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A Functional Genomic Approach to Develop More Rational Taxane Combination Therapies for Metastatic Breast Cancer
Background: About 40,000 American women with metastatic breast cancer (MBC) die each year because they fail to respond to current chemotherapeutic agents. Metastatic breast cancer is an incurable disease and novel therapeutic strategies are needed to combat this disease. The taxanes paclitaxel and docetaxel are among the most active chemotherapy agents used in the management of MBC. Although recent reports show that taxanes are active chemotherapeutic agents in MBC, the overall efficacy of these agents is far from ideal. Hence, this project is based on the development, design and application of RNA interference as a high throughput functional genomic method to identify and validate candidate genes that modulate the resistance of MBC cells to taxanes. Hypothesis: Increase in the sensitivity of invasive breast cancer cells to docetaxel and paclitaxel can be achieved through the targeted silencing of specific response modulator genes. Specific Aims: Specific Aim 1. Global RNAi Phenotype Profiling to Determine Genes that Modulate Breast Cancer Cell Sensitivity to Paclitaxel and Docetaxel. Specific Aim 2. In Vitro Confirmation and Functional Assessment of Paclitaxel and Docetaxel Modulating Genes in Metastatic Breast Cancer Cells. Specific Aim 3. In Vivo Confirmation and Functional Assessment of Paclitaxel and Docetaxel Modulating Genes in Metastatic Breast Cancer Cells Study Design: We have established a platform for identification of drug sensitizing genes using high throughput RNAi based silencing. HT-RNAi conditions will be established for two breast cancer cell lines and a HT-RNAi screen of breast cancer cells will be performed using a ?druggable genome? siRNA library that targets 6000 genes. Data generated from the screens will be analyzed to generate a ?hit list? of target genes involved in the sensitization to taxane treatment. After confirmation of screening results, validation of specific gene silencing by the siRNA will be conducted using either Western blot analysis or qRT-PCR. The in vitro results will be corroborated in vivo in a xenograft model to study the sensitivity of the tumors to taxanes using cell lines containing specific gene knockdown. Mice carrying xenografts will be treated with taxanes to determine if the specific gene knockdown has an effect on taxane sensitivity. Significance: With the completion of the proposed research, we expect to identify and validate the involvement of critical genes whose silencing can modulate the sensitivity of invasive breast cancer cells to the effects of paclitaxel and docetaxel. The functional data will contribute greatly to the understanding of the molecular mechanisms of taxane induced growth suppression. The identification of new molecular targets can lead to the discovery of innovative and rational molecular therapeutics, which when combined with a low dose chemotherapeutic can improve the quality of life for metastatic breast cancer patients.
Advanced or metastatic breast cancer (MBC) is currently the second leading cause of death in women after lung cancer in the US and it has been estimated that over 400,000 women worldwide die from the disease each year. In metastatic breast cancer the tumor is no longer confined within the breast and hence cannot be surgically removed. Unfortunately, metastatic breast cancer is an incurable disease and the therapeutic goals of chemotherapy are to minimize the symptoms and increase the quality and prolong the duration of the patient?s life. Over the past decade, the number of drugs that provide significant antitumor activity in breast cancer has increased with the development of new chemotherapeutic agents. Two chemotherapeutic agents that have shown much promise in treatment of metastatic breast cancer are paclitaxel (Taxol) and docetaxel (Taxotere), both members of a class of drug known as taxanes. Although many current regimens in the treatment of metastatic breast cancer include one of these taxanes, the overall response rate is between 30-60%, indicating that about half the patients that receive treatment for metastatic breast cancer do not respond. We hypothesis that there are genes in these breast cancer cells that modulate the response to taxanes and that targeting these genes pharmacologically could be the basis for development of new combination therapy strategies. Identification of these genes is important because they are potential targets and represent the ?Achilles heel? in metastatic breast cancer cells that don?t respond to paclitaxel or docetaxel. Thus, in order to improve the response to paclitaxel and docetaxel, we propose to delineate the molecular mechanisms of the action of the individual drugs and thereby identify potential novel drug sensitizing targets. To accomplish this task, we will incorporate an innovative functional genomics approach based on high-throughput RNA interference (HT-RNAi) analysis. RNA interference (RNAi) is a mechanism by which genes can be specifically silenced or ?switched off.? The use of global RNAi enables testing of the hypothesis that targeting specific genes can increase the responsiveness or sensitivity of breast cancer cells to taxanes. To test this hypothesis, we will use HT-RNAi to silence 6000 different genes in the human ?druggable genome? to determine if silencing these genes will synergize to increase taxane induced killing of breast cancer cells. The target genes will be validated and further characterized in breast cancer cell lines and in mouse models. The application of HT-RNAi represents an innovative functional genomic strategy to rapidly identify important genes which when silenced will increase the efficacy of taxanes in inhibiting proliferation and survival of breast cancer cells. The identification of new molecular targets can lead to the discovery of innovative and rational therapies that can improve the quality of life for metastatic breast cancer patients.