Molecular genetics of adrenocortical TUMORS and related disorders
Photo of Dr. Constantine Stratakis

Constantine Stratakis, MD, DSc, Head, Section on Endocrinology and Genetics

Dalia Batista, MD, Staff Fellow1

Kurt Griffin, MD, PhD, Staff Fellow1

John Bossis, PhD, Staff Scientist

Sosipatros Boikos, MD, Visiting Fellow

Anelia Horvath, PhD, Visiting Fellow

Ludmila Matyakhina, PhD, Visiting Fellow2

Sotirios Stergiopoulos, MD, Postdoctoral Fellow

Andrew Bauer, MD, Guest Researcher3

Audrey Robinson-White, PhD, Guest Researcher

Thalia Bei, PhD, Special Volunteer4

Isabelle Bourdeau, MD, Special Volunteer5

Jehan Riar, Special Volunteer

Nickolas Stathatos, MD, Special Volunteer6

Elise Meoli, BS, Predoctoral Fellow

Frank Weinberg, BS, Predoctoral Fellow

Margaret Ngyen, BS, Medical Student, Special Volunteer

Our work focuses on the genetic and molecular mechanisms leading to disorders that affect the adrenal cortex, with emphasis on disorders 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, such as familial hyperaldosteronism; adrenocortical and thyroid cancer; pituitary tumors; several 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, café-au-lait spots, and nevi). We have identified a defect on the regulatory subunit type 1-alpha (RI alpha) of protein kinase A (PKA), which is encoded by the PRKAR1A gene, as responsible for most CNC patients. Our projects rely on mouse models in which the prkar1a gene has been either knocked out or downregulated. Genome-wide screens for CNC-related disease are ongoing to identify new genes for the various disorders.

Carney complex genetics

We have collected data on families with Carney complex and related syndromes from a number of collaborating institutions worldwide. Through genetic linkage analysis, we have identified loci harboring genes for CNC on chromosomes 2 (2p16) and 17 (17q22-24); we are currently investigating  other possible loci for this genetically heterogeneous condition. Using state-of-the-art molecular cytogenetic techniques, we are investigating the participation of the genomic loci in the expression of CNC. For the cloning of the CNC-associated sequences from the 2p16 chromosomal region, we have constructed a comprehensive genetic and physical map of the 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, a central signaling pathway for many cellular functions and hormonal responses. We are now studying more patients with CNC for genotype-phenotype correlation studies, which are expected to shed light on the complex biochemical and molecular pathways regulated by PRKAR1A and PKA. We expect to identify new gene(s) by ongoing genome-wide searches for patients and families that do not carry PRKAR1A mutations.

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.

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, Aidan Carney J, 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.

Stratakis CA (updated March 2005) Carney complex. In: GeneReviews/ /at GeneTests: Medical Genetics Information Resource [database online]. Copyright, University of Washington, Seattle, 1997-2004. Available at http://www.genetests.org.

Effect of PRKAR1A on protein kinase A activity and endocrine tumor development

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 the cells’ growth, differentiation, and proliferation. We hypothesize that tumorigenicity of PRKAR1A-inactivating mutations relies on the switch from type-I PKA 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. In addition, we are seeking mutations of the PRKAR1A gene that would further establish the gene’s role as a general tumor suppressor in sporadic endocrine and nonendocrine tumors (thyroid adenomas and carcinomas, adrenocortical adenomas and carcinomas, ovarian carcinomas, melanomas, other benign and malignant pigmented lesions, and heart myxomas). Investigators within the NIH and around the world are providing the specimens on a collaborative basis. We found that the PRKAR1A gene and/or locus was altered in one out of five sporadic adrenal tumors that we investigated.

Perdigão PF, Stergiopoulos SG, De Marco L, Matyakhina L, Boikos S, Gomez RS, Pimenta FJGS, Stratakis CA. Molecular and immunohistochemical investigation of protein kinase A regulatory subunit type 1A (PRKAR1A) in odontogenic myxomas. Genes Chromosomes Cancer 2005;44:204-211.

Sandrini F, Stratakis CA. Clinical and molecular genetics of primary pigmented nodular adrenocortical disease. Arq Bras Endocrinol Metabol 2004;48:637-641.

Stergiopoulos SG, Abu-Asab MS, Tsokos M, Stratakis CA. Pituitary pathology in Carney complex patients. Pituitary 2005;7:73-82.

