Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies. Recent data have pointed to the persistence of quiescent ovarian cancer stem cells (OCSCs) not eliminated by chemotherapy and able to regenerate tumors as the main contributor to tumor relapse and metastasis. Downregulation of tumor suppressor DAB2IP significantly correlated with poor patient survival in OC. In the current study, we tested the hypothesis that DAB2IP is downregulated by EZH2 methylation in OC and targeting DAB2IP inhibits OCSCs, which would 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, which can be restored by EZH2 inhibition. 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 and increased (P<0.05) the percentage of ALDH(+) cells. Enforced overexpression of DAB2IP decreased (P<0.05) the number of ALDH(+) cells and inhibited (P<0.05) both spheroid and colony formation. Furthermore, elevated DAB2IP expression decreased (P<0.05) cisplatin IC50 of both OCSCs and OC cells and inhibited (P<0.05) cell migration capacity, suggesting DAB2IP plays a role in regulating OCSC function. OVCAR3 cells and DAB2IP-overexpressing OVCAR3 cells were further analyzed by RNA-sequencing and bioinformatics. Transcriptome analysis revealed that DAB2IP overexpression significantly (FDR < 0.05, fold change > 2) altered expression of 449 genes, including downregulation 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 analysis demonstrated activation of JNK/c-Jun as a possible downstream target of WNT5B. Collectively, our data reveal that DAB2IP, via noncanonical Wnt-mediated signaling pathway, plays a critical role in modulating OCSC properties. Based on these novel findings, we are testing novel combination treatment strategies targeting OCSCs and with the goal of inhibiting tumor relapse and overcoming chemoresistance.

Citation Format: Xingyue Zong, Ali Ozes, Weini Wang, Kenneth P. Nephew. Targeting EZH2/DAB2IP/Wnt axis in ovarian cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A81.