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ABSTRACT Extracellular matrix stiffness is enhanced in cancer and fibrosis; however, there is limited knowledge on how matrix mechanics modulate expression and signaling of the methyltransferase G9a. Here, we show that matrix stiffness and transforming growth factor (TGF)‐β1 signaling together regulate G9a expression and the levels of the histone mark H3K9me2. Suppressing the activity and expression of G9a attenuates TGFβ1‐induced alpha smooth muscle actin (αSMA) and N‐cadherin expression and cell morphology changes in mammary epithelial cells cultured on stiff substrata. Knockdown of G9a increases the expression of large tumor suppressor kinase 2 (LATS2) and decreases the nuclear localization of yes associated protein (YAP). Furthermore, inhibition of LATS promotes an increase in YAP nuclear localization and αSMA expression, while inhibition of YAP attenuates αSMA expression. Overall, our findings indicate that a G9a‐LATS‐YAP signaling cascade regulates mammary epithelial cell response to matrix stiffness and TGFβ1. 

ABSTRACT The methyltransferase enhancer of zeste homolog 2 (EZH2) regulates gene expression, and aberrant EZH2 expression and signaling can drive fibrosis and cancer. However, it is not clear how chemical and mechanical signals are integrated to regulate EZH2 and gene expression. We show that culture of cells on stiff matrices in concert with transforming growth factor (TGF)-β1 promotes nuclear localization of EZH2 and an increase in the levels of the corresponding histone modification, H3K27me3, thereby regulating gene expression. EZH2 activity and expression are required for TGFβ1- and stiffness-induced increases in H3K27me3 levels as well as for morphological and gene expression changes associated with epithelial–mesenchymal transition (EMT). Inhibition of Rho associated kinase (ROCK) proteins or myosin II signaling attenuates TGFβ1-induced nuclear localization of EZH2 and decreases H3K27me3 levels in cells cultured on stiff substrata, suggesting that cellular contractility, in concert with a major cancer signaling regulator TGFβ1, modulates EZH2 subcellular localization. These findings provide a contractility-dependent mechanism by which matrix stiffness and TGFβ1 together mediate EZH2 signaling to promote EMT. 

Abstract Mesenchymal–epithelial transition (MET) is essential for tissue and organ development and is thought to contribute to cancer by enabling the establishment of metastatic lesions. Despite its importance in both health and disease, there is a lack of in vitro platforms to study MET and little is known about the regulation of MET by mechanical cues. Here, hyaluronic acid‐based hydrogels with dynamic and tunable stiffnesses mimicking that of normal and tumorigenic mammary tissue are synthesized. The platform is then utilized to examine the response of mammary epithelial cells and breast cancer cells to dynamic modulation of matrix stiffness. Gradual softening of the hydrogels reduces proliferation and increases apoptosis of breast cancer cells. Moreover, breast cancer cells exhibit temporal changes in cell morphology, cytoskeletal organization, and gene expression that are consistent with mesenchymal–epithelial plasticity as the stiffness of the matrix is reduced. A reduction in matrix stiffness attenuates the expression of integrin‐linked kinase, and inhibition of integrin‐linked kinase impacts proliferation, apoptosis, and gene expression in cells cultured on stiff and dynamic hydrogels. Overall, these findings reveal intermediate epithelial/mesenchymal states as cells move along a matrix stiffness‐mediated MET trajectory and suggest an important role for matrix mechanics in regulating mesenchymal–epithelial plasticity. 

Abstract Transforming growth factor (TGF)‐β1 is a multifunctional cytokine that plays important roles in health and disease. Previous studies have revealed that TGFβ1 activation, signaling, and downstream cell responses including epithelial‐mesenchymal transition (EMT) and apoptosis are regulated by the elasticity or stiffness of the extracellular matrix. However, tissues within the body are not purely elastic, rather they are viscoelastic. How matrix viscoelasticity impacts cell fate decisions downstream of TGFβ1 remains unknown. Here, we synthesized polyacrylamide hydrogels that mimic the viscoelastic properties of breast tumor tissue. We found that increasing matrix viscous dissipation reduces TGFβ1‐induced cell spreading, F‐actin stress fiber formation, and EMT‐associated gene expression changes, and promotes TGFβ1‐induced apoptosis in mammary epithelial cells. Furthermore, TGFβ1‐induced expression of integrin linked kinase (ILK) and colocalization of ILK with vinculin at cell adhesions is attenuated in mammary epithelial cells cultured on viscoelastic substrata in comparison to cells cultured on nearly elastic substrata. Overexpression of ILK promotes TGFβ1‐induced EMT and reduces apoptosis in cells cultured on viscoelastic substrata, suggesting that ILK plays an important role in regulating cell fate downstream of TGFβ1 in response to matrix viscoelasticity. 

Abstract Epithelial‐mesenchymal transition (EMT) is a physiological process that is essential during embryogenesis and wound healing and also contributes to pathologies including fibrosis and cancer. EMT is characterized by marked gene expression changes, loss of cell–cell contacts, remodeling of the cytoskeleton, and acquisition of enhanced motility. In the late stages of EMT, cells can exhibit myofibroblast‐like properties with enhanced expression of the mesenchymal protein marker α‐smooth muscle actin and contractile activity. Transforming growth factor (TGF)‐β1 is a well‐known inducer of EMT and it activates a plethora of signaling cascades including extracellular signal‐regulated kinase (ERK). Previous reports have demonstrated a role for ERK signaling in the early stages of EMT, but the molecular impacts of ERK signaling on the late stages of EMT are still unknown. Here, we found that inhibition of the phosphorylation of ERK enhances focal adhesions, stress fiber formation, cell contractility, and gene expression changes associated with TGFβ1‐induced EMT in mammary epithelial cells. These effects are mediated in part by the phosphorylation state and subcellular localization of myocardin‐related transcription factor‐A. These findings indicate that the intricate crosstalk between signaling cascades plays an important role in regulating the progression of EMT and suggests new approaches to control EMT processes.