RNF43 induces the turnover of protease-activated receptor 2 in colon cancer.

Post-translational modification of G-protein coupled receptors (GPCRs) plays a central role in tissue hemostasis and cancer. The molecular mechanism of post-translational regulation of protease-activated receptors (PARs), a subgroup of GPCRs is yet understudied. Here we show that the cell-surface transmembrane E3 ubiquitin ligase ring finger 43 (RNF43) is a negative feedback regulator of PAR , impacting PAR -induced signaling and colon cancer growth. RNF43 co-associates with PAR , promoting its membrane elimination and degradation as shown by reduced cell surface biotinylated PAR levels and polyubiquitination. PAR degradation is rescued by R-spondin2 in the presence of leucine-rich repeat-containing G-protein-coupled receptor5 (LGR5). In fact, PAR acts jointly with LGR5, as recapitulated by increased β-catenin levels, transcriptional activity, phospho-LRP6, and anchorage-independent colony growth in agar. Animal models of the chemically induced AOM/DSS colon cancer of wt versus Par2/f2rl1 KO mice as also the 'spleen-liver' colon cancer metastasis, allocated a central role for PAR in colon cancer growth and development. RNF43 is abundantly expressed in the Par2/f2rl1 KO-treated AOM/DSS colon tissues while its level is very low to nearly null in colon cancer adenocarcinomas of the wt mice. The same result is obtained in the 'spleen-liver' model of spleen-inoculated cells, metastasized to the liver. High RNF43 expression is observed in the liver upon shRNA -Par2 silencing. "Limited-dilution-assay" performed in mice in-vivo, assigned PAR as a member of the cancer stem cell niche compartment. Collectively, we elucidate an original regulation of PAR oncogene, a member of cancer stem cells, by RNF43 ubiquitin ligase. It impacts β-catenin signaling and colon cancer growth.