Stratakis CA, Bertherat J, Carney JA. Mutations of perinatal myosin heavy chain. N Engl J Med 2004;351:2556-2558.

Tsilou ET, Cha CC, Sandrini F, Rubin BI, Shen D, Carney JA, Kaiser-Kupfer M, Stratakis CA. Eyelid myxoma in Carney complex without PRKAR1A allelic loss. Am J Med Genet 2004;130A:395-397.

Prkar1a +/- animal model and tissue-specific Prkar1a analysis

The Prkarla-knockout (KO) (-/-) mouse dies early in embryonic development because of heart and central nervous system abnormalities. S. McKnight (University of Washington, Seattle) created this animal model several years ago. Since the discovery of PRKAR1A’s involvement in CNC, Lawrence Kirschner in our laboratory, in collaboration with Heiner Westphal, developed a Prkar1a KO floxed by a lox-P system to generate, first, a novel Prkar1a +/– and, second, KOs 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). The heterozygote mouse develops several tumors reminiscent of the human disease (see Figure 3.2). Ongoing crosses with mice such as the transgenic GHRH-expressing mouse attempt to identify tissue-specific effects (in the case of the GHRH-expressing mouse, the pituitary).

Figure 3.2 Tailbone tumors from the Prkar1a+/– mouse and their histological analysis

Kirschner LS, Kusewitt DF, Matyakhina L, Towns II WH, Carney JA, Westphal H, Stratakis CA. A mouse model for the Carney complex tumor syndrome develops neoplasia in cyclic AMP-responsive tissue. Cancer Res 2005;65:4506-4514.

Antisense (AS) Prkar1a transgenic (TG) mouse model

Since 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 mouse carrying an antisense transgene for exon 2 of the mouse Prkar1a gene (X2AS) under the control of a regulable promoter. Along with completion of a pathologic examination, we have now determined the cAMP-stimulated kinase activity and immunohistochemistry of the TG mice at 6 to 8 months of age. Mice expressing the X2AS construct displayed normal reproductive behavior but showed marked differences in reproductive efficiency (presumably because of the in utero death of those expressing high levels of X2AS). As is seen in human CNC tumors, tissues from mice with the X2AS transgene showed higher cAMP-stimulated kinase activity. The mice exhibited several CNC-compatible histologic and clinical changes, including obesity attributed to subclinical Cushing’s syndrome (see Figure 3.3). Continuing observation of the animals and further studies may provide insight into the mechanisms leading to cAMP-related abnormal growth and proliferation in Cushing’s syndrome.

Figure 3.3 Mice with Prkar1A downregulation develop abdominal obesity; occasionally, abdominal fat content is found in inguinal hernias.

Griffin KJ, Kirschner LS, Matyakhina L, Stergiopoulos S, Robinson-White A, Lenherr S, Weinberg F, Claflin E, Batista D, Bourdeau I, Voutetakis A, Sandrini F, Meoli E, Bauer A, Cho-Chung YS, Bornstein SR, Carney JA, Stratakis CA. A transgenic mouse bearing an antisense construct of regulatory subunit type 1A of protein kinase A develops endocrine and other tumors: comparison to Carney complex and other PRKAR1A-induced lesions. J Med Genet 2004;41:923-931.

Griffin KJ, Kirschner LS, Matyakhina L, Stergiopoulos S, Robinson-White A, Lenherr S, Weinberg W, Claflin E, Meoli E, Cho-Chung YS, Stratakis CA. Down-regulation of regulatory subunit type 1A of protein kinase A leads to endocrine and other tumors. Cancer Res 2004;64:8811-8815.

Griffin KJ, Kirschner LS, Matyakhina L, Stergiopoulos S, Robinson-White A, Weinberg F, Meoli E, Bornstein SR, Stratakis CA. A mouse model for Carney complex. Endocr Res 2004;30:903-911.

PRKAR1A, the cell cycle, chromosomal stability, mitogen-activated protein kinases (MAPK), and other signaling pathways

This line of work aims to identify PRKAR1A-interacting mitogenic and other growth-signaling pathways in cell lines expressing PRKAR1A constructs and/or mutations. In addition, using classic and molecular cytogenetics, including fluorescent in situ hybridization (FISH), spectral karyotyping (SKY), and comparative genomic hybridization (CGH), we are studying 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 endocrine tumors of various types. To date, however, there is no convincing evidence that GNAS or GPCR activation, in the absence of additional genetic abnormalities, is involved in cancer. In a disease similar to CNC, individuals with McCune-Albright syndrome (MAS), who bear somatic GNAS 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 a constitutively active GPCR transgene or in other settings of increased cAMP signaling that lead to malignant transformation. Several 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.

