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Roles of BIK Apoptosis-inducing Protein in Breast Cancer
Background: The mRNA transcripts for BIK pro-apoptotic protein are occasionally detected in breast cancer tissues. In MCF-7 cells, antiestrogens induce both BIK mRNA and protein and kill cells by apoptosis in a BIK-dependent manner. In other commonly used cell lines (e.g., ZR75-1), BIK mRNA is expressed constitutively, but its protein is rapidly degraded by the proteasome. In these cells, MG-132 proteasome inhibitor induces BIK protein and kills cells by apoptosis in a manner partly dependent on BIK, suggesting critical roles of BIK in determination of survival of breast cancer cells. Hypothesis: Estrogen suppression of BIK mRNA expression or rapid proteasomal degradation of BIK protein is necessary for survival of breast cancer cells, suggesting that BIK functions as a tumor suppressor of breast cancer. Specific Aims: (1) To characterize molecular mechanisms of the antiestrogen induction of BIK mRNA in MCF-7 cells; (2) To determine mechanisms of regulation of the proteasomal degradation of BIK protein in ZR75-1 cells; and (3) To evaluate effects of BIK gene knockout on mammary carcinogenesis of MMTV-neu transgenic mice. Study Design: (1) Functions of human BIK gene promoter in MCF-7 cells will be characterized by (i) the transient transfection-based luciferase reporter assay to identify cis-elements involved in the antiestrogen-induced BIK mRNA expresssion and (ii) the DNA microarray-based parallel analysis of chromatin immunoprecipitation to efficiently determine antiestrogen-dependent protein recruitment to the promoter and chromatin modifications. (2) BIK amino acid residues involved in regulation of the proteasomal degradation of BIK protein will be determined by transiently transfecting synthetic BIK mutants into ZR75-1 cells and measuring their intracellular half-lives by S35 methionine pulse-chase method. (3) The MMTV-neu mice, which raise breast cancer at 23-28 weeks old, will be crossed with BIK knockout mice, which show no significant phenotype. The tumor latency, growth, and malignant features will be compared between the neu/BIK(+/+) and neu/BIK(-/-) animals by Kaplan-Meier analysis and histopathological approaches. Potential Outcome and Benefits of the Research: Information on the molecular mechanisms of regulation of BIK expression and its importance in mammary carcinogenesis will be obtained. It may provide important insights into therapeutic approaches to breast cancers such as novel drug targets.
Background: Apoptosis is a phenomenon that cells commit suicidal death under certain conditions. The majority of breast cancer cells are killed by apoptosis before they form a discernible mass of tumor. Most chemotherapeutic drugs kill cancer cells by inducing apoptosis, and the antiestrogens such as Tamoxifen and Faslodex also induce apoptosis in some breast cancer cells. BIK is an apoptosis-inducing protein that kills cells when expressed. Breast cancers occasionally express BIK, and the antiestrogens induce BIK expression to kill some breast cancer cells. However, many breast cancer cells seem to be able to escape from the BIK-induced apoptosis by rapidly degrading BIK protein. Hypothesis: BIK may suppress breast carcinogenesis. Estrogen reduces BIK expression in breast cancer cells by suppressing activation of BIK gene. Rapid degradation of BIK protein also contributes to the reduced expression of BIK in this malignancy. Specific Aims: (1) To characterize molecular mechanisms of activation of BIK gene by the antiestrogens in breast cancer cells. (2) To determine mechanisms of the rapid degradation of BIK protein in breast cancer cells. (3) To evaluate the importance of BIK gene in preventing breast cancer using laboratory mouse models. Study Design: (1) The gene function of BIK will be evaluated using breast cancer cell culture and techniques of molecular biology such as the reporter assay of gene activation. DNA microarray will also be used to evaluate recruitment of proteins to BIK gene and chemical modifications of the DNA-protein complex of BIK gene. (2) Mutations will be systematically introduced into BIK protein, and their rate of degradation in breast cancer cell culture will be evaluated. This analysis will determine which portions of BIK protein are important for regulation of BIK degradation. (3) A transgenic mouse strain that spontaneously raise breast cancers will be crossed with a BIK-deficient knockout strain, and the effects of lack of BIK on the latency, growth, and aggressiveness of breast cancers will be evaluated statistically and pathologically. Potential Outcome and Benefits of the Research: This study will provide information on the mechanisms of regulation of BIK expression in breast cancer cells and insights into the importance of BIK in prevention of breast cancer. Drugs designed based on the outcome of this study to specifically suppress degradation of BIK in breast cancers, for example, may lead to a novel therapeutic strategy to this malignancy.