Abstract B015: KDM6A, lysine demethylase, loss affects epithelial-to-mesenchymal related gene expression and breast cancer growth

Abstract

Abstract Epithelial-to-mesenchymal transition (EMT) or its reversal mesenchymal-to-epithelial transition (MET) are of critical importance throughout development and is implicated in wound healing, fibrotic disorders, and cancer metastasis. The ability of a cell to undergo EMT and MET, cellular plasticity, underlies tumor progression through multi-step metastasis and acquisition of chemotherapy resistance as alterations in the cellular and molecular level confer changes in motility, stemness properties, gene expression, and cellular dynamics. These changes facilitate invasion and dissemination of cancer cells. Epigenetic modifications leading to cancer cell plasticity are partially driven by histone modifying proteins including lysine (K)-specific demethylase 6A (KDM6A). KDM6A is a member of the COMPASS-like protein complex, and catalyzes the removal of methyl groups from H3K27me3, facilitating gene expression. KDM6A regulation of H3K27me3 has been associated with bivalent programs in gene expression, associates with oncogenic pathways, and contributes to low survival prognoses in breast cancer. There is critical need to elucidate the mechanisms of KDM6A suppression to understand its control on cellular plasticity. Previously we have shown that KDM6A is suppressed upon EMT. In this study, we sought to identify the regulatory mechanisms of KDM6A expression and how its activity is required to maintain epithelial cellular identity. To investigate the role of KDM6A in the context of EMT, KDM6A expression was compromised using CRISPR-Cas9 to determine the impact on H3K27me3, gene expression and cellular identity. Breast cancer cell lines were transduced with CRISPR-Cas9 and GFP via lentiviral transduction. KDM6A KO and control cell lines were generated via a CRISPR KO. We observed that the KDM6A suppression or inhibition profoundly impacts cellular identity, leading to gain of mesenchymal and stemness properties. Moreover, KDM6A was determined to be regulated through sub-cellular localization leading to partial sequestration of COMPASS-like protein complex outside the nucleus. These findings were validated via Western blot analysis of subcellular protein fractions of breast cancer cell lines and KDM6A KO cell lines and well as immunofluorescence and enzyme activity analysis. We conclude that KDM6A functions as a master regulator of epithelial cellular identity which is controlled by multi-factorial mechanisms including transcriptional suppression and altered protein localization. Citation Format: Charli Worth, Provas Das, Kelsey Johnson, Joseph Taube. KDM6A, lysine demethylase, loss affects epithelial-to-mesenchymal related gene expression and breast cancer growth [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B015.