Rho signaling, ROCK and mDia1, in transformation, metastasis and invasion
The Rho subgroup of the Rho GTPases consisting of RhoA, RhoB and RhoC induces a specific type of actin cytoskeleton and carry out a variety of functions in the cell. mDia and ROCK are downstream effectors of Rho mediating Rho action on the actin cytoskeleton; mDia produces actin filaments by nucleation and polymerization and ROCK activate myosin to cross-link them for induction of actomyosin bundles and contractility. mDia is potentially linked to Rac activation and membrane ruffle formation through c-Src-induced phosphorylation of focal adhesion proteins, and ROCK antagonizes this mDia action. Thus, cell morphogenesis, adhesion, and motility can be determined by the balance between mDia and ROCK activities. Though they are not oncogenes by themselves, overexpression of RhoA and RhoC are often found in clinical cancers, and RhoC has been repeatedly identified as a gene associated with metastasis. The Rho-ROCK pathway is implicated in Ras-mediated transformation, the amoeboid movement of tumor cells in the three-dimensional matrix, and transmigration of tumor cells through the mesothelial monolayer. On the other hand, the Rho-mDia1 pathway is implicated in Src-mediated remodeling of focal adhesions and migration of tumor cells. There is also an indication that the Rho pathway other than ROCK is involved in Src-mediated induction of podosome and regulation of matrix metalloproteases. Thus, Rho mediates various phenotypes of malignant transformation by Ras and Src through its effectors, ROCK and mDia.
1 Rho signaling; ROCK and mDia1
2 ROCK and mDia in cell migration
The works by the Grosse’s group and Yamana et al. thus clearly demonstrated the importance of Rho and its effector, mDia1, in cell migration, and suggested that this signaling collaborates with Rac and Cdc42 in this process. However, this view challenged the previous dogma that, among Rho GTPases, only Rac and Cdc42 are important and Rho is dispensable in cell migration. Furthermore, Arthur and Burridge demonstrated that p190Rho-GAP is activated by c-Src-dependent phosphorylation and the phosphorylated form is abundant in migrating cells and functions to down-regulate Rho [42, 43], further negating the role of Rho for migration. However, the role of Rho for cell migration is supported by a recent imaging study analyzing the localization of active Rho in migrating fibroblasts. Hahn and collaborators  developed a FRET biosensor for active Rho by conjugating a YFP-RhoA and CFP-Rho binding domain of Rhotekin, expressed in MEF cells, and examined localization of active Rho in cells during random migration or migration in the wound healing assay. Consistent with the previous findings , the Rho activity was minimal in the cell body. However, in addition to the high Rho activity in the tail of robust retraction, they found a sharp band of markedly higher Rho activity immediately adjacent to the front edge of cells with extending protrusion. This is consistent with the role of mDia1 proposed by the studies of the Grosse’s group and Yamana et al., and strengthens a significant role Rho plays for cell migration.
3 Rho signaling in metastasis and invasion
Given the roles of Rho GTPases in cell adhesion and migration, it is likely that they also play a role in tumor metastasis and invasion. Survey of genes over-expressed in clinical cancers and tumor cell lines showed frequent over-expression of RhoA and RhoC (see for review, ). Of the two, expression of RhoC was correlated with invasive phenotype of clinical cancers. Earlier, Suwa et al.  examined RhoA, RhoB and RhoC expression in 33 cases of pancreatic ductal adenocarcinoma, and found that the expression level of RhoC was higher in tumors than in non-malignant tissues, higher in metastatic lesions than in primary tumors, and correlated with perineural invasion and lymph node metastasis and poorer prognosis, whereas expression of either RhoA or RhoB did not show correlation with these clinicopathological findings. The RhoC gene was also identified experimentally as the gene involved in metastasis by genomic analysis of highly metastatic melanoma cells. Clark et al.  injected A375P human melanoma cells or B16F0 mouse melanoma cells intravenously into nude mice, dissected metastases occurring in the lung, expanded cells of the metastatic colonies in tissue culture and re-introduced into host mice. Repeating this procedure three times, they isolated cell populations with high metastatic potential from each cell line. By comparing gene expression between these populations and the parental populations with microarray analysis, they detected three genes that were highly expressed in all the metastatic tissues selected from both cell lines; they were fibronectin, RhoC and thymosin β4. The authors confirmed these genes derived from tumor cells and not surrounding lung tissues, and verified potential of RhoC as a metastasis gene by expressing exogenous RhoC in melanoma cells and examining lung metastasis. The selected metastatic cell population and the cells over-expressing exogenous RhoC did not show enhanced proliferation, but were more migratory and more invasive and exhibited elongated morphology, the properties suppressed by expressing dominant negative Rho mutant. This work thus confirmed experimentally the importance of RhoC in metastasis. More recently, analysis of microRNAs (miRNAs) expressed in breast cancer also identified RhoC as a metastasis-associated gene. Ma et al.  first detected 29 miRNAs that are differentially expressed between primary breast carcinomas and normal mammary tissues, then examined their expression in breast cancer cell lines with metastatic potential, and identified miR-10b as a candidate miRNA associated with metastasis. They then analyzed functions of this miRNA, and found that miR-10b regulates cell migration and invasion in vitro, and initiates tumor invasion and distant metastasis in vivo. Analyzing the mechanism by which miR-10b induces tumor invasion, the authors found that miR-10b directly inhibits translation of HOXD10, which results in release of HOXD10-mediated inhibition of expression of genes involved in cell migration including RhoC.
