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Directed movement of normal cells occurs when actin-related protein 2 and 3 complex (Arp2/3 complex) triggers the actin polymerization that forms lamellipodia immediately after binding to WAVE2. In order to determine whether the same mechanism correlates with liver metastasis from colorectal cancer, paired mirror sections of 154 cancer specimens (29 cases with liver metastasis and 125 cases without liver metastasis in which T factor, gender, primary tumor site, and age at operation were matched) were examined immunohistochemically for the localization of Arp2 and WAVE2. Expression of both Arp2 and WAVE2 was detected in the same cancer cells in 55 (35.7%) of the 154 cases, but not detected in the normal colonic epithelial cells. Univariate analysis showed that the colocalization was significantly predictive of liver metastasis (risk ratio [RR] 8.760. Likewise, histological grade (RR 2.46), lymphatic invasion (RR 9.95), and tumor budding (RR 4.00) were significant predictors. Among these, colocalization and lymphatic invasion were shown to be independent risk factors by multivariate analysis. Another 59 colorectal specimens were examined for mRNA expression of Arp2 by real time polymerase chain reaction. High mRNA levels of Arp2, that in situ hybridization revealed to be expressed by the cancer cells, were significantly associated with liver metastasis. However, its effect was absorbed by the influence of risk of the colocalization that is closely related to high expression of Arp2. These results indicate that the colocalization of Arp2 and WAVE2 is an independent risk factor for liver metastasis of colorectal carcinoma. (Cancer Sci 2007; 98: 992–999)
Actin filaments are the dominant structural components of lamellipodia, and actin is the most abundant protein in many eukaryotic cells. Regulation of lamellipodium formation is essential for embryonic development, wound healing, immune responses, and tissue development.(1–4) Since the discovery of the Arp2/3 complex about a decade ago, a broad range of biochemical and cell-biological studies have pointed to its pivotal role as a regulator of actin filament formation.(5–7)
One of the hallmarks of cancer cells is penetration into adjacent tissue, or invasion. While the majority of colorectal cancer cells form irregular ductal structures, highly metastatic colorectal cancer cells are frequently dissociated or dedifferentiated at the invasive front. This feature is called budding formation, indicating reduction of cell–cell adhesion. At the same time, cancer cells extend cell protrusions that are driven by actin polymerization at the leading edge, and thus co-ordinated inactivation of cell–cell adhesion and actin polymerization is required for active invasion and metastasis of colorectal cancer cells. It is possible that the Arp2/3 complex is one of the coordinators of both events in the downstream of cellular signaling, not only in normal cells but also in cancer cells.
The formation of cadherin-mediated cell junctions is accompanied by profound remodeling of the actin cytoskeleton. The Arp2/3 complex is involved in the regulation of cadherin-mediated cell–cell adhesion,(8–11) and is also linked to the proteins of the integrin family in various ways.(12–14) The composition of a large member of the integrin family proteins, and each of the members of the family appears to partly determine the specific reaction of each cell type to stimulation by the extracellular matrix. In a previous study of a human monocyte cell line (THP-1), we demonstrated that the Arp2/3 complex regulates integrin β-1-mediated attachment to fibronectin during chemotaxis induced by monocyte/macrophage chemoattractant protein 1 (MCP)-1 by binding to the Wiskott–Aldrich syndrome protein (WASP).(14)
Although many proteins are capable of regulating the function of the Arp2/3 complex in vitro, the members of the WASP/Scar/WASP family verprolin-homologous protein (WAVE) appear to be the most important.(15–17) The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe contain only one WASP/Scar protein, whereas Dictyostelium, Caenorhabditis elegans, and Drosophila melanogaster possess one WASP and one WASP/Scar protein.(2,18) Multicellular animals possess five WASP family proteins: WASP, N-WASP, and WAVE1–3 which are identical to Scar1–3. These proteins have a common C-terminal verprolin-homology domain that is responsible for Arp2/3 complex activation as well as for cell adhesion.(14,19–21)
Scar and WASP have clearly distinct functions in Drosophila. Scar, but not WASP, is indispensable for cytoplasmic organization in the blastoderm, axon development in the central nervous system, egg chamber structure, and adult eye morphology.(22) In mammals, the system may be more complicated, since WAVE1, WAVE2, WASP, and N-WASP appear to have specific functions during mammalian development that cannot be compensated by other family members.(23,24)
One of the five WASP family proteins, WAVE2, is essential for lamellipodial formation in directed migration, at least in mouse embryonic fibroblasts and endothelial cells.(25,26) In the present study we focused on the localization of Arp2 and WAVE2 in human colorectal cancer, since we have already reported that the Arp2/3 complex is involved in carcinogenesis.(27) We examined the localization of Arp2 and WAVE2 in colorectal cancer cells immunohistochemically, and the biological significance of their localization was assessed by correlation with clinical data, particularly in relation to liver metastasis.
