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DNA Double-Strand Break (DSB) Repair In Breast Cancer Cells And shRNA Approach To Inhibition Of DSB Repair As A Way To Fight Therapy Resistance
Background: Resistance to radiation or chemotherapy is a major obstacle to successful treatment. The most critical lesion following exposure to radiation, and many chemotherapeutic drugs is the DNA double-strand break (DSB). DSBs are repaired by two complementary pathways: homologous recombination (HR) and nonhomologous end joining (NHEJ). Objective/Hypothesis: We hypothesize that induction of DSB repair contributes to development of therapy resistance in breast cancer, and inhibition of both HR and NHEJ may be required to efficiently sensitize the cells. Our objective is to examine the status of DSB repair in breast cancer cells and identify the best targets for sensitization of the cells to therapy. Specific Aims: (1) Examine the efficiency and accuracy of HR and NHEJ pathways in normal and breast cancer cells. (2) Identify the genes from the HR and NHEJ pathways whose inactivation by shRNA provides the strongest inhibition of DSB repair. (3) Test how inactivation of both HR and NHEJ pathways affects the survival of breast cancer cells and normal breast cells following radiation or treatment with chemotherapeutic drugs. Study Design: Our laboratory has developed a fast and sensitive assay to assess the status of DSB repair in human cells. The reporter cassette contains modified versions of the genes for green fluorescent protein (GFP) and red fluorescent protein (DsRed) arranged so that repair of a DSB by HR generates red cells, while repair by NHEJ gives green cells. This system allows rapid quantification of the relative efficiencies of HR and NHEJ in multiple cell lines, and permits analysis of individual events to determine their accuracy. Furthermore, our laboratory has a technology to transfect multiple cell lines with high efficiency. Using these techniques, we will examine NHEJ and HR in a panel of breast cancer cells. We will then inhibit several components of the DSB repair pathways by shRNA and test how the inhibition of various combinations of genes from HR and NHEJ pathways affects the efficiency of repair and the sensitivity of the breast cells to therapeutic agents. Potential Outcomes: The proposed research will provide important knowledge on how the normal and malignant breast cells respond to therapy, and on the mechanisms of therapy resistance. In the future, genes identified in the course of this project will be used as targets for development of specific inhibitors, or gene therapy vectors to be used concomitantly with radiation or chemotherapeutic treatment.
Background: Radiation therapy is a cornerstone of breast cancer treatment that is used either alone or in combination with chemotherapeutic drugs. Unfortunately, radioresistance continues to be a problem due to failure of local therapy and development of metastases. Resistance to therapy is also a primary reason for the failure to treat advanced and metastatic disease. Therefore, there is an urgent need to find effective ways to fight therapy resistance. Hypothesis: Ionizing radiation and certain types of chemotherapy kill cells by generating double-stranded breaks in the DNA. We hypothesize the cells may become resistant to therapy by inducing their DNA break repair. Since DNA breaks are repaired by two complementary pathways called homologous recombination and nonhomologous end joining, we propose that inactivation of both pathways may be required to efficiently sensitize cells to therapy. Specific Aims: We plan to examine the status of DNA double-strand break repair in a wide collection of breast cancer cell lines and normal breast epithelial cells. We will then inhibit the genes involved in the two pathways of DNA break repair using a novel technique for gene inactivation called small hairpin RNA. This will allow to identify the gene combinations that suppress DNA repair and sensitize cancer cells to radiation or chemotherapeutic drugs. Study design: Our laboratory has unique tools and expertise to carry out this project. We have developed high throughput assay for the analysis of DNA double-strand break repair, and versatile assay for the analysis of cell death. Furthermore, we have expertise to transfect multiple cell types with high efficiency. These tools will enable us to perform comprehensive analysis of DNA double-strand break repair in a panel of breast cancer cell lines, and systematically identify the best gene targets to make cancer cells susceptible to radiation and chemotherapy. Potential Outcomes: In the future the genes identified as a result of this work will be used as targets for development of specific inhibitors, or gene therapy vectors to be used concomitantly with radiation or chemotherapeutic treatment. In summary, the proposed study is likely to identify a novel approach to therapeutics of breast cancer treatment, namely radiotherapy plus small hairpin RNA.