Source: Dissertation Abstracts International, Volume: 75-09(E), Section: B.

Adviser: Richard P. Lifton.

Cancer has long been recognized as a genetic disease. Sporadic tumors arise from the accumulation of somatic mutations, leading to the dysregulation of protein-coding genes and non-coding RNAs, allowing the acquisition of classical cancer hallmarks. As such, genomics is integral to the cancer gene discovery and whole-exome sequencing is an optimal tool for identifying novel driver genes in cancer. In collaboration, I applied this approach to different types of adrenocortical tumors -- aldosterone-producing adenomas (APAs), cortisol producing adrenal turners and adrenocortical cancer (ACC). We identified novel genes with previously unknown roles in adrenal tumor biology, and implicated mutations in these genes in the underlying pathogenesis of these adrenocortical tumors.

Constitutive production of aldosterone leads to hypertension, and aldosterone-producing adenomas are present in approximately 5% of patients referred to hypertension clinics. We identified somatic mutations in CACNA1D, encoding a voltage-gated calcium channel, in ∼8% of APAs. These mutations lie in S6 segments that line the channel pore and result in channel activation at less depolarized potentials. We inferred that these mutations cause increased Ca2+ influx in these cells, which is known to be the sufficient stimulus for aldosterone production and cell proliferation in adrenal glomerulosa. Interestingly, these mutations phenocopy recurrent mutations in the potassium channel KCNJ5, which were identified in approximately 40% of these tumors and result in cell depolarization and Ca2+ influx as well. We also identified de novo mutations in CACNA1D at the identical amino acid residues as APAs in two children with a previously undescribed Mendelian syndrome featuring primary aldosteronism and neuromuscular abnormalities. This finding is consistent with single CACNA1D mutations being sufficient for the formation of APAs, and implicates gain of function Ca2+ channel mutations in APAs and primary aldosteronism.

Adrenal tumors that constitutively produce cortisol cause Cushing syndrome, and we identified the identical somatic p.Leu206Arg mutation in PRKACA , encoding the catalytic subunit alpha of cAMP-activated protein kinase A (PKA), in 20.6% of cortisol-producing adrenal tumors, and 35% of adrenal adenomas causing overt Cushing syndrome. Leu206 directly interacts with the PKA inhibitory subunit encoded by PRKAR1A, and we demonstrate that the mutant PRKACA no longer binds PRKAR1A, resulting in constitutive catalytic activity and increased phosphorylation of downstream targets. Because PKA induces both cortisol production and cell proliferation, this mutation can explain the cardinal features of these tumors. We also identify 2 distinct groups of tumors defined by qualitative differences in the number of copy number variants, suggesting two distinct pathophysiologic mechanisms for adrenal Cushing tumors. Our findings suggest that these tumors commonly arise due to mutation in a single gene, or by more complex mechanisms involving typical cancer-related pathways.

Adrenocortical cancer is a rare and aggressive disease with poor clinical outcome. Here, we define the genomic landscape of ACC, and identify significantly mutated pathways. We found frequent mutation of TP53 and CTNNB1 in our ACC cohort, genes previously associated with this disease. We identified frequent amplification of TERT in ACCs, implicating telomerase dysregulation in these tumors. We also identified a recurrent homozygous deletion across ZNRF3, encoding the cell-surface transmembrane E3 ubiquitin ligase zinc and ring finger 3; ZNRF3 deletion was previously shown to increase stability of Wnt receptors and increase downstream Wnt signaling. ZNRF3 inactivating mutations and CTNNB1 activating mutations were identified in 19.5% of these tumors, suggesting Wnt activation is a main driver of tumorigenesis in ACC.

Our results identify novel cancer driver genes in adrenocortical tumors, and suggest novel targets for cancer therapy in patients with mutations in these genes. We hope that continued study of these tumors will lead to increased understanding of the disease pathogenesis and improve diagnosis, prognosis and therapy.