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    Research Grants Awarded

    Role Of Estrogen In Breast Cancer Gene Amplification

    Grant Mechanism:
    Investigator Initiated Research

    Scientific Abstract:
    Komen Grant 2007 Scientific Abstract (3911/5700 characters) Genetic instability and rearrangements, including gene amplification, is a hallmark of many cancers. Genes that amplify in breast cancer include the estrogen receptor and its co-receptor AIB1, as well as various oncogenes such as HER2 (Neu/ErbB2), Myc, EBAG9, EGFR, Int-2 and Myb. The most famous of these is HER2 gene amplification which occurs in ~25% of invasive breast cancers and in 50-60% of ductal carcinoma in situ. HER2 amplification and concomitant over-expression of this growth factor promotes cancer cell growth in a variety of tissue environments, acting as a metastasis-promoting factor. The extent of gene amplification and overexpression is correlated with the stage of disease. It would be desirable to prevent gene amplification, thereby moderating the aggressive growth of breast cancer cells. Our recent studies in a model system (Sciara DNA puffs) showed that a steroid hormone (ecdysone) triggers re-replication, consistent with an onionskin mechanism of nested replication forks. DNA breaks may occur at the nested replication forks to generate Double Minutes (DMs) and Homogeneously Staining Regions (HSRs) found in cancer cells with DNA amplification. Unlike the breakage-fusion-bridge model, the onionskin model is consistent with the head to tail tandem repeats found for the amplified HER2 gene in breast cancer. In our recent results, we found that a binding site for a steroid hormone (ecdysone) receptor occurs adjacent to the binding site for the origin recognition complex (ORC). We now wish to examine if this paradigm also applies to breast cancer. The research proposed here will test whether estrogen induces DNA amplification in breast cancer. The novel hypothesis that will be addressed is whether the estrogen receptor binds adjacent to replication origins and interacts with the replication machinery to induce re-replication leading to gene amplification. First, we will use the widely studied MCF-7 human breast cancer cells which are estrogen receptor positive. These cells will be grown in 2D cultures for short term or long term in the presence of estrogen. Another set of experiments will be performed with cells from breast cancer surgery (with the collaborative help of Drs. Theresa Graves and Shamlal Mangray) grown in 3D culture on matrigel, collagen or agarose. Our colleagues, Drs. Diane Hoffman-Kim and Jeffrey Morgan, are experienced with 3D cell culture and will serve as consultants for these experiments. Samples from the 2D and 3D culture experiments will be analyzed by comparative genomic hybridization (CGH) to identify loci with copy number increases at the genome-wide level, with collaborative help from Drs. Alexander Brodsky and Ben Raphael. The sites of DNA amplification will be confirmed by quantitative real time PCR and visualized by fluorescence in situ hybridization (FISH). Next, using quantitative real time PCR, we will see if estrogen will induce DNA amplification of the human c-Myc origin in which we will engineer an estrogen receptor binding site next to the c-Myc replication origin. Dr. Michael Leffak will collaborate in this experiment, and introduce the experimental construct into the genome of cultured human cells with the FRT/FLP integration system. Chromatin immunoprecipitation (ChIP) will analyze the occupancy of estrogen receptor (ER? isoform) at the c-Myc amplified origin. Also, we will use antibody against ER? to analyze if components of the replication machinery such as ORC co-immunoprecipitate with it, suggesting that ER? may act through this complex to induce re-replication. The results will provide a new paradigm for the roles of steroid hormones (such as estrogen) in cancer, indicating a direct mechanism for the steroid hormone receptor to induce DNA amplification at target genes. This will provide new avenues for diagnosis and therapy.

