154 Pituitary Adenomas Evade Apoptosis via Noxa Degradation in Cushing’s Disease

Abstract

INTRODUCTION: Cushing’s disease (CD) arising from ACTH-secreting pituitary adenomas is associated with severe morbidity and mortality. Pathogenic mechanisms of sporadic CD are poorly understood. Using novel syngeneic, pairwise transcriptomic analysis of surgical tissues, we identified transcriptional upregulation of PMAIP1 (encoding pro-apoptotic noxa) in CD adenomas compared to adjacent normal gland. METHODS: We investigated noxa protein levels by immunohistochemistry in 10 CD adenoma/normal pairs and by Western blotting in two syngeneic pairs of primary human cell lines from CD adenomas. We validated our key findings in ATT20 murine pituitary corticotroph tumor cells and in corticotroph-enriched murine cell lines mCortEFGF. Proteasomal-mediated degradation was assessed by cycloheximide chase assay. Bortezomib was used for proteasomal inhibition. Cell viability was assessed using CellTiterGlo (Promega). RESULTS: Despite PMAIP1 mRNA overexpression in CD, we found consistently low noxa protein in CD adenomas compared to surrounding normal glands but not in growth hormone secreting adenomas or prolactinomas. A cycloheximide assay demonstrated proteasomal-mediated noxa degradation mCortEFGF cells, which was rescued by proteasomal inhibition using bortezomib. Bortezomib treatment decreased cell viability and induced apoptosis in mCortEFGF cells, and also inhibited growth with apoptosis induction in CD adenoma primary cells. CONCLUSION: We identified proteasomal-mediated noxa degradation as a mechanism of escape from apoptosis in CD adenomas despite PMAIP1 transcriptional upregulation. Proteasomal inhibition using bortezomib rescued noxa protein levels and induced apoptosis in a dose-dependent fashion. Bortezomib, an FDA approved medication for mantle cell lymphoma, may represent a novel therapeutic target for patients with CD.