ins4 1429TT⫺⬎ GAAA Q442X L444P 1448del2 1449del11 F447S 1452del11* Q450X 1460⫹1 del14ins2 1460⫹1del11 1460⫹4del2

1461-2A⫺⬎C 1465del4 1466del12 Q453X 1472del5 1473del5 1484del8 1486del23* 1487ins6 1491 ins2 1498del8 1499ins8 R460X 1502del4 1508ins17 1509ins2 W471X 1521insG E473X 1536insG E477X 1539insG 1555insG 1583insC 1587CC⫺⬎G 1593insC 1606del29 1607delA

10

1kb 1630insC Q508X 1634delG 1639del2 1649insC 1650insC 1650insG 1650delC 1657insC 1657insT 1665delC 1666insT 1668insT R527X 1700AGG ⫺⬎GC G532C Q536X 1717delA 1766delT S555N S555R K557del 1782delA 1785insG 1798AGC⫺⬎T 1823del2

2248del3

A541T(GCA/ACA)

L432L(CTG/CTA) D418D(GAC/GAT)

15–20% of the MEN1 mutations are frame-shift and nonsense mutations, respectively, both of which cause truncation of menin. Splicing mutations usually recognized as a nucleotide change of the donor (GT) or the acceptor (AG) consensus sequence [128] account for 5–10% of the mutations. Large deletions encompassing the entire or partial gene regions have occasionally been demonstrated [12, 13, 48, 62]. Allele-specific expression analysis and Southern blotting proved to be useful for detection of this deletion. Missense mutations and in-frame insertions and deletions, which collectively account for 20–30% of the MEN1 mutations, are often difficult to interpret because they might represent benign polymorphism. The previously reported missense mutations preferentially occurred at the highly conserved regions among menin homologs of different species [84, 86–92]. However, the functional consequence of individual missense mutations identified in the patients cannot readily be deduced. Genotype-Phenotype Correlation To date, no obvious genotype-phenotype correlation has been established [10–12]. It is considered that no specific types and/or locations of mutations might be significantly correlated with the risk of occurrence of pituitary adenoma in a large series of MEN1 cases. No differences were observed for the frequency of pituitary disease, the sex ratio, the distribution of endocrine lesion, the rate of pituitary adenomas as initial lesion of MEN1, the size of the pituitary adenoma, the treatment of pituitary adenoma, and the outcome, between 197 MEN1 patients in whom genetic testing has been performed and 127 patients in whom genetic testing was not undertaken [12]. Each of three unrelated kindreds with the prolactinoma variant of MEN1 has been shown to harbor a distinct mutation of Y312X, R640X or another unidentified MEN1 mutation that showed linkage to 11q13 [18, 45]. The Y312X and R640X mutations do not appear to be basically different from other nonsense mutations causative of classical MEN1. In the year of 2000, a Japanese prolactinoma-dominant MEN1 family was shown to have germline mutation 357del4 [44], which has previously been reported to be associated with classical MEN1 [40, 41, 54, 77]. It is unlikely that particular MEN1 mutations are responsible for the prolactinoma variant of MEN1. Unknown Fig. 3. Distribution of germline MEN1 gene mutations identified in patients with MEN1 and related disorders. Closed and open boxes indicate protein-coding and non-coding regions of MEN1 gene exons, respectively. Mutations identified in each exon and its adjacent intron sequences are shown together in a box below. Benign polymorphisms are shown above. * and § indicate mutations identified in patients of FIHP and apparently sporadic parathyroid tumor, respectively. Mutation abbreviations follow standard nomenclature [117]. Broken lines below depict large deletions.

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genetic factors other than MEN1 mutation may contribute to a prolactinomadominant phenotype [129].

Roles of MEN1 Gene in Pituitary Tumorigenesis

Familial neoplastic syndromes like MEN1 give a useful opportunity to investigate the general mechanisms of tumorigenesis and to search for genes responsible for sporadic endocrine tumors. Like most inherited cancer syndromes, MEN1 is a hereditary autosomal-dominant disease, with variable penetrance in different kindreds. The MEN1 has long been presumed to be a tumor suppressor gene, since family members inherit a family MEN1 allele from one of their parents, leaving one normal MEN1 to function in somatic tissue. These individuals consequently develop tumors that arise from single cells in which a somatic mutational event has rendered the second MEN1 allele inactive. MEN1-associated pituitary tumors, most often prolactinomas, are part of a spectrum of disease. But such MEN1 kindreds are rare, and sporadic forms of the endocrine tumors are much more common than their inherited counterparts. For MEN1 kindreds, genetic testing for MEN1 germline mutations is now possible at an early age, and affected individuals can be monitored for the occurrence of endocrine tumors, while individuals with two normal alleles are not supposed to manifest the disease. Candidates for MEN1 mutation testing include patients with MEN1 and first-degree relatives of persons with MEN1. For molecular biologists studying sporadic pituitary tumors, MEN1 has long been a most promising candidate gene for investigation. Since MEN1 gene (menin gene) was discovered, several researchers began scanning for MEN1 somatic mutations in sporadic pituitary tumors of all types. In 1997, 39 sporadic pituitary adenomas were examined for MEN1 gene mutations and allelic deletions (loss of heterozygosity, LOH). Four sporadic pituitary adenomas showed a deletion of one copy of the MEN1 gene, and a specific MEN1 gene mutation in the remaining gene copy was detected in two of these tumors. The corresponding germline sequence was normal [130]. Similar studies followed [131–141]. Moreover, immunoblotting of menin protein and semiquantitative RT-PCR of transcriptional changes of the menin gene revealed that increased menin protein expression and increased MEN1 mRNA expression was almost always present in a large series of sporadic pituitary adenomas [138, 142]. All these studies suggest that somatic mutations in the MEN1 gene do not play a prominent role in the pathogenesis of sporadic pituitary adenomas and that inherited and sporadic forms of pituitary adenoma are different in terms of the genetic events that contribute to their development. Other loci associated with sporadic pituitary tumors must still be sought.

