Abstract 3343: Anomalous expression of claudin 16 in ovarian cancer: Role of PKC, PI3K and estrogen

Abstract

Abstract Background: We have demonstrated that 63% of primary epithelial ovarian cancer (EOC) anomalously overexpressed CLDN16 in the cell cytoplasm, regardless of tumor histological type or grade, thus suggesting that this is an early event in EOC development, and might serve as an EOC marker. The present work aimed to enlighten cellular mechanisms that modulate CLDN16 expression in EOC. Methods: PKA, PKC, and PI3K pathways affect CLDNs cellular localization and function, cancer development and progression. We investigated the effect of the pathways, and of intra-tumoral estrogen (0.5, 5, 50 and 500 nM), on CLDN16 expression in the serous and platinum-resistant EOC model, the OVCAR3 cell line. Immunofluorescence experiments were carried out to determine the cellular localization of CLDN16 in OVCAR3. CLDN16 expression was assessed by real-time RT-PCR, in the presence or absence of the pathway modulators cAMP (PKA activator, 10-6M), KT5720 (PKA inhibitor, 10-7M), PMA (PKC activator, 10-8M), and Wortmannin (WORT: PI3K activator, 10-7M). CLDN16 relative expression analyses followed the CT method of Pffalf, using the REST program (Version 2.0.13, 2009). Data, expressed as mean ± SE, were analyzed by Bonferroni and Dunnet multiple comparisons tests. Findings: OVCAR3 expressed CLDN16 exclusively in the cytoplasm compartment, resembling primary tumors, so being a reliable in vitro model to our expression study. While the PKA pathway did not affect CLDN16 expression in OVCAR3, the PKC pathway lead to an increase of the transcript by1.95-fold (PMA; p <0.001). Likewise, the PI3K pathway modulated CLDN16 expression, as seen by the 2.32-fold decrease on its expression in the presence of WORT (p <0.001). Whereas estrogen 0.5 and 5 nM did not change CLDN16 expression, but increased its expression by 6.57-fold at 50 nM. The stimulatory effect of estrogen was abolished at 500 nM, thus suggesting a biphasic effect of the hormone in CLDN16 expression in EOC. Estrogen effects in EOC result, in part, from the intra-tumoral production of the hormone by aromatases, as corroborated by the 10.31-fold inhibition of CLDN16 expression by the aromatase inhibitor anastrozole (IC50 2.5 x 10-4M). Conclusions: Our data strongly suggest that the expression of CLDN16 is stimulated by the PKC and PI3K, and intra-tumoral aromatase-produced estrogen in EOC. Whereas the anomalous cytoplasm overexpression of CLDN16 still puzzles us, we speculate that fragments of the molecule could be possibly secreted. If this is the case, screening for circulatory portions of the protein could serve as an EOC screening molecule, considering that it is overexpressed early in disease course. Despite the fact that more studies are necessary to elighten the mechanisms that control the expression and function of CLDN16 in EOC, our study opens a new avenue to overcome EOC dramatic epidemiological scenario, bringing hope to improve patients overall outcome and quality of life. Citation Format: Nayara G. Tessarollo, Marcela Paes, Murilo Cerri, Alice Herlinger, Klesia Madeira, Renata Daltoé, Ian Silva, Leticia Rangel. Anomalous expression of claudin 16 in ovarian cancer: Role of PKC, PI3K and estrogen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3343. doi:10.1158/1538-7445.AM2014-3343