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Molecular Genetics of Endocrine Tumors and Related Disorders (Z01-HD008920-01)

• Constantine Stratakis, MD, D(med)Sci, Head, Section on Endocrinology and Genetics
• Urania Dagalakis, NIH Summer Student Program
• Fabio Faucz, PhD, Visiting Scientist
• Anelia Horvath, PhD, Research Fellow
• Isaac Levy, PhD, Visiting Fellow
• Maya Lodish, MD, Assistant Clinical Investigator
• Charalampos Lyssikatos, MD, Research Associate
• Panagiotis Mastorakos, NIH Summer Student Program
• Spyridon Mastroyannis, NIH Summer Student Program
• Nima Miraftab, BS, Special Volunteer
• Maria Nesterova, PhD, Staff Scientist
• Niloofar Rezvani, PhD, Visiting Scientist
• Anya Rothenbuhler, MD, Visiting Scientist
• Emmanouil Saloustros, MD, Visiting Scientist
• Eva Szarek, BS, Visiting Scientist
• Paraskevi Xekouki, MD, Visiting Fellow
• Jennifer Gourgari, MD, Clinical Fellow
• Andreas Moraitis, MD, Clinical Fellow
• Edra London, PhD, Research Fellow
• Madelauz (Lucy) Sierra, BS, Research Assistant
• Jenna Shapiro, BS, Graduate Student
• Sophie (Sisi) Liu, BS, Graduate Student

The current project was proposed in SEGEN's first year of funding and was based on the hypothesis that studying hyperplasias of the adrenal cortex was likely to "identify molecular pathways involved in the first steps of tumor formation." This approach (in part forced by the reality of an NICHD-based Medical Genetics and Pediatric Endocrinology laboratory's lack of routine access to patients with endocrine cancers) has led to fruitful research over the last one-and-a-half decades: Our first studies led to the identification of the main regulator of the cAMP-signaling pathway, regulatory subunit-type 1A (R1a) of PKA (coded by the PRKAR1A gene on chromosome 17q22-24), as responsible for primary pigmented nodular adrenocortical disease (PPNAD) and Carney complex, a multiple endocrine neoplasia (MEN) whose main endocrine manifestation is PPNAD. We then focused on trying to delineate clinically the various types of primary bilateral adrenal hyperplasias (BAH). We described isolated micronodular adrenocortical disease (iMAD), a disorder likely to be inherited in an autosomal dominant manner that is unrelated to Carney complex or other MENs: the identification of PRKAR1A mutations in PPNAD led to the recognition that non-pigmented forms of BAHs existed, and a new nomenclature was proposed that we actually suggested first in 2008 that has since been in use worldwide. In 2006, a GWA study led to the identification of mutations in phosphodiesterase (PDE) genes, PDE11A -a dual specificity PDE, and PDE8B, a cAMP-specific PDE (coded by the PDE11A and PDE8B genes, respectively) in iMAD. Following the identification of cAMP/PKA involvement in PPNAD and iMAD, we and others found that increased cAMP levels and/or PKA activity and abnormal PDE activity may be found in most benign adrenal tumors (ADTs), including the common adrenocortical adenoma (ADA). We then found PDE11A and PDE8B mutations or functional variants in adrenocortical cancer (ACA), and other forms of adrenal hyperplasia like massive macronodular adrenocortical disease (MMAD), also known as ACTH-independent adrenocortical hyperplasia (MMAD/AIMAH). Germline PDE11A sequence variants may also predispose to testicular cancer (testicular germ cell tumors or TGCTs) and prostate cancer, indicating a wider role of this pathway in tumor formation on cAMP-responsive, steroidogenic, or related tissues. Ongoing work with collaborating NCI laboratories aims at clarifying the role of PDE in predisposition to these tumors. It is clear from these data, however, that there is significant pleiotropy of PDE11A and -8B defects. Histo-morphological studies that we perfomed on human adrenocortical tissues from patients with these mutations showed that iMAD is highly heterogeneous and, thus, likely to be caused by several genes of the cAMP/PKA-signaling pathway or its regulators and/or downstream effectors. Likewise, the G-protein coupled receptor (GPCR)-linked MMAD/AIMAH is a disease that includes a range of adrenal phenotypesfrom very similar to iMAD to the GNAS-caused primary bimorhic adrenocortical disease (PBAD) and McCune-Albright syndrome , caused by somatic mutations of the GNAS gene (coding for the G-protein stimulatory subunit alpha or Gsa). Although a few of the patients with MMAD/AIMAH have germline PDE11A, PDE8B, and somatic GNAS mutations others have germline fumarate hydratase (FH) , menin (29, 35, 36), and adenomatous polyposis coli (APC) mutations pointing to the range of possibilities and the pathways that may be involved. From these, particularly interesting is the connection with FH mutations associated with mitochondrial oxidation defects that have been linked to adrenomedullary tumors. This led us to investigate a disorder known as Carney Triad, the only known disease that has among its clinical manifestations both adreno-cortical (ADA, MMAD/AIMAH) and medullary tumors (pheochromocytomas [PHEOs] and paragangliomas [PGLs]), in addition to hamartomatous lesions in various organs (pulmonary chondromas, pigmented and other skin lesions) and a predisposition to gastrointestinal stromal tumors or sarcomas (GISTs) . A subgroup of patients with PHEOs, PGLs and GISTs were identified to harbor mutations in succinate dehydrogenase (SDH) subunits B, C and D (coded by the SDHB, SDHC and SDHD genes, respectively) ; they also rarely have adrenocortical lesions, ADAs and/or hyperplasia, and their disease is known as the dyad or syndrome of PGLs and GISTs or, as named by a group of pathologists and now in wide use, Carney-Stratakis syndrome (CSS).

