Abstract A57: Platinum induces IL-6-signaling mediated activation of ALDH1A1 and enriches the cancer stem cell population in ovarian cancer.
Authors: Wang Yinu , Mitra Anirban Kumar , Nephew Kenneth P. 
Published: May 2016

Purpose: While chemotherapy may succeed initially at decreasing the number of ovarian cancer (OC) cells, it leaves behind tumors enriched in ovarian cancer stem cells (OCSCs), which most likely drive chemoresistance and tumor relapse. An emerging model indicates that non-OCSCs may dedifferentiate and acquire stem cell properties under certain circumstances. Recent studies in OC implicate a critical role for aberrant cytokine interleukin-6 (IL-6) secretion and the IL-6 signaling pathway may play a critical role in converting non-CSCs to CSC. In the current study, we investigated the mechanism contributing to OCSC enrichment after platinum treatment in vivo. We hypothesize that platinum-induced IL-6 secretion activates STAT3 signaling-mediated expression of the cancer stem cell marker ALDH1A1, resulting in CSC enrichment. In addition, based on our previous study in OCSCs (Wang et al., 2014, Cancer Res.), we sought to examine the mechanism by which guadecitabine (SGI-110), a second generation hypomethylating agent HMA, inhibits OCSCs in OC xenograft residuals after platinum treatment.

Methods: OC cells (OVCAR4 and A2780) were cultured alone or co-cultured with normal omental fibroblasts (NOFs) or guadecitabine (100nM, 3days)-treated NOFs in the starving condition for 24h and then treated with 3h cisplatin (CDDP; respective IC50 doses of 27.4 and 14.7μM), guadecitabine, IL-6 (100ng/ml), neutralizing antibody (Nab) against IL-6 (IL-6-Nab, 200ng/mL) or IL-6+IL-6-Nab. IL-6 secretion levels were measured by ELISA. FACS was used to analyze OCSC population marked as aldehyde dehydrogenase (ALDH)+ cells, and western blot was used to examine ALDH1A1 and pSTAT3 levels in cells treated as described above. An ALDH1A1 promoter-luciferase reporter (pGL3-ALDH1A1-Luc; -1 to -1031) assay was used to determine whether IL-6 transactivates ALDH1A1 expression in OC cells. OC cell migration under the described conditions was determined by transwell migration assays.

Results: We found that CDDP induced (P&lt;0.05) IL-6 secretion by OCs up to 48h post-treatment and up to 96h by NOFs or by co-cultured cells, suggesting a role of the tumor microenvironment in platinum-induced IL-6 secretion. Guadecitabine inhibited (P&lt;0.05) CDDP-induced IL-6 secretion by NOFs alone or in co-culture with OVCAR4, suggesting an inhibitory role of the HMA on IL-6 signaling. By assaying FACS sorted ALDH+/- cells, we determined that ALDH+ cells express increased (P&lt;0.05) levels of the IL-6 receptor and secrete higher (P&lt;0.05) levels of IL-6 in the conditioned media compared to ALDH- cells. Treatment of OC cells with IL-6 enriched the percentage of ALDH+, which was inhibited by IL-6-Nab. Luciferase assay results revealed that IL-6 transactivated (p&lt;0.05) ALDH1A1 reporter gene expression in ALDH- cells, which was blocked by IL-6-Nab. Further, we observed that IL-6- or CDDP-treated OC cells increased (P&lt;0.05) ALDH1A1 and pSTAT3 protein expression, and guadecitabine treatment inhibited (P&lt;0.05) expression of ALDH1A1 and pSTAT3 in OCs. ALDH+ cells showed greater migration potential than ALDH- cells, and guadecitabine inhibited (P&lt;0.05) migration of both ALDH+ and ALDH- cells. Guadecitabine, alone or combination with IL-6-Nab, inhibited (P&lt;0.05) CDDP-induced OVCAR4 migration in co-culture with NOFs.

Conclusion: Our data indicate that IL-6 is a potent regulator of ALDH1A1 expression and the OCSC phenotype and further suggest an inhibitory effect of guadecitabine on the conversion of non-OCSCs to OCSCs. The data support a role for IL-6 in OCSC enrichment after platinum treatment and suggest that a combination approach of IL-6 neutralizing antibody with guadecitabine could represent a novel maintenance strategy after chemotherapy for eradicating OCSCs and preventing tumor recurrence.

Citation Format: Yinu Wang, Anirban Kumar Mitra, Kenneth P. Nephew. Platinum induces IL-6-signaling mediated activation of ALDH1A1 and enriches the cancer stem cell population in ovarian cancer. [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 A57.