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

Claudin-3和Claudin-4在包括卵巢癌在内的几种恶性肿瘤中经常过表达。 有趣的是，我们还发现这些基因在正常和恶性组织中倾向于协调表达，这表明了一种共同的调控机制。为了更好地了解这些基因的转录调控机制，我们对这些基因的启动子进行了系统的研究。我们发现CLDN 3和CLDN 4都需要SP1位点才能完全激活（CLDN 4中有两个关键的SP1位点，CLDN 3中有一个）。此外，我们还发现这两个启动子都是通过表观遗传过程进行调控的。表达高水平CLDN 3和/或CLDN 4的细胞具有相应启动子的低DNA甲基化和高组蛋白乙酰化。有趣的是，在CLDN 3的情况下，启动子的甲基化阻止了SP1结合，为非表达细胞中的CLDN 3沉默提供了机制。由于CLDN 3和CLDN 4在大部分卵巢癌中升高，导致其失调的机制可能代表卵巢肿瘤发生的一般途径。 在试图表征紧密连接蛋白-3和-4在卵巢癌中的作用时，我们使用了Illumina寡核苷酸阵列。 我们已经确定了基因的表达被改变的密封蛋白，并观察到几个基因已知参与血管生成，如IL-8，MMP 1，和几个趋化因子。功能测定已经证实，表达紧密连接蛋白-3和紧密连接蛋白-4的细胞在体外诱导共培养细胞的血管生成。这些发现已在体内小鼠背侧皮褶测定中得到验证。我们正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展这些发现，以验证它们在卵巢血管生成中的作用。最后，我们观察到claudins可以存在于卵巢癌患者的血液循环中，这表明这些蛋白质可能是卵巢癌早期检测的新标志物。我们正在研究这种可能性，开发更敏感的检测claudin。

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.