PPNAD appears to be less heterogeneous and is mostly caused by PRKAR1A mutations but up to 1/3 of patients with the classic features of PPNAD do not have PRKAR1A mutations, deletions or 17q22-24 copy-number variant (CNV) abnormalities. A subset of these patients may have defects in other molecules of the PKA holoenzyme and their study is important for understanding how PKA works and the tissue-specificity of each defect. About 1/4 of all patients with PPNAD do not have PRKAR1A defects. For patients with disorders that are yet to be molecularly elucidated, we continue the delination of the phenotypes and identification of the responsible genetic defects through a combination of genomic and transcriptomic analyses.

Animal model studies are essential for the investigation and confirmation of each of the identified new genes in disease pathogenesis. Furthermore, they provide insight for a function that can be tested quickly in human samples for confirmation of its relevance to human disease. One great example of that bench-to-bedside (and back) process in the last review cycle was the identification from a variety of animal experiments of the Wingless/int (Wnt)-signaling as one of the downstream effectors of tumor formation in the context of increased cAMP/PKA activity. Both our laboratory and our collaborators found somatic b-catenin (CTNNB1) mutations in large ADAs that formed in the background of PPNAD caused by germline PRKAR1A mutations. Our transcriptomic studies had previously identified WNT1-inducible signaling pathway protein 2 (WISP2) as the main molecule overexpressed in food-dependent Cushing syndrome caused by MMAD/AIMAH, and our recent micro-RNA studies showed that genes that regulate Wnt-signaling were major targets of micro-RNAs that were found dysregulated in both PPNAD and MMAD/AIMAH. Cells from tumors or other lesions from animals with R1a deficiency showed increased b-catenin expression and/or aberrant Wnt-signaling and similarities to adult stem cells or cancer stem cells in other models of ysregulated Wnt-signaling. However, it appears that b-catenin activation in R1a-deficient cells is an event preceded by yet unknown molecular abnormalities that take place within the still benign and R1a-haploinsufficient tissues in the early stages of tumor formation.

We continue the investigation of the pathways involved in early events in tumor formation in the adrenal cortex and/or the tissues affected by germline or somatic defects of the cAMP/PKA and related endocrine signaling defects employing animal models, transcriptomic and systems biology analyses. Understanding the role of the other PKA subunits in this process is essential.  An example of the combined use of whole genomic tools, transcriptomic analysis and mouse and zebrafish models to investigate the function of a gene or a pathway is the ongoing work in Carney Triad.

The discovery that neural crest, heart, adrenal -specific knockouts (KO) of R1a or other mice with R1a defects develop lesions caused by proliferation of stem cell-like, tissue-specific pluripotential cells (TSPCs) in adult tissues, such as the adult skeleton was an important one in the last 3-4 years. In our laboratory, we studied the bone and the adrenal cortex. Since it appears that various models of R1a deficiency feature the growth of lesions derived from TSPCs, we now focus on the characterization of these cells in adrenal and bone, the creation of the laboratory conditions (i.e. culture systems) to propagate these cells in vitro, study their growth and proliferation, and exploit their therapeutic potential and/or identify molecules that affect these cells for targeting the related tumors in humans. The establishment of an in vitro system to test cAMP/PKA responsive TSPCs is an essential goal and critical for our future therapeutical efforts.

Finally, we continue to accrue patients under a number of clinical protocols and the identification of unique patients, families with rare phenotypes, and/or explore (mostly on a collaborative basis) various aspects of endocrine and related diseases. Paramount to these investigations is the availability of modern genetic tools like CNV analysis, comparative genomic hybridization (CGH), WES and DSeq. As part of these clinical protocols, some clinical research is also being done: it consists mostly of observations of new associations and the description of novel applications or modifications and improvements of older diagnostic methods, tests or imaging tools. This is a particularly fruitful area of research especially for our clinical fellows who matriculate at our laboratory during their 2 year-long research time.

