Identification and characterization of the murine ortholog (brms1) of breast‐cancer metastasis suppressor 1 (BRMS1)

Abstract

We have cloned a novel metastasis-suppressor gene (BRMS1) by differential display, comparing metastatic human breast carcinoma cell line MDA-MB-435 to its metastasissuppressed human chromosome 11 microcell hybrid. Screening of a murine cDNA library led to the identification of a 1.4 kb cDNA with a sequence revealing 85% homology to human BRMS1 within the open reading frame. The predicted protein sequence for the murine ortholog is 95% identical, suggesting that it is strongly conserved across these 2 species. The cloned cDNA was used to screen a murine strain SV129 BAC library to obtain brms1 genomic DNA. Three Metastasis-suppressor genes are a relatively newly described group of genes that, by definition, suppress the spread of a primary tumor to a discontinuous site without altering its growth. 1 There are currently 8 genes for which functional suppression of metastasis has been described: Nm23, KiSS1, Kai1, TIMPs, Maspin, MKK4 and BRMS1. BRMS1 was identified by us using differential display, comparing the metastatic human breast carcinoma cell line MDA-MB-435 and its metastasis-suppressed microcell hybrid bearing full-length human chromosome 11. 2 Transfection of full-length BRMS1 cDNA into highly metastatic breast-cancer cell lines MDA-MB-435 and MDA-MB-231 suppressed their metastatic ability without affecting tumorigenicity, meeting the functional definition of metastasis suppression. That the BRMS1 gene maps to 11q13.1-13.2, a region frequently altered in late-stage breast carcinoma, 3 provides further impetus to determine the mechanism by which this candidate metastasis suppressor functions. Despite being a predominantly nuclear protein, BRMS1 restores homotypic gap junctional intercellular communication in MDA-MB-435 and MDA-MB-231 cells. 4,5 However, its normal physiologic role is not known.Determination of the normal physiologic role of BRMS1 might be assisted by the availability of a murine ortholog. Therefore, in the studies presented here, we describe the isolation of brms1, the structural characterization of the brms1 genomic locus and the functionality of brms1 (i.e., ability to suppress metastasis in a series of mammary carcinoma cell lines). That the human and murine orthologs are biologically and molecularly similar provides justification for developing a brms1 knockout mouse and use of murine mammary tumor models for mechanistic studies in the future.

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

“…This putative regulatory region shows no significant CpG island as defined by the CpG Island Searcher [28]. Upstream from the transcription start site of BRMS1 is an intergenic region preceded by β3GNT1 [4]. Our insilico searches revealed two strong CpG islands, one extending through the first exon of BRMS1 (−531 to +608), and another starting about 1683 bp upstream (−3477 to −2214) (Fig.…”

“…Breast Cancer Metastasis Suppressor 1 (BRMS1) was originally identified to suppress metastasis of highly metastatic human breast cancer cell lines when injected into nude mice [2][3][4]. Reduced expression of the BRMS1 mRNA is correlated with poor prognosis in breast cancer [5,6].…”

High level of messenger RNA for BRMS1 in primary breast carcinomas is associated with poor prognosis

“…Reintroduction of BRMS1 in cell lines derived from human melanoma, human and murine breast carcinomas correlated with decreased metastatic potential in in vivo metastasis assays. [12][13][14] The aim of this study was to clarify BRMS1 involvement in human mammary tumor progression, and the potential prognostic value of BRMS1 mRNA expression in breast carcinoma. BRMS1 gene expression was analyzed in tumor cells, in phenotypically normal epithelium surrounding the lesions and in metastatic cells from lymph nodes.…”

“…11 Reintroduction of BRMS1 in human and murine breast cancer cells as well as in human melanoma cells significantly reduced their metastatic potential to the lungs and to regional lymph nodes, without affecting primary tumor growth in athymic nude mice. [12][13][14] Rare metastases that developed had lost expression of BRMS1. 15 The step of the metastatic progression where BRMS1 is mainly involved need to be characterized.…”

“…BRMS1 protein, localized predominantly (>90%) to the nucleus, 13 is a conserved component of the mSin3 family of histone deacetylase (HDAC) complexes, 16 involved in transcriptional repression through local cromatin condensation. BRMS1 has been shown to restore gap junctional intercellular communication, 17 and recent data suggest that it might regulate second messenger phosphoinositide signaling.…”