Abstract A20: Claudin-4 regulates ovarian tumor cell response to the microenvironment

Interaction of tumor cells with extracellular matrix proteins of the peritoneal lining plays a critical role in the unique metastatic process of ovarian cancer. Although peritoneal attachment is known to be a key step in the progression of ovarian cancer, the underlying molecular mechanisms driving adhesion and the downstream changes in cell behavior that lead to poor treatment outcomes are not well understood. The objective of this study was to investigate the potential role of the transmembrane protein claudin-4 in regulating ovarian tumor cell interaction with and response to extracellular matrix proteins. Adhesion, apoptosis (cleaved caspase-3), proliferation (DNA content), and scratch assays were performed with ovarian tumor cells (OVCAR3, PEO4) cultured on different proteins found in the extracellular matrix of the peritoneal mesothelium (type I collagen, type IV collagen, fibronectin, and laminin) or a nonphysiologic cell adhesive (Cell-Tak). Number of cells attached within one hour, percent cells positive for apoptosis at 24 hours post treatment, cell number over time, and percent wound closure at 8 hours was measured in response to claudin-4 disruption (DFYNP mimic peptide) or loss of claudin-4 expression (shRNA-mediated gene silencing). Immunofluorescence of phosphorylated focal adhesion kinase (pFAK) was performed to examine formation of focal adhesions in response to claudin-4 disruption or loss of expression. Proximity ligation assays, immunoprecipitation, and immunofluorescence were performed to examine interaction of claudin-4 with tubulin. Results from these studies showed that ovarian tumor cells preferentially attach to type I collagen compared to the other matrix proteins and that disruption of claudin-4 inhibited this attachment. Attachment to type I collagen made tumor cells more resistant to apoptosis, more proliferative, and more migratory compared to tumor cells cultured on the other matrix proteins. In the presence of type I collagen, disruption of claudin-4 restored tumor cell apoptotic response to paclitaxel, induced mitotic arrest, reduced proliferation rate, and inhibited migration. The size of pFAK-containing focal adhesions was significantly smaller and fewer adhesions were present in ovarian tumor cells cultured on type I collagen treated with the claudin-4 disrupting peptide or with loss of claudin-4 expression compared to cells that express high levels of claudin-4. Additionally, we observed a direct interaction of claudin-4 with both alpha and beta tubulin that was dependent on stage of cell cycle. In conclusion, we have demonstrated a novel role for claudin-4 in regulating ovarian tumor cell response to the tumor microenvironment to promote tumor survival and growth. These observations have important therapeutic implications for inhibiting the survival and deadly spread of ovarian tumors through blocking the biologic activity of claudin-4. Citation Format: Douglas A. Hicks, Christopher Breed, Patricia G. Webb, Kristin L. Neal, Kian Behbakht, Heidi K. Baumgartner. Claudin-4 regulates ovarian tumor cell response to the microenvironment. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A20.