Here, we demonstrate autocrine and paracrine roles for IL-6 in regulation of ALDH1A1 expression in OC cells. shown altered DNA methylation in OCSC, we show here that IL-6 induces DNA methyltransferase 1 (DNMT1) expression and the hypomethylating agent (HMA) guadecitabine induced differentiation of OCSC and reduced but did not completely eradicate OCSC. IL-6 neutralizing antibody (IL-6-Nab) combined with HMA fully eradicated OCSC, and the combination blocked IL-6/IL6-R/pSTAT3Cmediated ALDH1A1 Hydroxyflutamide (Hydroxyniphtholide) expression and eliminated OCSC in residual tumors that persisted in vivo after Rabbit Polyclonal to IR (phospho-Thr1375) chemotherapy. We conclude that IL-6 signaling blockade combined with an HMA can eliminate OCSC after platinum treatment, supporting this strategy to prevent tumor recurrence after standard chemotherapy. and (4, 6). As a member of the ALDH family of detoxifying enzymes (8), ALDH1A1 has also been proposed as a functional regulator of OCSC. ALDH1A has been shown to be essential for oxidation of intracellular aldehydes (8) and is reported to play a key role in early differentiation of stem cells through oxidation of retinol to retinoic acid (9). Furthermore, therapies targeting ALDH1A1 appear to be a promising approach for eradicating CSCs and preventing chemoresistant tumor relapse (4). However, it has been recently recognized that differentiated cancer cells can acquire self-renewal and stemness properties under the influence of extrinsic factors found in the tumor microenvironment (TME) (10). Proinflammatory factors in the TME recently reported to play a regulatory role in CSC proliferation include IL-1, -6, and -23 (11) and the transcription factor NF-B (12). IL-6, a cytokine that stimulates cell proliferation and invasion, is enriched in OC-associated malignant ascites (12C14). Cancer associated fibroblasts (CAFs) in the ovarian TME serve as a reservoir for protumorigenic inflammatory cytokines, including IL-6 (15, 16). It has been demonstrated that CAF-cancer cell crosstalk plays a key role in OC progression (17), maintaining an optimal microenvironment for OC cell survival and proliferation. Furthermore, platinum-DNA damage induced secretion of IL-6 by OC cells and contributed to chemoresistance (18), suggesting an important connection between platinum activation of the IL-6 Hydroxyflutamide (Hydroxyniphtholide) signaling pathway and OC progression. In this regard, IL-6 has been hypothesized to create a protective niche, maintaining survival of residual tumor Hydroxyflutamide (Hydroxyniphtholide) cells and consequently contributing to tumor relapse (16). Epigenetic dysregulation that results from the reciprocal interplay between immune, stromal, and cancer cells plays a pivotal role in driving tumor initiation and tumor progression (19C22). Crosstalk between tumor cells and the microenvironment is mediated by both cell-to-cell contact and soluble substances, leading epigenetic alterations in both neoplastic and the surrounding nontumorigenic cells, including CAFs, and contributing to the formation of a cancer Hydroxyflutamide (Hydroxyniphtholide) favorable niche (19C21, 23). Extensive studies highlight that the epigenetic effects of chronic inflammation and immune cells on tumor cells to increase tumorigenesis risk. Inflammation cytokine IL-6, in the context of gastric cancer and colon cancer, induced upregulation of DNA methyltransferase 1 (DNMT1), leading to DNMT-mediated gene silencing and tumorigenesis (19, 24, 25). Altered DNA methylation has been associated with CSC phenotype maintenance (4) and has been linked to the undifferentiated phenotype of CSCs. We demonstrated that hypomethylating agents (e.g., guadecitabine, decitabine) inhibit stemness characteristics and tumor initiating capacity (4). In this regard, blocking IL-6 signaling in combination with a hypomethylating agent (HMA) may be a promising approach to disrupt crosstalk between tumor cells and their protective niche and to target OCSC. Early clinical trials using antibodies against human IL-6 (Siltuximab) or IL-6 receptor (IL-6R) (Tocilizumab) reported some activity as single agents (26), but convincing clinical activity has not yet been demonstrated (27), suggesting that rationally designed combinations should be investigated. Here, we demonstrate that treatment of OC cells with platinum- or IL-6C induced pSTAT3 signaling, which upregulated ALDH1A1 expression, increased stemness-associated genes and DNMT1 and enriched the population of ALDH+ cells. These cells displayed enhanced spheroid formation ability and increased resistance to platinum. Functional consequences of these molecular and cellular changes were further investigated using an in vivo model enriched in CSCs after platinum treatment. OCSC were targeted with an IL-6 neutralizing antibody (Nab) combined with the second-generation HMA guadecitabine. The combination treatment inhibited the stemness features of tumor cells persisting after chemotherapy and eradicated the ALDH+ population. These results support a combination between an epigenetic modulator and an antiCIL-6 antibody as a potentially novel strategy following chemotherapy with the goal of targeting surviving OCSC and preventing disease recurrence. Results IL-6 expression, OC progression, and reduced chemotherapy response. Inflammatory responses including IL-6Cmediated inflammation have been shown to contribute to OC progression.