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LABORATORY OF ENDOCRINOLOGY AND GENETICS Heritable Disorders Branch, NICHD, NIH, Bethesda, MD Head: CONSTANTINE STRATAKIS, MD BACKGROUND The goal of our work is to understand the genetic and molecular mechanisms leading to disorders that affect the adrenal cortex, with emphasis on those that are developmental, hered- itary and associated with adrenal hypoplasia or hyperplasia, multiple tumors and abnormali- ties 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 mul- tiple 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 the gonads, and is asso- ciated with a variety of other tumors, including myxomas and schwannomas, and skin pigmen- tation defects (lentigines, café-au-lait spots, and nevi). We identified the regulatory subunit type 1-a (RIa of protein kinase A (PKA), which is coded by the PRKAR1A gene, as the defect responsible for most CNC patients and phosphodiesterase-11A (PDE11A) mutated in patients with isolated adrenal hyperplasia and Cushing syndrome. Thus, a significant part of our work is currently focused on cyclic AMP (cAMP)/PKA-stimulated signaling pathways, PKA effects on tumor suppression and/or development, the cell cycle and chromosomal stability. These proj- ects are facilitated by prkar1a and pde11a gene mouse models in which the respective genes been knocked out. Genome-wide searches for other genes responsible for CNC and related dis- eases of the adrenal and pituitary glands are ongoing. RESEARCH 1. Carney complex genetics Families with Carney complex (CNC) and related syndromes have been collected from a number of collaborating institutions worldwide. Loci harboring genes for CNC on chromo- somes 2 (2p16) and 17 (17q22-24) were identified through genetic linkage analysis; possible other loci for this genetically heterogeneous condition are currently under investigation. With the application of state-of-the-art molecular cytogenetic techniques, the participation of these genomic loci in the expression of the disease is being investigated. A comprehensive genetic and physical map of the 2p16 chromosomal region has been constructed for the cloning of 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 this 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 of the cellular functions and hormonal responses. More patients with CNC are now being studied for genotype-phenotype correlation studies, which are expected to shed light into the complex biochemical and molecular pathways regulated by PRKAR1A and PKA. New gene(s) are expected to be identified by ongoing genome-wide searches for patients and families that do not carry PRKAR1A mutations. 2. PRKAR1A, protein kinase A activity and endocrine and other tumor devel- opment The functional and genetic consequences of PRKAR1A mutations are being investigated in LABORATORY OF ENDOCRINOLOGY AND GENETICS 156

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Page 1: LABORATORY OF ENDOCRINOLOGY AND GENETICS Heritable Disorders …molmedgp.med.uoc.gr/vml-mysite/arxeia/molmedgp-guide-2007/mol… · LABORATORY OF ENDOCRINOLOGY AND GENETICS Heritable

LABORATORY OF ENDOCRINOLOGY AND GENETICSHeritable Disorders Branch, NICHD, NIH, Bethesda, MD

Head: CONSTANTINE STRATAKIS, MD

BACKGROUND

The goal of our work is to understand the genetic and molecular mechanisms leading todisorders that affect the adrenal cortex, with emphasis on those that are developmental, hered-itary and associated with adrenal hypoplasia or hyperplasia, multiple tumors and abnormali-ties in other endocrine glands (especially the pituitary gland and to a lesser extent the thyroidgland). We have studied congenital adrenal hypoplasia caused by triple A syndrome and mul-tiple 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. CNCis a MEN syndrome affecting the pituitary, adrenal cortex, thyroid, and the gonads, and is asso-ciated with a variety of other tumors, including myxomas and schwannomas, and skin pigmen-tation defects (lentigines, café-au-lait spots, and nevi). We identified the regulatory subunittype 1-a (RIa of protein kinase A (PKA), which is coded by the PRKAR1A gene, as the defectresponsible for most CNC patients and phosphodiesterase-11A (PDE11A) mutated in patientswith isolated adrenal hyperplasia and Cushing syndrome. Thus, a significant part of our workis currently focused on cyclic AMP (cAMP)/PKA-stimulated signaling pathways, PKA effects ontumor suppression and/or development, the cell cycle and chromosomal stability. These proj-ects are facilitated by prkar1a and pde11a gene mouse models in which the respective genesbeen knocked out. Genome-wide searches for other genes responsible for CNC and related dis-eases of the adrenal and pituitary glands are ongoing.