Thus, there is substantial amount of evidence for involvement of RhoC in tumor metastasis. However, little information is available how RhoC mediates such an action. It remains unclear whether the metastatic potential of RhoC is due to specific localization or specific upstream or downstream signaling. Among RhoA, B and C, RhoB is known to localize to endosomes, while no distinct localization has been reported for RhoA and C . It was reported that RhoC expression leads to induction of angiogenic factors in breast epithelial cells . It was also reported that RhoC interacts with ROCK more effectively than RhoA and facilitates disruption of adherens junctions of epithelial cells . These mechanisms, particularly preferential binding to ROCK, may partly explain the mechanism of RhoC-mediated tumor invasion in vivo, given ROCK-mediated disruption of cell-cell junction as described above and ROCK-dependent transmigration and amoeboid movement of tumor cells as described below, though the morphology of RhoC-overexpressing cells is not consistent with the rounded morphology of ROCK-activated cells,
The earliest indication for involvement of ROCK in tumor invasion was obtained by transmigration experiment of tumor cells. In order for tumor cells to establish metastasis at sites distant from its origin and invade into tissues, they have to transmigrate through host cell layers such as the endothelial cell layer covering the blood vessels and the mesothelial cell layer covering the peritoneum. Starting with the finding that cultured rat MM1 hepatoma cells required serum stimulation and intact Rho activity to migrate through the mesothelial layer and establish tumor foci beneath the monolayer in vitro , Itoh et al.  examined involvement of ROCK in this process. They found that transfection of dominant active mutants of ROCK conferred MM1 cells the invasive activity independent of Rho and serum, whereas expression of a dominant negative ROCK mutant or treatment with a ROCK inhibitor, Y-27632, substantially attenuated invasiveness in vitro. Furthermore, continuous local infusion in vivo of Y-27632 markedly reduced dissemination and tumor nodule formation of MM1 cells injected into the peritoneal cavity of syngeneic rats. Thus, the work by Itoh et al. demonstrates that ROCK action is required not only for in vitro models of tumor invasion but also for tumor invasion in vivo. Given the work by Worthylake et al. on the role of ROCK in tail retraction discussed above, one obvious candidate of ROCK actions in this process is to retract tail while the cell body creeps beneath the monolayer, although there may be other ROCK actions that facilitate tumor invasion in vivo, one being ROCK-mediated tumor cell migration in the matrix (see below).
