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 SIN3.histone 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. BRMS1(L174D) 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(DeltaCC1)) 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(DeltaCC1), 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 SIN3.histone 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.

Fig. 1

Map of BRMS1:ARID4A interactions by yeast two-hybrid. The BRMS1 domain structure and the regions that were deleted or mutated are shown schematically. ARID4A was fused to the Gal4 activation domain and BRMS1 or BRMS1^mutant were fused to the Gal4 DNA binding domain as described in the Materials and Methods. The relative strength of interaction was estimated by color change: blue within 4 hr = ++, blue between 4–8 hr = +, and longer than 8 hr = −. The second coiled-coil domain of BRMS1 (AA 147–180, BRMS1^CC2) was found to be necessary and sufficient for interaction with ARID4A. Because deletion of AA 174–180 in the second coiled-coil domain (BRMS1^CC2:N) abolished ARID4A interaction, point mutants were generated in this region by site directed mutagenesis. The point mutant BRMS1^L174D was found to disrupt ARID4A interaction.

Fig. 2

ARID4A maintains indirect association with BRMS1^L174D but not BRMS1^ΔCC1. Co-immunoprecipitation (co-IP) of ARID4A with a mAb directed to the C-terminus of BRMS1 was performed with human breast cell lines expressing BRMS1 or BRMS1 mutants. Although BRMS1^L174D does not directly interact with ARID4A, it was able to co-IP ARID4A in both cell lines, suggesting that a SIN3:HDAC complex including these proteins is intact. Co-IP of ARID4A with BRMS1^ΔCC1, which interacts with ARID4A by Y2H was undetectable in MDA-MB-231 and significantly reduced in MDA-MB-435, suggesting that other BRMS1:protein interactions are necessary to enable the BRMS1:ARID4A interaction (Modeled in Figure 5). The bottom panels show whole cell lysates probed with the BRMS1 antibody. Each panel represents a single blot with three lanes removed that were samples containing additional BRMS1 mutants (unrelated to current study). The blots were otherwise not modified. ‘Parent’ represents the non-transduced cell lines and the numbers represent single-cell clones of the respective transduction. Although expression of the ΔCC1 and L174D mutants in MDA-MB-435 cells is not equal, the biological behaviors (i.e., metastasis, EGFR expression, etc.) replicate the findings in MDA-MB-231 transductants.

Fig. 3

BRMS1 mutants specifically down-regulate EGFR but do not repress basal transcription. A, basal transcription was measured by a dual luciferase reporter assay. BRMS1^L174D did not significantly repress transcription compared to pBIND vector control. All values are normalized to control and error bars represent standard experimental error (SEM) from three replicates of two independent experiments. B, immunoblot of serum free media shows differential down-regulation of OPN by BRMS1^mut (loading was normalized according to cell number as determined by a hemacytometer). C, immunoblot of whole cell lysate shows that although basal transcription and OPN expression is affected, the BRMS1^mut still specifically down-regulate EGFR which has been shown to affect downstream signaling (Vaidya et al. Submitted).

Fig. 4

BRMS1 mutants suppress metastasis. The table on the left shows the incidence and the mean number of lung metastases for each group. The data is shown graphically on the right with black dots representing the number of lung metastases from each mouse; the box represents the 10^th and 90^th percentile; and the black line is the mean for each group. The MDA-MB-231 parental cell line had 7 mice that had too many metastases too count and were conservatively assigned a value of 100.

Fig. 5

BRMS1 binding to the SIN3:HDAC complex is mediated by one or more complex components. BRMS1 and ARID4A are shown interacting with a SIN3:HDAC complex(es) that is not yet completely defined. The BRMS1^L174D is unable to directly interact with ARID4A, but both proteins remain associated via other interactions. The binding of BRMS1^L174D to the complex may change the overall composition (depicted by a change in shading) and/or a change in structure, but insufficiently to disrupt ARID4A:complex association. The BRMS1^ΔCC1 may no longer associate with the complex, even though it has the ability to directly interact with ARID4A. Again, the structure-function of the overall complex would determine selectivity of transcriptional regulation and/or metastasis suppression.