Abstract 1554: Claudin 3 (CLDN3) and claudin 4 (CLDN4) control the growth rate of human ovarian cancer cells in vivo

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Abstract Claudins are the major integral membrane proteins forming the backbone of tight junctions between epithelial cells. Using gene expression profiling, we and others have found that the genes coding for claudin-3 (CLDN3) and claudin-4 (CLDN4) are highly expressed in ovarian cancers. However, the role of the claudins and their functional importance for the growth of ovarian cancer remain unclear. We compared the in vivo growth rate of human ovarian cancer 2008 cells and sublines in which either CLDN3 or CLDN4 was knocked down, and investigated the mechanisms by which they control the growth rate of human ovarian cancers. Although no difference in growth rate was detected in vitro, strikingly the knockdown tumors grew much faster than the parental 2008 tumors. The tumor volume of the claudin knockdown 2008 xenografts was persistently larger than that of the parental 2008 tumors throughout the whole experimental period. To examine the basis for the difference in growth rate, 2008 and the CLDN3 and CLDN4 knockdown tumors were analyzed by immunohistochemistry for markers of tumor cell proliferation (Ki67), apoptosis (TUNEL) and vascular density (CD31). The effect on these parameters was similar for the CLDN3 and CLDN4 knockdown tumors. The CLDN3 and CLDN4 tumors, respectively, contained 2.9- and 6.8-fold more Ki67-positive cells than the parental 2008 tumors indicating that these knockdown cells possess higher proliferative capacity when grown in vivo. They contained 2.1- and 3.7-fold lower frequency of apoptotic cells suggesting that the death rate of tumor cells was decreased when either CLDN3 or CLDN4 was knocked down. The number of CD31-positive vessels per square millimeter was 130 and 136 in the 2008-CLDN3- and CLDN4- knockdown tumors but only 62 in the parental 2008 tumors reflecting a substantial effect of CLDN3 and CLDN4 on tumor vessel formation. A reduction in the transepithelial electrical resistance across a confluent monolayer confirmed that the knockdown of CLDN3 and CLDN4 impaired the formation of tight junctions. The effect of CLDN3 and CLDN4 knockdown on the expression of other proteins that participate in tight junction formation was determined by Western blot analysis but there were no significant changes in the expression levels of occludin, JAM-1, ZO-1 or E-cadherin when either CLDN3 or CLDN4 was knocked down. Interestingly, knockdown of CLDN3 reduced the expression of CLDN4 at both mRNA and protein level, and knockdown of CLDN4 had the same effect on CLDN3. Taken together, our results indicate that CLDN3 and CLDN4 control ovarian tumor growth in vivo through modulation of the birth rate and death rate of the tumor cells and vascular density within the tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1554. doi:10.1158/1538-7445.AM2011-1554