The role of alternative pre-mRNA splicing in breast cancer progression

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DESCRIPTION (provided by applicant): Recent work has documented that many changes in alternative pre-mRNA splicing associate with breast cancer. These observations raise the question whether changes in pre-mRNA splicing contribute to breast cancer? Another unanswered question is whether breast cancer-specific splicing events are the result of mis-regulations or whether they are the consequence of a general splicing defect in tumors? We have demonstrated that alternative splicing events associated with breast cancer are not caused by changes in the intrinsic ability of the spliceosome to remove introns. Here, we will carry out experiments to gain insights into the question whether alternative splicing contributes to breast cancer progression. In this application, we will characterize pre-mRNA splicing networks to test the hypothesis that aberrant expression of splicing regulators trigger breast cancer-specific alternative splicing. We will combine experimental analyses with bioinformatics to characterize breast cancer-specific alternative splicing. (1) Using high throughput sequencing approaches and computational analyses of EST databases we will generate and validate a comprehensive list of breast cancer-specific alternative splicing events. (2) Using real-time PCR and quantitative western blot analysis, we will test the hypothesis that splicing regulators are differentially expressed in breast cancer. We will combine these expression profiles with computational analyses of breast cancer-specific alternative splicing events and pre-mRNA splicing predictions to determine likely targets of splicing regulatory networks. The approaches taken integrate high-throughput data generation with bioinformatics and classical molecular biology methods to gain important new insights into splicing regulatory networks. The proposed experiments take advantage of the most up-to-date experimental and analytical approaches to generate a snapshot of gene expression unique to breast cancer, thus producing a wealth of data useful to the breast cancer research community. The described experiments will demonstrate whether alternative pre-mRNA splicing is a significant contributor to breast cancer biology. Introducing these molecular insights to analyses of tissues obtained from breast cancer patients of various stage groupings will indicate to what degree changes in alternative splicing contribute to breast cancer progression, thus, providing additional screening and outcome prediction possibilities. PUBLIC HEALTH RELEVANCE: Pre-mRNA splice variants have been identified for a large variety of breast cancer genes, suggesting that widespread aberrant and alternative splicing may be a consequence or even a cause of breast cancer. Here, we propose to evaluate to what degree alternative pre-mRNA splicing contributes to breast cancer progression. The value of the molecular insights gained is realized when applying such analyses to breast cancer patients of various stage groupings, as they are likely to provide additional screening and outcome prediction possibilities.