Molecular pathology of the pituitary

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.

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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

Proteasome p27 P p27

F-box

Nuclear import p27

Cyclin E

p27

p-Akt

CDK2

p27

p27

P p27

Ubiquitin

P Ser10

Nuclear export

Skp2 Cks1

Akt

?pK

P P

Jab1

MIF

Jab1 Nuclear export

Jab1 p27 P Thr187 Jab1 MIF

Thr157

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

Mean nuclear Jab1 staining (% strong  moderate)

*

GH CTH FPA N A

PR

L

FS

H ve atic t TS essi r tas g Me Ag

H

Mean nuclear P-p27 staining (% strongmoderate)

70 60 50 40 30 20 10 0

Thr187 P-p27 immunostaining 80 70 60 50 40 30 20 10 0

*

70 50

*

10 0

al

rm

No

GH CTH FPA PR N A

L

FS

H

*

rm

al

GH CTH FPA N A

PR

L

FS

H

H ve ic TS essi stat ta gr e g M A

Ki-67 immunostaining 

30

* *

Jab1 immunostaining 90

*

* *

No

Mean nuclear Ki-67 staining (% positive)

Mean nuclear p27 staining (% strongmoderate)

p27 immunostaining

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

4 3.5 3 2.5 2 1.5 1 0.5 0

* *

*

al

rm

No

GH CTH FPA PR N A

L

FS

H

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 G
q and G
11 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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