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Does RARbeta2 P2 Methylation Predict Short-Term Breast Cancer Risk?
Background: Recent studies suggest that that breast cancer incidence may be substantially reduced in high-risk women by tamoxifen-treatment, prophylactic oophorectomy, and/or prophylactic mastectomy. Although these reports are encouraging, current prevention strategies are expensive and can be associated with significant side effects. Biomarkers are needed to accurately predict short-term breast cancer risk, so that 1) women who are most likely to benefit from preventive therapy can be identified, and 2) response to chemoprevention can be accurately assesed.
Retinoids and the retinoic acid receptor-beta-2 (RARbeta2) are important regulators of normal mammary gland homeostasis. Loss of RARbeta2 expression is observed in both invasive breast cancers and the normal appearing epithelial cells adjacent to breast cancers. This has lead to the hypothesis that loss of RARbeta2 expression may provide an environment that promotes mammary carcinogenesis. Methylation of the RARbeta2 P2 promoter is thought to be the primary mechanism for loss of RARbeta2 expression during early breast carcinogenesis. The goal of this proposal is to test whether RARbeta2 P2 methylation in Random Peri-Areolar Breast Fine Needle Aspiration (rFNA) predicts the presence of atypical hyperplasia, and ultimately breast cancer risk, in a multi-institutional chemoprevention cohort.
Preliminary Studies: We observe in vitro that loss of RARbeta2 expression 1) promotes dysregulated growth, 2) apoptosis-resistance, and 3) loss of CBP expression. CBP is known to 1) regulates both proliferation and apoptosis, 2) play a critical role in retinoid-, estrogen-, prostaglandin-, and COX-2-signaling, and 3) is thought to participate in BRCA1-mediated repair. In patient samples we observe that RARbeta2 P2 methylated in 35/40 primary node-negative breast cancers and in 5/10 (50%) atypical rFNA clusters. In 4/5 of rFNA patient samples with atypical hyperplasia we observe a >50% reduction in CBP mRNA expression suggesting an in vivo link between RARbeta2 promoter methylation, CBP expression, and atypical hyperplasia.
Objective/hypothesis: The proposed study will test the hypothesis that the presence of RARbeta2 P2 promoter methylation in mammary epithelial cells obtained by rFNA predicts 1) cellular atypia (a surrogate marker of breast cancer risk) and 2) loss of CBP expression. In conducting this study the following Specific Aims will be pursued: Specific Aim I will test whether RARbeta2 P2 promoter methylation predicts mammary epithelial cell atypia in breast rFNA. The association between RARbeta2 P2 methylation will be tested by methylation-specific PCR. Specific Aim II will test whether RARbeta2 P2 methylation predicts loss of CBP expression in atypical mammary epithelial cells. Correlation between RARbeta2 P2 methylation and CBP expression in microdissected patient-matched atypical and normal cell clusters will be tested by methylation-specific PCR and semi-quantitative RT-PCR.
Relevance: Since RARbeta2 P2 methylation is a highly sensitive marker that we can detect in as few as 5 cells, we predict that the presence or absence of RARbeta2 P2 methylation in breast rFNA can be used as a sensitive and biologically relevant test for both 1) breast cancer risk and 2) response to chemoprevention agents. We are part of a 6 institution group that has been trained to perform breast rFNA and has longstanding expertise in developing and testing new prevention agents. If RARbeta2 P2 methylation proves to be a 1) marker of cellular atypia and 2)surrogate marker of breast cancer risk, we can then readily use this biomarker in our on-going prevention efforts as a predictor of response to prevention drugs (e.g. cox-2 inhibitors), dietary changes (e.g. low-fat, high-vegetable diet), and complementary agents (e.g. flax seed or fish oil).
Background: Clinical breast exams and mammograms are not always able to detect early breast changes and have limited sensitivity in women with dense breast tissue. Many times the women who are at the highest risk for breast cancer are the most difficult to evaluate with breast exam and mammogram. There is need for a low cost “breast Pap-smear” to monitor high-risk women for the presence of pre-cancerous breast cells. Such a test would be extremely valuable because it could evaluate women for both 1) breast cancer risk and 2) response to prevention agents.
Breast random fine needle aspiration (rFNA) is a research tool that has been developed by our group to sample cells from the entire breast of high-risk women. rFNA 1) works best in dense breast tissue, 2) is inexpensive, and 3) can be performed rapidly (15 mins) in a doctor’s office using simple clinic supplies (syringe, lidocane, and the preservative used for cervical “Pap-smears”). Most importantly, rFNA can function as a “breast Pap-smear” in its ability to test for pre-cancerous changes in the whole breast.
Our group currently uses breast rFNA as a research tool to test for breast cancer risk in high-risk women. We observe that the presence of abnormal breast cells (atypia) in breast rFNA is associated with a 4-X higher risk of developing breast cancer. Our group has also used breast rFNA to test for response to prevention agents. However, rFNA has limitations. If a prevention agents causes breast cells to stop growing, the number of cells obtained by rFNA is greatly reduced. This can sometimes make it difficult to determine whether the prevention agent 1) merely reduced the total number of cells or 2) whether it truly eliminated the pre-cancerous cells. In this grant we propose to improve the sensitivity and specificity of breast rFNA by coupling it with a test for a vitamin A-based marker that can be detected in as few as 5 cells.
Preliminary Data: Vitamin A signaling is important for keeping breast cells normal. We and others have shown that that vitamin A signaling is lost very early in the development of breast cancer due to a loss of expression of the retinoic acid receptor-beta2 (RARbeta2). Promoters function as “on/off” switches for genes. Methylation of a promoter prevents the gene from being turned on. Methylation of the RARbeta2 P2 promoter is an important mechanism for shutting off RARbeta2 in early breast cancer. In Preliminary Data, we observe that RARbeta2 P2 methylation can 1) be detected in as few as 5 cells and 2) is present in 40% of atypical hyperplasia and in 80% of node-negative, primary breast cancers.
In the laboratory, we have also shown that one way the RARbeta2 keeps cells normal is by maintaining the level of a protein called CBP. CBP is important regulator of cell growth and death and is also important for the action of a broad range of prevention agents including those that control estrogen-, vitamin A-, prostaglandins- (e.g. fish oil and flax seed), and cox-2-signaling (Celebrextm and Vioxxtm). We predict that RARbeta2 P2 methylation will result in loss of CBP expression in breast tissue from high-risk women and can be used in the future as a biologically relevant marker of response to prevention agents.
Hypothesis: In this grant, we propose to investigate whether the presence of RARbeta2 P2 promoter methylation predicts breast cancer risk in high-risk women. We will specifically test the hypothsis that the presence of RARbeta2 P2 methylation in rFNA predicts 1) atypical hyperplasia (a surrogate marker of breast cancer risk) and 2) loss of CBP expression. Through this study, we will have access to over 1,300 women at risk for breast cancer in three prevention clinics.
Relevance: Since RARbeta2 P2 methylation can be detected in as few as 5 cells, we predict that the presence or absence of RARbeta2 P2 methylation in breast rFNA can be used as a highly sensitive way to test for both 1) breast cancer risk and 2) response to chemoprevention agents. We are part of a 6 institution group that has been trained to perform breast rFNA and has longstanding expertise in developing and testing new prevention agents. If RARbeta2 P2 methylation proves to be a sensitive marker for the presence of precancerous cells, we can then add this test to our on-going prevention efforts as a predictor of response to prevention drugs (e.g. cox-2 inhibitors), dietary changes (e.g. low-fat, high-vegetable diet), and complementary agents (e.g. flax seed or fish oil).