RhoGTPases exerted its cytoskeletal impact through activation of their specific effectors. In this section, we will review the effectors that have been shown to be involved in HCC metastasis, namely WAVE2, PAK1 and ROCK.
There are five members in the WASP family, which can be further divided into two different sub-groups: WASP (WASP, N-WASP) and WASP family verprolin-homologous protein (WAVE1, WAVE2 and WAVE3). The WASP family acts as downstream effectors of Cdc42 and Rac. WASP binds and activates the Arp2/3 complex, which in turn catalyses and nucleates actin polymerization (68). Among the five members of the WASP family, only WAVE2 has been reported to be associated with HCC and therefore will be further discussed herein. WAVE2 has been reported to be associated with metastasis in colorectal cancer, melanoma, as well as HCC (68). It has been demonstrated in melanoma cells that WAVE2 is the main downstream effector of Rac1 in melanoma invasion and metastasis (69). Knockdown of WAVE2 remarkably suppresses membrane ruffles, cell migration and invasion in vitro, and reduces pulmonary metastasis of melanoma cells in C57BL/6 mice (69). Furthermore, colocalization of Arp2 and WAVE2 is associated with colorectal metastasis to liver and lymphatic invasion (70). From a study on 112 HCC samples, immunohistochemistry demonstrated that 71 of 112 HCC cases had WAVE2 overexpression, and overexpression of WAVE2 was associated with aggressive HCC features including the presence of venous invasion and multiple tumour nodules, absence of tumour capsules, poor cellular differentiation and shortened survival (71).
P21-activated protein kinase 1 is a downstream effector shared by Cdc42 and Rac. PAK1 is involved in the regulation of cell morphology and cell movement. PAK1 was found to be overexpressed in breast and colorectal cancers (72, 73). Stable expression of the kinase-active form of PAK1 enhances the mobility but not the proliferation of MCF7 cells, a breast cancer cell line (74). Reversely, stable expression of the kinase-inactive form of PAK1 suppresses breast cancer cell invasion by inhibiting the formation of focal adhesions and stress fibres (75). PAK1 is overexpressed in HCC at both mRNA and protein levels (76). Clinicopathologic correlation has revealed that overexpression of PAK1 mRNA is closely associated with features of aggressive HCC including the presence of venous invasion, poor cellular differentiation, advanced tumour stages and shortened disease-free survival (76). PAK1 is also highly expressed in H2M as compared with H2P (76). Stable overexpression of PAK1 in HepG2 hepatoma cells enhances cell migration and spreading through the regulation of stress fibres while knockdown of PAK1 in H2M cells suppresses HCC cell migration. Consistent with the findings in breast cancer, PAK1 has no major effect on cell proliferation in HCC (76).
Rho-kinase is a RhoA-GTP binding protein and a direct effector of RhoA (77–79). ROCK is a serine/threonine kinase that phosphorylates a wide repertoire of downstream targets involved in the cytoskeleton rearrangement. ROCK1 and ROCK2 are the two family members of the ROCK family (80). Overexpression of ROCK1 and ROCK2 was demonstrated in testicular and bladder cancers at the protein level (35, 36). In addition, ROCK inhibitor (Y27632) treatment was sufficient to abolish chemotactic migration in pancreatic cancer cell lines (81). Although ROCK1 and ROCK2 are highly homologous, sharing many common targets and cellular functions, only the ROCK2 protein is significantly overexpressed in HCC (82). Overexpression of ROCK2 is closely associated with the formation of a tumour microsatellite, which is an indicator of intrahepatic metastasis (82). Using constitutively active/inactive ROCK constructs, short hairpin RNA targeting ROCK and ROCK-specific inhibitors, the functional roles of ROCK in HCC invasion and metastasis have been elucidated by different research groups (24, 39, 40, 82, 83). Both ROCK1 and ROCK2 have been reported to participate in the activation of myosin and the polymerization of actin filaments in HCC cells. ROCK inhibitor, Y27632, suppresses actin reorganization, focal adhesion formation and myosin activity, and therefore suppresses cell migration and invasion in different HCC cell lines (24). Reversely, stable transfection of dominant-active ROCK1 or ROCK2 in rat hepatoma cells and human HCC cells significantly enhanced HCC cell motility and invasiveness (39, 40, 82). In a model using the injection of Li7 cells, a human HCC cell line, into the livers of SCID mice, Y27632 and stable expression of dominant-negative ROCK1 significantly reduced the incidence of macroscopic and microscopic intrahepatic metastasis and suppressed infiltrative tumour boundary (39, 83). In another model using the injection of MMP1 cells, a rat hepatoma cell line, into the peritoneal cavities of Donryu rats, Y27632 reduced RhoA-induced ascites and nodule formation and the frequency of tumour cell dissemination into the peritoneal cavity (40). In a model using the implantation of solid tumours into the livers of nude mice, our group showed that stable knockdown of ROCK2 remarkably suppressed different invasive features of the tumours, especially the incidence of tumour microsatellite formation, which is consistent with the clinical pathological correlation in human HCC (82). Intriguingly, almost all of the findings reported by different groups indicate that both ROCK1 and ROCK2 are more involved in the metastatic aspect instead of the tumour-proliferative aspect (24, 39, 40, 82, 83). The ROCK inhibitor shows no significant impact on HCC cell proliferation in vitro (24). In ROCK inhibitor-treated animals, HCC cells overexpressed with dominant-negative ROCK1, and HCC cells with ROCK2 knocked down all, resulting in a slight decrease of primary liver tumour size; however, these differences were mild and statistically insignificant (39, 82, 83).