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molecular genetics of adrenocorticaltumors

and relateddisorders

Constantine Stratakis, MD, DSc, Head, Section on Endocrinology and Genetics Dalia Batista, MD, Staff Fellow^a Kurt Griffin, MD, PhD, Staff Fellow^a Ludmila Matyakhina, PhD, Visiting Fellow^b Anelia Horvath, PhD, Postdoctoral Fellow Sotirios Stergiopoulos, MD, Postdoctoral Fellow Andrew Bauer, MD, Guest Researcher^c Audrey Robinson-White, PhD, Guest Researcher Thalia Bei, PhD, Special Volunteer^d Isabelle Bourdeau, MD, Special Volunteer^e Nickolas Stathatos, MD, Special Volunteer^f Elise Meoli, BS, Predoctoral Fellow Frank Weinberg, BS, Predoctoral Fellow Margaret Ngyen, BS, Medical Student, Special Volunteer Anna Binstock, NIH Summer Student Jehan Riar, NIH Summer Student Jennifer Siegel, NIH Summer Student

Our goal is to understand the genetic and molecular mechanisms leading to disorders that affect the adrenal cortex, with emphasis on those that are developmental, hereditary, and associated with adrenal hypoplasia or hyperplasia, multiple tumors, and abnormalities in other endocrine glands (especially the pituitary gland and, to a lesser extent, the thyroid gland). We have studied congenital adrenal hypoplasia caused by triple A syndrome and multiple endocrine deficiencies, familial hyperaldosteronism, adrenocortical and thyroid cancer, pituitary tumors and multiple endocrine neoplasia (MEN) syndromes affecting the pituitary, thyroid, and adrenal glands, and Carney complex (CNC), an autosomal dominant disease. CNC is a MEN syndrome affecting the pituitary, adrenal cortex, thyroid, and gonads and is associated with a variety of other tumors, including myxomas and schwannomas and skin pigmentation defects (lentigines, cafè-au-lait spots, and nevi). We have identified the regulatory subunit type 1-alpha (RI-alpha ) of protein kinase A (PKA), which is encoded by the PRKAR1A gene, as the gene responsible for most CNC patients. Thus, a significant part of our work now focuses on PKA-stimulated signaling pathways, PKA effects on tumor suppression and/or development, the cell cycle, and chromosomal stability. Our projects make use of Prkar1a-specific animal models. Carney complex genetics Bauer, Bei, Bourdeau, Griffin, Matyakhina, Stergiopoulos, Stratakis; in collaboration with Bertherat, Carney, Kirschner We have been collecting families with CNC and related syndromes from several collaborating institutions worldwide and, through genetic linkage analysis, have identified loci harboring genes for CNC on chromosomes 2 (2p16) and 17 (17q22-24)s. We are currently investigating possible other loci for this genetically heterogeneous condition and, with the application of state-of-the-art molecular cytogenetic techniques, the participation of these genomic loci in the expression of the disease. For the cloning of the CNC-associated sequences, we have constructed a comprehensive genetic and physical map of the 2p16 chromosomal region. Studies in cultured primary tumor cell lines (established from our patients) identified a region of genomic amplification in CNC tumors in the center of the map. The PRKAR1A gene on 17q22-24, the gene responsible for CNC in most cases of the disease, appears to undergo loss of heterozygosity in at least some CNC tumors. PRKAR1A is also the main regulatory subunit of protein kinase A (PKA), a central signaling pathway for many cellular functions and hormonal responses. We are conducting more genotype-phenotype correlation studies in patients with CNC, studies that are expected to shed light on the complex biochemical and molecular pathways regulated by PRKAR1A and PKA. Perhaps the most important development in that area is the identification of a new type of adrenal hyperplasia not caused by PRKAR1A mutations (see Figure 3.3). Bossis I, Voutetakis A, Matyakhina L, Pack S, Abu-Asab M, Bourdeau I, Griffin KJ, Courcoutsakis N, Stergiopoulos S, Batista D, Tsokos M, Stratakis CA. A pleiomorphic GH pituitary adenoma from a Carney complex patient displays universal allelic loss at the protein