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A Mouse Model of MEN1 (Men1 Knockout Mice)

A mouse model of MEN1, which develops multiple endocrine tumors, has been developed very recently [143]. In mice, the Men1 gene is localized on chromosome 19 and has exon-intron organization similar to that of the human gene (MEN1). Men1 demonstrates 97% identity and 98% similarity to MEN1 at the amino acid level and ubiquitously expressed in all tissues and stages of mouse development. To pursue an understanding of tumorigenesis upon loss of menin function, a mouse model was generated with homologous recombination of the mouse homolog Men1. The heterozygous phenotype of menin inactivation in mice is strikingly similar to that of the human disorder of MEN1. As early as 9 months of age, pancreatic islets showed a range of lesions from hyperplasia to insulinproducing islet cell tumors, and parathyroid adenomas were also frequently observed. Larger, more numerous tumors involving pancreatic islets, parathyroids, thyroid, adrenal cortex, and pituitary were seen by 16 months of age. All of the tumors showed loss of the wild-type Men1 allele, further supporting its role as a tumor suppressor gene. It remains to be elucidated, however, whether loss of the wild-type Men1 allele is both necessary and sufficient for tumor formation, or other clonal genetic events are required. Detection of additional genes that are activated or repressed in the early stages of these lesions will be instrumental in understanding the role of menin in this endocrine tumor syndrome. Other Animal Models of Tumor Suppressor Gene Loss in Pituitary Neoplasia for Understanding Tumorigenesis of Human Pituitary Tumor In 1992, heterozygous mice in which one Rb (retinoblastoma) gene allele was deleted by homologous recombination developed pituitary tumors arising from the pituitary intermediate lobe [145]. These tumors produced corticotroph markers, arose as multiple neoplastic foci in the intermediate lobe, and continued to proliferate into invasive pituitary lesions. But the role of Rb gene in human pituitary pathogenesis remains unknown. At least two studies failed to detect any LOH of Rb in human pituitary adenomas [146, 147], which is also located at chromosome 13q as MEN1 gene. Rb allelic loss in a high percentage of malignant or highly invasive pituitary tumors was observed and no LOH at the Rb locus in 4 benign adenomas was present [148]. There was no evidence of point mutations in any of 20 pituitary tumors examined. These data suggest that, while chromosome 13q is a frequent target for allelic deletion in invasive pituitary tumors, Rb expression remains intact and that there may be other tumor suppressor gene loci near Rb gene region.

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Another potential mouse knockout model of human pituitary tumorigenesis was discovered during the characterization of mice lacking of 18Ink4C and p27Kip 1. Both of these nucleoproteins are members of a broad class of cellcycle elements that act to regulate cyclin function during the cell cycle and specifically during G1 progression. Functional cyclin holoenzymes consist of cyclin-regulating subunits, along with catalytic cyclin-dependent kinases (CDKs), initiate a series of nuclear signals to move a cell through Go1 and to the S-phase of cell cycle. One kind of regulation is controlled by CDK inhibitors that are activated to suppress cell proliferation in response to a wide variety of growth inhibitory signals. The Ink4 family of CDK inhibitors includes p15, p16, p18 and p19, while the CIP1/KIP1 family has p21, p27 and p57. Both p18 and p27 knockout mice display larger body size, retinal dysplasia, multiorgan hypertrophy and hyperplasia, and finally, pituitary hyperplasia and tumorigenesis. Double knockout mice lacking both p18 and p27 were recently generated and developed pituitary tumors much more rapidly than mice with only a single gene knockout [149]. In this regard, clinical investigators began screening human pituitary tumors for mutations in this class of cellcycle regulating genes [150–152]. These reports suggest that it is most unlikely that inactivating somatic mutations at the p27 and p18 loci are primary pathogenetic events in human pituitary tumorigenesis.

Conclusion

The discovery of the MEN1 gene may promote future understanding of normal cell growth and of neoplasia, particularly in endocrine tissues including pituitary. The gene has now been subjected to intense study with powerful methods, and new therapies directed at certain sporadic or hereditary pituitary tumors could be developed from this knowledge [2]. It will be expected that a specific mutation of the MEN1 gene is not related to a specific clinical phenotype. Two main issues remain to be resolved. First, some MEN1 probands/families share no germline MEN1 mutations even after full sequence analysis of exons and intronic borders. In such cases, a search of deletions, either at larger scale or intragenic by quantitative PCR, Southern blot, or fluorescence in situ hybridization will assess the syndromic (clinical) diagnosis of MEN1. Secondly, 5⬘ regulatory and promoter regions remain unknown, despite extensive studies and evidence of complex 5⬘ alternative splicing. Promoter mutations or methylation may represent alternative pathways of MEN1 gene inactivation. Multicenter studies in MEN1 and related diseases should represent the right way to establish the full panel of MEN1 mutations and correlate each type of mutation with a specific functional domain of the

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protein and consequently better understanding of the clinical expression of the disease, and evaluation of prognosis related to the putative malignant evaluation of some neuroendocrine tumors related to the MEN [13]. It is evident that the mechanisms responsible for the initiation and progression of pituitary tumors are multifactorial. While the initiating genetic defect occurs at the level of a single mutated cell, it is likely that hypothalamic and intrinsic intrapituitary factors are either permissive for and/or facilitators of tumor progression. It is clear that we have made significant progress regarding the molecular events that characterize both early and progressive changes in pituitary tumorigenesis. The challenge will be to translate these emerging findings into meaningful prognostic markers that are useful in clinical management [153]. Inactivation of Rb, MEN1, and CDK inhibitors could drive cell proliferation in the pituitary through the same mechanisms seen in heritable neoplasia syndromes and knockout models of pituitary neoplasia. Basic pituitary research that elucidates the details of these signaling pathways and the factors that regulate them will continue to discover compelling novel candidate gene(s) for study in primary pituitary adenomas, as investigators make efforts toward a unifying theory of human pituitary tumorigenesis [144]. References 1 2 3 4

