There are multiple modes of individual tumor cell invasion with differing sensitivities to ROCK inhibition. Cells that migrate through 3-dimensional extracellular matrix with a rounded morphology are more dependent upon ROCK activity, whereas cells that invade using elongated actin-rich protrusions are relatively insensitive to ROCK inhibition. However, both invasion modes are dependent upon the contractile force generated by myosin ATPase activity, indicating that regulators of actomyosin function in addition to ROCK are involved. Cdc42 is a member of the Rho BMS-214778 GTPase protein family that plays key roles in actomyosin cytoskeletal organization and cell migration through effector proteins including the 1542705-92-9 myotonic dystrophy kinase-related Cdc42-binding kinases a and b. Both ROCK and MRCK belong to the AGC kinase family, and MRCK can be further classified into the myotonic dystrophy protein kinase subfamily. MRCKa and MRCKb are 190 kDa multi-domain proteins expressed in a wide range of tissues, with,80 sequence identity across their kinase domains. ROCK and MRCK kinases share,45�C50 sequence identity homology over the N-terminal kinase domains, which is reflected in their shared abilities to phosphorylate a similar set of substrates of the myosin light chain phosphatase complex ). However, the C-terminal regulatory regions of ROCK and MRCK are distinctly different. Importantly, it has been observed that actomyosin contractility required for the invasion of cells with elongated mesenchymal morphology is dependent on Cdc42-MRCK signaling. In such cells, which were largely resistant to ROCK inhibition alone, siRNA-mediated knockdown of MRCK had some effect on inhibiting invasion while the combination of MRCK knockdown along with ROCK inhibition more effectively inhibited invasion and caused cells to adopt a spherical, non-blebbing morphology. These data indicate that during elongated mesenchymal invasion, ROCK and MRCK regulate independent and co-operative pathways that collaborate in a non-compensatory manner. Given that there appears to be considerable plasticity in the abilities of tumor cells to interchange between elongated and rounded modes of tumor cell invasion in response to varying environmental circumstances, one potential anti-invasion strategy would be to simultaneously target ROCK and MRCK activity in order to inhibit multiple invasion modes and to counteract tumor cell adaptability. Further data supporting the strategy of simultaneous ROCK and MRCK inhibition comes from organotypic cell culture systems used to examine ECM invasion by co-cultures of squamous cell carcinoma and cancer-associated stromal fibroblasts. SCC cells form an epidermal-like layer when grown on a three-dimensional collagen matrix, within which embedded CAFs a