kinase A regulatory subunit 1A (PRKARIA) locus. J Med Genet 2004;41:596-600. Bourdeau I, Lacroix A, Schurch W, Caron P, Antakly T, Stratakis CA. Primary pigmented nodular adrenocortical disease: paradoxical responses of cortisol secretion to dexamethasone occur in vitro and are associated with increased expression of the glucocorticoid receptor. J Clin Endocrinol Metab 2003;88:3931-3937. Courcoutsakis NA, Patronas NJ, Cassarino D, Griffin K, Keil M, Ross JL, Carney JA, Stratakis CA. Hypodense nodularity on computed tomography: novel imaging and pathology of micronodular adrenocortical hyperplasia associated with myelolipomatous changes. J Clin Endocrinol Metab 2004;89:3737-3738. Gunther DF, Bourdeau I, Matyakhina L, Cassarino D, Kleiner DE, Griffin K, Courkoutsakis N, Abu-Asab M, Tsokos M, Keil M, Carney JA, Stratakis CA. Cyclical Cushing syndrome presenting in infancy: an early form of primary pigmented nodular adrenocortical disease, or a new entity? J Clin Endocrinol Metab 2004;89:3173-3182. Matyakhina L, Pack S, Kirschner LS, Pak E, Mannan P, Jaikumar J, Taymans SE, Sandrini F, Carney JA, Stratakis CA. Chromosome 2 (2p16) abnormalities in Carney complex tumours. J Med Genet 2003;40:268-277. PRKAR1A effects on protein kinase A activity and endocrine tumor development Stergiopoulos, Matyakhina, Robinson-White, Stratakis; in collaboration with Bertherat, Cho-Chung We are investigating the functional consequences of PRKAR1A mutations in cell lines established from CNC patients and their tumors. We measure both cAMP and PKA activity in the cell lines, along with the expression of the other subunits of the PKA tetramer. We have established stable transfectants of antisense PRKAR1A constructs in mouse endocrine and other cell lines that are commercially available; we study these cell lines for the effects of PRKAR1A silencing on their growth, differentiation, and proliferation. We hypothesize that the tumorigenicity of PRKAR1A-inactivating mutations relies on the switch from type-I PKA (based almost exclusively on endocrine cells in PRKAR1A) to type-II PKA activity; cell lines with an antisense PRKAR1A construct are believed to be a representative model of the in vivo situation in CNC patients. We are also looking, in sporadic endocrine and nonendocrine tumors (thyroid adenomas and carcinomas, adrenocortical adenomas and carcinomas, ovarian carcinomas, melanomas and other benign and malignant pigmented lesions, and heart myxomas), for mutations of the PRKAR1A gene that would further establish its role as a general tumor suppressor. Using specimens provided collaboratively from a variety of investigators within the NIH and around the world, we found that the PRKAR1A gene and/or locus are altered in one out of five sporadic adrenal tumors that we investigated (see Figure 3.4). Bertherat J, Sandrini F, Matyakhina L, Bei T, Stergiopoulos S, Papageorgiou T, Bourdeau I, Kirschner LS, Vincent-Dejean C, Perlemoine K, Gisquel C, Bertagna X, Stratakis CA. Molecular and functional analysis of PRKAR1A and its locus (17q22-24) in sporadic adrenocortical tumors: 17q losses, somatic mutations, and protein kinase A expression and activity. Cancer Res 2003;63:5308-5319. Bossis I, Voutetakis A, Bei T, Sandrini F, Griffin KJ, Stratakis CA. Protein kinase A and its role in human neoplasia: the Carney complex paradigm. Endocr Relat Cancer 2004;11:265-280. Prkar1a^+/- animal model and tissue-specific Prkar1a analysis Bauer, Griffin, Stergiopoulos, Claflin, Meoli, Weinberg, Stratakis; in collaboration with Kirschner, Westphal The Prkar1a-knockout (KO) (-/-) mouse, a model created several years ago by S. McKnight (University of Washington, Seattle), dies early in embryonic development because of heart and central nervous system abnormalities. Since the discovery of PRKAR1A’s involvement in CNC, Lawrence Kirschner, in collaboration with Heiner Westphal, developed a Prkar1a knockout floxed by a lox-P system to generate, first, a novel Prkar1a^+/- and, second, knockouts of the Prkar1a gene in a tissue-specific manner after crossing the new mouse