Bossis I, Stratakis CA. PRKAR1A: normal and abnormal functions. Endocrinology 2004;145:5452-5458.

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.

Pack SD, Qin LX, Pak E, Wang Y, Ault DO, Mannan P, Jaikumar S, Stratakis CA, Oldfield EH, Zhuang Z, Weil RJ. Common genetic changes in hereditary and sporadic pituitary adenomas detected by comparative genomic hybridization. Genes Chromosomes Cancer 2005;43:72-82.

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.

Genetic investigations into other adrenocortical diseases and tumors

Using general and pathway-specific microarrays on a variety of adrenocortical tumors, including single adenomas and massive macronodular adrenocortical disease (MMAD), we aim to identify genes with important functions in adrenal oncogenetics. We also examine specific candidate genes (such as INHA, TP53, and other tumor suppressors and oncogenes) for their roles in adrenocortical tumors and development and work to identify, by positional cloning, additional genes with a role in inherited adrenocortical and related diseases.

Elphinstone MS, Gordon RD, So A, Stratakis CA, Stowasser M. Genomic structure of the mouse and human genes for protein kinase A regulatory subunit R1-beta (PRKAR1B) on 7p22: no evidence for mutations in familial hyperaldosteronism type II in a large affected kindred. Clin Endocrinol (Oxf) 2004;61:716-723.

Longui CA, Lemos-Marini SH-V, Figureido B, Mendona BB, Castro M, Liberatore R Jr, Watanabe C, Lancelotti CLP, Rocha MN, Melo MB, Monte O, Calliari LEP, Guerra J Jr, Baptista MTM, Sbragia-Neto L, Latronico AC, Moreira A, Tardelli AMD, 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, Bossis I. Genetics of the adrenal gland. Rev Endocr Metab Disord 2004;5:53-68.

Genetic investigations on pituitary tumors, other endocrine neoplasias, and related syndromes

In collaboration with several other investigators at the NIH and elsewhere, we are focusing on the genetics of CNC- and adrenal-related endocrine tumors (see Figure 3.4), including childhood adrenocortical cancer, testicular tumors, and thyroid and pituitary masses related (or unrelated) to PRKAR1A mutations. 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).

Figure 3.4 CT scan showing bilateral adrenocortical hyperplasia (white arrows) in a patient with hereditary leiomyomatosis and renal
cancer syndrome.

Bauer AJ, Stratakis CA. The lentiginoses: cutaneous markers of systemic disease and a window to new aspects of tumorigenesis. J Med Genet 2005[E-pub ahead of print].

Brooks BP, Kleta R, Stuart C, Tuchman M, Bjornson B, Russel L, Chanoine J-P, Tsagarakis S, Kalsner LR, Stratakis CA. Genotypic heterogeneity and clinical phenotype in triple A syndrome: a review of the NIH experience 2000-2004. Clin Genet 2005;68:215-221.

Stathatos N, Bourdeau I, Espinosa AV, Saji M, Vasko VV, Burman KD, Stratakis CA, Ringel M. KiSS-1/GPR54 metastasis suppressor pathway increases MCIP-1 expression and chronically inhibits calcineurin activity. J Clin Endocrinol Metab 2005;90:5432-5440.

Stratakis CA. Applications of genomic medicine in endocrinology and post-genomic endocrine research. Hormones 2005;4:38-44.

Stratakis CA, Marx SJ. Multiple endocrine neoplasias in the era of translational medicine. Horm Metab Res 2005;37:343-346.

Clinical investigations in the diagnosis and treatment of adrenal and pituitary tumors

Patients with adrenal tumors (see Figure 3.4) and other types of Cushing’s syndrome (and occasionally other pituitary tumors) come to the NIH Clinical Center for diagnosis and treatment. Our ongoing investigations focus on (1) the prevalence of ectopic hormone receptor expression in adrenal adenomas and massive macronodular adrenocortical disease; (2) the diagnostic use of high-sensitivity magnetic resonance imaging for the earlier detection of pituitary tumors; and (3) the diagnosis, management, and post-operative care of children with Cushing’s disease and other pituitary tumors.

