51 BVES Orchestrates Pr61α-PP2A Mediated C-Myc Degradation

Abstract

Background: Blood vessel epicardial substance (BVES) is a tight-junction associated protein that regulates epithelial-to-mesenchymal transition (EMT). BVES is underexpressed in ulcerative colitis (UC), and the BVES promoter is hypermethylated in colitis-associated cancer (CAC). We have recently presented that Bves mice have greater tumor burden and higher grade dysplasia in the AOM/DSS model of CAC. Here we defined the mechanism by which BVES regulates EMT and CAC. Methods: After tumor induction using AOM/DSS, intratumoral RNA was isolated from Bves and WT mice and the transcriptome defined via RNAseq. Tumors were stained by IHC for c-myc and phospho-S62-c-myc and scored using an index based on positive cells per HPF. C-myc protein analysis was conducted in HEK 293T, HCT116, and SW480 cell lines. Enteroid cultures were isolated from 4-week old mice and harvested after 72 hours for RNA and protein. Cells were treated with 100ug/ml of cycloheximide 72 hours after transfection. Polyhis-tagged ubiquitin was co-transfected with HA-c-myc with empty vector or V5-BVES. Ubiquitin was pulled down using Ni-NTA agarose and c-myc was blotted. Transepithelial resistance (TER) was measured daily for 7 days. WST-1 values were recorded daily for 3 days post-plating. Results: RNA-seq analysis using Ingenuity Pathway identified that the c-myc transcriptional network was disrupted in AOM/ DSS Bves tumors. IHC analysis of c-myc and phopsho-S62c-myc showed an increase in Bves tumors (c-myc 2.6± 0.1 vs. 1.8± 0.1 P<0.0001 and phospho-S62-c-myc 2.8± 0.2 vs. 2.1± 0.1 per tumor HPF P<0.01). Bves enteroids had 10-fold upregulation of c-myc protein and a 38-fold increase in the c-myc transcriptional target ornithine decarboxylase. Knocking down BVES in 293T, HCT116, and SW480 cells increased c-myc protein levels by 25%. Cycloheximide treatment after BVES knockdown doubled c-myc half-life. Further, transfection of BVES increased poly-ubiquitylated c-myc suggesting BVES promotes c-myc degradation. Lastly, immunopreciptation demonstrated that BVES and c-myc exist in complex. A yeast-two-hybrid screen identified PR61α, a regulatory subunit of the PP2A family of phosphatases known to promote c-myc degradation, interacts with BVES. We confirmed the BVES:PR61α interaction by directed Y2H and co-IP. PR61α knockdown blocked BVESinduced reduction of c-myc, suggesting BVES requires PR61α to promote c-myc destruction. Caco-2 cell differentiation requires post-translational downregulation of c-myc. When PR61α was knocked down, BVES was unable to increase TER in Caco-2 cells. Further, c-myc promotes proliferation, and PR61α knockdown prevented BVES-induced reduction of WST1. Conclusions: BVES requires PR61α to regulate TER and proliferation, potentially through directing the c-myc downregulation.