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Suppression of BTG2 Expression in Breast Cancer
BTG2, an immediate early gene inhibits cellular proliferation and its expression is reduced in mammary glands during pregnancy when a massive expansion of mammary epithelial cells occurs. BTG2 mRNA was expressed in immortalized human mammary epithelial cells but suppressed or undetectable in breast cancer cells; re-expression of BTG2 in breast cancer cells inhibited growth. Consistent with these observations, BTG2 protein was absent in >50% of human breast tumors but present in normal breast tissue. Analysis of mice that spontaneously develop mammary tumors demonstrated the loss of BTG2 protein. These observations led to the hypothesis that loss of BTG2 expression may promote carcinogenesis of the breast. The BTG2 gene localizes to chromosomal locus 1q32 and loss of heterozygosity at 1q23-32 is one of the lesions associated with breast cancer development. However, PCR amplification of both BTG2 exons and splice sites revealed no mutations or nucleotide polymorphisms. Hypermethylation of DNA, which silences gene expression plays a critical role in carcinogenesis in multiple tumor types and preliminary results indicate that promoter hypermethylation may be one of the mechanisms by which BTG2 is silenced in breast cancer. Hypotheses: (1) Degree of BTG2 promoter methylation may correlate with breast tumor progression. (2) BTG2 is a suppressor of mammary tumor growth. Specific Aims: Aim 1: To characterize BTG2 promoter methylation in breast cancer. Aim 2: To determine whether BTG2 expression can suppress tumorigenesis in mouse mammary tumor models. Methylation status of the BTG2 promoter will be characterized in breast cancer cell lines by southern anaysis and bisulfite modification of DNA/ sequence analysis. We have currently acquired about >150 paraffin embedded human breast tumors with matched normal glands. For each specimen, age of patient and associated tumor characteristics and clinical follow-up are available. Methylation status of the BTG2 promoter will be determined in these samples. In aim 2, we will test whether reexpression of BTG2 in breast cancer cell lines can prevent tumorigenicity and metastasis in mice. These observations will be validated in trangenic mice overexpressing BTG2 in the mammary gland; they will be bred with mice which give rise to spontaneous mammary tumors (eg: C(3) T antigen model; Her2/neu model) to determine whether reexpression of BTG2 can suppress tumorigenesis Regional patterns of DNA methylation are among the earliest and most frequent events known to occur in human cancers. Determining BTG2 promoter methylation status in the breast could constitute a potentially important marker that can be screened in small biopsies using a rapid and sensitive assay to predict risk, prognosis, and could possibly provide a target for prevention. The accompanying study will help us assess whether BTG2 mediated tumor suppression would provide molecular targets for mechanism-based treatment strategies.
Breast cancer, the most commonly diagnosed cancer in females, is the second leading cause of death in women. One out of every eight women will develop breast cancer in her lifetime; in 2003, approximately 211,300 women in the United States were diagnosed with invasive breast cancer. The B-cell translocation gene-2 (BTG-2) encodes for a protein that has growth inhibitory activity. In agreement with this concept, BTG2 mRNA could not be detected in breast cancer cells and forced expression of BTG2 in these cells inhibited growth. Furthermore, BTG2 protein was absent in >50% of human breast tumors tested but present in the adjacent normal breast tissue. Analysis of mice that develop mammary tumors demonstrated that loss of BTG2 protein occurs very early during the tumorigenic process. Thus suppression of BTG2 expression may be an important step in the development of breast cancer. The objectives of this proposal are to uncover the mechanisms, which lead to the suppression of BTG2 expression in breast cancer, and to determine whether re-expression of BTG2 can suppress the incidence of mammary tumors in mouse models. The outcome of this grant will provide insight into the role of BTG2 in breast cancer and provide the basis for future studies that will help us determine whether this pathway might constitute a potentially important marker that can be used to screen small tissue biopsies using a rapid and sensitive assay to predict risk and prognosis. Moreover, it could also provide molecular targets for mechanism-based prevention and treatment strategies that would be effective in patients with breast cancer.