Caveolin‐1 down‐regulation is required for Wnt5a‐Frizzled 2 signalling in Ha‐Ras^V12‐induced cell transformation

Abstract

Caveolin‐1 (Cav1) is down‐regulated during MK4 (MDCK cells harbouring inducible Ha‐Ras V12 gene) transformation by Ha‐RasV12. Cav1 overexpression abrogates the Ha‐RasV12‐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‐RasV12‐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‐RasV12, 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‐RasV12‐ and MK4+I‐CM‐induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down‐regulation, either by Ha‐RasV12 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‐RasV12‐Wnt5a‐Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha‐RasV12‐driven cell transformation.

Oncogenic KRAS cells use Wnt signalling and cell dormancy to override homeostatic cell elimination mechanisms in adult pancreas.

“…By inhibiting porcupine, Wnt ligands cannot be secreted and therefore cannot signal to cells. In MDCK cells, overexpression of RasV12 induces expression and secretion of Wnt5a in a porcupine-dependent manner ( 60 ), suggesting porcupine inhibition in our experiments blocks secretion of endogenous Wnt, including Wnt5a. The proportion of non-extruded siMyc 1+2-FP-RasV12 cells significantly reduced in the presence of WNT-974 (Fig.…”

“…Moreover, Wnt5a activates cancer cell dormancy in metastatic niches ( 43, 44 ). In MDCK cells, overexpression of RasV12 induces expression and secretion of Wnt5a in a porcupine-dependent manner ( 51 ), suggesting porcupine inhibition in our experiments blocks endogenous Wnt secretion, including Wnt5a. We assessed the effect of Wnt5a treatment on normal-RasV12 cell-cell interactions by pre-treating GFP-RasV12 cells with recombinant Wnt5a (or PBS) before mixing with normal MDCK cells in 1:50 ratios (recombinant Wnt5a was maintained for the entire experiment).…”