Abstract MIP-045: CLAUDIN–4 PROMOTES ATTACHMENT OF OVARIAN TUMOR CELLS TO THE EXTRACELLULAR MATRIX AND FACILITATES SURVIVAL AND MIGRATION

Dissemination of chemoresistant tumor cells throughout the peritoneal cavity is a major contributing factor to poor patient outcomes in ovarian cancer. Although it is known that attachment of tumor cells to the peritoneal mesothelium is a key step in the metastatic process of ovarian cancer, the underlying molecular mechanisms driving this important stage of tumor progression are not well understood. The objective of this study was to investigate the potential role for the transmembrane protein claudin–4 in ovarian tumor cell attachment, cell survival and migration. Adhesion, apoptosis (cleaved caspase–3) and scratch assays were performed with ovarian tumor cells (OVCAR3, PEO4) cultured on MeT5A mesothelial cell monolayers, different proteins found in the extracellular matrix of the peritoneal mesothelium (type I collagen, type IV collagen, fibronectin, and laminin) or a non–physiological cell adhesive (Cell–Tak). Number of cells attached within one hour, percent cells positive for apoptosis at 24 hours post treatment with apoptotic stimuli, and percent closure of the wound created by a scratch 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 also performed to examine formation of focal adhesions in response to claudin–4 disruption or loss of expression. Results from these studies showed that ovarian tumor cells preferentially attach to type I collagen and that disruption of claudin–4 inhibited this attachment. Attachment to type I collagen made tumor cells more resistant to apoptosis and disruption of claudin–4 rescued apoptotic response. Additionally, movement of tumor cells on type I collagen–coated slides was inhibited with treatment of the cells with the claudin–4 disrupting peptide or with loss of claudin–4 expression. This inhibition was not seen with other matrix proteins tested. 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. In conclusion, we have demonstrated a novel role for claudin–4 in ovarian tumor cell attachment to the extracellular matrix through interactions with type I collagen and formation of focal adhesions for tumor cell survival and migration. These observations have important therapeutic implications for inhibiting the survival and deadly spread of ovarian tumors through blocking the biological activity of claudin–4. Citation Format: Douglas A. Hicks, Carly E. Galimanis, Patricia G. Webb, Kian Behbakht, Heidi. K. Baumgartner. CLAUDIN–4 PROMOTES ATTACHMENT OF OVARIAN TUMOR CELLS TO THE EXTRACELLULAR MATRIX AND FACILITATES SURVIVAL AND MIGRATION [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-045.