Resident National Institutes of Health Bethesda, Maryland, United States
Introduction: No medical therapies exist for Cushing’s disease (CD) despite advances in treatment paradigms. Our objective was to identify targetable mechanisms of CD tumorigenesis.
Methods: Formalin-fixed, paraffin-embedded human CD adenomas (n=5) were sectioned and stained using multiplexed immunohistochemistry (mIHC). Association between R17 and protein phosphatase 2A (PP2A) was assessed using proximity ligation assays (PLA, Sigma). We overexpressed R17 or GFP controls in murine corticotroph (mCort) cell lines and assessed for cell-cycle changes (Click-IT EdU) and cell proliferation (OneGlo; n = 3).
Results: R17 protein abundance was identified in the CD adenoma compartment by mIHC but not in the adjacent normal pituitary gland (Figure 1). PP2A protein abundance was also restricted to the adenoma compartment and colocalized with R17 (Figure 1). PLA staining confirmed close interaction between R17 and PP2Ac within CD adenomas (Figure 2). mCortR17 cells showed increased phosphorylation of PP2A targets EGFR and ERK. mCortR17 cells showed accelerated cell cycle progression compared to mCortGFP (S-phase 16.4% versus 24.1%, P < 0.001) with increased proliferation (two-way ANOVA P < 0.001). PP2A agonists Fingolimod or DT061 led to proliferation arrest in mCortR17 cells (Figure 3).
Conclusion : We identified increased R17 expression as an underlying mechanism of tumor hyperproliferation in CD adenomas, which was reversible using protein phosphatase agonists in vitro. Our findings uncover a new therapeutic target for patients with CD.
