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Identification of novel regulators of tamoxifen resistance in breast cancer cells
Background: Targeting the estrogen signaling pathway dramatically improves long-term disease-free and overall survival for women with estrogen receptor-positive breast cancer. Tamoxifen, a selective estrogen receptor modulator (SERM), is the most widely used anti-estrogen. However, only 40-50% of patients with estrogen receptor (ER) positive breast cancer benefit from tamoxifen treatment and 30-50% acquire resistance and the disease progresses. Objective: The objective of this proposal is identification and characterization of novel genes/pathways and small molecules that regulate resistance to anti-estrogen therapy in breast cancer cells. Specific Aims: To elucidate mechanisms underlying the SERM resistance of breast cancer cells we aim to 1) identify regulators of tamoxifen resistance by applying a genome-wide siRNA library and libraries of heterocyclic small molecules to a cellular model of development of breast cancer cell tamoxifen tolerance; 2) characterize the pathways that the identified genes and small molecules act upon; 3) determine the status of these candidate genes in clinical samples of human breast cancer; and 4) determine the effectiveness of the identified small molecules in a tamoxifen resistant xenograft breast cancer model. Study Design: An established endocrine-sensitive and tamoxifen-resistant MCF-7(TAM-R) cell line will be transfected with the siRNA library targeting 24,373 genes, in the presence of tamoxifen. High content imaging will be used to determine cell proliferation and cell cycle phenotypes for each gene, and results will be verified for both tamoxifen-dependence and multiple siRNA sequences for the gene of interest. Genes whose downregulation selectively reduces cell proliferation in combination with tamoxifen will be selected for further study using a variety of cell biological methods. Similarly, this assay system will also be used to screen large compound libraries for synergy with tamoxifen. We will characterize the signaling pathways that the identified genes and small molecules act upon using gene expression array and phosphoproteomics approaches. The importance of the selected modulators (RNAi constructs and small molecules) for alleviation of tamoxifen resistance will be confirmed in vivo with tumorigenicity assays. To complement these studies, clinical samples of human breast cancer patients will be analyzed for the expression levels of promising regulators of tamoxifen responsiveness. Potential Outcomes and Benefits of the Research: These studies will provide insight into new signaling pathways which are involved in breast cancer progression. Importantly, these studies can provide identification of molecules critical for several aspects of breast cancer treatment: new protein drug targets for in vitro screening, biomarkers for characterization of tumors, and small molecules that can be used as tool compounds to study pathways or as potential therapeutic agents.
The first evidence that hormones play a critical role in the progression of breast cancers was discovered more than 100 years ago by the British surgeon George Beatson, who found that removal of a woman?s ovaries (which synthesize hormones) led to a dramatic response in her metastatic breast cancer. Further studies found that only 1 in 3 women with advanced breast cancers responded to this type of surgery, but this procedure nevertheless remained an important option for breast cancer treatment for the next six decades. Research by Elwood Jensen in the 1960s helped to clarify which patients would benefit from hormone-based treatment strategies: breast cancers that expressed the protein receptor for the hormone estrogen (ER) were more likely to be responsive. The drug most commonly used for treatment of these ER-positive breast cancers is tamoxifen, which has been widely used for the last 20 years. Studies and clinical trials suggest that hundreds of thousands of women are alive today because of taxmoxifen treatment. Despite the success of this drug, many hurdles remain to improved treatments for breast cancer. An estimated 40% of ER-positive breast cancers are unresponsive to tamoxifen treatment, and some breast cancers develop resistance to tamoxifen. The genes and pathways responsible for tamoxifen resistance are coming into focus, and recently cell culture models using breast cancer cell lines with increased amounts of growth factor receptors have been reported. Our proposal seeks to address tamoxifen resistance using two strategies. First, we propose to test nearly all of the genes in the human genome (approximately 25,000) to see which ones can be targeted to restore tamoxifen sensitivity in a cellular model of tamoxifen resistance. Finding the right drug often means finding the right target, and this research seeks to discover genes that can be used in rational drug design programs. Second, we propose to screen large collections of drug-like compounds in the presence of tamoxifen to find leads for potential combination therapies. The genes found in these studies will be correlated with gene abundance in tissue samples from human breast cancers to verify their role. In addition, compounds found to synergize with tamoxifen will be tested in breast cancer mouse models for efficacy. These two approaches ? functional genomics and chemical biology ? could lead to a greater molecular understanding of why some tumours are treatable compared to others, and form the basis for the development of next generation breast cancer therapeutics.