Abstract POSTER-BIOL-1304: Cleavage of the cell adhesion molecule nectin-4 from the surface of ovarian cancer cells: a mechanism for cancer progression and a novel serum biomarker

Abstract

Purpose of the Study: The mechanisms by which ovarian cancer cells are released from the primary tumor, seed throughout the peritoneal cavity, attach to local organs, and then invade these organs, are not understood. A unique aspect of ovarian cancer progression is the collection of ascites fluid in the abdominal cavity, which contains free-floating tumor cells in the form of single cells and multicellular aggregates. In previous studies, we showed that the cell adhesion molecule Nectin-4 is highly expressed in ovarian cancer tumors, whereas its expression in normal tissues is limited; making Nectin-4 an attractive target for therapy. Nectins are a family of four cell adhesion molecules that are important in the formation and maintenance of cell junctions. Other studies have suggested that the extracellular domain of Nectins can be proteolytically cleaved by the A Disintegrin And Metalloprotease 17 (ADAM17) to release a soluble fragment. We hypothesize that this cleavage step may be important in regulating Nectin function and increases the likelihood of cancer dissemination. We have shown that the soluble fragment of Nectin-4 (sN4) is detected in the sera of 50% of ovarian cancer patients, but is not detected in the sera of women with benign gynecological conditions. The purpose of this study was to determine how Nectin-4 is cleaved from the surface of ovarian cancer cells and the role that Nectin-4 plays in the aggregation, migration, and proliferation of ovarian cancer cells. Experimental Procedures: In the first series of experiments, NIH:OVCAR5, a human ovarian cancer cell line that expresses moderate levels of Nectin-4, was transfected with shRNA targeting Nectin-4, resulting in the partial knockdown of Nectin-4 expression. The cells were then tested in several functional assays. In the second series of experiments, NIH:OVCAR5 cells were transfected with full length Nectin-4 and the level of sN4 in the spent media in the presence of various inhibitors of metalloproteases was quantified by ELISA and Western blotting. We also tested these cells for their rate of proliferation. Summary of Data: NIH:OVCAR5 cells that expressed low levels of Nectin-4 required a substantially longer time to form multicellular aggregates, migrate, and proliferate. NIH:OVCAR5 cells that expressed high levels of Nectin-4 shed sN4 following 3 hours of stimulation with phorbol myristate acetate (PMA). A broad spectrum inhibitor of matrix metalloproteases (MMPs) and ADAMs completely inhibited Nectin-4 shedding, while inhibitors of ADAM17 and/or ADAM10 inhibited Nectin-4 shedding by 30-100%. NIH:OVCAR5 cells that expressed high levels of Nectin-4 proliferated more rapidly than the parental cell line. Conclusions: This study suggests that Nectin-4 promotes cell-cell adhesion (important in the formation of multicellular aggregates), yet it can be cleaved from the surface of cells to allow cells to disaggregate (important in invasion). Additional functional studies leading to a better understanding of the role of Nectin-4 overexpression and shedding in ovarian cancer are currently underway. Understanding the role of Nectin-4 in ovarian cancer progression is critical in order to facilitate its development as a novel therapeutic target for ovarian cancer. This project was funded by the Minnesota Ovarian Cancer Alliance. Citation Format: Kristin L.M. Boylan, Petra C. Buchanan, Adam Meyer, Bruce Walcheck, Amy P.N. Skubitz. Cleavage of the cell adhesion molecule nectin-4 from the surface of ovarian cancer cells: a mechanism for cancer progression and a novel serum biomarker [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1304.