model with mice that express the cre protein in the respective endocrine tissues (adrenal cortex, anterior lobe of the pituitary, and the thyroid gland). Kirschner has now moved to Ohio State University and is independently pursuing the creation of these three animal models while collaborating with us on the characterization of the novel Prkar1a^+/- mouse. Antisense Prkar1a transgenic mouse model Griffin, Stergiopoulos, Claflin, Meoli, Weinberg, Stratakis; in collaboration with Bornstein, Kirschner Given that the Prkar1a^+/- model created by McKnight et al. was not known to develop any tumors, we hypothesized that a different system would have a better chance of reproducing the human disease caused by a haploinsufficient PRKAR1A gene. Thus, in addition to the model described above, we created a transgenic (TG) mouse carrying an antisense (AS) transgene for exon 2 of the mouse Prkar1a gene (X2AS) under the control of a regulable promoter. We have now completed an assessment of cAMP-stimulated kinase activity, pathologic examination, and immunohistochemistry of the TG mice at six to eight months of age. Mice expressing the X2AS construct displayed normal reproductive behavior but showed marked differences in reproductive efficiency (presumably because those expressing high levels of X2AS died in utero). As is seen in human CNC tumors, tissues from mice with the X2AS transgene showed higher cAMP-stimulated kinase activity. Although the mice did not have tumors in endocrine tissues at this age, they exhibited some CNC-compatible histologic changes. Continuing observation of these animals and further studies may provide insight into the mechanisms leading to cAMP-related abnormal growth and proliferation (see Figure 3.3) in this syndrome. PRKAR1A, the cell cycle, chromosomal stability, mitogen-activated protein kinases (MAPK), and other signaling pathways Matyakhina, Meoli, Shiferaw, Robinson-White, Stratakis; in collaboration with Bornstein, Grimberg, Papadopoulos We aim to identify PRKAR1A-interacting mitogenic and other growth-signaling pathways in cell lines expressing PRKAR1A constructs and/or mutations. In addition, we are studying, by classic and molecular cytogenetics, including fluorescent in situ hybridization (FISH), spectral karyotyping, and comparative genomic hybridization, chromosomal stability in both human and mouse cell lines in which PRKAR1A has been inactivated. Genes implicated in cyclic nucleotide-dependent signaling have long been considered likely candidates for endocrine tumorigenesis. Somatic activating mutations in a number of G protein-coupled receptors (GPCRs) and the gene encoding a subunit of the stimulatory G protein (GNAS1) lead to increased cAMP production and are responsible for several types of endocrine tumors. To date, however, there is no convincing evidence that GNAS1 or GPCR activation, in the absence of additional genetic abnormalities, is involved in cancer. In McCune-Albright syndrome, a disease with similarities to CNC, individuals who bear somatic GNAS1 mutations in their endocrine glands may be predisposed to developing some cancers. Activation of additional pathways and/or other changes appear to be required for the in vitro transformation of 3T3 or FRTL5 cells by constitutively active GPCR transgenes or in other settings of increased cAMP signaling that lead to malignant transformation. We recently showed that the MAPK ERK1/2 pathway is activated in haploinsufficient PRKAR1A cell lines. Other genes that regulate PKA function and increase cAMP-dependent proliferation and related signals may be altered in the process of endocrine tumorigenesis initiated by a mutant PRKAR1A, a gene with important functions in the cell cycle and chromosomal stability. In collaboration with Vassilios Papadopoulos, we have identified proteins that are directly bound to PRKAR1A (RI-alpha) and regulate its function, including PAP7, a novel protein. It is generally accepted that compartmentalization of PKA function is mediated by anchoring proteins; almost all PKA anchoring proteins known to date, however, are