PAR-1 plays a major role in the progression of melanoma. We and others have shown that PAR-1 is over-expressed in metastatic melanoma cell lines and tumors as compared to non-metastatic cell lines and dysplastic nevi (8
). We, therefore, silenced PAR-1 by utilizing both shRNA as well as in vivo
liposomal-delivery of siRNA and found a significant decrease in both tumor growth and metastasis, further establishing the central role PAR-1 plays in melanoma progression (7
). Interestingly, the addition of thrombin or hirudin in our studies revealed no differences in the effects of PAR-1 activity on melanoma cells beyond that of basal levels probably due to activation of PAR-1 by means other than thrombin. We cannot exclude the possibility that activation of PAR-1 in our system could occur in an autocrine manner.
In order to fully understand how PAR-1 is involved in melanoma growth and metastasis, we utilized cDNA microarray profiling on PAR-1 silenced cells. Based on these results, we have found a link between PAR-1 expression and the gap junctional intracellular communication molecule, Connexin 43, in melanoma. This is the first time that Connexin 43 has been identified to be regulated by PAR-1in human melanoma.
The role of Connexin 43 in cancer is controversial. Studies have shown that in several cancers, Cx-43 acts as a tumor suppressor gene with loss of Cx-43 contributing to metastasis (37
). Conversely, expression of Cx-43 has also been shown to increase tumor metastasis in breast cancer as well as in gliomas through increase attachment and communication with the vascular endothelium (15
Previous studies using murine melanoma cells report increase coupling of melanoma cells expressing higher levels of Cx-43 to the vascular endothelial cells (23
). In fact, the ability of tumor cells to metastasize appears to correlate with the ability of tumor cells to communicate with endothelial cells (24
). Nevertheless, studies analyzing the early steps in melanoma progression found a decrease in Cx-43 in human melanoma cells (19
). Our findings, however, show high levels of Cx-43 protein expression in metastatic melanoma cell lines and that loss of PAR-1 expression results in the loss of Cx-43. Our finding does not support the role of Cx-43 acting as a tumor suppressor gene in malignant melanoma. Its expression level was high in the metastatic A375SM and C8161 cell lines, but was dramatically reduced in these cells transduced with PAR-1 shRNA. Our data suggest another possible mechanism by which PAR-1 contributes to invasion and metastasis in melanoma, namely by regulating Connexin 43.
After identifying Cx-43 as a possible gene target of PAR-1, we validated our cDNA microarray results to determine if there was indeed a decrease in Cx-43 protein levels. This validation is essential as cDNA microarrays only analyze the mRNA, thereby necessitating further studies to determine if actual protein levels are decreased. Therefore, Western blot analyses on both PAR-1 silenced metastatic melanoma cell lines were performed. These studies revealed a significant decrease in Cx-43 protein levels after PAR-1 silencing, thus confirming that Cx-43 was being regulated by PAR-1 at the protein level. We, therefore, sought to determine the mechanism by which PAR-1 was affecting Cx-43 expression. Promoter analyses revealed that the Cx-43 promoter activity was significantly inhibited by PAR-1 shRNA, suggesting that PAR-1 regulates Cx-43 expression at the transcriptional level.
Based on these results, we determined whether PAR-1 was affecting the binding of transcription factors that were previously shown to be required for maximal promoter activity; AP-1 and SP-1 (35
). Although PAR-1 did not affect the expression of these transcription factors, it did affect binding of SP-1, c-Jun and c-Fos to the promoter of Cx-43. As both transcription factors (SP-1 and AP-1) act as positive regulators of Cx-43, our data provide a novel mechanism for the regulation of Cx-43 expression by PAR-1.
The link between PAR-1 affecting binding of AP-1 and SP-1 to the Cx-43 promoter was further strengthened by performing mutation analyses on these transcription factor binding sites and determining the effects on Cx-43 promoter activity after PAR-1 silencing. With mutations in both SP-1 and AP-1 sites, there is significantly less PAR-1 induction of the Cx-43 promoter as seen in the NT transduced cells (PAR-1 positive) after the transcription factor binding sites were mutated. Furthermore, the promoter activity in PAR-1 silenced cells is further decreased after promoter mutagenesis. This allowed us to conclude that the mechanism for the regulation of Cx-43 by PAR-1 occurs through differential binding of AP-1 and SP-1 to the promoter of Cx-43.
Previous studies have found that increased Connexin 43 levels have an effect on tumor cell attachment to cells including endothelial cells (21
). These studies support our findings showing that with decreased Cx-43 expression, via PAR-1 shRNA, there was a significant decrease in melanoma cell attachment to endothelial cells. To ascertain that the changes seen in attachment of PAR-1 silenced cells to endothelial cells was truly a result of decreased Cx-43 expression, lentiviral Cx-43 shRNA was utilized to silence Cx-43 in A375SM and C8161 cell lines. As with PAR-1 silenced cells, silencing of Cx-43 also caused a reduction in binding of melanoma cells to HUVEC. Further proof that PAR-1 was regulating attachment of melanoma cells to endothelial cells via Cx-43 was obtained when PAR-1 was re-expressed in PAR-1-silenced C8161 melanoma cells. Rescuing PAR-1 resulted in an increase in Cx-43 expression. This also resulted in an increase of melanoma cell attachment to endothelial cells as compared to PAR-1 silenced cells or control. HDMECs were utilized in these experiments to illustrate that these effects on attachments were also seen in microvessel endothelial cells and was therefore, not an artifact of using HUVECs. Finally, to illustrate that Cx-43 gap junctions are present between melanoma cells and microvessel endothelial cells, dye transfer assays were utilized to show functional gap junction formations (Supplemental Figure 1
). We therefore concluded that attachment of melanoma cells to endothelial cells was in part due to the expression of Connexin 43.
PAR-1 regulation of Connexin 43 in melanoma cells might have direct impacts on melanoma metastasis. The ability of tumor cells to metastasize appears to correlate with the ability of tumor cells to communicate with endothelial cells (24
). The metastatic cascade is complex and involves expression and silencing of a myriad of genes. It has been argued that Cx-43 is lost in the early phases of melanoma progression in which melanocytes, but not melanoma cells, were able to communicate with keratinocytes through connexins (19
). However, these findings did not include studies on melanoma cells en route to the metastatic organ. Once melanoma cells have reached the vasculature, they must arrest and extravasate through the vascular endothelium in the metastatic organ. In this process (tumor cell diapedesis), Connexin 43 plays an important role in melanoma progression. Previous studies have found this increase in Cx-43 is not only crucial for communication between tumor cells and endothelial cells, but also plays a role in tumor cell adherence and diapedesis (21
). Studies have also shown the importance of Cx-43 in enhancing angiogenesis in vivo
) which correlate with our previous findings of decreased blood vessel formation and angiogenesis after treating tumor-bearing mice with liposomal delivered PAR-1 siRNA (7
Our data indicate that Cx-43 is not a tumor suppressor gene in melanoma. Rather, it functions to enhance attachment and diapedesis of circulating melanoma cells to the vascular endothelium. Moreover, upregulation of Cx-43 allow for the establishment of intracellular communication between the tumor microenvironment and the metastatic tumor cells allowing for the passage of ions and second messengers which further enhances the metastatic process. Herein, we report that upregulation of PAR-1 expression seen in melanoma progression, mediates high levels of Cx-43 expression through increased binding of SP-1 and AP-1 transcription factors to the Connexin 43 promoter.