Abstract
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, β1 and β6 subunits was detected by quantitative PCR and immunoblotting in MCF-7 cells. Integrin β6 mRNA was remarkably upregulated by transforming growth factor (TGF)-β1 treatment, and protein expression was prominently increased by additional exposure to TNC. Immunofluorescent labeling demonstrated integrin αvβ6 accumulation in focal adhesions after TNC treatment, especially in combination with TGF-β1. The α2 and β1 subunits were mainly localized at cell–cell contacts, αv being found near cell cluster surfaces. Immunoprecipitation showed increase in αvβ1 heterodimers, but not α2β1, after TNC treatment. Activated β1 subunits detected by an antibody against the Ca2+-dependent epitope colocalized with αv in focal adhesion complexes, associated with FAK phosphorylation at tyrosine 925. Neutralizing antibodies against αv and β1 blocked EMT-like change caused by TNC alone. In addition, anti-αv and combined treatment with anti-β1 and anti-αvβ6 inhibited TGF-β1/TNC-induced EMT, whereas either of these alone did not. Integrin subunits αv, β1 and β6, but not α2, bound to TNC immobilized on agarose beads in a divalent cation-dependent manner. Treatments with neutralizing antibodies against β1 and αvβ6 reduced αv subunit bound to the beads. Immunohistochemistry of these receptors in human breast cancer tissues demonstrated frequent expression of β6 subunits in cancer cells forming scattered nests localized in TNC-rich stroma. These findings provide direct evidence that binding of αvβ6 and αvβ1 integrins to TNC as their essential ligand induces EMT-like change in breast cancer cells.
Highlights
Tenascin-C (TNC) is an extracellular matrix glycoprotein formed by hexamers of 200–400 kDa subunits
We reported that TNC addition to culture medium induces epithelial–mesenchymal transition (EMT)-like change in human breast cancer MCF-7 cells, accompanied by SRC activation and focal adhesion kinase (FAK) phosphorylation.[18]
Substantial mRNA expression of av, a2, b1, and b6 subunits was validated by quantitative PCR (qPCR) (Figure 1a), and these subunits were readily detected (Figure 2a) in immunoblot analysis of TGFb1/TNC-treated MCF-7 cells
Summary
Tenascin-C (TNC) is an extracellular matrix glycoprotein formed by hexamers of 200–400 kDa subunits. Expression of TNC is rare in normal adult tissues, high levels are observed in pathological states featuring tissue remodeling, such as inflammation, wound healing and cancer.[1,2,3] In breast cancer tissues, TNC demonstrates upregulated production by both tumor and stromal cells.[4] Immunohistochemistry has shown TNC expression to be indicative of a poorer patient outcome in invasive ductal carcinoma cases.[5] Low expression levels of miR-335, allowing high. TNC expression, are linked to lung metastasis by human breast cancer.[6] In a mouse model, TNC knockdown causes dramatic inhibition of lung metastasis and colonization by breast cancer cells.[7] In vitro studies have shown that TNC promotes proliferation and migration of cancer cells[8,9,10,11] and increases their expression of matrix metalloproteinases.[11,12]
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