The SWI/SNF ATPases Are Required for Triple Negative Breast Cancer Cell Proliferation

Abstract

The Brahma (BRM) and Brahma-related Gene 1 (BRG1) ATPases are highly conserved homologues that catalyze the chromatin remodeling functions of the multi-subunit human SWI/SNF chromatin remodeling enzymes in a mutually exclusive manner. SWI/SNF enzyme subunits are mutated or missing in man cancer types, but are overexpressed without apparent mutation in other cancers. Here, we report that that both BRG1 and BRM are overexpressed in most primary breast cancers independent of the tumor’s receptor status. Knockdown of either ATPase in a triple negative breast cancer cell line reduced tumor formation in vivo and cell proliferation in vitro. Fewer cells in S phase and an extended cell cycle progression time were observed without any indication of apoptosis, senescence or alterations in migration or attachment properties. Combined knockdown of BRM and BRG1 showed additive effects in the reduction of cell proliferation and time required for completion of cell cycle, suggesting that these enzymes promote cell cycle progression through independent mechanisms. Knockout of BRG1 or BRM using CRISPR/Cas9 technology resulted in loss of viability, consistent with a requirement for both enzymes in triple negative breast cancer cells.

The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation

“…Western blot analysis confirmed the knockdown of BRG1 in both cell lines (Figure 1F). This observation was reproduced in other triple negative breast cancer lines (MDA-MB-468 and HDQ-P1) that were treated with a previously validated pool of siRNAs targeting BRG1 [22, 27] (Figure 1G–1H). ADAADi (Active DNA-dependent ATPase A Domain inhibitor), a minor product generated by the bacterial APH (3′)-III enzyme that encodes for aminoglycoside resistance, inhibits the ATPase activity of the SWI2/SNF2 family of ATPases [28, 29] and increases the chemosensitivity of triple negative breast cancer cells to clinically relevant therapeutic drugs [30].…”

“…To support unlimited growth, cancer cells exhibit higher rates of glucose metabolism, protein synthesis and de novo lipid synthesis [25, 26]. We surveyed these pathways by metabolic labeling in MDA-MB-231 triple negative breast cancer cells in the presence of a scrambled sequence shRNA or shRNA targeting BRG1 [21, 22, 27]. Glucose uptake and protein synthesis were not affected in MDA-MB-231 BRG1 knockdown cells (Figure 1A–1C).…”

“…Re-expression or over-expression of wildtype or catalytically inactive BRG1 is negligible in some cell types if Brahma (BRM), the closely related ATPase that can act as the catalytic subunit in mammalian SWI/SNF complexes in a manner that is mutually exclusive with BRG1, is expressed [38–40]. In addition, we have previously shown that knockdown of either BRG1 or BRM in triple negative breast cancer cell lines results in increased expression of the remaining ATPase [22] and that both BRG1 and BRM contribute to triple negative cell proliferation [30]. Therefore we re-established BRG1 expression in MDA-MB-231 cells after expressing shRNA against both BRG1 and BRM [22] by transient transfection with plasmid vectors.…”

“…Meta-analyses of cancer genome-sequencing data estimates that nearly 20% of human cancers harbor mutations in one or more SWI/SNF genes [17–20]. We and others reported that knockdown of BRG1 reduces cell proliferation in both breast epithelial and cancer cells in vitro [21–23] and attenuates tumor growth in a xenograft model [21, 22]. However, the underlying mechanisms remained unknown.…”

SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells

“…In the shSMARCA4 MCF-10A cells, the SMARCA4 homolog SMARCA2 (also known as BRM) still exists, and we previously reported that MCF-10A cells are not viable when both SMARCA4 and SMARCA2 are simultaneously knocked down (Cohet et al 2010). Total removal of SMARCA4 by CRISPR/Cas9 in the MDA-MB-231 human metastatic breast cancer cell line and genetic ablation in primary myoblasts also resulted in cell death Wu et al 2015). These findings suggest that the importance of SMARCA4 in cell viability may preclude observing more severe effects on genome organization.…”