Binding of αvβ1 and αvβ6 integrins to tenascin-C induces epithelial-mesenchymal transition-like change of breast cancer cells

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 alpha v, alpha 2, beta 1 and beta 6 subunits was detected by quantitative PCR and immunoblotting in MCF-7 cells. Integrin beta 6 mRNA was remarkably upregulated by transforming growth factor (TGF)-beta 1 treatment, and protein expression was prominently increased by additional exposure to TNC. Immunofluorescent labeling demonstrated integrin alpha v beta 6 accumulation in focal adhesions after TNC treatment, especially in combination with TGF-beta 1. The alpha 2 and beta 1 subunits were mainly localized at cell-cell contacts, av being found near cell cluster surfaces. Immunoprecipitation showed increase in alpha v beta 1 heterodimers, but not alpha 2 beta 1, after TNC treatment. Activated beta 1 subunits detected by an antibody against the Ca2+ -dependent epitope colocalized with av in focal adhesion complexes, associated with FAK phosphorylation at tyrosine 925. Neutralizing antibodies against av and beta 1 blocked EMT-like change caused by TNC alone. In addition, anti-alpha v and combined treatment with anti-beta 1 and anti-alpha v beta 6 inhibited TGF-beta 1/TNC-induced EMT, whereas either of these alone did not. Integrin subunits av, beta 1 and beta 6, but not alpha 2, bound to TNC immobilized on agarose beads in a divalent cation-dependent manner. Treatments with neutralizing antibodies against beta 1 and alpha v beta 6 reduced av subunit bound to the beads. Immunohistochemistry of these receptors in human breast cancer tissues demonstrated frequent expression of beta 6 subunits in cancer cells forming scattered nests localized in TNC-rich stroma. These findings provide direct evidence that binding of alpha v beta 6 and alpha v beta 1 integrins to TNC as their essential ligand induces EMT-like change in breast cancer cells.