5 6 7 8

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101 Knapp JI, Heppner C, Hickman AB, Burns AL, Chandrasekharappa SC, Collins FS, Marx SJ, Spiegel AM, Agarwal SK: Identification and characterization of JunD missense mutants that lack menin binding. Oncogene 2000;19:4706–4712. 102 Heppner C, Bilimoria KY, Agarwal SK, Kester MB, Whitty L, Guru SC, Chandrasekharappa SC, Collins FS, Spiegel AM, Marx SJ, Burns AL: The tumor suppressor protein menin interacts with NF-␬B proteins and inhibits NF-␬B-mediated transactivation. Oncogene 2001;20: 4917–4925. 103 Kaji H, Canaff L, Lebrun JJ, Goltzman D, Hendy GN: Inactivation of menin, a Smad3-interacting protein blocks transforming growth factor type-␤-signaling. Proc Natl Acad Sci USA 2001;98:3837–3842. 104 Ohkura N, Kishi M, Tsukada T, Yamaguchi K: Menin, a gene product responsible for multiple endocrine neoplasia type 1, interacts with the putative tumor metastasis suppressor nm23. Biochem Biophys Res Commun 2001;282,1206–1210. 105 Yaguchi H, Ohkura N, Tsukada T, Yamaguchi K: Menin, the multiple endocrine neoplasia type 1 gene product, exhibits GTP-hydrolyzing activity in the presence of the tumor metastasis suppressor nm23. J Biol Chem 2002;277:38197–38204. 106 Sukhodolets KE, Hickman AB, Agarwal SK, Sukhodolets MV, Obungu VH, Novotny EA, Crabtree JS, Chandrasekharappa SC, Collins FS, Spiegel AM, Burns AL, Marx SJ: The 32-kilodalton subunit of replication protein A interacts with menin, the product of the MEN1 tumor suppressor gene. Mol Cell Biol 2003;23:493–509. 107 Gallo A, Cuozzo C, Esposito I, Maggiolini M, Bonofiglio D, Vivacqua A, Garramone M, Weiss C, Bohmann D, Musti AM: Menin uncouples Elk-1, JunD and c-Jun phosphorylation from MAP kinase activation. Oncogene 2002;21:6434–6445. 108 Rahmani M, Peron P, Weitzman J, Bakiri L, Lardeux B, Bernuau D, Morelli A, Falchetti A, Martineti V, Becherini L, Mark M, Friedman E, Brandi ML: Functional cooperation between JunD and NF-␬B in rat hepatocytes. Oncogene 2001;20:5132–5142. 109 Yakicier MC, Irmak MB, Romano A, Kew M, Ozturk M: Smad2 and Smad4 gene mutations in hepatocellular carcinoma. Oncogene 1999;18:4879–4883. 110 Mulder KM: Role of Ras and Mapks in TGF-␤ signaling. Cytokine Growth Factor Rev 2000; 11:23–35. 111 Sowa H, Kaji H, Canaff L, Hendy GN, Tsukamoto T, Yamaguchi T, Miyazono K, Sugimoto T, Chihara K: Inactivation of menin, the product of the MEN1 gene, inhibits the commitment of multipotential mesenchymal stem cells into the osteoblast lineage. Biol Chem 2003;278: 21058–21069. 112 Roymans D, Willems R, Vissenberg K, De Jonghe C, Grobben B, Claes P, Lascu I, Van Bockstaele D, Verbelen JP, Van Broeckhoven C, Slegers H: Nucleoside diphosphate kinase ␤ (Nm23-R1/NDPK␤) is associated with intermediate filaments and becomes upregulated upon cAMP-induced differentiation of rat C6 glioma. Exp Cell Res 2000;261:127–138. 113 Gustavson KH, Jansson R, Öberg K: Chromosomal breakage in multiple endocrine adenomatosis (types I and II). Clin Genet 1983;23:143–149. 114 Tomassetti P, Cometa G, Del Vecchio E, Baserga M, Faccioli P, Bosoni D, Paolucci G, Barbara L: Chromosomal instability in multiple endocrine neoplasia type 1. Cytogenetic evaluation with DEB test. Cancer Genet Cytogenet 1995;79:123–126. 115 Itakura Y, Sakurai A, Katai M, Ikeo Y, Hashizume K: Enhanced sensitivity to alkylating agent in lymphocytes from patients with multiple endocrine neoplasia type 1. Biomed Pharmacother 2000;54(suppl):187–190. 116 Poisson A, Zablewska B, Gaudray P: Menin interacting proteins as clues toward the understanding of multiple endocrine neoplasia type 1. Cancer Lett 2003;189:1–10. 117 Beaudet AL, Tsui LC: A suggested nomenclature for designating mutations. Hum Mutat 1993; 2:245–248. 118 Bear JC, Briones-Urbina R, Fahey JF, Farid NR: Variant multiple endocrine neoplasia I (MEN1): Further studies and non-linkage to HLA. Hum Hered 1985;35:15–20. 119 Matsuno A, Teramoto A, Yamada S, Kitanaka S, Tanaka T, Sanno N, Osamura Y, Kirino T: Gigantism in sibling unrelated to multiple endocrine neoplasia: Case report. Neurosurgery 1994; 35:952–956.