While many molecular mechanisms including Rho signaling underlying tumor invasion have been thought out based on the results of in vitro motility studies in the two dimensional culture, tumor cells actually invade into the three dimensional (3D) space where extensive fibrillar network of extracellular matrix (ECM) proteins such as collagen restricts their movement. This in vivo situation led to the proposal that the invasion of tumor cells requires coordination of cell adhesion/motility and proteolytic degradation of ECM substrates, a concept supported by many in vitro and in vivo model studies . However, application of inhibitors targeted to ECM-degrading proteases, particularly matrix metalloproteases (MMPs), provided only weak beneficial effects in tumor models in vivo in intact animals as well as in clinical trials in humans, raising a possibility of alternative mode of cell invasion in the face of MMP inhibition. Friedl and collaborators  examined this issue by studying the behavior of HT1080 fibrosarcoma cells over-expressing MT1-MMP (HT1080/MT1 cells) invading the 3D-collagen matrix in vitro. They observed that, without protease inhibition, the HT1080/MT1 cells adhere the collagen matrix in an integrin-dependent manner, produce tube-like defects in the matrix by proteolysis and migrate in the formed tubes. This is consistent with the classic motility-proteolysis coordination concept. They, however, found that, when proteolytic activity was inhibited, this proteolysis-dependent mesenchymal movement was converted to the amoeboid movement, in which cells adapt spherical round shape and pass through the fibrillar network by changing the shape of their bodies by propulsive squeezing along preformed fiber strands. They suggested that this mesenchymal-amoeboid transition is a supramolecular plasticity tumor cells can adopt in tissue invasion and escape from abrogation of proteolysis. Sahai and Marshall  also examined the behavior of several tumor cell lines in the 3D matrix and found that some tumor cell lines migrate through the 3D matrix in the rounded form, i.e. by contraction of their bodies. The round form of migration Sahai and Marshall found apparently corresponds to the amoeboid movement described by the Friedl group. Sahai and Marshall further analyzed signal transduction therein and found that the Rho-ROCK pathway is a major driving force for this mode of migration, and suggested that inhibition of both proteases and ROCK may be beneficial for inhibition of tumor invasion. Sahai and collaborators then extended this work by showing that cells with rounded morphology pushed away the collagen in front of them for invasion and this deformation was dependent on myosin phosphorylation and ROCK activity . They found that the actomyosin bundles are formed in a ROCK-dependent manner in the cell cortex perpendicular to the direction of migration just behind the invading edge, and suggest that the contraction of the cell cortex by these actomyosin bundles causes the cell body to move forward by pushing the collagen matrix away. A more recent report from this group demonstrated that localization of ROCK in the above actomyosin bundles and their contraction is dependent on PDK1 but not on its kinase activity , thus raising an interesting possibility for interaction of PI-3-kinase pathway and the Rho-ROCK pathway in tumor invasion.
4 Rho signaling and malignant transformation
Following these initial observations, Sahai and Treisman  then examined downstream signaling of Rho involved in transformation. They first used several point mutants in the effector loop of Val14RhoA and examined their synergism with active ΔNRaf in focus formation. Comparing the results obtained in this experiment with specificity of each mutant in binding to Rho effectors including ROCK, PKN, mDia2, citron and Rhophilin and activity of each mutant in other Rho actions such as stress fiber formation and serum response factor activation, they concluded that ROCK is involved in this process. They then used the ROCK inhibitor, Y-27632, and found that Y-27632 indeed inhibits transformation induced by activated Ras, H-Ras-R12, and co-expresison of ΔNRaf and V14RhoA . On the other hand, although co-expression of active ROCK mutant with ΔNRaf induced significant enhancement of transformation induced by ΔNRaf alone, the extent of enhancement was much lower than that achieved by co-expression of V14-RhoA and ΔNRaf. These results indicate that ROCK is required for Rho-mediated enhancement of Ras-induced transformation but that it is not the sole Rho effector in this process. The Treisman’s group further examined possible involvement of ezrin, a ROCK substrate, in this process, and found that expression of T567A ezrin mutant insensitive to ROCK phosphorylaitn interfered with transformation induced by the active Ras mutant . Sahai et al.  further examined intracellular localization of ROCK in Ras-transformed cells and found that the majority of ROCK is sequestered in an inactive pool by sustained ERK-MAP kinase activity under active Ras. They suggested that this may be one of the mechanisms for dissolution of stress fibers seen in Ras transformants. However, they presented no explanation how requirement for transformation and down-regulation for dissolution of stress fibers of ROCK can occur at the same time.
