Abstract 4219: Claudin-3 and claudin-4 regulate ovarian cancer growth rate and metastatic potential through effect on integrin and β-catenin signaling

Abstract

Abstract Claudins (CLDNs) are the major structural proteins of tight junctions between epithelial cells and aberrant expression of CLDNs in cancer cells is common. We discovered that constitutive knockdown of CLDN3 or CLDN4 in human ovarian cancer cells using shRNAi vectors increased their in vivo growth rate by 2.3- and 3.7-fold, respectively. In this study we examined the effect of knocking down CLDN3 and CLDN4 on the adhesion, migration and invasion and explored underlying mechanisms. Knockdown of CLDN3 or CLDN4 caused a 2.5- and 2.2-fold decrease in adhesion but increased migration by 1.4- and 1.9-fold, and invasion by 2.9- and 3.8-fold when measured in a Boyden chamber assay. To examine the effect of CLDN3 and CLDN4 knockdown on the metastatic process, we molecularly engineered the parental, CLDN3 knockdown (CLDN3KD) and CLDN4 knockdown (CLDN4KD) cells to express the DsRed fluorescent protein and then injected one million cells into the tail vein of nu/nu mice. Mice injected with either CLDN3KD or CLDN4KD cells had significantly more metastases to the lungs and other organs by day 42 compared with mice injected with the parental cells when examined under Olympus OV100 fluorescence microscope. Quantification of total lung metastatic burden in the same sets of mice by measurement of RFP fluorescence in lung homogenates showed that there was a 1.7- and 2.4-fold increase in tumor burden, respectively, in the lungs of CLDN3KD and CLDN4KD injected mice. We next asked whether the motile and invasive phenotype of the CLDN3KD and CLDN4KD cells was the result of alteration in formation of αvβ3 complex, one of the integrins that plays an important role in tumorigenicity and metastasis. Both flow cytometric analysis and deconvoluting microscopic analysis demonstrated a marked reduction in αvβ3 expression in the CLDN3KD and CLDN4KD cells. Suppression of E-cadherin expression is regarded as one of the main molecular events responsible for dysfunction in cell-cell adhesion, and the loss of E-cadherin is a well-known prerequisite for tumor cell invasion. Interestingly, we found that knockdown of CLDN3 and CLDN4 expression in the ovarian carcinoma cells was accompanied by a 55% and 95% decrease, respectively, in E-cadherin protein levels. Measurement of E-cadherin mRNA levels by qRT-PCR showed a 60% and 83% reduction in the CLDN3KD and CLDN4 cells, respectively. In association with the decrease in E-cadherin, β-catenin signaling was augmented as evidenced by a marked increase in nuclear active (de-phosphorylated) β-catenin, activation of TOPflash promoter activity and the expression of the β-catenin/TCF-regulated target genes cyclin D1 and c-myc. Our findings demonstrate that CLDN3 and CLDN4 have large effects on migration, invasion and metastasis of ovarian cancer cells and suggest that the enhanced aggressiveness is associated with diminished αvβ3 signaling and elevated β-catenin activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4219. doi:1538-7445.AM2012-4219