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120 Yamada S, Yoshimoto K, Sano T, Takada K, Itakura M, Usui M, Teramoto A: Inactivation of the tumor suppressor gene on 11q13 in brothers with familial acrogigantism without multiple endocrine neoplasia type 1. J Clin Endocrinol Metab 1997;82:239–242. 121 Tanaka C, Yoshimoto K, Yamada S, Nishioka H, Ii S, Moritani M, Yamaoka T, Itakura M: Absence of germ-line mutations of the multiple endocrine neoplasia type 1 (MEN1) gene in familial pituitary adenoma in contrast to MEN1 in Japanese. J Clin Endocrinol Metab 1998;83:960–965. 122 Gadelha MR, Prezant TR, Une KN, Glick RP, Stanley F, Ii M, Vaisman M, Melmed S, Kineman RD, Frohman: Loss of heterozygosity on chromosome 11q13 in two families with acromegaly/gigantism is independent of mutations of the multiple endocrine neoplasia type I gene. J Clin Endocrinol Metab 1999;84:249–256. 123 Gadelha MR, Une KN, Rhode K, Vaisman M, Kineman RD, Frohman LA: Isolated familial somatotropinomas: Establishment of linkage to chromosome 11q13.1–11q13.3 and evidence for a potential second locus at chromosome 2p16–12. J Clin Endocrinol Metab 2000;85: 707–714. 124 Stock JL, Warth MR, Teh BT, Coderre JA, Overdorf JH, Baumann G, Hintz RL, Hartman ML, Seizinger BR, Larsson C, Aronin N: A kindred with a variant of multiple endocrine neoplasia type 1 demonstrating frequent expression of pituitary tumors but not linked to the multiple endocrine neoplasia type 1 locus at chromosome region 11q13. J Clin Endocrinol Metab 1997; 82:486–492. 125 Ackermann F, Krohn K, Windgassen M, Buchfelder M, Fahlbush R, Paschke R: Acromegaly in a family without a mutation in the menin gene. Exp Clin Endocrinol Diabetes 1999; 107:93–96. 126 Agarwal SK, Debelenko LV, Kester MB, Guru SC, Manickam P, Olufemi SE, Skarulis MC, Heppner C, Crabtree JS, Lubensky IA, Zhuang Z, Kim YS, Chandrasekharappa SC, Collins FS, Liotta LA, Spiegel AM, Burns AL, Emmert-Buck MR, Marx SJ: Analysis of recurrent germline mutations in the MEN1 gene encountered in apparently unrelated families. Hum Mutat 1998; 12:75–82. 127 Emmert-Buck MR, Debelenko LV, Agarwal S, Kester MB, Manickam P, Zhuang Z, Guru SC, Olufemi SE, Burns AL, Chandrasekharappa SC, Lubensky IA, Liotta LA, Skarulis MC, Spiegenl AM, Marx SJ, Collins FS: 11q13 allelotype analysis in 27 Northern American MEN 1 kindreds identifies two distinct founder chromosomes. Mol Genet Metat 1998;63:151–155. 128 Padgett RA, Grabowski PJ, Konarska MM, Seiler S, Sharp PA: Splicing of messenger RNA precursors. Annu Rev Biochem 1986;55:1119–1150. 129 Yoshimoto K: Multiple endocrine neoplasia type 1: From bedside to benchside. J Med Invest 2000;47:108–117. 130 Zhuang Z, Ezzat SZ, Vortmeyer AO, Weil R, Oldfield EH, Park WS, Pack S, Huang S, Argarwal SK, Guru SC, Manickam P, Debelenko LV, Kester MB, Olufemi SE, Heppner C, Crabtret JS, Burns AL, Spiegel AM, Marx SJ, Chandrasekharappa SC, Collins FS, Emmert-Buck MR, Liotta LA, Asa SL, Lubensky IA: Mutations of the MEN1 tumor suppressor gene in pituitary tumors. Cancer Res 1997;57:5446–5451. 131 Prezant TR, Levine J, Melmed S: Molecular characterization of the men 1 tumor suppressor gene in sporadic pituitary tumors. J Clin Endocrinol Metab 1998;83:1388–1391. 132 Tanaka C, Kimura T, Yang P, Moritani M, Yamaoka T, Yamada S, Sano T, Yoshimoto K, Itakura M: Analysis of loss of heterozygosity on chromosome 11 and infrequent inactivation of the MEN1 gene in sporadic pituitary adenomas. J Clin Endocrinol Metab 1998;83:2631–2634. 133 Asa SL, Somers K, Ezzat S: The MEN-1 gene is rarely down-regulated in pituitary adenomas. J Clin Endocrinol Metab 1998;83:3210–3312. 134 Farrell WE, Simpson DJ, Bicknell J, Magnay JL, Kyrodimou E, Thakker RV, Clayton RN: Sequence analysis and transcript expression of the MEN1 gene in sporadic pituitary tumors. Br J Cancer 1999;80:44–50. 135 Weil RJ, Nortmeyer AO, Huang S, Boni R, Lubensky IA, Pack S, Marx SJ, Zhuang Z, Oldfield EH: 11q13 allelic loss in pituitary tumors in patients with multiple endocrine neoplasia syndrome type 1. Clin Cancer Res 1998;4:1673–1678. 136 Wenbin C, Asai A, Teramoto A, Sanno N, Kirino T: Mutations of the MEN1 tumor suppressor gene in sporadic pituitary tumors. Cancer Lett 1999;142:43–47.