RESEARCH

1. Carney complex geneticsFamilies with Carney complex (CNC) and related syndromes have been collected from a

number of collaborating institutions worldwide. Loci harboring genes for CNC on chromo-somes 2 (2p16) and 17 (17q22-24) were identified through genetic linkage analysis; possibleother loci for this genetically heterogeneous condition are currently under investigation. Withthe application of state-of-the-art molecular cytogenetic techniques, the participation of thesegenomic loci in the expression of the disease is being investigated. A comprehensive geneticand physical map of the 2p16 chromosomal region has been constructed for the cloning of theCNC-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 inthe center of this map. The PRKAR1A gene on 17q22-24, the gene responsible for CNC in mostcases 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 signalingpathway for many of the cellular functions and hormonal responses. More patients with CNCare now being studied for genotype-phenotype correlation studies, which are expected to shedlight into the complex biochemical and molecular pathways regulated by PRKAR1A and PKA.New gene(s) are expected to be identified by ongoing genome-wide searches for patients andfamilies that do not carry PRKAR1A mutations.

2. PRKAR1A, protein kinase A activity and endocrine and other tumor devel-opment

The functional and genetic consequences of PRKAR1A mutations are being investigated in

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cell lines established from CNC patients and their tumors. Both cAMP and PKA activity aremeasured in these cell lines, along with the expression of the other subunits of the PKAtetramer (see Figure 1). It is hypothesized that tumorigenicity of PRKAR1A-inactivating muta-tions relies on the switch from type-I PKA (based almost exclusively on endocrine cells onPRKAR1A) to type-II PKA activity; cell lines with an antisense PRKAR1A construct are believedto be a representative model of the in vivo situation in CNC patients. In addition to the above,mutations of the PRKAR1A gene, that would further establish its role as a general tumor sup-pressor, are being sought in sporadic endocrine and non-endocrine tumors (thyroid adenomasand carcinomas, adrenocortical adenomas and carcinomas, ovarian carcinomas, melanomasand other benign and malignant pigmented lesions, myxomas in the heart and other sites)-these specimens are provided on a collaborative basis from a variety of investigators within theNIH and around the world.

3. Prkar1a +/- and antisense (AS) Prkar1a transgenic animal models Since the discovery of PRKAR1A's involvement in CNC, Dr. Kirschner in our laboratory,

in collaboration with Dr. Westphal (NICHD, NIH) developed a Prkar1a KO floxed by a lox-Psystem to generate, first, a novel Prkar1a +/- and, second, KO's of the Prkar1a gene in a tissue-specific manner after crossing this new mouse model with mice that express the cre protein inthe respective endocrine tissues (adrenal cortex, anterior lobe of the pituitary and the thyroidgland). The heterozygote mouse develops several tumors reminiscent of the human disease.Ongoing crosses with mice such as the transgenic GHRH-expressing mouse attempt to identi-fy tissue-specific effects (in the case of the GHRH-expressing mouse, the pituitary), or specificsignaling events (like involvement of the p53 and Rb proteins in prkara1a-related tumorigene-sis). In addition to the model described above, we created a transgenic mouse carrying an anti-sense transgene for exon 2 of the mouse Prkar1a gene (X2AS) under the control of a regulablepromoter. As human CNC tumors, tissues from mice with the X2AS transgene showed highercAMP-stimulated kinase activity.These mice had several CNC-compatible histologic and clini-cal changes, including obesity attributed to sub-clinical Cushing syndrome.

4. PRKAR1A, the cell cycle, chromosomal stability, mitogen-activated proteinkinases (MAPK) and other signaling pathways

This line of work aims at identifying PRKAR1A - interacting mitogenic and other growthsignaling pathways in cell lines expressing PRKAR1A contructs and/or mutations. In addition,chromosomal stability in both human and mouse cell lines in which PRKAR1A has been inac-tivated is being studied by classic and molecular cytogenetics, including fluorescent in situhybridization (FISH), spectral karyotyping (SKY) and comparative genomic hybridization(CGH). Genes implicated in cyclic nucleotide-dependent signaling have long been consideredlikely candidates for endocrine tumorigenesis. Somatic activating mutations in a number of G-protein coupled receptors (GPCRs) and the gene coding for a subunit of the stimulatory G pro-tein (GNAS) lead to increased cAMP production and are responsible for a number of endocrinetumors of various types. Several genes that regulate PKA function and increase cAMP-depend-ent proliferation and related signals may be altered in the process of endocrine tumorigenesisinitiated by a mutant PRKAR1A, a gene with important functions on the cell cycle and on chro-mosomal stability.

