Claudin-3 and -4 are frequently overexpressed in several malignancies, including ovarian cancer. Interestingly, we also found that these genes tended to be coordinately expressed in both normal and malignant tissues, suggesting a common mechanism of regulation. In order to better understand the mechanisms of transcriptional regulation, we systematically studied the promoters of these genes. We have found that both CLDN3 and CLDN4 require SP1 sites for full activation (there are two crucial SP1 sites in CLDN4 and one in CLDN3). In addition, we have found that both promoters are regulated through epigenetic processes. Cells that express high levels of CLDN3 and/or CLDN4 have low DNA methylation and high histone acetylation of the corresponding promoter(s). Interestingly, in the case of CLDN3, methylation of the promoter prevented SP1 binding, providing a mechanism for CLDN3 silencing in non-expressing cells. Because both CLDN3 and CLDN4 are elevated in a large fraction of ovarian cancer, the mechanisms leading to their deregulation may represent a general pathway in ovarian tumorigenesis. In an attempt to characterize the roles on claudin-3 and -4 in ovarian cancer, we have used Illumina oligonucleotide arrays. We have identified genes that are altered by expression of claudins and observed several genes known to be involved in angiogenesis such as IL-8, MMP1, and several chemokines. Functional assays have confirmed that cells expressing claudin-3 and claudin-4 induce angiogenesis of co-cultivated cells in vitro. These findings have been validated in an in vivo mouse dorsal skinfold assay. We are planning to extend these findings by inhibiting putative downstream targets such as IL-8 and other chemokines to verify their roles in ovarian angiogenesis. Finally, we have observed that claudins can be present in the circulation of women with ovarian cancer, suggesting that these proteins may represent new markers of early detection of ovarian cancer. We are investigating this possibility by developing more sensitive tests for claudin detection.
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