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137 Fukino K, Kitamura Y, Sanno N, Teramoto A, Emi M: Analysis of the MEN1 gene in sporadic pituitary adenomas from Japanese patients. Cancer Lett 1999;144:85–92. 138 Poncin J, Stevenaert A, Beckers A: Somatic MEN1 gene mutation does not contribute significantly to sporadic pituitary tumorigenesis. Eur J Endocrinol 1999;140:573–576. 139 McCabe CJ, Gittoes NJL, Steppard MC, Franklyn JA: Increased MEN1 mRNA expression in sporadic pituitary tumors. Clin Endocrinol 1999;50:727–733. 140 Bergman L, Boothroyd C, Palmer J, Grimmond S, Walters M, Teh B, Shepherd J, Hartley L, Hayward N: Identification of somatic mutations of the MEN1 gene in sporadic endocrine tumors. Br J Cancer 2000;83:1003–1008. 141 Asteria C, Anagni M, Persani L, Beck-Peccoz P: Loss of heterozygosity of the MEN1 gene in a large series of TSH-secreting pituitary adenoma. J Endocrinol Invest 2001;24:796–801. 142 Wrocklage C, Gold H, Hack W, Buchfelder M, Fahlbush R, Paulus W: Increased menin expression in sporadic pituitary adenomas. Clin Endocrinol 2000;56:589–594. 143 Crabtree JS, Scacheri PC, Ward JM, Garrett-Beal L, Emmert-Buck MR, Edgemon KA, Lorang D, Libbutti SK, Chandrasekharappa SC, Marx SJ, Spiegel AM, Collins FS: A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors. Proc Natl Acad Sci USA 2001;98:1118–1123. 144 Alexander JM: Tumor suppressor loss in pituitary tumors. Brain Pathol 2001;11:342–352. 145 Jacks T, Fazeli A, Schmitt EM, Bronson RT, Goodell MA, Weinberg RA: Effects of an Rb mutation in the mouse. Nature 1992;359:295–300. 146 Cryns VL, Alexander JM, Klibanski A, Arnold A: The retinoblastoma gene in human pituitary tumors. J Clin Endocrinol Metab 1993;77:644–646. 147 Zhu J, Leon SP, Beggs AH, Busque L, Gilliland DG, Black PM: Human pituitary adenomas show no loss of heterozygosity at the retinoblastoma gene locus. J Clin Endocrinol Metab 1994;78:922–927. 148 Pei L, Melmed S, Scheithauer B, Kovacs K, Benedict WF, Prager D: Frequent loss of heterozygosity at the retinoblastoma susceptibility gene (RB) locus in aggressive pituitary tumors: Evidence for a chromosome 13 tumor suppressor gene other than RB. Cancer Res 1995;55: 1613–1616. 149 Franklin DS, Godfrey VL, Lee H, Kovalev GI, Schoonhoven R, Chen-Kiang S, Su L, Xiong Y: CDK inhibitors p18 (INK4c) and p27 (Kip1) mediate two separate pathways to collaboratively suppress pituitary tumorigenesis. Genes Dev 1998;12:2899–2911. 150 Tanaka C, Yoshimoto K, Yang P, Kimura T, Yamada S, Moritani M, Sano T, Itakura M: Infrequent mutations of p27Kip 1 gene and trisomy 12 in a subset of human pituitary adenomas. J Clin Endocrinol Metab 1997;82:3141–3147. 151 Ikeda H, Yoshimoto T, Shida N: Molecular analysis of p21 and p27 genes in human pituitary adenomas. Br J Cancer 1997;76:1119–1123. 152 Dahia PL, Aguiar RC, Honegger J, Fahlbush R, Jordan S, Lowe DG, Lu X, Clayton RN, Besser GM, Grossman AB: Mutation and expression analysis of the p27/kip 1 gene in corticotrophinsecreting tumours. Oncogene 1998;16:69–76. 153 Farrell WE, Clayton RN: Molecular pathogenesis of pituitary tumors. Front Neuroendocrinol 2000;21:174–198.

Toru Kameya, MD Pathology Division, Shizuoka Cancer Center Hospital and Research Institute 1007 Shimonagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777 (Japan) Tel. ⫹81 55 9895222, Fax ⫹81 55 9895329, E-Mail [email protected]

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Author Index

Arzt, E. 96 Asa, S.L. 1 Bonner, S. 34 Borboli, N. 34 Bossis, I. 253 Carney, J.A. 253 Clayton, R.N. 186 Courkoutsakis, N. 253 Egashira, N. 20 Ezzat, S. 1 Farrell, W.E. 186 Grossman, A.B. 34, 63 Gueorguiev, M. 34

Kapranos, N. 217 Kola, B. 63 Kontogeorgos, G. IX, 205, 217 Korbonits, M. 34, 63 Kovacs, K. IX, 217

Saeger, W. 110 Sanno, N. 20 Sano, T. 127 Stalla, G.K. 96 Stergiopoulos, S. 253 Stratakis, C.A. 253

Lamberts, S.W.J. 235 Lloyd, R.V. 146

Takekoshi, S. 20 Teramoto, A. 20 Torre, N.G. de la 133 Tsukada, T. 265 Turner, H.E. 133

Matyakhina, L. 253 Melmed, S. VII, 175 Miyai, S. 20 Morris, D.G. 63 Mus¸at, M. 34 Nanzer, A. 63 Osamura, R.Y. 20

Hofland, L.J. 235 Horvath, E. 217

Paez-Pereda, M. 96 Patronas, N. 253

Kagawa, N. 127 Kameya, T. 265

Renner, U. 96 Rong, Q.Z. 127

Vax, V.V. 34 Voutetakis, A. 253 Wass, J.A.H. 133 Yamada, S. 127 Yamaguchi, K. 265 Yamazaki, M. 20 Yu, R. 175

292

Subject Index

Activin activity decrease, gonadotroph adenoma 78 pituitary tumorigenesis, role 99 Adenoma, see also specific cell types angiogenesis, see Angiogenesis clonality, see Clonality, pituitary tumors frequency by type 26, 27 functioning versus nonfunctioning 24, 25 growth factors and cytokines epidermal growth factor 99–101 fibroblast growth factor 101 gp130 cytokines 102, 103 intrinsic growth factor network 104–106 leukemia inhibitory factor 103, 104 nerve growth factor 101, 102 overview 96, 97 therapeutic prospects 106 transforming growth factor alpha 99–101 transforming growth factor beta family 97–99 hormonal factors, tumorigenesis corticotroph adenomas 64–70 gonadotroph adenomas 77–79 hypothalamic versus pituitary site 186, 187 lactotroph tumorigenesis 79–82 local autocrine and paracrine effects 84, 96, 97

nonfunctioning adenomas 77–79 somatotroph adenomas 70–77 thyrotroph tumorigenesis 82–84 nonfunctioning adenomas, see Nonfunctioning pituitary adenomas null cell adenomas 26, 27 proliferation markers, see specific markers rodent models 29, 30 transcription factor regulation 25, 26 Adrenocorticotropic hormone (ACTH) adenomas, see Corticotroph receptor underexpression, corticotroph adenomas 67 somatostatin, modulation of release 245 Androgen receptor, X-chromosome inactivation analysis 189 Angiogenesis pituitary adenoma growth factor regulation 136–138 hormonal regulation 138–141 morphologic vascular changes 134 pituitary tumorderived transforming gene regulation 140 therapeutic targeting 141, 142 tumor behavior effects 134, 135 tumor growth, role 133, 134 Annexin-1 expression, corticotroph adenomas 70 Apoptosis assays 121, 122, 221, 222 Bcl-2 regulation 121, 220, 221 Caenorhabditis elegans genes 218

