Abstract 5207: The NF-κB and EGFR pathways mediate LPA-induced VEGF-VEGFR-2 signaling and cell invasion in epithelial ovarian cancer

Abstract

Abstract Objective: Lysophosphatidic acid (LPA) regulates the epithelial ovarian cancer (EOC) microenvironment by stimulating multiple downstream effectors. We have previously shown that the VEGF-VEGFR-2 signaling axis is involved in LPA-induced EOC invasion; however, the mechanism by which LPA controls VEGF signaling remains unclear. Thus, in this study, we propose to identify and characterize the signaling pathways by which LPA regulates VEGF signaling. Methods: Signaling pathways commonly involved in cancer progression were profiled with the Superarray RT2 Profiler PCR array in an EOC cell line (SKOV3) upon LPA (20 μM) stimulation. Deregulated pathways were validated by RT-PCR. The role of specific pathways in LPA-induced VEGF signaling was further assessed with commercially available inhibitors targeting each pathway. The effect of those inhibitors on LPA-induced VEGF and VEGFR-2 mRNA expression was analyzed by RT-PCR 6 hours post treatment. ELISAs were done to measure the concentration of VEGF secreted in conditioned media and Western blotting was performed to examine VEGFR-2 levels. In addition, Matrigel invasion assays were conducted to assess the biological effect of pathway inhibition on LPA-induced EOC invasion. Finally, RT-PCR and gelatin zymography was performed to evaluate the effect of inhibition on metalloproteinases. Results: Of the genes examined in the initial array, LPA significantly enhanced the expression of TNF, EGFR and NF-κB. Other molecules such as IL-8 and BRCA1 were also upregulated, but the changes were not significant. Among TNF, EGFR and NF-κB inhibitors, NF-κB and EGFR inhibition significantly decreased LPA-induced mRNA expression of VEGFR-2, VEGF121, and VEGF165 (p<0.05). Additionally, VEGFR-2 and VEGF levels were reduced by treatment with either inhibitor in the setting of LPA stimulation, with VEGFR-2 levels reaching that of baseline. Finally, invasion assays showed that NF-κB and EGFR inhibitors decreased LPA-induced invasion, in a concentration dependent manner, to that of control cells (p<0.05). This decrease appears to be due to decreased metalloproteinase activity, specifically MMP-1 and MMP-2. When cells were simultaneously treated with both EGFR and NF-κB inhibitors, mRNA expression of NF-κB, EGFR, MMP-1 and MMP-2 were significantly reduced (p<0.02). Of note, the NF-κB and EGFR inhibitors did not alter the invasiveness of SKOV3 cells or the expression of VEGF and VEGFR-2 in the absence of LPA, suggesting that the NF-κB and EGFR pathways are activated upon LPA treatment, which subsequently induces VEGF and VEGFR-2 expression. Conclusion: Our results show that the LPA regulation of VEGF-VEGFR-2 signaling and EOC invasion is under the dual control of the NF-κB and EGFR pathways. The identification of these pathways and their importance in EOC invasion reveals potential therapeutic targets for regulating metastatic dissemination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5207.