Abstract TMEM-030: INTERLEUKIN–6 (IL6) AND LEUKEMIA INHIBITORY FACTOR (LIF) ARE FUNCTIONALLY REDUNDANT IN PROMOTING OVARIAN CANCER GROWTH AND REQUIRE DUAL BLOCKADE FOR THERAPEUTIC EFFECT
Authors: McLean Karen , Tan Lijun , Buckanovich Ronald J. 
Published: June 2017

PURPOSE: 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. Microarray analysis demonstrates the upregulation of the cytokines IL6 and LIF in CA-MSC versus control MSC. The purpose of this work is to characterize the role of cytokines interleukin-6 (IL6) and leukemia inhibitory factor (LIF) secreted by CA-MSC in ovarian cancer tumorigenesis.

EXPERIMENTAL PROCEDURES: We have isolated and established cultures of CA-MSC. Healthy donor adipose-derived MSC were used as controls (Invitrogen). Ovarian cancer cells were treated with MSC-conditioned media or recombinant IL6 and/or LIF (EBioscience). Cytokine function was inhibited with IL6 blocking antibody, LIF blocking antibody, both antibodies or the JAK2 inhibitor ruxolitinib for dual blockade. Phosphorylated STAT3 and total STAT3 immunoblotting was performed by standard methods. 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). For in vivo studies, NSG mice were injected with OVCAR3 ovarian cancer cells without or with CA-MSC, and then either mock treated or treated with IL6 blocking antibody (tocilizumab, Genentech), LIF blocking antibody or ruxolitinib. Tumor size was monitored both during treatment and at sacrifice, and tumors were analyzed.

RESULTS: Treatment of ovarian cancer cells with either recombinant IL6 or LIF alone leads to STAT3 phosphorylation, suggesting functional redundancy in stimulation of this pathway. 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. The JAK2 inhibitor ruxolitinib inhibits cytokine-mediated upregulation of phospho-STAT3. 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. Ruxolitinib blocks both LIF and IL6 mediated increase in cancer stem cells. In vivo, the pro-tumorigenic effect of CA-MSC is abrogated by dual blockade with ruxolitinib but not by treatment with anti-IL6 antibody alone or anti-LIF antibody alone. Ruxolitinib treated tumors demonstrate decreased phospho-STAT3, indicating on-target activity.

CONCLUSIONS: IL6 and LIF are secreted by CA-MSC in the ovarian cancer tumor microenvironment. Both cytokines upregulate phospho-STAT3 and increase the percentage of cancer stem cells in epithelial tumor cells. Functional inhibition of these effects cannot be achieved in vitro or in vivo with blockade of either cytokine individually; dual blockade with compounds such as the JAK2 inhibitor ruxolitinib is necessary. Thus the shared downstream signaling molecules of the JAK/STAT pathway may be preferred targets for novel anticancer therapeutics for ovarian cancer.

Citation Format: Karen McLean, Lijun Tan and Ronald J. Buckanovich. INTERLEUKIN–6 (IL6) AND LEUKEMIA INHIBITORY FACTOR (LIF) ARE FUNCTIONALLY REDUNDANT IN PROMOTING OVARIAN CANCER GROWTH AND REQUIRE DUAL BLOCKADE FOR THERAPEUTIC EFFECT [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 TMEM-030.