Caveolin-1 down-regulation is required for Wnt5a-Frizzled 2 signalling in Ha-RasV12-induced cell transformation

Caveolin-1 (Cav1) is down-regulated during MK4 (MDCK cells harbouring inducible Ha-Ras(V12) gene) transformation by Ha-Ras(V12). Cav1 overexpression abrogates the Ha-Ras(V12)-driven transformation of MK4 cells; however, the targeted down-regulation of Cav1 is not sufficient to mimic this transformation. Cav1-silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction-related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I-CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha-Ras(V12)-inducing MK4 cells increased exosome-like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I-CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I-CM (MK4+I-EXs). Wnt5a, a downstream product of Ha-Ras(V12), was markedly secreted by MK4+I-CM and MK4+I-EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha-Ras(V12)- and MK4+I-CM-induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down-regulation, either by Ha-Ras(V12) or targeted shRNA, increased frizzled-2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I-EXs in MDCK cells. These data suggest that Cav1-dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha-Ras(V12)-Wnt5a-Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha-Ras(V12)-driven cell transformation.