In addition to Ras, Rho signaling may also be implicated in transformation by Src. v-Src is the oldest oncogene, and its proto-oncogene, c-Src, is amplified in a variety of clinical cancers and its activity often correlates with their invasive potency . Src kinases, both v-Src and c-Src, are inactive in a soluble non-myristylated form, indicating that they must be directed to a specific subcellular structure(s) in order to induce transformation. Src exhibits a variety of discrete subcellular distribution including plasma membrane, adhesion plaques, cell-cell contact and perinuclear membranes. Earlier, Hamaguchi and Hanafusa  used various Src mutants, and found correlation between cytoskeletal association and transforming activity. Liebl and Martin  prepared chimera molecules in which v-Src was conjugated with motifs targeting to a specific subcellular site, and found that v-Src and not c-Src targeted to adhesion plaques could induce transformation phenotype, though the malignant phenotype by this chimera was not exactly the same as that induced by wild type v-Src. Given their own findings that translocation of v-Src to the cell periphery is important for its transformation activity, Frame and collaborators examined mechanism of peripheral translocation using temperature-sensitive mutant of v-Src . They found that v-Src accumulated in the perinuclear region at the restrictive temperature, and moves to the periphery upon the shift to the permissive temperature. In Swiss3T3 fibroblasts, this translocation of v-Src from the perinuclear pool to the periphery required serum stimulation. They found that, on serum addition, v-Src associates with serum-induced (Rho-mediated) actin stress fibers and accumulates in focal adhesions, and that intact actin filaments are required, while microtubules are dispensable, for this translocation. They then examined structural and catalytic requirement of Src for this translocation and found that the intact SH3 domain is essential for this translocation, while the myristylation and kinase activity are dispensable [79, 80]. They further found that stimulation of Swiss3T3 cells expressing GFP-Src construct with LPA, platelet-derived growth factor (PDGF) and bradykinin, stimuli known to activate Rho, Rac and Cdc42, respectively, translocated Src-GFP to focal adhesions, membrane ruffles and filopodia, respectively . This translocation was mimicked by co-expression of dominant active Rho GTPases, V14-RhoA, V12-Rac1 and V12-Cdc42, and inhibited by expression of dominant negative mutants of each GTPases. Intriguingly, localization of Src-GFP to lamellipodia and filopdia was suppressed by inhibition with N17-Rac1 and N17-Cdc42, but in both cases Src-GFP then accumulated in focal adhesions. Moreover, treatment with a specific ROCK inhibitor, Y-27632, not only suppressed of Rho-mediated accumulation of Src-GFP in focal adhesions but also that in lamellipodia and filopodia induced by PDGF and bradykinin, respectively. These results indicate that Src is originally recruited to focal adhesions in a Rho and ROCK-dependent manner, and then moves to focal complexes in lamellipodis or filopodia upon remodeling of focal adhesions to focal complexes induced by Rac or Cdc42. The Frame’s group  more recently reported that Src in the prinuclear region and during the transit to the periphery associates with endosomes, as originally observed by Kaplan et al. . The Src-containing endosomes partly overlap with those containing RhoB that resides in the endosome, and the peripheral translocation of Src is impaired in MEF cells prepared from Rho-B-/- mice, the defect rescued by re-expression of RhoB in the knockout cells. They also reported that, when cellular F-actin abolished by treatment with cytochalasin D reappear with the washout of the drug, clouds of F-actin become associated with the RhoB/Src-containing endosomes, and suggested that such actin structure may function to propel the endosomes in the cell. The Frame’s group  also examined translocation of Src related kinase, Yes and Fyn, and found that their translocation to the periphery also requires intact actin filaments. Interestingly, Fyn is localized to RhoD and not RhoB-containing endosomes, and this selective localization to the RhoD endosomes is dependent on palmitoylation of the N-terminal region of Fyn. Thus, the Frames group has carried out extensive study on the translocation mechanism of Src and Src-related kinases, and has found an important link between Src, actin and Rho GTPases (Fig. 8b). Curiously, however, they have discussed their results mainly in relation to Src-induced disassembly of focal adhesions, and not addressed how critical the pathway they defined is in Src-induced cell transformation. They have not examined, either, effector mechanism for actin filament assembly required for translocation of Src and Src-related kinases. Given the requirement of Rho in this process, a strong candidate is the mDia family of proteins. As discussed above, Yamana et al.  already reported that depletion of mDia1 resulted in impaired accumulation of c-Src in focal adhesions of migrating C6 rat glioma cells. Whether the mDia1-mediated mechanism also operates for elicitation of Src-induced malignant transformation should be explored in future studies.
As we review in this article, Rho signaling consisting of Rho, mDia and ROCK is apparently involved in elicitation of various phenotypes of tumor cells, transformation, motility, transmigration and invasion in vivo, and circumferential evidence has accumulated for strong relation of this signaling to oncogenic actions of Ras and Src. Naturally, such information raises many questions. They include; how does the Rho-ROCK pathway contribute to Ras-induiced transformation? Does it facilitate signaling pathway(s) other than the Raf-Erk pathway under Ras? Is there any Rho-mediated pathway other than ROCK functioning in Ras-induced transformation? Is the action of Rho signaling in transformation independent of its action in invasion or do they represent different aspects of the same action? Does Rho signaling function not only in Src-mediated motility and invasion but also in transformation induced by this oncogene? Are the actions of Rho signaling for Ras and Src separate and independent or are they intimately connected in elicitation of transformed phenotype by each oncogene? Finally, how much does Rho signaling contribute to tumorigenesis in intact animals and in clinical cancers? We are now in a stage where we can answer some or all of these questions. Clarifying these questions is hoped to provide an insight into how tumor cells integrate various signaling pathways including Ras, Src and Rho for expression of their malignancy.
This work was supported in part by Grants-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan
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