Claudin-4-dependent mechanisms of high-grade serous ovarian cancer progression.

In The Cancer Genome Atlas, Claudins-3, -4, and -7 are highly expressed in high-grade serous ovarian cancer (HGSOC). While claudins-3 and -7 are often expressed at tight junctions, claudin-4 (CLDN4) appears to be cytoplasmic, both in normal cells where it is expressed rarely and in a model of HGSOC, often used to study cellular interactions in this disease, OVCAR3 cells. CLDN4 expression reduces response to paclitaxel and conveys worse overall survival. We, therefore, wanted to further elucidate CLDN4-dependent mechanisms of cancer progression and therapeutic response. To address CLDN4 biology, we utilized immunohistochemical (IHC) analysis, loss-of-function studies, and several nonbiased approaches, including RNA-sequencing and proteomics. The IHC showed CLDN4 protein localized in the Golgi compartment with GM130 and around the cells at sites of cell-cell contact. Knocking down CLDN4 by 80% in OVCAR3 cells led them to undergo a transition from a more epithelial morphology to a mesenchymal phenotype (EMT). RNA-Seq analysis supports this morphologic interpretation by showing an increase in mRNA for genes coding for mesenchymal proteins such as fibronectin-1 (FN1), CD44, VIM, TNC, MMP2, and SNAI2 and a decrease in epithelial genes such as E-cadherin (CDH1), CD24, KRT18, and KRT19. RNA-Seq analysis of non-membrane protein kinases showed a 2-fold increase in PLAU, MSN, CAV1, CAV2, and EPHA2 and a 2-fold decrease in IGFBP2 and ERBB2, results that correlate well with the sensitivity of cancer cells to the BCR-ABL inhibitor, dasatinib. To determine the proteins that associate with CLDN4, we performed biotin ligase analysis (Bio-ID) fusing biotin protein ligase to CLDN4 and introducing the gene for the fusion protein into both OVCAR3 and OVCAR8 cells. Of the 36 proteins repeatedly more highly biotinylated in the presence of CLDN4-biotin ligase, 20 were identified as plasma membrane proteins and eight as Golgi localized by Gene Ontology analysis. Notably, we discovered a tyrosine protein kinase, YES1, to be biotinylated in both cell lines. YES1 is a poor prognostic indicator in HGSOC and promotes signal propagation following receptor tyrosine kinase activation, which contributes to a plethora of downstream outputs, including cell-cell adhesion and cell survival. These findings imply that CLDN4 expression is related to EMT and knockdown of CLDN4 could increase the sensitivity HGSOC to inhibitors such as dasatinib (BCR-ABL) or saracatinib (c-Yes). Citation Format: Patricia Webb, Margaret C. Neville, Heidi Baumgartner-Wilson, Miriam D. Post, Andrew Goodspeed, Benjamin G. Bitler. Claudin-4-dependent mechanisms of high-grade serous ovarian cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A30.