C-CBL phosphorylation status influences colorectal cancer cell survival in a Wnt-dependent manner
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Hyperactive Wnt signaling is the seminal event in colorectal cancer (CRC) pathogenesis, where β-catenin serves as a key Wnt mediator enhancing CRC cell proliferation and survival. c-Cbl is a unique E3 ligase, which degrades both mutant and active (tumorigenic) β-catenin. c-Cbl phosphorylation at tyrosine 731 (Y731) regulates its binding and down regulation of β-catenin specifically in the presence of Wnt ligand (Wnt-on state). Since aberrant Wnt signaling activation is found in almost all cases of human CRC, it would be critical to understand the influence of c-Cbl phosphorylation on CRC cell survival. We hypothesized that c-Cbl phosphorylation regulates CRC cell survival in a Wnt dependent manner, a state that is mediated through mutations in β-catenin or adenomatosis polyposis coli (APC). Cbl phosphorylation was examined in a panel of Wnt-off cells with wild-type β-catenin and APC CRC cell line (RKO cell line) and Wnt-on cell lines with mutant APC (Wnt-on- DLD1, HCT15 cell line) or mutant β-catenin (HCT116) using phospho-specific antibodies to c-Cbl tyrosine residues at 700 (Y700), 731 and 774 (Y774) positions. Biological significance of specific phosphorylation sites was evaluated with phospho-inactive mutants of c-Cbl (Y700F, Y731F and Y774F) using both the MTT cell proliferation assay and the non-adherent colony formation assay. Potential meditators of c-Cbl were examined using immunoblotting. Here we show that c-Cbl was phosphorylated at all three major phosphorylation sites (Y700, Y731 and Y774) in both Wnt-off and Wnt-on CRC cell lines. However, the amount of phosphorylation was reduced in Wnt-on CRC cell lines (DLD1, HCT116 and HCT15) compared to Wnt-off (RKO) cell line. Wild-type c-Cbl significantly enhanced survival in RKO cell lines and reduced survivability in DLD1 cell lines. In contrast to the effect of wild-type c-Cbl, Y731F increased CRC cell survival and non-adherent colony forming units. Our preliminary data suggests that c-Cbl Y731 mutation regulates CRC survival through β-catenin. c-Cbl is heavily phosphorylated in CRC cell lines, where wild-type c-Cbl significantly inhibits cell survival in Wnt-on and enhances cell survival in Wnt-off CRC cell lines. Furthermore, our data indicates that Y731 influences CRC survival and colony formation only in Wnt-on cell lines. Though further validation is required, this dichotomy in the effect of c-Cbl phosphorylation on CRC survival being mediated by Wnt status can be further explored as a potentially novel therapeutic target in mutant CRC tumors, which represent more than 90% of CRC cases in humans.