ROCK isoforms differentially modulate cancer cell motility by mechanosensing the substrate stiffness

Abstract

Tumors are characterized by extracellular matrix (ECM) remodeling and stiffening. The importance of ECM stiffness in cancer is well known. However, the biomechanical behavior of tumor cells and the underlying mechanotransduction pathways remain unclear. Here, we used polyacrylamide (PAA) substrates to simulate tissue stiffness at different progress stages of breast cancer in vitro, and we observed that moderate substrate stiffness promoted breast cancer cell motility. The substrate stiffness directly activated integrin β1 and focal adhesion kinase (FAK), which accelerate focal adhesion (FA) maturation and induce the downstream cascades of intracellular signals of the RhoA/ROCK pathway. Interestingly, the differential regulatory mechanism between two ROCK isoforms (ROCK1 and ROCK2) in cell motility and mechanotransduction was clearly identified. ROCK1 phosphorylated the myosin regulatory light chain (MRLC) and facilitated the generation of traction force, while ROCK2 phosphorylated cofilin and regulated the cytoskeletal remodeling by suppressing F-actin depolymerization. The ROCK isoforms differentially regulated the pathways of RhoA/ROCK1/p-MLC and RhoA/ROCK2/p-cofilin in a coordinate fashion to modulate breast cancer cell motility in a substrate stiffness-dependent manner through integrin β1-activated FAK signaling. Our findings provide new insights into the mechanisms of matrix mechanical property-induced cancer cell migration and malignant behaviors. STATEMENT OF SIGNIFICANCE: Here, we examined the relationship between substrate stiffness and tumor cellular motility by using polyacrylamide (PAA) substrates to simulate the stages in vivo of breast cancer. The results elucidated the different regulatory roles between the two ROCK isoforms in cell motility and demonstrated that stiff substrate (38 kPa) mediated RhoA/ROCK1/p-MLC and RhoA/ROCK2/p-cofilin pathways through integrin β1-FAK activation and eventually promoted directional migration. Our discoveries would have significant implications in the understanding of the interaction between cancer cells and tumor microenvironments, and hence, it might provide new insights into the metastasis inhibition, which could be an adjuvant way of cancer therapy.

Keywords: Cell motility; Integrin β1; Mechanotransduction; ROCK isoforms; Substrate stiffness.