bound to type II-PKA. PAP7 may be the first PKA type-I-specific anchoring protein. Liu J, Matyakhina L, Han Z, Sandrini F, Bei T, Stratakis CA, Papadopoulos V. Molecular cloning, chromosomal localization of human peripheral-type benzodiazepine receptor and PKA regulatory subunit type 1A (PRKAR1A)-associated protein PAP7, and studies in PRKAR1A-mutant cells and tissues. FASEB J 2003;17:1189-1191. Robinson-White A, Hundley TR, Shiferaw M, Bertherat J, Sandrini F, Stratakis CA. Protein kinase-A activity in PRKAR1A-mutant cells, and regulation of mitogen-activated protein kinases ERK1/2. Hum Mol Genet 2003;12:1475-1484. Genetic investigations on other adrenocortical diseases and tumors Bei, Bourdeau, Farrell, Stergiopoulos, Stratakis; in collaboration with Bertherat, Bossis, Brooks, Carney, Chan, Hammer, Lacroix, Libutti, Stowasser, Torpy, Voutetakis Our work, most of which we carry out collaboratively, aims to (1) identify genes with important functions in adrenal oncogenetics by using general and pathway-specific microarrays to analyze a variety of adrenocortical tumors, including single adenomas and massive macronodular adrenocortical disease (MMAD); (2) examine specific candidate genes (such as INHA, TP53, and other tumor suppressors and oncogenes) for their roles in adrenocortical tumors and development; and (3) identify by positional cloning additional genes with a role in inherited adrenocortical and related diseases. Bourdeau I, Antonini SR, Lacroix A, Kirschner LS, Matyakhina L, Lorang D, Libutti SK, Stratakis CA. Gene array analysis of macronodular adrenal hyperplasia confirms clinical heterogeneity and identifies several candidate genes as molecular mediators. Oncogene 2004;23:1575-1585. Brooks BP, Kleta R, Caruso RC, Stuart C, Ludlow J, Stratakis CA. Triple-A syndrome with prominent ophthalmic features and a novel mutation in the AAAS gene: a case report. BMC Ophthalmol 2004;4:7. Longui CA, Lemos-Marini SH, Figueiredo B, Mendonca BB, Castro M, Liberatore R Jr, Watanabe C, Lancellotti CL, Rocha MN, Melo MB, Monte O, Calliari LE, Guerra-Junior G, Baptista MT, Sbragia-Neto L, Latronico AC, Moreira A, Tardelli AM, Nigri A, Taymans SE, Stratakis CA. Inhibin alpha-subunit (INHA) gene and locus changes in paediatric adrenocortical tumours from TP53 R337H mutation heterozygote carriers. J Med Genet 2004;41:354-359. Stratakis CA. Genetics of adrenocortical tumors: gatekeepers, landscapers and conductors in symphony. Trends Endocrinol Metab 2003;14:404-410. Genetic investigations on pituitary tumors, other endocrine neoplasias, and related syndromes Bei, Bourdeau, Bauer, Farrell, Stergiopoulos, Stathatos, Stratakis; in collaboration with Francis, Marx, Ringel We are also investigating the genetics of CNC- and adrenal-related endocrine tumors, including childhood adrenocortical cancer, testicular tumors, and thyroid and pituitary masses related (or unrelated) to PRKAR1A mutations (see Figure 3.5). As part of our work, we have described novel genetic abnormalities in thyroid tumors. In addition, we are identifying the genetic defects in patients with CNC-related syndromes (the lentigenoses, i.e., Peutz-Jeghers syndrome and others). We are conducting the work largely collaboratively with a number of investigators at the NIH and elsewhere. Stratakis CA, Matyakhina L, Courkoutsakis N, Patronas N, Voutetakis A, Stergiopoulos S, Bossis I, Carney JA. Pathology and molecular genetics of the pituitary gland in patients with the ‘complex of spotty skin pigmentation, myxomas, endocrine overactivity and schwannomas’ (Carney complex). Front Horm Res 2004;32:253-264. Weeks DC, Walther MM, Stratakis CA, Hwang JJ, Linehan WM, Phillips JL. Bilateral testicular adrenal rests after bilateral adrenalectomies in a cushingoid patient with von Hippel-Lindau disease. Urology 2004;63:981-982. Clinical investigations in the diagnosis and treatment of adrenal and pituitary tumors Griffin, Bourdeau, Batista, Stratakis; in collaboration with Keil, Patronas Patients with adrenal tumors and other types of Cushing syndrome (and occasionally other