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, hereditary, and associated with adrenal hypoplasia or hyperplasia, multiple tumors, and abnormalities in other endocrine glands, such as the pituitary gland. In this context, our laboratory has studied congenital adrenal hypoplasia caused by Allgrove syndrome (or triple A syndrome) and multiple endocrine deficiencies (APECED syndrome), familial hyperaldosteronism, adrenocortical and thyroid cancer, pituitary tumors, multiple endocrine neoplasia syndromes (MEN 1, Cowden disease) affecting the pituitary, thyroid, and adrenal glands, and Carney complex, an autosomal dominant disease that affects the adrenal cortex. Recently, our laboratory identified the regulatory subunit type 1-a(RIaof protein kinase A (PKA) that is encoded by the PRKAR1A gene, the gene responsible for almost half of the patients with Carney complex. Since then, our laboratory has focused on PKA-stimulated signalling pathways, PKA effects on tumor suppression and/or development, the cell cycle and chromosomal stability, and PKA-dependent hormonal interactions.

Carney Complex Genetics
Bauer, Bei, Bourdeau, Griffin, Matyakhina, Stratakis; in collaboration with Carney, Kirschner, Bertherat
Carney complex is the only inherited form of adrenal gland-dependent Cushing’s syndrome; it is also a multiple endocrine neoplasia affecting the pituitary, thyroid, and gonads, and it is associated with a variety of other tumors, including myxomas (heart and other myxomas) and schwannomas (it is one of three conditions associated with inherited schwannomas; the other two are neurofibromatosis and isolated schwannomatosis), and with skin pigmentation defects (lentigines, café-au-lait spots, and nevi).
Families with Carney complex (CNC) and related syndromes have been collected from a number of collaborating institutions worldwide. Genetic linkage analysis identified loci harboring genes for CNC on chromosomes 2 (2p16) and 17 (17q22-24); possible other loci for this genetically heterogeneous condition are currently under investigation by using state-of-the-art molecular cytogenetic techniques. We have constructed a comprehensive genetic and physical map of the 2p16 chromosomal region 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 instability in the center of the map (Fig. 5).

Fig. 5

Deletions of the 17q22-24 PRKAR1A region in sproradic adrenal tumors. (A) Interphase FISH with the BAC 321-G-8 containing the PRKARIA gene in an adrenal adenoma showed cells with one signal of the BAC (white spots). (B) FISH with the BAC 321-G-8 (white spots) and an a-satellite probe specific for chromosome 17 (white spots with arrow) to an adrenal carcinoma showed cells with one signal for each probe suggesting loss of chromosome 17 from the centromere to the 17q22-24 region. (C) A control interphase FISH for the same carcinoma with BAC RP3-526I14 from chromosome 22 showed the expected two signals. (D) Likewise, FISH with BAC 588-h-15 from chromosome 6 (white spots) to an adenoma that showed loss for 17q22-24, showed the expected two signals. (E) Normal adrenal cells hybridized with BAC 321-G-8 showed two copies of the probe. (F) Hybridization of BAC 321-G-8 (harboring the PRKAR1A gene) to another carcinoma detected two copies of the probe in all cells showing that this particular tumor had not undergone losses of the 17q22-24 region; these data were consistent with the LOH (by polymorphic markers) analysis for this tumor.

Tumor studies led to the identification of the PRKAR1A gene on 17q22-24 as the gene responsible for CNC in approximately 40 percent of the cases of the disease. It was determined that PRKAR1A functions as a novel tumor suppressor gene in CNC-associated tumors; it is also the main regulatory subunit of protein kinase A (PKA), a central signalling pathway for many of cellular functions and hormonal responses. More patients with CNC are now the subject of genotype-phenotype correlation studies, which are expected to shed light into the complex biochemical and molecular pathways regulated by PRKAR1A and PKA.

Effects of PRKAR1A on Protein Kinase A Activity and Endocrine Tumor Development
Matyakhina, Sandrini, Stergiopoulos, Robinson-White, Stratakis; in collaboration Cho-Chung, Bertherat
The functional consequences of PRKAR1A mutations are under investigation in cell lines established from CNC patients and their tumors. Both cAMP and PKA activity are measured 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 commercially available mouse endocrine and other cell lines; we study the 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 a switch from type-I PKA (based almost exclusively on endocrine cells on 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. In addition to the above, we are seeking mutations of the PRKAR1A gene, which would further establish the gene’s role as a general tumor suppressor, in sporadic endocrine and non-endocrine tumors (thyroid adenomas and carcinomas, adreno-cortical adenomas and carcinomas, ovarian carcinomas, melanomas and other benign and malignant pigmented lesions, and heart myxomas). We receive specimens on a collabora-tive basis from a variety of investigators within the NIH and around the world [Dr. Sarlis (NIDDK), Dr. Eng (Ohio State University), Dr. Melmed (UCLA), Dr. Tanaka (Tokio University), Dr. Lacroix (University of Montreal)].

