Tenascin C to regulate the function of sHER3, an endogenous inhibitor of neuregulin signaling.

Abstract

e23002 Background: HER3 expression is associated with poor prognosis in melanoma patients but the mechanistic basis has not been determined. An endogenous secreted isoform of HER3, p85-sHER3, has been shown by us to inhibit neuregulin-1 mediated, Akt-dependent breast and ovarian cancer cell growth in vitro. Here we demonstrate that p85-sHER3 also regulates neuregulin-dependent melanoma cell migration via an Akt- independent, tenascin C-dependent mechanism. Methods: Immunoblot analysis and RT-PCR were used to study HER3 and p85-sHER expression. Cell proliferation and chamber assays were conducted to determine the effect of p85-sHER3 on melanoma cell growth and migration. Mass spectrometry (MS) was performed to identify sHER3-interacting proteins. CRSPR in vitro editing methods were used to knock out tenascin C (TNC) expression. Results: HER3 and p85-sHER3 were expressed in 5 of 6 human melanoma-derived cell lines tested, with exception of SK-MEL-103 which did not express either gene product. Exogenous addition of 10 nM p85-sHER3 inhibited growth in 3 cells lines, although Akt phosphorylation was not reduced. Protein extracts from M14-MEL cell conditioned media were characterized by MS, and identified p85-sHER3 and TNC. Their association was confirmed by co-immunoprecipitation. sHER3 was further shown to inhibit neuregulin stimulated cell migration in 2 cell lines which exhibited high levels of TNC. CRSPR knockout of TNC in these cell lines (but not in control lines) eliminated sHER3 inhibition of neuregulin stimulated melanoma cell migration. Conclusions: Our results suggest that p85-sHER3, predominantly secreted by HER3-expressing melanoma cells, inhibits melanoma cell proliferation and migration via an Akt-independent mechanism, which is likely achieved through interaction(s) with TNC. TNC promotes an intermediate adhesive state favoring cell migration and this state appears to be mediated by Rho-associated kinase signaling. The Rho inhibitor, CCG-203971 has been shown to inhibit melanoma cell migration. Together, these results suggest the existence of a TNC and HER3-dependent signaling pathway in melanoma that regulates cell migration, and therefore may be correlated with poor patient survival.