Batista D, Courkoutsakis NA, Oldfield EH, Griffin KJ, Keil M, Patronas NJ, Stratakis CA. Detection of ACTH-secreting pituitary adenomas by magnetic resonance imaging (MRI) in children and adolescents with Cushing disease. J Clin Endocrinol Metab 2005;90:5134-5140.

Frank GR, Speiser PW, Griffin KJ, Stratakis CA. Safety of medications used in pediatric endocrinology: adrenal. Pediatr Endocrinol 2004;Rev 2(Supplement 1):134-145.

Matyakhina L, Freedman RJ, Bourdeau I, Stergiopoulos S, Chidakel A, Walther M, Abu-Asab M, Tsokos M, Keil M, Toro J, Linehan WM, Stratakis CA. Hereditary leiomyomatosis associated with bilateral, massive, macronodular adrenocortical disease and atypical Cushing syndrome: a clinical and molecular genetic investigation. J Clin Endocrinol Metab 2005;90:3773-3779.

Merke DP, Giedd JN, Keil MF, Mehlinger SL, Wiggs E, Holzer S, Rawson E, Vaituzis AC, Stratakis CA, Chrousos GP. Children experience cognitive decline despite reversal of brain atrophy one year following resolution of Cushing syndrome. J Clin Endocrinol Metab 2005;90:2531-2536.

Stergiopoulos S, Torpy DJ, Stratakis CA. Primary aldosteronism: pathophysiology, diagnosis and management. In: Schwarz AE, Pertsemlidis D, Gagner M, eds. Endocrine Surgery, Chapter 34. New York: Marcel Dekker, 2004;4297-4304.

Clinical and molecular investigations of pediatric genetic syndromes

In collaboration with a number of other investigators at the NIH and elsewhere, we are working on pediatric genetic syndromes seen in our clinics and wards.

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. BMC Ophthalmology 2004;4:7.

Lee JS, Tartaglia M, Gelb BD, Fridrich K, Sachs S, Stratakis CA, Muenke M, Robey PG, Collins MT, Slavotinek A. Phenotypic and genotypic characterization of Noonan-like/multiple giant cell lesion syndrome. J Med Genet 2005;42:e11.

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.

Stratakis CA, Rennert OM. Turner syndrome: an update. Endocrinologist 2005;15:27-36.

Tilkeridis C, Bei T, Garantziotis S, Stratakis CA. Association of a COL1A1 polymorphism with lumbar disk disease in young military recruits. J Med Genet 2005;42:e44.

1Clinical Associate, Pediatric Endocrinology Training Program, NICHD

2Institute of Cytology and Genetics, Russian Academy of Sciences, Siberian Department, Novosibirsk, Russia

3Walter Reed Army Medical Center, Pediatric Endocrinology Program, Bethesda, MD

4University of Thessaly, Larissa, Greece

5University of Montreal, Montreal, Canada

6Endocrinology Training Program, Washington Hospital Center, Washington, DC

7William Farrell, PhD, former Guest Researcher

Collaborators

Jérôme Bertherat, MD, PhD, Service des Maladies Endocriniennes et Métaboliques, Hôpital Cochin, Paris, France

Stephan Bornstein, MD, PhD, Universität Düsseldorf, Düsseldorf, Germany

Brian Brooks, MD, PhD, Ophthalmic Genetics and Clinical Services Branch, NEI, Bethesda, MD

J. Aidan Carney, MD, PhD, Mayo Clinic, Rochester, MN

Wai-Yee Chan, PhD, Laboratory of Clinical Genomics, NICHD, Bethesda, MD

Yoon S. Cho-Chung, MD, PhD, Basic Research Laboratory, NCI, Bethesda, MD

Adrian Clark, MD, PhD, St. Bartholomew’s Hospital, London, UK

Nickolas Courkoutsakis, MD, PhD, Diagnostic Radiology, Warren G. Magnuson Clinical Center, Bethesda, MD

Gary Francis, MD, PhD, Uniformed Services University of the Health Sciences, 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, 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 G. 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, Department of Internal Medicine, Ohio State University, Columbus, OH

Michael Stowasser, MD, University of Queensland, Brisbane, Australia

David Torpy, MD, University of Queensland, Brisbane, Australia

Heiner Westphal, MD, PhD, Laboratory of Mammalian Genes and Development, NICHD, Bethesda, MD

For further information, contact stratakc@cc1.nichd.nih.gov.

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