Effect of protein C inhibitor on renal cell carcinogenesis and on tumor invasion, growth and metastasis

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Protein C inhibitor (PCI), a member of the serine protease inhibitor (serpin) family, regulates the anticoagulant protein C pathway and inhibits urinary plasminogen activator (uPA), a mediator of tumor cell invasion. We recently demonstrated that PCI expression is significantly decreased in renal carcinoma cells derived from renal proximal tubular epithelial cells, and that PCI suppresses in vitro Matrigel invasion of renal carcinoma cells by inhibiting uPA. Furthermore, we also suggested that decreased expression of PCI in renal cell carcinoma (RCC) is mainly caused by methylation of the promoter region of PCI gene. In the present study, we elucidated the detailed mechanism of decreased expression of PCI induced by PCI gene methylation in RCC, and evaluated the effect of human PCI and its inactive derivatives on tumor cell invasion in vitro, and on tumor growth and metastasis in vivo using human breast cancer (MDA-231) cells. Hydrogen bisulfite DNA sequence showed that four out of thi … More rteen CpG sequence located near transcription initiation site are specifically methylated in PCI gene of RCC. Luciferase reporter gene assay using methylated PCI gene fragment showed that transcriptional activity of PCI gene promoter fragment is decreased by methylation of these four CpG sequence near transcription initiation site. On the other hand, the in vitro invasiveness of MDA-231 cells expressing intact PCI (MDA-PCI) was significantly decreased as compared to MDA-231 cells expressing inactive R354APCI (MDA-R354APCI) or the N-terminal fragment of PCI (MDA-NTPCI). Further, in vivo growth and metastatic potential of MDA-PCI, MDA-R354APCI and MDA-NTPCI cells in severe combined immunodeficient (SCID) mice were significantly decreased as compared to MDA-Mock cells. Overall, these studies showed that, decreased PCI expression in RCC is mainly caused by specific CpG methlation of PCI gene promoter, and in addition to its reactive site-dependent action, PCI also regulates tumor growth and metastasis independently of its protease inhibitory activity. Less