pituitary tumors) come to the NIH Clinical Center for diagnosis and treatment. Ongoing investigations focus on the prevalence of ectopic hormone receptor expression in adrenal adenomas and MMAD; the diagnostic use of high-sensitivity magnetic resonance imaging for the earlier detection of pituitary tumors; and the diagnosis, management, and post-operative care of children with Cushing's syndrome and other pituitary tumors. Patronas N, Bulakbasi N, Stratakis CA, Lafferty A, Oldfield EH, Doppman J, Nieman LK. Spoiled gradient recalled acquisition in the steady state technique is superior to conventional postcontrast spin echo technique for magnetic resonance imaging detection of adrenocorticotropin-secreting pituitary tumors. J Clin Endocrinol Metab 2003;88:1565-1569. Clinical and molecular investigations of pediatric genetic syndromes Griffin, Batista, Stratakis; in collaboration with Keil, Raygada, Rennert Largely through collaboration with several investigators at the NIH and elsewhere, we are investigating pediatric genetic syndromes that are seen in our clinics and wards. Ruf RG, Xu PX, Silvius D, Otto EA, Beekmann F, Muerb UT, Kumar S, Neuhaus TJ, Kemper MJ, Raymond RM Jr, Brophy PD, Berkman J, Gattas M, Hyland V, Ruf EM, Schwartz C, Chang EH, Smith RJ, Stratakis CA, Weil D, Petit C, Hildebrandt F. SIX1 mutations cause branchio-oto-renal syndrome by disruption of EYA1-SIX1-DNA complexes. Proc Natl Acad Sci USA 2004;101:8090-8095. ^aClinical Associate, Pediatric Endocrinology Training Program ^bInstitute of Cytology and Genetics, Russian Academy of Sciences, Siberian Department, Novosibirsk, Russia ^cWalter Reed Army Medical Center, Pediatric Endocrinology Program, Bethesda, MD ^dUniversity of Thessaly, Larissa, Greece ^eUniversity of Montreal, Canada ^fEndocrinology Training Program, Washington Hospital Center, Washington, DC COLLABORATORS Jèrôme Bertherat, MD, PhD, Service des Maladies Endocriniennes et Métaboliques, Hôpital Cochin, Paris, France Stephan Bornstein, MD, PhD, University of Düsseldorf, Germany Ioannis Bossis, PhD, The Johns Hopkins University, Baltimore, MD Brian Brooks, MD, PhD, Ophthalmic Genetics and Clinical Services Branch, NEI, Bethesda, MD J. Aidan Carney, MD, PhD, Mayo Clinic, Rochester, MN Adrian Clark, MD, PhD, St. Bartholomew’s Hospital, London, UK Nickolas Courkoutsakis, MD, PhD, Diagnostic Radiology, Warren Grant Magnuson Clinical Center, Bethesda, MD Yoon S. Cho-Chung, MD, PhD, Basic Research Laboratory, NCI, Bethesda, MD Wai-Yee Chan, PhD, Laboratory of Clinical Genomics, NICHD, Bethesda, MD Gary Francis, MD, PhD, Walter Reed Army Medical Center, Bethesda, MD Adda Grimberg, MD, Children’s Hospital of Philadelphia, Philadelphia, PA Gary Hammer, MD, PhD, University of Michigan, Ann Arbor, MI Friedhelm Hildebrandt, MD, University of Michigan, Ann Arbor, MI Peter Hornsby, PhD, University of Texas Health Science Center, San Antonio, TX Meg Keil, RN, PNP, Developmental Endocrinology Branch, NICHD, Bethesda, MD Lawrence Kirschner, MD, PhD, James Cancer Hospital, Ohio State University, Columbus, OH André Lacroix, MD, PhD, Centre Hospitalier de l’Université de Montréal, Canada Stephen Libutti, MD, Center for Cancer Research, NCI, Bethesda, MD Stephen Marx, PhD, Surgery Branch, NCI, Bethesda, MD Maximilian Muenke, MD, PhD, Medical Genetics Branch, NHGRI, Bethesda, MD Vassilios Papadopoulos, PhD, Georgetown University Medical Center, Washington, DC Nickolas Patronas, MD, Diagnostic Radiology, Warren Grant Magnuson Clinical Center, Bethesda, MD Margarita Raygada, PhD, Laboratory of Clinical Genomics, NICHD, Bethesda, MD Owen M. Rennert, MD, Laboratory of Clinical Genomics, NICHD, Bethesda, MD Matthew Ringel, MD, PhD, Ohio State University, Columbus, OH Michael Stowasser, MD, University of Queensland, Australia David Torpy, MD, University of Queensland, Brisbane, Australia Antonis Votetakis, MD, Gene Therapy and Therapeutics Branch, NIDCR, Bethesda, MD Heiner Westphal, MD, PhD, Laboratory of Mammalian Genes and Development, NICHD, Bethesda, MD For further information, contact stratakc@mail.nih.gov