Abstract
Abstract Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies. The persistence of quiescent OC stem cells (OCSCs) not eliminated by chemotherapy plays a key role in tumor relapse and metastasis. In the current study, we tested the hypothesis that targeting DAB2IP will inhibit OCSCs and prevent disease recurrence. Subpopulations of CSC and non-CSC were isolated from OC cell lines by fluorescence-activated cell sorting (FACS) based on aldehyde dehydrogenase (ALDH) activity, a consistent CSC marker. Expression of DAB2IP in ALDH(+) cells was lower (P<0.05) compared to non-CSC ALDH(-) cells. Chromatin immunoprecipitation (ChIP) analysis demonstrated enrichment (P<0.05) of H3K27me3 at DAB2IP promoter loci in CSC compared to non-CSC. Furthermore, inhibiting EZH2 decreased H3K27me3 and increased (P<0.05) DAB2IP expression in OC cells, demonstrating that DAB2IP downregulation in CSC was due to EZH2. Knocking out DAB2IP using CRISPR/Cas9 system in OC cell lines upregulated (P<0.05) expression of stemness-related genes. Enforced overexpression of DAB2IP decreased (P<0.05) the number of ALDH(+) cells and inhibited (P<0.05) spheroid formation. Transcriptome analysis revealed that DAB2IP overexpression significantly (FDR < 0.05, fold change > 2) altered expression of 449 genes, including down-regulation of ALDH1A1, LGR5, PROM1 and TWIST1, markers strongly associated with CSC phenotypes. Ingenuity Pathway Analysis for upstream regulators of differentially expressed genes revealed WNT-signaling as a dominant pathway mediating the anti-OCSC effects of DAB2IP. Based on RNA-seq analysis, WNT5B expression decreased (P<0.05) by 3.83 fold, indicating that DAB2IP may negatively regulate Wnt signaling pathway by repressing WNT5B. Furthermore, WNT5B recombinant protein significantly increased (P<0.05) the OCSC population, and Reverse Phase Protein Array (RPPA) analysis demonstrated activation of non-canonical WNT signaling via C-JUN as a possible downstream target of WNT5B. Collectively, our data revealed that EZH2-regulated DAB2IP is a critical tumor suppressor regulating cancer stemness-related phenotypes, via inhibiting WNT5B-induced C-JUN activation, which could be abolished by RAC1 inhibitor. Based on these novel findings, we are testing novel combination treatment strategies targeting OCSCs, with the goal of inhibiting tumor relapse and overcoming chemoresistance. Importantly, we showed that co-administration of EZH2 inhibitor and RAC1 inhibitor inhibits OCSC survival in vitro, impedes tumor growth and augments chemoresponse in vivo. These findings establish that targeting EZH2/DAB2IP/C-JUN axis could be considered as adjuvant therapy for platinum-based chemotherapy to prevent OC recurrence. Citation Format: Xingyue Zong, Ali Ozes, Weini Wang, Fang Fang, Kenneth P. Nephew. Targeting EZH2/DAB2IP/C-JUN axis in ovarian cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 498.
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