Animal Models of PRKAR1A
Bauer, Griffin, Claflin, Shiferaw, Stratakis; in collaboration with Westphal, Kirschner
The PRKAR1A -knock-out (KO) (-/-) mouse, created several years ago by S. McKnight of the University of Washington, Seattle, dies of heart and central nervous system abnormalities on day nine of embryonic development. Since the discovery of PRKAR1A ’s involvement in CNC, our laboratory has been developing conditional PRKAR1A KOs in endocrine tissues (adrenal cortex, anterior lobe of the pituitary, and thyroid). The first analysis of a one-year-old mouse expressing an antisense PRKAR1A transgene is ongoing.

PRKAR1A , the Cell Cycle, Chromosomal Stability, and Other Signalling Pathways
Matyakhina, Shiferaw, Robinson-White, Stratakis; in collaboration with Papadopoulos
Genes implicated in cyclic nucleotide–dependent signalling 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 a number of endocrine tumors of various types. To date, however, there is no convincing evidence that, in the absence of additional genetic abnormalities, GNAS1 or GPCR activation is involved in cancer. Individuals with McCune-Albright syndrome (MAS) who bear somatic GNAS1 mutations in their endocrine glands may be predisposed to developing some cancers (MAS exhibits similarities to CNC). However, 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 signalling that lead to malignant transformation. Thus, 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 functions important to both the cell cycle and chromosomal stability. Our work aims to identify these interactions of PRKAR1A by studying mitogenic and other growth-signalling pathways in cell lines expressing the antisense PRKAR1A constructs referred to above. In addition, chromosomal stability in both human and mouse cell lines in which PRKAR1A has been inactivated is under investigation with classic and molecular cytogenetics, including fluorescent in situ hybridization (FISH), spectral karyotyping (SKY), and comparative genomic hybridization (CGH). More recently, we have also been investigating proteins that are directly bound to PRKAR1A (RIa) and regulate its function; we are particularly interested in the novel protein PAP7, which was identified by a collaborating laboratory. Compartmentalization of PKA function is thought to be mediated by anchoring proteins; almost all the PKA-anchoring proteins known to date, however, are bound to type IIPKA. PAP7 may be the first PKA type-I–specific anchoring protein.

Genetic Investigations on Other Adreno-cortical Diseases and Tumors
Bourdeau, Sandrini, Farmakidis,* Stratakis; in collaboration with Libutti, Chan, Carney, Stowasser, Torpy, Lacroix, Bertherat
The goal of our work is to apply general and pathway-specific microarrays to a variety of adrenocortical tumors in order to identify genes with important functions in adrenal oncogenetics; to examine specific candidate genes (such as TP53 and other tumor suppressors and oncogenes) for their roles in adrenocortical tumors and development; and to identify by positional cloning additional genes with a role in inherited adrenocortical diseases. We undertake a large part of our work collabora-tively, as is evident from the accompanying list of collaborating investigators and institutions. As part of this work, we have recently accomplished the following: identification of a novel TP53 mutation in a cohort of adreno-cortical tumors from Southern Brazil with important implications for p53’s function in adrenocortical tumor suppression; completion of a genome-wide screen for the identification of gene(s) responsible for inherited adrenocortical aldosteronomas (familial hyperaldosteronism type II - FH-II) that led to the specification of a locus for FH-II on chromosome 7 (7p22); initiation of a genome-wide screen for the identification of a syndrome composed of familial paragangliomas and adrenal, gastric stromal, and pulmonary tumors; and identifica-tion of new mutations in the APECED and Allgrove syndrome genes leading to congenital adrenal hypoplasia.

Genetic Investigations on Other Endocrine Neoplasias and Related Syndromes
Bauer, Bourdeau, Francis, Sandrini, Stratakis; in collaboration with Marx, Haddad, Blancato, Meck, Eng
We are also working, largely collaboratively, with a number of other investigators at the NIH and elsewhere on the genetics of CNC- and adrenal-related endocrine tumors, including childhood adrenocortical cancer and thyroid and pituitary tumors. As part of this work, we described novel genetic abnormal-ities in thyroid tumors. We are also identifying the genetic defects in patients with CNC-related syndromes (the lentigenoses, i.e., Peutz-Jeghers syndrome and others).

Clinical Investigations in the Diagnosis and Treatment of Adrenal and Pituitary Tumors
Griffin, Bourdeau, Stratakis; in collaboration with Keil, Patronas
Ongoing investigations of NIH Clinical Center patients with adrenal tumors and other types of Cushing’s syndrome (and occasionally other pituitary tumors) are studying the prevalence of ectopic hormone receptor expression in adrenal adenomas and massive macronodular adrenocortical disease; 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 disease and other pituitary tumors.