LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells.

LIM kinases 1 and 2 (LIMK1/2) are centrally positioned regulators of actin cytoskeleton dynamics. Using siRNA-mediated knockdown or a novel small molecule inhibitor, we show LIMK is required for path generation by leading tumor cells and nontumor stromal cells during collective tumor cell invasion. LIMK inhibition lowers cofilin phosphorylation, F-actin levels, serum response factor transcriptional activity and collagen contraction, and reduces invasion in three-dimensional invasion assays. Although motility was unaffected, LIMK inhibition impairs matrix protein degradation and invadopodia formation associated with significantly faster recovery times in FRAP assays indicative of reduced F-actin stability. When LIMK is knocked down in MDA-MB-231 cells, they lose the ability to lead strands of collectively invading cells. Similarly, when LIMK activity is blocked in cancer-associated fibroblasts, they are unable to lead the collective invasion of squamous carcinoma cells in an organotypic skin model. These results show that LIMK is required for matrix remodeling activities for path generation by leading cells in collective invasion.

BCL6 suppresses RhoA activity to alter macrophage morphology and motility.

BCL6 is a potent transcriptional repressor that plays important roles in germinal center formation, T helper cell differentiation and lymphomagenesis and regulates expression of several chemokine genes in macrophages. In a further investigation of its role in macrophages, we show that BCL6 inactivation in primary bone marrow-derived macrophages leads to decreased polarization, motility and cell spreading accompanied by an increase in peripheral focal complexes, anchored F-actin bundles and cortical F-actin density. These changes were associated with excess RhoA activation. C3 transferase inhibition of RhoA activity reverted the adhesion structure phenotype, which was not affected by Rho kinase inhibitors, suggesting that other downstream effectors of Rho maintain this Bcl6(-/-) phenotype. Excess RhoA activation in BCL6-deficient macrophages is associated with a decrease in the p120RasGAP (RASA1)-mediated translocation of p190RhoGAP (GRLF1) to active RhoA at the plasma membrane and a reduction in cell surface expression of the CSF1R that has been reported to recruit RasGAP to the plasma membrane. Reconstitution of BCL6 expression in Bcl6(-/-) macrophages results in complete reversion of the morphological phenotype and a significant increase in cell surface CSF1R expression whereas overexpression of the CSF1R corrects the polarization and adhesion structure defects. These results demonstrate that BCL6 suppresses RhoA activity, largely through upregulation of surface CSF1R expression, to modulate cytoskeletal and adhesion structures and increase the motility of macrophages.