Abstract GMM-062: TARGETING OVARIAN CANCER STEM CELLS THROUGH THE TUMOR SUPPRESSOR DAB2IP-MEDIATED WNT SIGNALING PATHWAY
Authors: Xingyue Zong, Ali R Ozes, Kenneth P Nephew
Journal: Clinical Cancer Research (2019)

Abstract Ovarian cancer (OC) recurrence after tumor eradication by chemotherapy invariably heralds poor outcome. Recent data point to persistence of quiescent cancer stem cells (CSCs) not eliminated by chemotherapy and able to regenerate tumors as the main contributor to tumor relapse. Downregulation of tumor suppressor gene (TSG) DAB2IP, a member of the Ras GTPase-activating protein family, significantly correlated with poor patient survival in high-grade serous (HGS) OC. Furthermore, loss of DAB2IP in prostate and colon cancer enriched CSC population, suggesting a key role for DAB2IP in modulating cancer stemness. In the current study, we tested the hypothesis that targeting DAB2IP would inhibit CSCs in OC and prevent disease recurrence. Subpopulations of CSC and non-CSC were isolated from Kuramochi, OVCAR3 and COV362 HGSOC cell lines by fluorescence-activated cell sorting (FACS) based on aldehyde dehydrogenase (ALDH) activity, a consistent CSC marker. It was previously demonstrated by us and others that ALDH(+) cells share characteristics of normal stem cells such as the ability to form anchorage-independent multicellular aggregates (spheroids), undergo membrane efflux, express stem cell restricted transcription factors, and generate tumors in vivo. DAB2IP was examined in the FACS-sorted cells using qPCR and western blot. Expression of DAB2IP in ALDH(+) cells was lower (P&amp;lt;0.05) compared to non-CSC ALDH(-) cells. Chromatin immunoprecipitation (ChIP) analysis revealed greater (P&amp;lt;0.05) enrichment of H3K27me3 at DAB2IP promoter loci in CSC than non-CSC, suggesting that DAB2IP downregulation in CSC is caused by EZH2 methylation. Enforced overexpression of DAB2IP decreased (P&amp;lt;0.05) the number of ALDH(+) cells, inhibited (P&amp;lt;0.05) the ability of these cells to form spheroids (14-day incubation under stem cell conditions) and decreased (P&amp;lt;0.05) colony formation. Furthermore, elevated DAB2IP expression decreased (P&amp;lt;0.05) cisplatin IC50 of both OCSCs and HGSOC cells and inhibited (P&amp;lt;0.05) cell migration capacity (Bowden chamber transwell assay), suggesting DAB2IP plays a role in regulating OCSC function. Mechanistically, decreased expression of stemness-related genes in DAB2IP-overexpressing OCSCs was also observed, indicating potential key effectors downstream of DAB2IP. Moreover, dual luciferase reporter assay showed that ALDH1A1 transcription level was significantly repressed (P&amp;lt;0.05) in DAB2IP-overexpressing cells. OVCAR3 cells and DAB2IP-overexpressing OVCAR3 cells were further analyzed by RNA-sequencing and bioinformatics. This transcriptome analysis revealed that DAB2IP overexpression resulted in significantly (FDR &amp;lt; 0.05, fold change &amp;gt; 2) altered expression of 449 genes, including markers strongly associated with CSC phenotypes, including down-regulation of ALDH1A1, LGR5, PROM1, TWIST1 and ATP-binding cassette transporters. Differentially expressed genes were subjected to Ingenuity Pathway Analysis (IPA) for upstream regulators. IPA identified Wnt-signaling pathways as top upstream regulators of these differentially expressed genes, suggesting that Wnt-signaling is a dominant pathway mediating the anti-OCSC effects of DAB2IP. In addition, treating cells with Wnt inhibitor decreased (P&amp;lt;0.05) the CSC population, colony formation ability and ALDH1 expression. Collectively, our data reveals that DAB2IP plays a critical role in modulating CSC properties via Wnt-mediated signaling pathway, suggesting novel combination treatment strategy targeting OCSCs and thus impacting tumor relapse and chemoresistance in OC. Citation Format: Xingyue Zong, Ali R Ozes, Kenneth P Nephew. TARGETING OVARIAN CANCER STEM CELLS THROUGH THE TUMOR SUPPRESSOR DAB2IP-MEDIATED WNT SIGNALING PATHWAY [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-062.