Expression and roles of claudin-3 and claudin-4 in ovarian cancer

Claudin-3和-4经常在包括卵巢癌在内的几种恶性肿瘤中过度表达。有趣的是，我们还发现这些基因倾向于在正常和恶性组织中协调表达，这表明了一种共同的调控机制。为了更好地理解转录调控机制，我们对这些基因的启动子进行了系统的研究。我们发现CLDN3和CLDN4都需要SP1位点才能完全激活(CLDN4中有两个关键的SP1位点，CLDN3中有一个)。此外，我们发现这两个启动子都是通过表观遗传过程进行调控的。高表达CLDN3和/或CLDN4的细胞DNA甲基化程度低，相应启动子的组蛋白乙酰化程度高(S)。有趣的是，在CLDN3的情况下，启动子的甲基化阻止了SP1的结合，为非表达细胞中的CLDN3沉默提供了一种机制。由于CLDN3和CLDN4在很大一部分卵巢癌中都表达升高，导致它们失控的机制可能代表了卵巢肿瘤发生的一般途径。 为了研究claudin-3和claudin-4在卵巢癌中的作用，我们使用了Illumina寡核苷酸阵列。我们已经确定了通过Claudins的表达而改变的基因，并观察到了几个已知参与血管生成的基因，如IL-8、MMP1和几种趋化因子。功能分析证实，表达claudin-3和claudin-4的细胞在体外可诱导共培养细胞血管生成。这些发现已经在活体小鼠背部皮褶试验中得到了验证。我们计划通过抑制假定的下游靶点，如IL-8和其他趋化因子来扩大这些发现，以验证它们在卵巢血管生成中的作用。最后，我们观察到，在卵巢癌患者的血液循环中可以出现Claudins，这表明这些蛋白可能是卵巢癌早期检测的新标记物。我们正在通过开发更灵敏的Claudin检测测试来研究这种可能性。

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.