Alterations of BRMS1-ARID4A Interaction Modify Gene Expression but Still Suppress Metastasis in Human Breast Cancer Cells

Abstract

The BRMS1 metastasis suppressor interacts with the protein AT-rich interactive domain 4A (ARID4A, RBBP1) as part of SIN3⅐histone deacetylase chromatin remodeling complexes. These transcriptional co-repressors regulate diverse cell phenotypes depending upon complex composition. To define BRMS1 complexes and their roles in metastasis suppression, we generated BRMS1 mutants (BRMS1 mut ) and mapped ARID4A interactions. BRMS1L174D disrupted direct interaction with ARID4A in yeast two-hybrid genetic screens but retained an indirect association with ARID4A in MDA-MB-231 and -435 human breast cancer cell lines by co-immunoprecipitation. Deletion of the first coiled-coil domain (BRMS1 ⌬CC1 ) did not disrupt direct interaction in yeast two-hybrid screens but did prevent association by co-immunoprecipitation. These results suggest altered complex composition with BRMS1 mut . Although basal transcription repression was impaired and the pro-metastatic protein osteopontin was differentially down-regulated by BRMS1 L174D and BRMS1 ⌬CC1 , both down-regulated the epidermal growth factor receptor and suppressed metastasis in MDA-MB-231 and -435 breast cancer xenograft models. We conclude that BRMS1 mut , which modifies the composition of a SIN3⅐histone deacetylase chromatin remodeling complex, leads to altered gene expression profiles. Because metastasis requires the coordinate expression of multiple genes, down-regulation of at least one important gene, such as the epidermal growth factor receptor, had the ability to suppress metastasis. Understanding which interactions are necessary for particular biochemical/ cellular functions may prove important for future strategies targeting metastasis.The ability of a cancer cell to complete all steps of the metastatic cascade requires diverse tumor-host interactions that are dependent on the coordinate expression of specific genes both intrinsically and extrinsically (1-3). The metastasis suppressor BRMS1 3 has been shown to regulate the expression of multiple genes leading to the suppression of metastasis in multiple model systems, including human breast carcinoma (4, 5), melanoma (6), and ovarian carcinoma (7), without preventing orthotopic tumor growth. Specifically, down-regulation of the pro-metastatic genes osteopontin (OPN) and urokinase-type plasminogen activator has been linked to BRMS1 expression (8, 9). Gap junctional intercellular communication is restored by BRMS1 through a change in connexin expression (10). Microarray and proteomic analyses have also been performed showing multiple changes in gene and protein expression when BRMS1 was introduced (11-13). Clinically, loss of BRMS1 protein has been correlated with progesterone receptor expression and inversely correlated with HER2 expression in breast cancer patients (14).BRMS1 has been proposed to regulate transcription of genes by interaction with a large SIN3⅐HDAC chromatin remodeling complex through interaction with the protein AT-rich interactive domain 4A (ARID4A) that suppresses basal transcription in vivo using a Gal...

Structural insights into the assembly of the histone deacetylase-associated Sin3L/Rpd3L corepressor complex

“…We propose that a model for BRMS1 dimerization based on the structure of the Sds3 dimerization domain is likely to be physiologically relevant. Indeed, previous genetic and biochemical studies have shown that BRMS1 could recruit ARID4A/B to the complex in a manner dependent on intact CC1 and CC2, raising the possibility that CC1 and CC2, which are in close proximity in the Sds3-based model for the dimer, form a recruiting platform for these types of interactions (37). However, it has been shown that Sds3 is unable to suppress metastasis in the absence of BRMS1 (38).…”

Epigenetic silencing contributes to the loss of BRMS1 expression in breast cancer

“…40 µg of total protein was loaded onto a 10% SDS-PAGE gel and transferred to PVDF membrane. All membranes were blocked overnight at 4°C and immunoblotting was done with mouse anti-BRMS1 antibody (at 1:2,500) [27] or with mouse anti-β-actin antibody (1:30,000). Anti-mouse HRP conjugated secondary antibody was used for detection and blots were developed with SuperSignal enhanced chemiluminescence substrate (Pierce, Rockford, IL) and exposed using a Fuji LAS3000 imager.…”

Breast Cancer Metastasis: Advances Through the Use of In Vitro Co-Culture Model Systems

“…For Breast Cancer Metastasis Suppressor-1 (BRMS1), the clinical data reporting it to be a metastasis suppressor protein in breast cancer tumour samples is also conflicting (Kelly, Buggy et al 2005;Hicks, Yoder et al 2006;Lombardi, Di Cristofano et al 2007). Again, its role in mouse models is clearer, where higher expression in breast cancer xenografts clearly resulted in reduced metastasis (Hedley, Vaidya et al 2008;Hurst, Xie et al 2008;Phadke, Vaidya et al 2008). The stage at which BRMS1 suppresses metastasis is less clear, as it appears to affect a number of steps in the process of metastasis (Stafford, Vaidya et al 2008).…”