Carney complex genetics

We have collected families with CNC and related syndromes from several collaborating institutions worldwide. Through genetic linkage analysis, we identified loci harboring genes for CNC on chromosomes 2 (2p16) and 17 (17q22–24) and are currently searching for other possible loci for this genetically heterogeneous condition. With the application of state-of-the-art molecular cytogenetic techniques, we are investigating the participation of these currently identified genomic loci in the expression of the disease and have constructed a comprehensive genetic and physical map of the 2p16 chromosomal region for cloning the CNC-associated sequences from this 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 (subunit type 1-α) of PKA, a central signaling pathway for many cellular functions and hormonal responses. We have increased the number of CNC patients in genotype-phenotype correlation studies, which are expected to provide insight into the complex biochemical and molecular pathways regulated by PRKAR1A and PKA. We expect to identify new genes by ongoing genome-wide searches for patients and families who do not carry PRKAR1A mutations.

PRKAR1A, protein kinase A activity, and endocrine and other tumor development

We are investigating the functional and genetic consequences of PRKAR1A mutations in cell lines established from CNC patients and their tumors. We measure both cAMP and PKA activity in these cell lines, along with the expression of the other subunits of the PKA tetramer. In addition, we are seeking mutations of the PRKAR1A gene in sporadic endocrine and non-endocrine tumors (thyroid adenomas and carcinomas, adrenocortical adenomas and carcinomas, ovarian carcinomas, melanomas and other benign and malignant pigmented lesions, and myxomas in the heart and other sites)—mutations that would further establish the gene's role as a general tumor suppressor. Many investigators within the NIH and around the world provide specimens on a collaborative basis.

Prkar1a ^+/− and antisense (AS) Prkar1a transgenic animal models

In collaboration with Heiner Westphal, Lawrence Kirschner, while in our laboratory, developed a Prkar1a knockout mouse floxed by a lox-P system for the purpose of generating, first, a novel Prkar1a ^+/- and, second, knockouts of the Prkar1a gene in a tissue-specific manner after crossing the new mouse model with mice expressing the cre protein in the adrenal cortex, anterior lobe of the pituitary, and thyroid gland (Kirschner et al., Cancer Res 2005;65:4506). The heterozygote mouse develops several tumors reminiscent of the equivalent human disease. Ongoing crosses with mice such as the transgenic GHRH–expressing mouse attempt to identify tissue-specific effects (the pituitary in the case of the GHRH–expressing mouse) or specific signaling events (such as involvement of the p53 and Rb proteins in Prkara1a-related tumorigenesis). We also created a transgenic mouse carrying an antisense transgene for exon 2 of the mouse Prkar1a gene (X2AS) under the control of a regulatable promoter. As in human CNC tumors, tissues from mice with the X2AS transgene showed elevated cAMP-stimulated kinase activity. The mice had several CNC-compatible histologic and clinical changes, including obesity attributed to subclinical Cushing syndrome.

PRKAR1A, the cell cycle, and other signaling pathways

We work to identify PRKAR1A-interacting mitogenic and other growth-signaling pathways in cell lines expressing PRKAR1A constructs and/or mutations. 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 in chromosomal stability. Recently, we found an interaction with the mTOR pathway in both human and mouse cells with altered PKA function.

Phosphodiesterase (PDE) genes in endocrine and other tumors

In patients who did not exhibit CNC or PRKAR1A mutations but presented with bilateral adrenal tumors similar to those in CNC, we found inactivating mutations of the PDE11A gene, which encodes phosphodiesterase-11A that regulates PKA in the normal physiologic state. Phosphodiesterase 11A is a member of a 22 gene–encoded family of proteins that break down cyclic nucleotides controlling PKA. PDE11A appears to act as a tumor suppressor such that tumors develop when its action is abolished. In what proved to be the first cases in which mutated PDE was observed in a genetic disorder predisposing to tumors, we found pediatric and adult patients with bilateral adrenal tumors. Recent data indicate that PDE11A sequence polymorphisms may be present in the general population. The finding that genetic alterations of such a major biochemical pathway may be associated with tumors in humans raises the reasonable hope that drugs that modify PKA and/or PDE activity may eventually BE developed for use in both CNC and patients with other, non-genetic, adrenal tumors—and perhaps other endocrine tumors. Most recently, we identified a patient with a PDE8B mutation and Cushing syndrome, with the PDE8B transcript and protein seemingly expressed widely in the endocrine system.

