Soft Matrices Downregulate FAK And Its Downstream Small GTPases To Promote Growth Of Tumor-repopulating Cells

Tumor-repopulating cells(TRCs)are a tumorigenic sub-population of cancer cells that drives tumorigenesis.We functionally define these soft-fibrin-gel-selected melanoma cells as tumor-repopulating cells(TRCs)based on their high efficiency in repopulating tumors in mice.TRCs are more resistant to anti-tumor drugs,and TRCs maintain long-term survival and growth in vivo.We have recently reported that soft fibrin matrices maintain TRCs growth by promoting histone 3 lysine 9(H3K9)demethylation and Sox2 expression and that Cdc42 expression influences H3K9 methylation.However,the underlying mechanisms of how soft matrices induce H3K9 demethylation remain elusive.Here we find that focal adhension kinase(FAK)is expressed in much lower amount and has lower activity in mouse melanoma cells and human cancer cells in soft matrices where TRCs are selected and grown than the unselected cells cultured on 2D surfaces.Importantly,silencing FAK promotes Sox2 expression and proliferation of control melanoma cells in stiff fibrin matrices,whereas overexpressing FAK suppresses Sox2 expression and reduces growth of TRCs in soft fibrin matrices.These data suggest that silencing FAK leads to increases in TRCs growth,rather than decreases as other researchers found in earlier papers.Silencing FAK in control melanoma cells decreases H3K9 methylation,whereas overexpressing FAK in TRCs enhances H3K9 methylation.Overexpressing small GTPases Cdc42 or RhoA in the presence of FAK knockdown restores H3K9 methylation levels,and overexpression of Cdc42 restores H3K9 methylation inhibited by RhoA knockdown.And silencing Cdc42,RhoA promotes Sox2 expression and proliferation of control melanoma cells in stiff fibrin matrices,whereas overexpressing each gene suppresses Sox2 expression and reduces growth of TRCs in soft fibrin matrices.In conclusion,our findings suggest that low FAK mediated by soft fibrin matrices downregulates H3K9 methylation through reduction of Cdc42 and RhoA,which in turn promotes TRCs growth.