5. Phosphodiesterase (PDE) genes in endocrine and other tumorspatients that did not have CNC or PRKAR1A mutations but had bilateral adrenal tumors

like those in CNC, inactivating mutations of the PDE11A gene (Figure 2) that regulates PKA inthe normal, physiologic state were found. Phosphodiesterase 11A is a member of a 22-gene fam-

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ily of proteins that break down cyclic nucleotides that control PKA. PDE11A appears to act as atumor suppressor, so that when its action is abolished tumors develop: this was shown in pedi-atric and adult patients with bilateral adrenal tumors. This is the first time that any PDE isfound mutated in a genetic disorder predisposing to tumors. The finding that genetic alter-ations of such a major biochemical pathway may be associated with tumors in humans raisesthe reasonable hope that eventually drugs that modify PKA and/or PDE activity will be devel-oped for use in both CNC and general patients with other, non-genetic, adrenal and perhapsother endocrine tumors.

6. Genetic investigations on other adrenocortical diseases, neoplasias andrelated syndromes.

This work aims at (a) using general and pathway-specific microarrays on a variety ofadrenocortical tumors, including single adenomas and massive macronodular adrenocorticaldisease (MMAD) (Figure 3), to identify genes with important functions in adrenal oncogenet-ics; (b) examining specific candidate genes for their roles in adrenocortical tumors and devel-opment; (c) identifying additional genes with a role in inherited adrenocortical and related dis-eases, such as for example Allgrove syndrome. Additional work in our laboratory is being doneon the genetics of CNC- and adrenal- related endocrine tumors, including childhood adreno-cortical cancer, testicular tumors, thyroid and pituitary masses related or unrelated toPRKAR1A mutations. As part of this work, novel genetic abnormalities in thyroid and otherendocrine tumors have been described. The genetic defects in patients with related syndromes(the lentigenoses, i.e., Peutz-Jeghers syndrome and others, the Carney Triad) are also beingidentified. This is work that is done largely collaboratively with a number of other investigatorsat the NIH and elsewhere.

REPRESENTATIVE PUBLICATIONS

1. Horvath A, Boikos S, Giatzakis C, Robinson-White A, Groussin L, Griffin KJ, Stein E,Levine E, Delimpasi G, Hsiao HP, Keil M, Heyerdahl S, Matyakhina L, Libe R, Fratticci A,Kirschner LS, Cramer K, Gaillard RC, Bertagna X, Carney JA, Bertherat J, Bossis I, StratakisCA. A genome-wide scan identifies mutations in the gene encoding phosphodiesterase 11A4(PDE11A) in individuals with adrenocortical hyperplasia. Nat Genet. 2006;38:794-800.

2. Kirschner LS, Kusewitt DF, Matyakhina L, Towns II WH, Carney JA, Westphal H & CAStratakis. A mouse model for the Carney complex tumor syndrome develops neoplasia in cyclicAMP-responsive tissue. Cancer Res 65:4506-14, 2005.

3. Tilkeridis C, Bei T, Garantziotis S & CA Stratakis. Association of a COL1A1 polymor-phism with lumbar disk disease in young military recruits. J Med Genet 42(7):e44, 2005.

4. Brooks BP, Kleta R, Stuart C, Tuchman M, Jeong A, Stergiopoulos SG, Bei T, BjornsonB, Russell L, Chanoine JP, Tsagarakis S, Kalsner L, Stratakis CA. Genotypic heterogeneity andclinical phenotype in triple A syndrome: a review of the NIH experience 2000-2005. ClinGenet. 2005;68:215-21.

5. Griffin KJ, Kirschner LS, Matyakhina L, Stergiopoulos S, Robinson-White A, Lenherr S,Weinberg W, Claflin E, Meoli E, Cho-Chung YS & CA Stratakis. Down-regulation of regulatorysubunit type 1A of protein kinase A leads to endocrine and other tumors. Cancer Res 64:8811-5, 2004.

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GROUP MEMBERS

Constantine Stratakis, MD, DSc, HeadSosipatros Boikos, MD, Visiting Fellow Anelia Horvath, PhD, Visiting Fellow,Christopher Giatzakis, PhD, Visiting FellowAndrew Bauer, MD, Guest Researcher,Audrey Robinson-White, PhD, ResearcherHui-Pin Hsiao, MD, ResearcherElizabeth Levine, Student fellowVirginia Kamvissi, MD, Special VolunteerMaria Lopez-Iglesias, MD, Special VolunteerGeorgia Delimpassi, Graduate Student, Special VolunteerYianna Patronas, Student, Special VolunteerEirini Bibaki MSc student

FUNDING

NIH International Program

CONTACT

Constandinos A Stratakis Section on Endocrinology & GeneticsBuilding 10, Room 9D42, MSC 1830,Bethesda, MD, 20892, USAtel. 301-496-6683 Fax 301-480-0378 e-mail: [email protected]

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