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    Epigenetic Consequences of BRCA1: The Key to its Role as a Tumor Suppressor Gene in Human Breast Cancer

    Scientific Abstract:
    Epigenetic consequences of BRCA1: the key to its role as a tumor suppressor gene in human breast cancer Background: BRCA1 was identified nearly a decade ago as a gene responsible for many families with inherited predisposition to breast and ovarian cancer. As a tumor suppressor gene, loss of function in mammary epithelial cells is a critical early step in the development of breast cancer. Although mutations in BRCA1 have not been observed in sporadic breast cancers, diminished expression has been reported. Multiple functions have been attributed to BRCA1 in important cellular processes. However, the specific functions of BRCA1 that mediate its role as a tumor suppressor gene in breast cancer have not been conclusively identified. Evidence from varied experimental approaches suggests that regulation of gene expression is a key aspect of BRCA1’s functions in breast cancer. We have observed that one of the most distinctive features of breast cancers arising in BRCA1 mutations carriers, the lack of estrogen receptor (ER) expression, is associated with specific changes in CpG methylation, an epigenetic mechanism of gene repression. Other investigators have reported additional notable characteristics in BRCA1-linked breast cancers that relate to alterations in CpG island methylation and X-chromosome inactivation. Objective/Hypothesis: To pursue the novel hypothesis that dysregulation of gene expression by epigenetic mechanisms underlies BRCA1’s role as a tumor suppressor gene in breast cancer. Specific Aims: 1. Determine whether changes in the level of BRCA1 expression in a mammary epithelial cell line results in reciprocal changes in the level of CpG methylation at the HIN-1 and ER genes. 2. Compare mammary epithelial cells lacking expression of BRCA1 with cells expressing either wild type BRCA1 or mutant forms of BRCA1 in terms of both global CpG methylation and patterns of histone H3 acetylation, using a high-throughput microarray. 3. Test the hypothesis that alterations in CpG methylation that correlate with BRCA1 expression are also evident in sporadic breast cancers that are deficient in BRCA1 expression. Study Design: In vitro cell culture experiments will utilize the MCF10A cell line. This is an immortalized human mammary epithelial cell line that expresses low levels of BRCA1, lacks ER expression, and is hypermethylated at the ER CpG island. Stable transfectants expressing an anti-BRCA1 siRNA, and therefore lacking expression of BRCA1 protein, will be generated. By transfection of these cells with BRCA1 cDNA expression constructs we can determine the effect of BRCA1 (wild type and mutant) on CpG methylation and histone H3 acetylation. CpG methylation will be evaluated by 3 methods: bisulfite treatment, followed by PCR amplification and sequence analysis; comparative hybridization to a CpG island microarray; and analysis of genomic content of methylated cytosine via digestion with methylation-sensitive restriction enzymes followed by single nucleotide extension. The same CpG island microarray will be used to profile changes in histone H3 acetylation by competitive hybridization with DNA isolated by chromatin immunoprecipitation. To address aim 3, we will evaluate sporadic breast cancers by immunohistochemistry and western blot for expression of BRCA1 (as well as ER and HIN-1), isolate genomic DNA, and perform the three assays of CpG methylation described above. Relevance: Characterization of the specific functions of BRCA1 that mediate its role as a mammary tumor suppressor gene, apart from its myriad of functions in cells, could serve as the basis for the development of a functional assay that could be used in conjunction with genetic testing to evaluate genetic alterations of uncertain significance, and thereby distinguish pathogenic mutations from benign polymorphisms. Further, such knowledge could lead to novel strategies for prevention in mutation carriers.

    Lay Abstract:
    Project Title: Epigenetic consequences of BRCA1: the key to its role as a tumor suppressor gene in human breast cancer Background: BRCA1 was identified nearly a decade ago as a gene responsible for many of the families with inherited predisposition to breast and ovarian cancer. As a typical tumor suppressor gene, loss of function from the normal copy of BRCA1 in breast cells is a critical early step in the development of breast cancer in women who carry a mutation in BRCA1. Although mutations in BRCA1 have not been observed in sporadic (i.e. non-inherited) breast cancers, diminished expression has been reported. BRCA1 is expressed in many tissues throughout the body, and multiple functions have been attributed to BRCA1 in important cellular processes. However, the specific functions of BRCA1 that mediate its role as a tumor suppressor gene in breast cancer have not been conclusively identified. Evidence from varied experimental approaches suggests that regulation of gene expression is a key aspect of BRCA1’s functions in breast cancer. We have observed that one of the most distinctive features of breast cancers arising in BRCA1 mutations carriers, the lack of estrogen receptor (ER) expression, is associated with specific changes in CpG methylation, a so-called ‘epigenetic’ mechanism of gene repression. Other investigators have reported additional notable characteristics in BRCA1-linked breast cancers that relate to alterations in epigenetic regulation of gene expression, specifically CpG methylation at another gene locus and defective X-chromosome inactivation. Objective/Hypothesis: To pursue the novel hypothesis that dysregulation of gene expression by epigenetic mechanisms underlies BRCA1’s role as a tumor suppressor gene in breast cancer. Specific Aims and Study Design: We will test this hypothesis through experiments with an established immortalized human mammary epithelial cell line (MCF10A), as well as patient specimens. First, we will suppress the expression of the endogeneous BRCA1 in MCF10A cells using the powerful siRNA approach. Specifically, cells will be transfected with an expression plasmid that directs the production of small RNA molecules that will bind to and inactivate the normal BRCA1 messenger RNA within the cell. This approach has been successfully used in the past for inhibiting the expression of multiple genes, including BRCA1. We can then selectively restore expression of normal or mutant forms of BRCA1. These cells will then be examined by sensitive and robust methods analyzing for epigenetic changes in CpG methylation. We will look at selected specific genes for which there are prior data implicating BRCA1 as an epigenetic regulator of expression, and will perform global analyses of CpG methylation and histone H3 acetylation using a novel CpG island microarray. Once we have identified epigenetic changes in CpG methylation the correlate with BRCA1 status, we will examine sporadic breast cancers lacking BRCA1 for similar alterations. Relevance and Potential Impact: Characterization of the specific functions of BRCA1 that mediate its role as a breast cancer tumor suppressor gene, apart from its myriad of functions in cells, could serve as the basis for the development of a functional assay that could be used in conjunction with genetic testing to evaluate genetic alterations of uncertain significance, and thereby distinguish pathogenic mutations from benign polymorphisms. Further, such knowledge could lead to novel strategies for prevention in women who are carriers of a BRCA1 mutation.