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We have shown that the colocalization of Arp2 and WAVE2 is an independent risk factor of liver metastasis. Metastasis is a multistep process by which cancer cells spread to other organs. The first step involves loosening of cell–cell adhesion.(35,36) The second step is the formation of lamellipodial protrusions that allows directional cell movement. Arp3 and WAVE2 were colocalized at the advancing margin of a colon cancer cell (SW620) in this study. Because the mechanism of the formation of lamellipodium that Arp2/3 complex is activated by binding with WAVE2 was demonstrated by using non-cancerous cells,(37,38) we think this mechanism is also involved in the second step leading to the liver metastasis of colorectal cancer.
The colocalization of Arp2 and WAVE2 was significantly associated with tumor budding, and indeed, cancer cells forming tumor budding frequently show colocalization of Arp2 and WAVE2 at the margin of invasion. The presence of tumor budding is considered strong evidence of high metastatic potential.(39,40) Cancer cells with budding formation have loose cell–cell adhesion. Our data suggest that these cells have the potential to move aggressively.
In our immunohistochemical staining for colon cancer tissue, distinct colocalization of Arp2 and WAVE2 was not detected in normal epithelial cells, though the immunoreaction of either Arp2 or WAVE2 was seen focally. In a previous study,(27) we detected immunoreaction of Arp2 and Arp3 in macrophages, lymphocytes, and endothelial cells. Macrophages were shown to express both Arp2 and WAVE2 in this study. Because these cells are thought to function while migrating in tissues, cells that coexpress Arp2 and WAVE2 may have greater ability to move freely than cells that do not. We think that immunohistochemical colocalization of Arp2 and WAVE2 by cancer cells partly explains their ability to migrate actively.
We carried out a Western blot analysis to determine whether the Arp2 protein level was elevated in the tissue specimens of metastatic cases, and we found no difference in expression level between the cancer tissue of liver metastatic cases and non-metastatic cases (data not shown). We therefore examined the paired colon cancer cell lines SW480 (derived from the primary site) and SW620 (derived from the metastatic site). But a distinct difference in the expression of either Arp2 or WAVE2 was not detected by Western blot analysis. Though quantification of the protein level was not examined, the result may be due to the fact that no more than 10–20% of cancer cells show colocalization.
The level of expression of Arp2 mRNA was high in cases that coexpressed WAVE2. Another study demonstrated lower mRNA expression of Arp2/3 complex in gastric cancer tissue than in normal gastric mucosa.(41) Poorly-differentiated cancer cells show almost complete loss of cell–cell adhesion and polarity, and diffusely invade into the surrounding normal tissue. This invasive pattern is likely to be different from that of well-differentiated carcinoma, which forms a well-defined mass lesion. Since poorly-differentiated cancers do not form clear lamellipodial extensions, they may possess other mechanisms of cell movement. However, our results of the level of Arp2 mRNA expression cannot exclude the probability of the influence of the stromal cells because the specimens include abundant stromal cells that can express Arp2 mRNA. Various types of cancer cell lines should be examined for the expression level of Arp2 mRNA.
In the present study of colorectal cancer cells we focused on the Arp2/3 complex and attempted to correlate its presence with clinical features, including metastasis. In colorectal cancer cells with high metastatic potential, the Arp2/3 complex appears to be activated by binding to WAVE2. In well-differentiated adenocarcinomas, such as the majority of colon cancers, the function of Arp2/3 complex is likely to be maintained or enhanced. As far as we know, no Arp2 or Arp3 gene abnormalities have been reported in cancer, and our own examinations of several human cell lines have failed to reveal any mutations (data not shown). High mRNA expression of WAVE2 may serve to regulate cell movement in colon cancer. A highly metastatic melanoma cell line has been reported to show higher expression of WAVE2, at both the protein and mRNA levels than its parental cell line, and WAVE2 RNAi was found to reduce its metastatic potential.(42)
We do not have data that can explain how a high level of Arp2 mRNA is associated with colocalization. We hypothesize that a high Arp2 mRNA level might be the increase of the activated form of WAVE2. The majority of WAVE2 in the cell is complexed with Abl-interactor-1 (Abi1), Nck-associated protein-1 (Nap1), a small protein HSPC300, and p53-inducible protein-121 (PIR121)/Sra1. However, it is not clear how activated WAVE2 interacts with the Arp2/3 complex on the membrane of the lamellipodia. This interaction may be a key to explaining the association between high Arp2 mRNA levels and the colocalization of Arp2 and WAVE2.
The present study demonstrates that the colocalization of Arp2 and WAVE2 is an independent risk factor for liver metastasis in colorectal cancer. The result is considered to be reliable because the function of the interaction of Arp2/3 complex and WAVE2 has been established as the signal to form lamellipodial protrusion to induce cell migration, and because it is associated with tumor budding and irregular invading growth (INF) that are established features of aggressive colorectal cancer. Further studies designed to selectively block the interaction of Arp2 and WAVE2 in cancer cells may yield means of preventing invasion and metastasis.