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To reduce cancer mortality, understanding of mechanisms of cancer metastasis is crucial. We have established six rat hepatocellular carcinoma (HCC) cell lines, which exhibit differing metastatic potential to the lung after inoculation into the tail veins of nude mice. In the present experiment, we investigated the process of cell attachment to metastatic sites and possible regulating factors. One hour after inoculation, two of two HCC cell lines with high metastatic potential and one of two HCC cell lines with low metastatic potential exhibited many attached cells in the lung. One day after inoculation, lung metastatic foci were observed only with highly-metastatic cells with elevated connexin 43 (Cx43) expression as assessed by cDNA array analysis. Furthermore, 24 or 48 h after transfection of an siRNA targeting Cx43, in vitro invasion and migration were suppressed by 68% (P < 0.001) and 36% (P < 0.05) compared with control-siRNA transfected cells, despite no differences in cellular morphology, cell proliferation or apoptotic activity. Moreover, the number of metastatic nodules per lung area in nude mice was significantly (P < 0.01) reduced. In conclusion, suppression of Cx43 expression in tumor cells reduced in vitro migration and invasion capacity and in vivo metastatic ability so that Cx43 has potential as a molecular target for prevention of cancer metastasis with Cx43 overexpressing tumors. (Cancer Sci 2012; 103: 860–867)
Despite significant improvements in diagnosis, surgical techniques, general patient care and local and systemic adjuvant therapies, most deaths from cancer are due to metastases that are resistant to conventional therapies. To reduce cancer mortality, an understanding of the mechanisms of cancer metastasis and the development of treatments focusing on prevention and/or care of metastases are a high priority.
We have established six rat hepatocellular carcinoma (HCC) cell lines from an in vivo rat HCC model,[2-4] in which combination treatment of N-nitrosomorpholine and diethylnitrosamine induces liver tumors metastasizing to the lung. These cell lines (HSU-C1, -C2, -C5F, -C6, -N1 and -L2) show similar in vitro doubling times but widely differing metastatic ability after inoculation into the tail vein or transplantation into the subcutis of nude mice. In particular, the N1 and L2 lines are particularly metastatic to the lung when inoculated into the tail vein, as compared with the other lines. All these lines express the glutathione S-transferase placental form (GST-P), known to be upregulated during rat hepato-carcinogenesis, from preneoplastic liver cell foci to hepatocellular carcinoma.[5-7] Thus, immunohistochemistry for rat GST-P can readily visualize the cells in mouse tissue.
Metastasis is a multi-step process, involving angiogenesis, invasion, detachment, circulation, adhesion and extravasation. The genes and factors responsible for promotion or suppression of metastasis are multiple for each step and reasons for differences in metastatic potential remain to be elucidated.
It has been reported that cancer cells can be detected in peripheral blood or bone marrow of patients before metastatic lesions become clinically evident.[9-11] In one experimental analysis of micrometastasis using LacZ gene-tagged Lewis lung carcinoma cells, <0.1% of injected cells generated metastatic foci. Metastasis or attachment of circulating-tumor cells to the distant organ might thus be considered as a very rare event.
In the present experiment, using rat HCC cells with different metastatic potential, we analyzed factors that might play a critical role in attachment of circulating tumor cells to metastatic sites as possible targets for suppression of metastasis.
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The present results demonstrated many anchored cells to be present in the lungs 1 h after intravenous inoculation of HSU cell lines with high metastatic potential and one of two HSU cell lines with low metastasis potential. A few cells of the other low metastatic potential were also observed in the lung capillaries. Interestingly, 24 h after inoculation, most of the attached cells in the lung had disappeared. Moreover, no anchored inoculated cells were noted in other organs such as the kidney, liver and spleen at any time point. These findings are consistent with our former study, in which metastatic nodules were not observed except in the lungs and regional lymph nodes at a later time point.
Overall, it can be said that only a limited number of cells are able to survive in the target organs, in line with the idea of seed and soil proposed by Paget over 100 years ago. This concept has been emphasized in more recent studies focused on the tumor-microenvironment. However, the mechanisms and reasons for the limited number of cells that can attach and grow, among the enormous number of circulating tumor cells, remain unclear.
