We also show that this pre-metastatic step was drastically reduced in cells stably transfected with DLC1. when the tumors reached the same size as those produced by DLC1-unfavorable cells, prior to the onset of morbidity due to excessive tumor size. To detect dissemination of the tumor cells to liver and lung, a highly sensitive qPCR assay using human Alu sequences that is increasingly used for detection of metastases, was employed (6,9C11). The Alu primers show high specificity for human DNA, and the assay detects human DNA as low as 500 fg/20 systems Erlotinib HCl for cell migration and invasion have been widely used to assess metastatic potential of cancer cells, but they may not fully reproduce the microenvironment of the metastasis target (3). Based on cell invasion observations, we undertook this study, in which we show that cells from subcutaneous tumors derived from two DLC1-negative HCC cell lines disseminated to liver and lung of nude mice. We also show that this pre-metastatic step was drastically reduced in cells stably transfected with DLC1. Dissemination of human HCC cells underscores the role of DLC1 deficiency in the acquisition of invasiveness and tumor cell migration to secondary sites and the remarkable ability of the DLC1 protein to block this process. Our results are consistent with the ability of DLC1 to suppress lung metastasis of breast cancer cells (6). Of note, lung is the most frequent site for extrahepatic metastases in patients with HCC (19,20). The basis Rabbit Polyclonal to BRP44 for organ tropism, one of the most critical aspects of metastatic process remains elusive (3). The absence of microscopic metastases in either liver or lung in our experiments might be due to the relatively short time allowed for metastases to form, as the mice were sacrificed before tumors reached a very large size. The dormancy of the colonizing tumor cells at both organs may be an alternative explanation (21). The existing genomic and functional evidence implicates DLC1 in the process of metastasis. Loss of chromosome 8p is associated with greater metastatic potential in various models of HCC (22,23). A high frequency of LOH of markers located in the vicinity of DLC1 is common in HCC but not in dysplastic liver nodules (24), and recently LOH at 8p22 containing DLC1 was associated with worse survival of early stage HCC patients after resection and may be a useful prognostic marker for this subgroup of patients (25). DLC1 is consistently down-regulated in highly invasive HCC cell lines and metastatic subclones compared to non-metastatic ones (26). While genomic deletions have been detected in HCC cell lines and primary HCC, Erlotinib HCl promoter hypermethylation and histone deacetylation, however, are the predominant mechanisms for down-regulation or silencing of DLC1 in various cancers including HCC (5). Loss of DLC1 expression in 7703K cells is due to heavy promoter hypermethylation, while Focus cells exhibit partial methylation as well as loss of DNA copy number on 8p, where DLC1 resides (27,28). Ectopic restoration of DLC1 expression in Focus and 7703K cells decreased RhoA activity, impeded cell motility associated with a significant Erlotinib HCl reduction of actin stress fibers and focal adhesion molecules and an increase in cell rounding. The RhoGAP activity of DLC1 is apparently responsible for this response in HCC cells (29,30). Rho proteins are implicated in the control of actin cytoskeleton organization and focal adhesion assembly and are important components of the neoplastic process and metastasis (31,32). RhoGAPs stimulate the GTPase activity of Rho proteins, converting them from the active GTP-bound form to the inactive GDP bound state (33). DLC1 is a GAP specific for RhoA and Cdc42 and was recently also shown to have GAP activity for RhoB and RhoC, thus expanding the spectrum of targets of DLC1 in tumor development (34,35). Using a Rho A biosensor, it has recently been demonstrated in lung cancer.
We also show that this pre-metastatic step was drastically reduced in cells stably transfected with DLC1
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