293

Apoptosis (continued) caspase cascade 218, 219 DNA laddering 222 hormonal regulation 122, 123 mitochondria mediation 221 morphological changes human pituitary studies 224, 225, 227 overview 221, 222 necrosis comparison 218 p27, role 42, 43 p53, role 230 pathway classification 219, 220 pituitary assay studies 228–230 pituitary cell line experiments 222, 223 Arginine vasopressin (AVP), receptor overactivity, corticotroph adenomas 66 Bax, immunocytochemistry, human pituitary 230 Bcl-2 apoptosis regulation 121, 220, 221 immunocytochemistry, human pituitary 230 Bone morphogenetic proteins (BMPs) BMP-4, role, lactotroph tumorigenesis 82 pituitary development, role 2, 3 pituitary tumorigenesis, role 99 Carney complex (CNC) hormonal derangements 254 pituitary findings adenoma hormone staining 255, 256 clonality, pituitary adenomas 188 comparative genomic hybridization findings, pituitary tumors 213, 259 electron microscopy 258, 259 morphology 255 tumor types 259–261 protein kinase A gene mutations 254, 255, 261 Caspase apoptosis cascade 218, 219 detection, human pituitary 231 Catenin, growth hormone adenoma downregulation genetic alterations 130, 131 immunohistochemistry 129, 130

Subject Index

intermediate filament/adhesion molecule abnormalities 129 Cell cycle adenoma proliferation markers, see specific markers checkpoints 36, 110 cyclin-dependent kinase regulation 37–39, 110 dysregulation, pituitary tumorigenesis CDK2-p27 pathway 42–51 CDK4-Rb-INK4 pathway 40–42 Krüppel-like factor-6 54 p53 53 peroxisome proliferator activated receptor gamma 53, 54 pituitary tumor transforming gene 53 p53, regulation 39, 40 phases 35, 36, 110 pituitary tumor transforming gene overexpression, mitotic disruption 177, 179, 183 separin regulation 39 Clonality, pituitary tumors assays loss of heterozygosity 189, 190, 199 polymerase chain reaction microsatellite analysis 190, 191 primer extension preamplification 190, 191 sampling bias 199 Southern blot 190, 191 X chromosome inactivation analysis 189 Carney complex pituitary adenomas 188 corticotroph adenomas 192, 193 hypothalamic versus pituitary site, tumorigenesis 186, 187 monoclonality, normal tissue 193, 194 morphology independence 201 multiclonal pituitary tumors adenoma examples 188, 192, 193 recurrent tumors 194–199 multiple endocrine neoplasia type 1 pituitary adenomas 188, 200 Comparative genomic hybridization (CGH) Carney complex findings, pituitary tumors 213, 259

294

pituitary tumor studies 149–151, 206, 207, 211–213 principles 149, 206 Corticotroph adenoma clonality 192, 193 somatostatin modulation, adrenocorticotropic hormone release 245 somatostatin receptor expression 244, 245 DNA microarray analysis, adenoma 158 excess adrenocorticotropic hormone secretion, mechanisms, tumorigenesis adrenocorticotropic hormone receptor underexpression 67 annexin-1 expression 70 arginine vasopressin receptor overactivity 66 corticotropin-releasing hormone and receptor overactivity 65, 66 cortisol deficiency 67, 68 glucocorticoid receptor dysregulation 68 11␤-hydroxysteroid dehydrogenase expression 68–70 overview 64 in situ hybridization studies, adenomas 148 lineage development 5, 6 Corticotropin-releasing hormone (CRH), overactivity, corticotroph adenomas 65, 66 Cortisol, deficiency, corticotroph adenomas 67, 68 CUTE, proopiomelanocortin expression, role 6 Cyclic AMP response element binding protein (CREB) activation, somatotroph adenoma 74 postdifferentiation pituitary cell proliferation regulation 11 Cyclin cell proliferation analysis, cyclin D 112 types and functions 37

Subject Index

Cyclin-dependent kinase (CDK) activation 37 cell cycle regulation 37–39, 110 dysregulation, pituitary tumorigenesis CDK2-p27 pathway 42–51 CDK4-Rb-INK4 pathway 40–42 inhibitors 38, 39, 110, 111 substrates 38 DNA microarray pituitary tumor studies 157–159 principles 155–157 specificity, studies 159, 160 DNA topoisomerase II␣, adenoma cell proliferation analysis 117, 118 Dopamine angiogenesis inhibition, adenomas 138 D2 receptor knockout, prolactinoma development 187 receptor expression, lactotroph tumorigenesis 80 E-cadherin, growth hormone adenoma downregulation genetic alterations 130, 131 immunohistochemistry 129, 130 intermediate filament/adhesion molecule abnormalities 129 Epidermal growth factor (EGF), pituitary tumorigenesis, role 99–101 Estrogen receptor (ER) gonadotroph modulation 10, 11 isoforms gonadotroph adenoma 78, 79 lactotroph tumorigenesis 81, 82 pituitary expression, development 7–9 Ets-1, Pit-1 synergism 9 Fibroblast growth factor (FGF) angiogenesis regulation, adenomas, FGF-2 137, 138 intrinsic growth factor network, pituitary 104 pituitary development, role 3 pituitary tumorigenesis, role 101

