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

Abstract

Breast Cancer Metastasis Suppressor 1 (BRMS1) suppresses metastasis of human breast cancer, ovarian cancer and melanoma in athymic mice. Studies have also shown that BRMS1 is significantly downregulated in some breast tumors, especially in metastatic disease. However, the mechanisms which regulate BRMS1 expression are currently unknown. Upon examination of the BRMS1 promoter region by methylation specific PCR (MSP) analysis, we discovered a CpG island (-3477 to -2214), which was found to be hypermethylated across breast cancer cell lines. A panel of 20 patient samples analyzed showed that 45% of the primary tumors and 60% of the matched lymph node metastases, displayed hypermethylation of BRMS1 promoter. Furthermore, we found a direct correlation between the methylation status of the BRMS1 promoter in the DNA isolated from tissues, with the loss of BRMS1 expression assessed by immunohistochemistry. There are several studies investigating the mechanism by which BRMS1 suppresses metastasis; however thus far there is no study that reports the cause(s) of loss of BRMS1 expression in aggressive breast cancer. Here we report for the first time that BRMS1 is a novel target of epigenetic silencing; and aberrant methylation in the BRMS1 promoter may serve as a cause of loss of its expression.

Fig. 1

BRMS1 mRNA levels are decreased in tumorigenic and/or metastatic breast cancer cell lines. RNA was isolated from breast cancer cell lines, cDNA was generated and subjected to real-time PCR. The C_T values of BRMS1 were normalized to GAPDH and fold change in BRMS1 was calculated by comparing cell lines to normal breast RNA (defined as 100%)

Fig. 2

CpG island identified upstream of BRMS1 is responsible for silencing in MDA-MB-231 cells. (a) Schematic of BRMS1 in which CpG islands were identified using CpG island searcher. The transcription initiation site (TIS) is marked at +1 with one island lying within the first exon of BRMS1 (−531 to +608) while the other is 1683 bp upstream (−3477 to −2214). E1 and E2 are exon 1 and 2 respectively and ATG is translation start site for BRMS1. (b) MDAMB-231 cells were treated with 5-aza-2′-deoxycytidine (5-aza-dC) at two concentrations of 0.5 and 1.0 µM and assayed for BRMS1 expression by western blotting. Beta-actin was used as loading control. The densitometric change in the expression of BRMS1 is depicted as the IDV normalized to untreated MDA-MB-231 cells

Fig. 3

CpG island B is hypermethylated with increasing metastatic potential of breast cancer cell lines. (a) DNA was isolated and subjected to bisulfite modification and subsequent MSP analysis. A band at 109 bp was generated for both unmethylated (U) and methylated (M) reactions. Lack of the M bands in the lanes of MCF 10A and MCF10AT was a clear indication of no hypermethylation. Control represents the chemically methylated DNA.U = Unmethylated primers; M = Methylated primers. (b) Cells were treated with either 0.5 or 1.0 µM 5-aza-2′-deoxycytidine to demethylate the BRMS1 CpG island. The hypomethylated phenotype is characterized by the appearance of the ‘U’ amplicon in the MCF10CA, MDA-MB-231 and MDA-MB-435 cells concomitant with a decreased intensity of the ‘M’ amplicon. U = Unmethylated primers; M = Methylated primers. (c) Modified DNA was subjected to BGS which targeted −2460 to −2337 bp of the CpG island B. Four representative sequenced clones are shown here. Open circle represents unmethylated and closed circle depicts methylated CG residues

Fig. 4

Methylation of BRMS1 is correlated with metastatic breast tumors. (a) A cohort of matched-patients breast tumors were analyzed using MSP. Cells were isolated using laser capture microdissection from which DNA was extracted and subjected to MSP. All twelve matched patient samples examined are shown here. U = Unmethylated primers; M = Methylated primers. (b) One set of matched patient tissues was examined by BGS. A representative of 10 sequenced clones is shown here. The open circle represents unmethylated CG residues while the closed circle depicts methylated CG residues