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

概要

Introduction
Tenascin-C (TNC), known as an extracellular matrix protein, is highly expressed both in breast cancer cells and cancer associated fibroblasts (CAFs). However, the role of TNC on CAFs remains unclear. Here, we examined the effects of TNC on fibroblasts.

Methods
We used human mammary fibroblasts (HMFs) and human dermal fibroblasts (HDFs) in vitro, and human breast cancer tissues in vivo.

Results
TNC induced the morphological change of HMFs from spindle-shaped to star-shaped morphology, indicating myofibroblasts transformation. Therefore, we examined the expression of CAFs markers. TNC significantly increased the protein expression of a-smooth muscle actin (a-SMA) and calponin, and the number of a-SMA and/or calponin expressing cells with actin stress fibers. Next, we examined the altered function of HMFs treated with TNC. TNC promoted contraction of collagen gel mediated by HMFs. TNC also increased the cell number of HMFs. Interestingly, TNC significantly up-regulated its own synthesis in HMFs, indicating the formation of positive feedback loop. On the other hand, mRNA levels of collagen type I/III and fibronectin remained unchanged after the treatment of TNC. In human breast cancer tissues, immunofluorescence revealed a-SMA and/or calponin expressing myofibroblasts in TNC-positive cancer stroma. We further examined the functional receptor of TNC in HMFs. The protein expressions of integrin av and 81 subunits were significantly increased after the treatment of TNC. Furthermore, TNC promoted the formation of integrin av81 heterodimer at focal adhesion. Silencing of integrin av or 81, using siRNA, suppressed the expression of a-SMA and calponin induced by TNC. Thus, heterodimer formation of integrin av81 was important for TNC-induced myofibroblast transition. Actually, integrin av and 81 double-positive fibroblasts were increased in human breast cancer tissues. We finally investigated the molecular signaling pathway associated with the TNC-induced myofibroblast transition. Because transforming growth factor 8 (TGF-8)/SMAD is important for myofibroblast differentiation, we examined theses cascades. TNC increased the phosphorylated SMAD2/3 and the nuclear translocation of SMAD2/3 in HMFs. Inhibition of TGF-8 receptor or SMAD3 suppressed TNC-induced expression of a-SMA and calponin. Furthermore, nuclear SMAD2/3 positive fibroblasts were increased in TNC-positive human breast cancer stroma, while these cells were hardly observed in normal breast tissues. We finally examined the difference of the response to TNC between mammary and skin fibroblasts. TNC increased the expressions of a-SMA and calponin in HDFs. However, absolute expressions of a-SMA and calponin were considerably lower in HDFs, compared to HMFs. Furthermore, myofibroblasts in the skin infiltrating lesions of breast cancer lacked the expression of calponin. Thus, the response of fibroblasts to TNC differs between mammary and dermal tissues.

Conclusion
TNC forms a positive feedback by increasing its own synthesis and increases the formation of integrin av81 heterodimer to activate TGF-8 signaling, leading to highly contractile myofibroblasts transformation. TNC may contribute to the formation of stiff breast cancer stroma.