295

Flow cytometry cell cycle analysis 111 ploidy analysis 111 Fluorescence in situ hybridization (FISH) comparison with karyotyping 205, 206 pituitary tumor findings 207–213 principles 149 sample types 206 techniques utilizing fluorescence in situ hybridization 206, 207 Folliculostellate (FS) cells features 21, 136 growth factor secretion 137 GATA-2 adenoma regulation 25, 26 gonadotroph differentiation, role 11 pituitary development, role 9 Ghrelin, dysregulation, somatotroph adenoma 75 Glucocorticoid receptor (GR), dysregulation, corticotroph adenomas 68 Gonadotroph adenoma somatostatin modulation, gonadotropin release 246 somatostatin receptor expression 245 excess gonadotropin secretion, mechanisms, tumorigenesis activin activity decrease 78 estrogen receptor isoforms 78, 79 gonadotropin isoform alterations 78 gonadotropin-releasing hormone, receptor overactivity 77, 78 leptin/leptin receptor dysregulation 79 overview 77 lineage development 9–11 Growth hormone adenoma, see Somatotroph Growth hormone-releasing hormone (GHRH), overactivity, somatotroph adenoma 71, 72 Growth hormone secretagogue receptors, overactivity, somatotroph adenoma 74, 75 Gs␣, mutations, somatotroph adenoma 72, 73

Subject Index

11␤-Hydroxysteroid dehydrogenase, expression, corticotroph adenomas 68–70 Inhibin, pituitary tumorigenesis, role 99 In situ hybridization (ISH), see also Fluorescence in situ hybridization applications 146 pituitary tumor studies 147–149 polymerase chain reaction combination 149 principles 146, 147 Insulin-like growth factor-I (IGF-I), receptor deficiency, somatotroph adenoma 76, 77 Interleukin-6 (IL-6), pituitary tumorigenesis, role 102, 103 Karyotyping comparison with fluorescence in situ hybridization 205, 206 pituitary tumor findings 207 Ki-67, adenoma cell proliferation analysis 113–115 Krüppel-like factor 6 (KLF6), dysregulation, pituitary tumorigenesis 54 Lactotroph adenoma DNA microarray analysis 158 prolactin secretion modulation, somatostatin 243, 244 somatostatin analog treatment 244 somatostatin receptor expression 242, 243 estradiol control 105, 106 hormonal derangement, tumorigenesis bone morphogenetic protein-4 82 dopamine receptor expression 80 estrogen receptor isoforms 81, 82 overview 79, 80 pituitary adenylate cyclase activating peptide 82 prolactin feedback 81 prolactin-releasing peptide 81 vasoactive intestinal polypeptide 82 lineage development 6–9

296

Lanreotide, see Somatostatin Laser capture microdissection (LCM) disadvantages compared with manual microdissection 154, 155 pituitary tumor studies 155 principles 150–152 systems 152, 154 technique 152–154 Leptin, dysregulation, gonadotroph adenoma 79 Leukemia inhibitory factor (LIF) angiogenesis regulation, adenomas 138 pituitary tumorigenesis, role 103, 104 Lhx genes, pituitary development, role 4 Loss of heterozygosity (LOH), clonality assay 189, 190, 199 Mammosomatotroph, lineage development 6–9 Matrix metalloproteinase-9 (MMP9), pituitary adenoma expression 119 Mib-1, adenoma cell proliferation analysis 114, 115 Microarrays, see DNA microarray; Tissue microarray Multiple endocrine neoplasia type 1 (MEN-1) clinical presentation 266, 267 clonality, pituitary adenomas 188, 200 epidemiology 266 familial isolated somatotropinoma 275 fluorescence in situ hybridization findings, pituitary adenomas 210, 211 history of study 266 MEN1 gene detection rate, mutations 275–277 genotype-phenotype correlation 279, 280 inactivating mutations 270 knockout mouse 281 locus 270 menin product functions, protein interactions 272–274 mutation distribution, types 277–279 sporadic pituitary tumor mutations 280 structure 270–272

Subject Index

tissue distribution, expression 271, 272 pituitary lesion pathology 267–269 prospects for study 282, 283 variants 275 Nerve growth factor (NGF), pituitary tumorigenesis, role 101, 102 Nonfunctioning pituitary adenomas (NFPAs) DNA microarray analysis 158 excess gonadotropin secretion mechanisms, tumorigenesis activin activity decrease 78 estrogen receptor isoforms 78, 79 gonadotropin isoform alterations 78 gonadotropin-releasing hormone, receptor overactivity 77, 78 leptin/leptin receptor dysregulation 79 overview 77 features 24, 25 growth hormone combination, adrenocorticotropic hormone 27, 29 hormone expression, nonfunctioning adenomas 26, 27 Octreotide, see Somatostatin p16 adenoma cell proliferation analysis 115 dysregulation, pituitary tumorigenesis 41 p18 dysregulation, pituitary tumorigenesis 41, 42 knockout mouse 282 p27 adenoma cell proliferation analysis 116 apoptosis, role 42, 43 cell adhesion, role 43 cell cycle regulation 42 dysregulation, pituitary tumorigenesis 49–51 knockout mouse 282 subcellular compartmentalization 46–48 transcriptional regulation 48, 49 translational regulation 48 ubiquitin-proteasome system, regulation 43–46

297

p53 adenoma cell proliferation analysis 117 cell cycle regulation 39, 40 dysregulation, pituitary tumorigenesis 53 immunocytochemistry, human pituitary 230, 231 Pax-6, pituitary development, role 5 Peroxisome proliferator activated receptor gamma (PPAR-␥), dysregulation, pituitary tumorigenesis 53, 54 Pit-1 activation, somatotroph adenoma 74 adenoma regulation 25, 26 hormone expression regulation 7–9 isoforms 6, 7 pituitary development, role 6 Pituitary adenoma, see Adenoma; see also specific cell types Pituitary adenylate cyclase activating peptide (PACAP) apoptosis inhibition 122, 123 lactotroph tumorigenesis, role 82 Pituitary gland cell types 1, 2, 20, 21 development adenohypophysial cytodifferentiation 12, 13 adenohypophysis 2, 21, 22 corticotroph lineage 5, 6 early development signaling 2–5 gonadotroph lineage 9–11 immunohistochemistry, functional differentiation 24 intermediate lobe 22 Pit-1, role 6–9 postdifferentiation cell proliferation regulation 11, 12 transcription factors, overview 24, 25 hormones and regulation 1, 2 lobes 21 secretory granules, anterior pituitary 21 vasculature 135, 136 Pituitary tumor transforming gene (PTTG) aneuploidy, role 176 angiogenesis regulation, adenomas 140, 176 apoptosis induction 175

