Comprehensive analysis on the whole Rho-GAP family reveals that ARHGAP4 suppresses EMT in epithelial cells under negative regulation by Septin9.

Epithelial to mesenchymal transition (EMT) is a fundamental biological process that occurs during development and tumorigenesis. The Rho family of GTPases (Rho-family) is a well-characterized regulator of actin cytoskeleton that gives rise to EMT-associated cell activities. Meanwhile, there are in total at least 66 different Rho-GTPase-activating proteins (Rho-GAPs), which, as an upstream regulator, inactivate specific members of the Rho-family in a cell context-dependent manner. However, molecular roles of individual Rho-GAPs are poorly understood, particularly regarding their involvements in EMT. Here, based on comprehensive screening on the whole Rho-GAP family, we identified specific Rho-GAPs that are responsible for the maintenance of epithelial cell phenotypes, suppressing EMT in human mammary epithelial cells. Specifically, we revealed that at least two Rho-GAPs, that is, ARHGAP4 and SH3BP1, critically regulate the cell morphology. Among them, we focused on ARHGAP4 and demonstrated with multidisciplinary approaches that this specific Rho-GAP regulates epithelial/mesenchymal-selective marker expression, cell proliferation, migration, 3D morphogenesis, and focal adhesion/stress fiber-driven physical force generation in a manner reminiscent of the EMT process. Furthermore, we identified Septin9 with proteomic analyses as a negative regulator of ARHGAP4, which promotes the occurrence of EMT by activation of the FAK/Src signaling pathway. These findings shed light on the novel Rho-GAP-associated pathway in the EMT process under development and tumorigenesis.