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BRMS1 Regulates Osteopontin Expression in Metastatic Breast Cancer
Alterations in chromosome 11 are a hallmark of late stage, metastatic breast carcinomas. BRMS1 (Breast cancer Metastasis Suppressor 1) was identified by differential display comparing metastatic breast carcinoma cell line with its metastasis-suppressed chromosome 11 hybrid. Metastatic human breast carcinoma cell lines express very low levels of BRMS1 in contrast to non-metastatic (tumorigenic) breast carcinoma cells. Constitutive expression of BRMS1 into multiple metastatic breast carcinoma cell lines of human and mouse origin, resulted in significant suppression of metastasis. Analysis of the upstream region of genomic BRMS1 (that encompasses the putative promoter) reveals a CpG island. This raises the possibility of regulation of BRMS1 expression by epigenetic modification (CpG island methylation and/or histone deacetylation).
In parallel, to elucidate the mechanism(s) underlying the ability of BRMS1 to suppress metastasis, microarray analysis was performed with metastatic cells and their respective counterparts constitutively expressing BRMS1. The microarray analysis revealed ~20-fold lower expression of osteopontin (OPN) mRNA in the BRMS1-expressing cells. OPN, an acidic glycoprotein rich in aspartate, glutamate and serine and is detected in the sera of late stage breast and colorectal carcinoma patients.
Relating the loss of expression of BRMS1 in metastatic breast carcinoma with increased OPN expression forms the primary objective of this proposal to study the regulation and mechanism of action of BRMS1.
Based on the observation of (1) putative epigenetic modification of BRMS1, (2) decreased OPN levels in BRMS1-expressing cells this proposal will test the central hypothesis that loss of expression of BRMS1 by metastatic breast carcinomas will be reflected in higher OPN expression by the tumor. This hypothesis will be tested in three Specific Aims:
Specific Aim 1: Will test the hypothesis that BRMS1 expression is regulated by epigenetic modification.
This will include analysis of various human breast carcinoma cell lines displaying a range of metastatic potential for evidence of epigenetic regulation of BRMS1 expression. This will address methylation of CpG islands in the promoter region of BRMS1.
Specific Aim 2: To characterize the suppression of OPN expression by BRMS1
Understanding the mechanism of suppression of OPN by BRMS1 offers an approach to understand the normal function of BRMS1, which has gone awry in metastatic cells. Characterization of OPN expression will be addressed using reporter-based assays with the promoter of OPN in the background of BRMS1, Chromosomal immunoprecipitation (ChIP) assay and electromobility shift assay (EMSA).
Specific Aim 3: Will encompass experiments to test the hypothesis that expression of BRMS1 in clinical specimens correlates inversely with the level of OPN expression by the tumor. Specifically, we will examine clinical specimens to address the issue.
The proposed research represents a critical step towards understanding the regulation of BRMS1 gene. The study becomes clinically very relevant when put in perspective of osteopontin, a molecule whose level is elevated in late stage, advanced breast carcinoma patients. The diagnosis of epigenetic modification of BRMS1 and the suppression of OPN brought about by BRMS1 offers a new window into understanding the suppression of breast cancer metastasis by BRMS1. It allows an approach towards better control of metastatic disease.
The proposal is aimed at understanding the regulation and mechanism of action of a novel gene that suppresses metastasis of breast carcinoma.
The gene BRMS1 (Breast cancer Metastasis Suppressor 1) is found in significantly low levels in metastatic breast carcinoma cells (in culture). This would lead to the premise that the expression of BRMS1 is ‘silenced’ in metastatic breast cancer. The BRMS1 gene is found to display molecular signatures that are indicative of possible control of expression of BRMS1.
This proposal aims to diagnose, confirm and characterize the ‘silencing’ of BRMS1 expression.
BRMS1 also suppresses the expression of a molecule, osteopontin (OPN), detected in circulation of advanced breast carcinoma patients. We will characterize the suppression of OPN by BRMS1.
Putting together the facts that 1) BRMS1 is ‘silenced’ in metastatic breast carcinomas, 2) OPN levels are higher in metastatic breast carcinoma and 3) BRMS1 affects OPN expression, form the hypothesis of this proposal – silencing of BRMS1 gene will be reflected as higher OPN levels.
The proposed research represents a critical step towards understanding the regulation of BRMS1 gene. The study becomes clinically very relevant when put in prespective of osteopontin, a molecule whose level is elevated in late stage, advanced breast carcinoma patients. The diagnosis of ‘silencing’ of BRMS1 and the suppression of OPN brought about by BRMS1 offers a new window into understanding the mechanism of action of BRMS1. It allows an approach towards better control of metastatic disease.