Genetic investigations into other adrenocortical diseases and related tumors

Through collaborations, we (1) apply general and pathway-specific microarrays to a variety of adrenocortical tumors, including single adenomas and massive macronodular adrenocortical disease (MMAD), to identify genes with important functions in adrenal oncogenetics; (2) examine candidate genes for their roles in adrenocortical tumors and development; and (3) identify additional genes that play a role in inherited adrenocortical and related diseases, such as Allgrove syndrome.

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

In collaboration with several other investigators at the NIH and elsewhere, we are investigating the genetics of CNC- and adrenal-related endocrine tumors, including childhood pituitary tumors, related or unrelated to PRKAR1A mutations. As part of this work, we have identified novel genetic abnormalities in other endocrine glands.

Genetic investigations into other endocrine neoplasias and related syndromes; hereditary paragangliomas and related conditions

As part of a collaboration with other investigators at the NIH and elsewhere (including an international consortium organized by our laboratory), we are studying the genetics of a rare syndrome that predisposes to adrenal and other tumors, the Carney Triad, and related conditions (associated with gastrointestinal stromal tumors, or GIST). In the course of our work, we identified a patient with a new syndrome, known as the paraganglioma and gastrointestinal stromal tumor syndrome (or Carney-Stratakis syndrome), for which we found mutations in the genes encoding succinate dehydrogenase (SDH) subunits B, C, and D. In another patient, we found a novel germline mutation of the PDFGRA gene.

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

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 (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 syndrome and other pituitary tumors.

Clinical and molecular investigations into other pediatric genetic syndromes

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

Additional Funding

• INSERM, Paris, France: Co-Principal Investigator, Clinical and molecular genetics of Carney complex, 06/2003-present (480,000 Euros/year).
• NIH Bench to Bedside Award 2010: "Adrenal hyperplasia in patients with PCOS" 07/2010-06/2012 ($135K/year)

Publications

• Xekouki P, Pacak K, Almeida M, Wassif CA, Rustin P, Nesterova M, de la Luz Sierra M, Matro J, Ball E, Azevedo M, Horvath A, Lyssikatos C, Quezado M, Patronas N, Ferrando B, Pasini B, Lytras A, Tolis G, & CA Stratakis. Succinate dehydrogenase (SDH) D subunit (SDHD) inactivation in a growth-hormone-producing pituitary tumor: A new association for SDH? J Clin Endocrinol Metab 2012 97:E357-66.
• Almeida MQ, Azevedo M, Xekouki P, Horvath A, Bimpaki E, Collins M, Bhattacharyya N, Karaviti LP, Jeha GS, Cheadle C, Watkins T, Bourdeau I, Nesetrova M, & CA Stratakis. Activation of cyclic AMP signaling leads to different pathway alterations in lesions of the adrenal cortex caused by germline PRKAR1A defects versus those due to somatic GNAS mutations. J Clin Endocrinol Metab 2012 97:687-693.
• Lodish M, Dunn SV, Sinaii N, Keil MF, & CA Stratakis. 97(5):1483-91, 2012. Recovery of the hypothalamic-pituitary- adrenal axis in children and adolescents after surgical cure of Cushing's disease. J Clin Endocrinol Metab. 2012 97:1483-91.
• Carney JA, Ho J, Kitsuda K, Young WF Jr, & CA Stratakis. Massive neonatal adrenal enlargement due to cytomegaly, persistence of the transient cortex, and hyperplasia of the permanent cortex: findings in Cushing syndrome associated with hemihypertrophy. Am J Surg Pathol 2012 36:1452-1463.
• Zhuang Z, Yang C, Lorenzo F, Merino M, Fojo T, Kebebew E, Popovic V, Stratakis CA, Prchal JT, Pacak K. Somatic HIF2A gain-of-function mutations in paraganglioma with polycythemia. N Engl J Med. 2012 367:922-30.

Collaborators

• Jérôme Bertherat, MD PhD, Service des Maladies Endocriniennes et Mètaboliques, Hôpital Cochin, Paris, France
• Stephan Bornstein, MD PhD, Universität Dresden, Dresden, Germany
• Isabelle Bourdeau, MD, Universitè of Montrèal, Montrèal, Canada
• J. Aidan Carney, MD PhD, Mayo Clinic, Rochester, MN
• Nickolas Courkoutsakis, MD PhD, University of Thrace, Alexandroupolis, Greece
• Meg Keil, RN PNP, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
• Lawerence Kirschner, MD PhD, James Cancer Hospital, Ohio State University, Columbus, OH
• Andre Lacroix, MD PhD, Centre Hospitalier de l'Universitè de Montrèal, Montrèal, Canada
• Nickolas Patronas, MD, Diagnostic Radiology, Clinical Center, NIH, Bethesda, MD
• Margarita Raygada, PhD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
• David Torpy, MD, University of Queensland, Brisbane, Australia

Contact

For more information, email stratakc@mail.nih.gov or visit segen.nichd.nih.gov.

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