Abstract A70: Ovarian cancer mesenchymal stem cells secrete leukemia inhibitory factor (LIF) and interleukin-6 (IL6) to activate epithelial cancer cell STAT3 signaling and increase cancer stem cells

Abstract

Abstract Background: We have previously isolated mesenchymal stem cells from the microenvironment of tumors from ovarian cancer patients (cancer-associated MSC, CA-MSC). We found that CA-MSC promote tumorigenesis in vitro and in vivo by increasing cancer stem cells (CSC) in the epithelial tumor cells. The purpose of this work is to identify and characterize tumor microenvironment signals from mesenchymal stem cells to epithelial ovarian cancer cells that promote tumorigenesis, specifically investigating the role of the cytokines leukemia inhibitory factor (LIF) and interleukin-6 (IL6). Experimental Procedures: We have isolated and established cultures of CA-MSC. Healthy donor adipose-derived MSC were used as controls (Invitrogen). RNA was isolated, RT-PCR was performed, and array analysis for expressed RNA transcripts carried out using the Human MSC PCR Array (SABiosciences). Cells were treated with recombinant LIF and IL6 (EBioscience). Cells were treated with the STAT3 inhibitor Stattic (Sigma) or the JAK2 inhibitor TG10209 (gift from Dr. Moshe Talpaz, University of Michigan). Immunoblotting was performed by standard methods using commercially available antibodies. Sphere assays were performed using low-adherence plates and X-Vivo20 serum free media (Lonza). Flow cytometry was carried out using the Aldefluor assay for aldehyde dehydrogenase activity (Stem Cell Technologies). Results: The treatment of epithelial ovarian cancer cells with CA-MSC conditioned media results in increased numbers of cancer stem cells (CSC), as measured by sphere assays and flow cytometry. This is associated with upregulation of phospho-STAT3 expression in the epithelial cancer cells. Microarray analysis demonstrates the upregulation of the cytokines LIF and IL6 in CA-MSC versus control MSC. This upregulation was validated by qRT-PCR. Immunoblotting demonstrates variable LIF and IL6 protein levels in CA-MSC. Treatment of ovarian cancer cells with either recombinant IL6 or LIF alone leads to STAT3 phosphorylation, suggesting functional redundancy. Either LIF or IL6 treatment results in an increase the percentage of cancer stem cells in ovarian cancer cell lines, as measured using both sphere assays and aldehyde dehydrogenase activity quantified by flow cytometry. Neither LIF nor IL6 blocking antibodies alone can prevent upregulation of phospho-STAT3, but combined therapy with both LIF and IL6 blocking antibodies prevents STAT3 phosphorylation. Importantly, the JAK2 inhibitor TG10209 blocks both LIF and IL6 mediated induction of STAT3 signaling and prevents the cytokine-mediated increase in cancer stem cells in all cell lines tested. Conclusions: Both LIF and IL6 are secreted by CA-MSC in the ovarian cancer microenvironment. Both LIF and IL6 upregulate phospho-STAT3 and increase the percentage of cancer stem cells in the epithelial cell population, which may promote tumorigenesis. Because of the redundancy in LIF and IL6 signaling pathways, therapeutic approaches targeting specifically one of these cytokines (such as the anti-IL6 antibody siltuximab) may be ineffective due to continued signaling through the other cytokine. Thus the shared downstream signaling molecules of the JAK/STAT pathway may be preferred targets for novel anticancer therapeutics. Citation Format: Karen McLean, Lijun Tan, Ronald J. Buckanovich. Ovarian cancer mesenchymal stem cells secrete leukemia inhibitory factor (LIF) and interleukin-6 (IL6) to activate epithelial cancer cell STAT3 signaling and increase cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A70.