Targeted Inhibition of c-KIT Activation Sensitizes Adrenocortical Tumor Cells to Apoptosis

Abstract

Primary Pigmented Nodular Adrenocortical Disease (PPNAD), a type of micro nodular adrenal hyperplasia (MAH) that occurs mostly in the context of Carney Complex or rarely in an isolated form is caused by germline inactivating mutations in PRKAR1A, the main regulatory subunit of cAMP dependent Protein Kinase (PKA). In addition, defects in phosphodiesterase genes 11A (PDE11A), 8B (PDE8B) and abnormalities of cAMP signaling are known to occur in a small fraction of MAH and massive macronodular adrenocortical disease (MMAD) respectively, suggesting that dysregulation of PKA is a key player in the genesis of these lesions. The only current treatment option is adrenalectomy resulting in adrenal insufficiency. In search of therapeutic targets to develop alternative strategies to treat this disease, we identified for the first time tumor specific over expression and constitutive activation of c-KIT in PPNAD compared to cortisol-producing adenomas (CPA), MMAD and aldosterone-producing adenomas (APA). Next, we investigated the mutational status of the signature exons in KIT gene and found no mutations. Real-time PCR analysis confirmed up regulation of KIT mRNA. Interestingly, KIT over-expression was recapitulated in adrenals from Prkar1a knockout mice reminiscent of that seen in human patients suggesting PKA specific dysregulation of KIT expression. To better characterize these changes at the biochemical level we used an adrenocortical cell line (CAR 47.01) derived from a patient with mutant PRKAR1A and compared it to the H295R adrenocortical cancer line. Consistent with the alterations seen in tumors, CAR47.01 cells exhibited enhanced expression and activation of c-KIT which was further enhanced by the treatment with forskolin, a PKA activator. This up regulation was markedly rescued by reintroduction of PRKAR1A suggesting that PKA functions upstream of c-KIT and the alterations in c-Kit are secondary to changes in PKA. Knockdown of PKAC using siRNA specific to PKAC significantly reduced c-KIT protein levels. Furthermore, in vitro kinase assay demonstrated c-KIT to be a direct phosphorylation target of PKA. The biological and functional consequences of this phosphorylation are currently being explored in vivo. To explore whether over expressed c-KIT is a therapeutic target, the cells were treated with imatinib mesylate, a pharmacological KIT inhibitor resulted in reduced cell viability and induced growth arrest and apoptosis in a time and dose-dependent manner. In the CAR47.01 cells, imatinib treatment induced down regulation of phospho-c-KIT and phospho-Akt, an immediate downstream phosphorylation target of KIT without altering total c-KIT and Akt levels. Interestingly imatinib treatment at 100mg/kg for 7days in a mouse xenograft model induced tumor stasis. Taken together, our data provide novel insights on PKA specific regulation of KIT and indicate KIT as a promising potential target for therapeutic intervention in adrenocortical neoplasms.