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Supplementary Methods and Figure Legend from Overexpression of Protease-Activated Receptor-1 Contributes to Melanoma Metastasis via Regulation of Connexin 43

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posted on 2023-03-30, 18:53 authored by Gabriel J. Villares, Andrey S. Dobroff, Hua Wang, Maya Zigler, Vladislava O. Melnikova, Li Huang, Menashe Bar-Eli
Supplementary Methods and Figure Legend from Overexpression of Protease-Activated Receptor-1 Contributes to Melanoma Metastasis via Regulation of Connexin 43

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ARTICLE ABSTRACT

Protease-activated receptor-1 (PAR-1) is a key player in melanoma metastasis with higher expression seen in metastatic melanoma cell lines and tissue specimens. cDNA microarray and Western blot analyses reveal that the gap junctional intracellular communication molecule connexin 43 (Cx-43), known to be involved in tumor cell diapedesis and attachment to endothelial cells, is significantly decreased after PAR-1 silencing in metastatic melanoma cell lines. Furthermore, Cx-43 promoter activity was significantly inhibited in PAR-1–silenced cells, suggesting that PAR-1 regulates Cx-43 at the transcriptional level. Chromatin immunoprecipitation studies showed a reduction in the binding of SP-1 and AP-1 transcription factors to the promoter of Cx-43. Both transcription factors have been shown previously to be required for maximal Cx-43 promoter activity. These results were corroborated by mutating the AP-1 and SP-1 binding sites resulting in decreased Cx-43 promoter activity in PAR-1–positive cells. Moreover, as Cx-43 has been shown to facilitate arrest of circulating tumor cells at the vascular endothelium, melanoma cell attachment to endothelial cells was significantly decreased in PAR-1–silenced cells, with this effect being abrogated after PAR-1 rescue. Herein, we report that up-regulation of PAR-1 expression, seen in melanoma progression, mediates high levels of Cx-43 expression. As both SP-1 and AP-1 transcription factors act as positive regulators of Cx-43, our data provide a novel mechanism for the regulation of Cx-43 expression by PAR-1. Indeed, Cx-43 expression was restored following PAR-1 rescue in PAR-1–silenced cells. Taken together, our data support the tumor promoting function of Cx-43 in melanoma. [Cancer Res 2009;69(16):6730–7]

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