Resident National Institutes of Health Bethesda, Maryland, United States
Introduction: Common mechanisms underlying canonically mutated and ‘wildtype’ Cushing’s disease remain unknown. The role of PPP1R17, an endogenous inhibitor of tumor-suppressor phosphatase PP2A in the embryonic context remains unknown in the pituitary. Here, we elucidated mechanistically, the effect of PPP1R17 on the phopshoproteome of CD adenomas.
Methods: We quantitatively profiled the proteome and phosphoproteome of three pairs of CD adenomas and their syngeneic adjacent normal pituitary glands with TMT labeling and mass spectrometry (LC-MS/MS). We validated the mechanisms with murine corticotroph cells (mCort) overexpressing R17 (mCortR17) or GFP (mCortGFP). Elevated total and phosphprotein expression was assessed using R packages by performing cross sample normalization, noise filtering and differential protein abundance or phosphorylation analyses. Pathway enrichment analysis was performed on the KEGG database using ShinyGo 0.76.2.
Results: We identified 37 proteins differentially phosphorylated in CD adenomas compared to adjacent normal pituitary glands, including the AP2-associated protein kinase 1 (Aak1) and the apoptotic chromatin condensation inducer Acin1. Of these 37 proteins, 30 (81%) were also differentially phosphorylated in mCortR17 versus mCortGFP cells, indicating that differential phosphorylation in CD is driven by R17 hyperactivity. Of the 35 proteins overabundant in CD adenomas, 15 (43%) overlapped with mCortR17, including the Myc-associated protein (Max) and Presenilin 1 (Psen1). Proteins enriched in both human CD and mCort datasets were involved in NOTCH signaling and neurotransmitter release, while phospho-proteome profiling highlighted R17-mediated perturbation of RNA polymerase II transcription termination and mRNA processing.
Conclusion : We first discovered a heavily dysregulated phosphoproteome in CD adenomas compared to syngeneic normal glands. We found that we could replicate a the signature phosphoproteomic profile by overexpressing PPP1R17 in-vitro. These data suggest the central role of phosphatase (PP2A) inihibition in CD adnenomas, and suggests potential anti-tumor targets.
