Abstract A65: Nodal contributes to cisplatin resistance in ovarian cancer cells.

Abstract

Abstract The capacity of ovarian tumors to grow and propagate is dependent on a small subset of tumor cells, termed cancer stem-like cells, which contribute to drug resistance, and metastasis. Nodal, an embryonic morphogen has been found to sustain stem cell pluripotency and cellular plasticity, but in cancer cells its expression promotes cancer stem cell renewal, tumor growth, invasion, angiogenesis and metastasis. The role of Nodal in the development of recurrent chemoresistant ovarian cancer (OC) has not been previously investigated. The aim of this study was to investigate whether Nodal can modulate resistance of OC cell line A2780s to cisplatin. Methods. Human OC cell line A2780s was cultured in DMEM/F12 medium supplemented with 10% FBS and treated with cisplatin. Cytotoxity of cisplatin was determined by MTT assay (Roche) and clonogenic assay. Nodal expression after cisplatin treatment was determined by digital PCR and immunofluorescence staining. To increase Nodal signaling, we used a Nodal expression vector (versus an empty pcDNA3.3 vector; pcDNATM3.3-TOPO cloning kit; Invitrogen). We also employed recombinant human Nodal (rhNodal; R&D). Transfection was performed with Lipofectamine (Invitrogen) as per manufacture instructions. Cell cycle analysis after cisplatin treatment and expression of cancer stem cell marker CD133 were determined using flow cytometry analysis (FACS). In vitro sphere limiting diluting assay was applied to measure ovarian cancer stem cell (OCSC) self-renewal. Results. The expression of Nodal increased significantly in A2780s cells after 24h cisplatin treatment (20,000 copies/1ug RNA versus 800 copies/1ug RNA in untreated cells) and retained for 96h after cisplatin withdrawal (120,000 copies/1ug RNA). Overexpression of Nodal rendered A2780s cells more resistant to cisplatin (IC50 2.9ug/ml) compared to control cells (IC50 1.3ug/ml). Treatment of A2780s cells with rhNodal (100ng for 24h) during cisplatin treatment increased the ability of ovarian cancer cells to form spheres. FACS analysis revealed that cisplatin along with rhNodal treatment increased the population of CD133 positive cancer stem cells in A2780s cell line (5.85% versus 0,65% in untreated cells and 1.75% after cisplatin treatment alone). Treatment of A2780s cells with rhNodal prevented cell arrest in S phase after cisplatin treatment (19.4% of cells in S phase after rhNodal+cisplatin versus 63.6% - after cisplatin alone). Conclusison. Our findings demonstrate that Nodal may contribute to cisplatin resistance in OC cells and tumor initiation capacity after drug therapy, and may hold promise as a therapeutic target to prevent chemoresistant recurrence. Citation Format: Olena Bilyk, Lynne-Marie Postovit. Nodal contributes to cisplatin resistance in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A65.