CBX7 binds TWIST-1's E-box to inhibit TWIST-1 function and curtail tumorigenecity and metastatic potential in ovarian cancer.

Introduction: Ovarian cancer metastasizes via the transcoelomic route and yields overwhelmingly numerous micrometastases. This clinical presentation limits the value of standard of care and contributes to patient mortality. Whereas EMT is required in the acquisition of a migratory phenotype, MET has been shown to occur on secondary sites leading to the establishment of metastasis. The nature of the ovarian cancer cells that result after MET is, however, yet to be fully characterized. Whether these secondary epithelial ovarian cancer cells (sEOC) are required to repeat EMT in order to generate additional metastatic sites is not known. The objectives of this study are twofold: to determine if full MET occurs on secondary sites, and to determine mechanisms that confer to disseminated ovarian cancer cells the ability to rapidly spread and form the characteristic micrometastatic disease. Materials and Methods: Ovarian cancer spheroids were derived from three epithelial ovarian cancer (EOC) cell lines (OVCAR3, R182, R2615). sEOC were derived from the spheroids by plating in tissue culture-treated plates. Gene signature pattern was determined by RNAseq. mRNA and protein levels were quantified by RT-qPCR and Western blot, respectively. TWIST-1 activity was measured by a luciferase reporter system. Promoter binding was determined by anti-CBX7 chromatin IP followed by PCR. CBX7 was knocked out (KO) using CRISPR-Cas9. Tumorigenic potential was quantified by measuring resulting intraperitoneal burden (g) in nude mice. Results: PCA and clustering analysis of RNA-seq data showed sEOC do not fully recapitulate the molecular signature of the primary EOC cells from which they are derived. Despite displaying an epithelial morphology, sEOC maintain a high expression of the classical mesenchymal effector, TWIST-1. TWIST-1 is, however, transcriptionally inactive in these cells as it is inhibited by the PcG protein, CBX7. KO of CBX7 in sEOC was sufficient to promote a mesenchymal morphology, significantly promote TWIST-1-induced transcription of mir-199A (p = 0.004, wt vs. CBX7KO), and induce higher protein levels of FOXC2. CBX7 precludes TWIST-1 from accessing its promoter by binding at or near the E box. Finally, KO of CBX7 was able to significantly enhance tumorigenicity and increase tumor load and metastatic potential in vivo (p=0.033, wt vs CBX7 KO). Conclusion: We demonstrate that full MET does not occur in ovarian cancer cells at secondary metastatic sites. Although sEOC display an epithelial morphology, they do not achieve the full molecular signature of the primary EOC cells from which they are derived. sEOC maintain high levels of TWIST-1 whose activity is inhibited by CBX7. This regulation allows secondary tumors to achieve an epithelial morphology while conferring the advantage to achieve a prompt reversal to a mesenchymal morphology and gain in tumorigenic potential upon perturbation of CBX7. This may explain ovarian cancer’s rapid progression and characteristic micrometastatic disease. Citation Format: Juanni Li, Ayesha B Alvero, Sudhakar Nuti, Roslyn Tedja, Cai M. Roberts, Mary Pitruzzello, Yimin Li, Qing Xiao, Sai Zhang, Yaqi Gan, Gil Mor, Gang Yin. CBX7 binds TWIST-1’s E-box to inhibit TWIST-1 function and curtail tumorigenecity and metastatic potential in ovarian cancer [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 A02.