We have been studying the role of Cx32 and Cx43 in rat liver carcinogenesis over many years.[14-17] Moreover, data have accumulated suggesting that these molecules play an important role in cancer metastasis.[18-25] Therefore, we have intensively given attention to the expression of connexins. The present microarray analysis revealed expression of Cx26 and Cx32, major connexins in the liver, to be extremely low in all HSU cell lines. However, Cx43, a minor connexin in the liver, was highly expressed in metastatic HSU cell lines (N1 and L2) compared with low metastatic HSU cell lines (C1, C5F and C6) and in normal liver tissue. Connexins are components of gap junction channels, which mediate direct cell-to-cell communication by allowing passage of small molecules under about 1000 Da. Six connexin subunits form hemichannels and by pairing of two hemichannels the gap junction is constructed. More than 20 different mammalian connexins have been identified. The roles of connexins in cancer are still controversial. Most of the studies suggest that they are protective against tumor onset. For example, mice and rats lacking or with reduced Cx32 are more susceptible to chemical- and radiation-induced liver[14, 16, 27, 28] and lung carcinogenesis. The sharing of ions, amino acids, second messengers and various metabolites through gap junctions is thought to maintain homeostasis between cells. Glutathione has high permeability through gap junction channels,[30, 31] and its antioxidant properties protect cells from reactive oxygen species. Thus, glutathione is considered to protect against DNA damage, as well as detoxifying carcinogens.
In the present experiment, interference in Cx43 expression with an siRNA significantly suppressed invasion and migration of L2 cells, accompanied by reduction of active MMP-9 secretion in vitro, and also suppressed lung metastasis after inoculation into tail veins of nude mice. Although reports indicating relations between Cx45 and metastasis are limited, upregulation of Cx43 is associated with invasion, intravasation, extravasation and metastasis of melanoma cells,[18, 19] mammary gland carcinoma cells,[19, 20] and glioma cells. Indeed, Cx43-transfected malignant glioma cells showed elevated invasive capacity associated with higher MMP-2 and MMP-9 secretion, compared with mock-transfected cells. Moreover, several studies have suggested that upregulation of MMP-9 is related to the metastasis in human hepatocellular carcinoma.[33, 34]
On the other hand, there is opposing evidence that reduced expression of Cx43 correlates with invasion and metastasis in lung cancer cell and overexpression of Cx43 was found to reduce cell adhesion and/or migration of breast cancer cells.[24, 25]
It is of considerable interest that only a limited number of circulating tumor cells can survive and form metastatic foci among the enormous numbers inoculated into veins. Clonal cultured cells should share exactly the same genetic characteristics. Variation in metastasizing potential may thus not be simply explained by an additional gene alteration. Among more than 20 connexins, Cx43 is commonly found in endothelial cells, osteoblastic cells and heart. Highly metastatic HCC cells that aberrantly express Cx43 might form gap junction channels with endothelial cells, which might be beneficial for HCC cell survival. Further studies regarding the expression and detailed localization of Cx43 in HCC metastatic foci and effects on the angiogenesis would be needed in our system. Cytoplasmic dye transfer between metastatic tumor cells that express Cx43 and endothelium has been reported for melanoma and mammary carcinoma. Upregulation of Cx43 may also occur in tumor cell to endothelial cell contact areas. Generally, cells express more than one connexin, and gap junction channels can thus potentially contain more than one type. Homomeric heterotypical channels are formed by the pairing of two different types of connexons, which each consist of uniform connexins. Such heterotypical channels have been recognized in Xenopus oocytes.[38, 39] Heterotypic pairing of Cx26 and Cx43 did not result in electrical coupling in paired Xenopus oocytes. Another kind of mixed channel, a heteromeric channel formed by non-uniform connexins within one connexon also has been identified with a number of combinations, including Cx26-Cx30, Cx26-Cx32, Cx43-Cx37, Cx43-Cx40, Cx43-Cx45, Cx43-Cx56 and Cx46-Cx50. Interestingly, co-expression of Cx26 and Cx43 in mouse neuroblastoma cells did not result in formation of heteromeric channels, but rather a reduction in the total junctional conductance to around 10% of that in cells expressing a single connexin. In our rat HCC cells, the Cx26 level is decreased, while Cx43, Cx40 and Cx45 levels are increased. Variation in the ratios of Cxs on cells and aberrant functions of heterotypic and/or heteromeric channels might offer one explanation of the mechanisms underlying limited tumor cell attachment to endothelial cells and different potentials for invasion and metastasis among tumor cells.
In conclusion, the potential for colonization of circulating tumor cells within the first 24 h after inoculation was here found to differ among four rat HCC cell lines with different metastasizing ability. cDNA array analysis revealed that highly-metastatic cells express elevated Cx43 and silencing of Cx43 in highly metastatic tumor cells with an siRNA reduced in vitro migration and invasion ability and in vivo metastatic ability. Our results suggest that Cx43, at least in part, may regulate tumor cell invasion and metastasis. Thus, Cx43 can be a molecular target for suppression of cancer metastasis by Cx43 overexpressing tumors.