Subject Index

binding factor 177 dysregulation, pituitary tumorigenesis 53 knockout mouse phenotype 180, 181, 183 mitotic disruption 177, 179, 183 overexpression, tumors 176, 177 prospects for study 183 protein-interacting partners 180 regulation of expression 179, 180 P-LIM, pituitary development, role 4 Polymerase chain reaction (PCR) in situ polymerase chain reaction 149 microsatellite analysis, tumor clonality 190, 191 Primer extension preamplification (PEP), clonality assay 190, 191 Prolactin angiogenesis inhibition, adenomas 139 dysregulation, lactotroph tumorigenesis prolactin feedback 81 prolactin-releasing peptide 81 prolactinoma, see Lactotroph regulation of expression, Pit-1 cells 7–9 secretion modulation, somatostatin 243, 244 Proliferating cell nuclear antigen (PCNA), adenoma cell proliferation analysis 113 PROP-1 adenoma regulation 26 pituitary development, role 5 Protein kinase A (PKA) Carney complex gene mutations 254, 255, 261 regulatory subunit inactivation, somatotroph adenoma 73, 74 Protein kinase C (PKC), inhibition and apoptosis induction, adenoma cell lines 223 Proteomics pituitary tumor studies 164, 165 principles 161–163 Ptx1, pituitary development, role 4 Ptx2, pituitary development, role 4 Retinoblastoma protein (Rb) cyclin-dependent kinase substrate 38 dysregulation, pituitary tumorigenesis 41 knockout mouse 281

298

RNA interference (RNAi) efficacy/specificity factors 165, 166 mechanisms 165 pituitary studies 167 Rpx/HesX-1, pituitary development, role 4, 5 Separin, cell cycle regulation 39 Somatostatin adrenocorticotropic hormone adenoma somatostatin modulation, adrenocorticotropic hormone release 245 somatostatin receptor expression 244, 245 analogs 237 angiogenesis inhibition, adenomas 139, 140 cortistatin homology 236, 237 gonadotroph adenoma somatostatin modulation, gonadotropin release 246 somatostatin receptor expression 245 growth hormone adenoma somatostatin analog treatment 239–242 somatostatin receptor expression 238, 239 prolactinoma prolactin secretion modulation, somatostatin 243, 244 somatostatin analog treatment 244 somatostatin receptor expression 242, 243 receptors binding potency, analogs 237 deficiency, somatotroph adenoma 75, 76 expression and functional significance, subtypes, pituitary 237, 238 scintigraphy, adenoma diagnostics 247 subtypes 236 structure 236 thyrotroph adenoma somatostatin analog treatment 246, 247 somatostatin receptor expression 246

Subject Index

Somatotroph adenoma DNA microarray analysis 158 in situ hybridization studies 147, 148 somatostatin analog treatment 239–242 somatostatin receptor expression 238, 239 E-cadherin/catenin downregulation, adenoma genetic alterations 130, 131 immunohistochemistry 129, 130 excess growth hormone secretion mechanisms, tumorigenesis cyclic AMP response element binding protein activation 74 ghrelin dysregulation 75 growth hormone receptor deficiency 76 growth hormone-releasing hormone and receptor overactivity 71, 72 growth hormone secretagogue receptor overactivity 74, 75 Gs␣ mutations 72, 73 insulin-like growth factor I receptor deficiency 76, 77 overview 70, 71 Pit-1 activation 74 protein kinase A regulatory subunit inactivation 73, 74 somatostatin receptor deficiency 75, 76 thyrotropin-releasing hormone receptor abnormalities 74 familial isolated somatotropinoma 275 intermediate filament/adhesion molecule abnormalities, adenoma 129 lineage development 6–9 ultrastructural classification, adenomas 127, 128 Sonic hedgehog (Shh), pituitary development, role 3 Southern blot, clonality assay 190, 191 Steroidogenic factor-1 (SF-1), gonadotroph differentiation, role 9, 10 Thyroid hormone, receptor defects, thyrotroph tumorigenesis 84

299

Thyroid-stimulating hormone (TSH) adenoma, see Thyrotroph defects, thyrotroph tumorigenesis 83, 84 Thyrotroph adenoma DNA microarray analysis 158, 159 somatostatin analog treatment 246, 247 somatostatin receptor expression 246 hormonal derangement, tumorigenesis overview 82, 83 thyroid hormone receptor defects 84 thyroid-stimulating hormone 83, 84 thyrotropin-releasing hormone 82, 83 lineage development 6–9 Thyrotroph embryonic factor (TEF), thyroid-stimulating hormone expression activation 9 Thyrotropin-releasing hormone (TRH) defects, thyrotroph tumorigenesis 82, 83 receptor abnormalities, somatotroph adenoma 74 Tissue microarray (TMA) pituitary tumor studies 161 principles 160, 161 validation 161

Subject Index

Tissue plasminogen activator (tPA), pituitary adenoma expression 119 Tpit, corticotroph differentiation, role 5 Transforming growth factor alpha (TGF-␣), pituitary tumorigenesis, role 99–101 Transforming growth factor beta (TGF-␤), pituitary tumorigenesis, role 97–99 Ubiquitin-proteasome system, overview, p27 regulation 43–46 Urokinase, pituitary adenoma expression, receptor 119 Vascular endothelial growth factor (VEGF), angiogenesis regulation, adenomas 137–139 Vasoactive intestinal polypeptide (VIP), lactotroph tumorigenesis, role 82 Wnt, pituitary development, role 3 X chromosome inactivation analysis, clonality assay 189 Zn-15, Pit-1 synergism 7

300