    Lay Abstract:
    Komen Grant 2007 Public Abstract (Clinical Impact)(5219/5700 characters) Our nation is facing an epidemic of breast cancer. Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer death among women in the United States. The already alarming numbers that one in nine women will have breast cancer has now sky-rocketed to one in six. Further improvements in diagnosis and treatment are needed. Research is the foundation upon which such improvements are built. A link between basic research and translational research is the focus of this grant application. Certain genes (oncogenes) encode proteins that push the cell into division which runs out of control in cancer. When extra copies are made of these genes (gene amplification), then they can code for even more protein, thus enhancing further the bad effects of these genes. Several genes are known to amplify in breast cancer. One of these is the HER2 gene, which encodes a growth factor receptor that promotes growth of cancer cells in a variety of tissue environments, acting as a metastasis-promoting factor. Moreover, cancer cells with HER2 amplification commonly show resistance to the anti-estrogen reagent tamoxifen, resulting in endocrine therapy unresponsiveness. Recent spectacular advances, however, by the biotechnology company Genentech have been able to target HER2 positive cells for destruction. Specifically, the monoclonal antibody Herceptin recognizes the extracellular domain of HER2 where it binds and leads to cell death. There can, however, be cardiac side effects of Herceptin. Another negative feature is that Herceptin cannot pass the blood/brain barrier, and 25-45% patients treated with this antibody have secondary metastasis to the brain. In addition, Herceptin resistance can develop, leading to the spread of disease. Other oncogenes (c-myc, etc.) can also undergo DNA amplification and thus promote cancer, but they have been less studied than HER2 gene amplification. The focus of this grant application is to understand the mechanism leading to gene amplification in breast cancer cells. Elucidation of this mechanism will allow the development of better means of early diagnosis before DNA re-replication has led to high levels of DNA amplification. Moreover, knowledge of the mechanism of DNA amplification can lead to better and more refined therapies. The steroid hormone estrogen plays an important role in the growth and differentiation of the normal mammary gland. Estrogen is also involved in the development and progression of breast cancer, though the detailed mechanism is unknown. Estrogen can turn on gene expression and thus production of the proteins encoded by these genes. The current thinking in the field is that estrogen stimulates the expression of genes that govern the cell cycle, thereby pushing the cancer cell into cell division. However, our recent research from a model system suggests that a steroid hormone receptor can bind adjacent to a replication origin, and therefore may interact directly with the replication machinery. Our data indicated that a steroid hormone can induce gene amplification in which re-replication creates extra copies of the gene. This in turn will also increase production of the protein encoded by the amplified gene. Hormonal induction of gene amplification is a new paradigm for how hormones work, and we wish to see if it applies to human breast cancer. We propose to test if estrogen induces DNA amplification in MCF-7 cells grown in 2D cultures and in primary breast cancer cells from surgical specimens grown in 3D cultures. The results will identify sites of in the genome where DNA amplification is induced by estrogen. These sites may provide new targets for diagnosis and therapy. Also, we will make a genetic construct where the estrogen receptor will bind directly adjacent to the c-Myc replication origin to ask if estrogen will induce amplification of this engineered DNA in cultured cells. We will analyze if the estrogen receptor associates in a complex with the pre-replication machinery, suggesting its mechanism of action for DNA amplification. Our study will provide a whole new way of thinking about how steroid hormones may cause cancer, not only for breast cancer, but also for other hormonally sensitive cancers (e.g., ovarian cancer and prostate cancer). Moreover, this study will serve as a foundation for new methods of prevention, diagnosis and treatment of breast cancer. Our results may serve as a cautionary note for the over-exposure of women to hormones such as in birth control pills, hormone replacement therapy, through food (e.g., hormone fed dairy and beef cattle) and hormone mimics (e.g., the breakdown of plastics in microwave ovens). New methods of diagnosis could develop to measure estrogen receptor binding at the HER2 locus before gene amplification is measurable or cancer is detectable. Our results could lead to new therapies where designer drugs block the interaction of the estrogen receptor with the replication machinery. Identification of other co-amplified genes besides HER2 will serve as new targets for breast cancer cell drugs to block the action of their encoded proteins.