RESUMO
Tenascin-C (TNC), a large hexameric extracellular glycoprotein, is a pleiotropic molecule with multiple domains binding to a variety of receptors mediating a wide range of cellular functions. We earlier reported that TNC induces epithelial-mesenchymal transition (EMT)-like change in breast cancer cells. In the present study, we clarified TNC receptor involvement in this process. Among integrins previously reported as TNC receptors, substantial expression of αv, α2, ß1 and ß6 subunits was detected by quantitative PCR and immunoblotting in MCF-7 cells. Integrin ß6 mRNA was remarkably upregulated by transforming growth factor (TGF)-ß1 treatment, and protein expression was prominently increased by additional exposure to TNC. Immunofluorescent labeling demonstrated integrin αvß6 accumulation in focal adhesions after TNC treatment, especially in combination with TGF-ß1. The α2 and ß1 subunits were mainly localized at cell-cell contacts, αv being found near cell cluster surfaces. Immunoprecipitation showed increase in αvß1 heterodimers, but not α2ß1, after TNC treatment. Activated ß1 subunits detected by an antibody against the Ca(2+)-dependent epitope colocalized with αv in focal adhesion complexes, associated with FAK phosphorylation at tyrosine 925. Neutralizing antibodies against αv and ß1 blocked EMT-like change caused by TNC alone. In addition, anti-αv and combined treatment with anti-ß1 and anti-αvß6 inhibited TGF-ß1/TNC-induced EMT, whereas either of these alone did not. Integrin subunits αv, ß1 and ß6, but not α2, bound to TNC immobilized on agarose beads in a divalent cation-dependent manner. Treatments with neutralizing antibodies against ß1 and αvß6 reduced αv subunit bound to the beads. Immunohistochemistry of these receptors in human breast cancer tissues demonstrated frequent expression of ß6 subunits in cancer cells forming scattered nests localized in TNC-rich stroma. These findings provide direct evidence that binding of αvß6 and αvß1 integrins to TNC as their essential ligand induces EMT-like change in breast cancer cells.
