Tenascin-C Induces Phenotypic Changes in Fibroblasts to Myofibroblasts with High Contractility through the Integrin αvβ1/Transforming Growth Factor β/SMAD Signaling Axis in Human Breast Cancer

Abstract

Tenascin-C (TNC) is strongly expressed by fibroblasts and cancer cells in breast cancer. To assess the effects of TNC on stromal formation, we examined phenotypic changes in human mammary fibroblasts treated with TNC. The addition of TNC significantly up-regulated α-smooth muscle actin (α-SMA) and calponin. TNC increased the number of α-SMA- and/or calponin-positive cells with well-developed stress fibers in immunofluorescence, which enhanced contractile ability in collagen gel contraction. The treatment with TNC also significantly up-regulated its own synthesis. Double immunofluorescence of human breast cancer tissues showed α-SMA- and/or calponin-positive myofibroblasts in the TNC-deposited stroma. Among several receptors for TNC, the protein levels of the αv and β1 integrin subunits were significantly increased after the treatment. Immunofluorescence showed the augmented colocalization of αv and β1 at focal adhesions. Immunoprecipitation using an anti-αv antibody revealed a significant increase in coprecipitated β1 with TNC in lysates. The knockdown of αv and β1 suppressed the up-regulation of α-SMA and calponin. The addition of TNC induced the phosphorylation of SMAD2/3, whereas SB-505124 and SIS3 blocked myofibroblast differentiation. Therefore, TNC enhances its own synthesis by forming a positive feedback loop and increases integrin αvβ1 heterodimer levels to activate transforming growth factor-β signaling, which is followed by a change to highly contractile myofibroblasts. TNC may essentially contribute to the stiffer stromal formation characteristic of breast cancer tissues.