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Micro-RNA Regulation Of Oncogenes And Tumor Suppressors In Breast Cancer
Investigator Initiated Research
Background/Rationale: Micro-RNAs (miRNAs) are small non-coding RNAs that can have diverse effects on cell proliferation, differentiation, and apoptosis. They are generally believed to function by binding to imperfectly complimentary sequences in the 3?UTR of target genes and cause suppression of translation. While some prior studies have identified a number of miRNAs that are either up or down-regulated in breast cancer, these prior studies are neither comprehensive nor do they provide direct functional evidence for the role of specific miRNAs in breast cancer.
Objective/Hypothesis: Targets of miRNAs in breast cancer cells may include oncogenes and tumor suppressor and thus miRNAs may directly alter tumor phenotypes. In this proposal we take three complimentary approaches to identify miRNA:oncogene or miRNA:tumor suppressor pairs that play a direct role in the initiation or maintenance of breast cancer. We seek to both identify miRNAs that can alter breast cancer phenotypes, and secondly determine whether inhibition of particular miRNAs can affect a reversion of the malignant phenotype.
Specific Aims and Study Design: 1) Understand how miRNAs regulate the oncogene ERBB2 and tumor suppressor TP53 whose amplification and inactivation, respectively, account for alterations in the majority of human breast cancers. We have recently identified several miRNAs that can regulate ERBB2 and other miRNAs that can regulate TP53 expression. Using conditional expression of these miRNAs in human breast epithelial cells or transgenic mouse model systems we propose to study the direct effects of these miRNAs on breast tumor formation and regression.
2) Identify novel miRNAs that can play a direct role in breast cancer formation or resistance to chemotherapeutics. We have generated a human miRNA expression library that is comprised of approximately 375 unique miRNAs in retroviral vectors. Individual miRNAs can be expressed in human breast epithelial cells using this approach and are appropriately processed and functional. We will perform high-throughput screens in human breast epithelial cells to identify miRNAs that can cooperate with oncogenes for full malignant transformation or identify miRNAs that can render human epithelial cells resistant to common chemotherapeutics used in breast cancer treatment such as taxanes or anthracyclines.
3) Determine whether mutations in the 3?UTR of TP53 in primary human breast cancers are of functional significance. Deregulated expression of oncogenes and tumor suppressors may occur through either altered expression of miRNAs that target a particular gene or through the mutation of miRNA binding-sites in the 3?UTR of target genes. We have identified a number of mutations that occur in primary human breast cancer in the TP53 3?UTR. Using luciferase reporter constructs in human breast epithelial cell lines, will determine if the TP53 3?UTR mutations identified in primary cancers can alter translation regulation.
Proposal Impact on Human Breast Cancer: The importance of miRNAs in regulating cancer phenotypes has only recently been appreciated and a comprehensive functional approach to identify miRNAs relevant to human breast cancer biology has thus far not been reported. We have generate a human miRNA expression library that we will use to test the functional importance of specific miRNAs. Our library is more comprehensive than any currently reported in the literature and should allow for the identification of novel miRNAs that can play a direct role in breast tumor biology. We anticipate that miRNAs identified in our functional screens will be 1) important in breast tumor formation, 2) may serve as novel diagnostic or prognostic markers for human breast cancer and 3) may be targets for novel therapeutics. A more complete understanding of miRNA function in breast cancer, as described in this project, should lead to immediate translation of these findings to the reduction of the incidence and mortality associated with human breast cancer.
Background and Hypothesis: Micro-RNAs (miRNAs) are small genes that do not code for proteins yet can have a big impact on how other genes are regulated. They are generally believed to function by binding to the ends of other gene transcripts and altering the amount of protein that is produced. Only recently has it been realized that miRNAs can affect various aspects of cancer biology including the growth rate and aggressiveness of tumor cells. Approximately 500 different human miRNAs have been identified, yet the function of all but a few of these remains entirely unknown. Since miRNAs expressed in human breast cancers may regulate oncogene and tumor suppressor genes, they may directly regulate breast cancer initiation, aggressiveness and resistance to chemotherapy. While some prior studies have identified a number of miRNAs that are either up or down-regulated in breast cancer, these prior studies are neither comprehensive nor do they provide direct functional evidence for the role of specific miRNAs in breast cancer. In this proposal we take three complimentary approaches to identify miRNA:oncogene or miRNA:tumor suppressor pairs that play a direct role in the initiation or maintenance of breast cancer. Unlike prior studies that examined the relative abundance of a subset of miRNAs in breast cancers, we propose to systematically test nearly all human miRNAs for their ability to participate in breast tumor formation or resistance to chemotherapy using experimental model systems. To carry out these experiments we have cloned ~375 human miRNAs such they can be introduced systematically into human or mouse breast cells and observe their effects on a) cell proliferation b) ability to cause tumors in mice and c) the ability to cause resistance to the commonly used chemotherapeutic drugs taxol and adriamycin. In addition to our ability to express these miRNAs and look for tumor formation, our experimental systems also allow us to turn off their expression in animal models and see if they are required for tumor maintenance. To date, no systematic functional analysis of miRNAs in breast cancer has been reported. We feel we are uniquely suited to carry out these experiments since we have cloned a large fraction of human miRNAs and have access to appropriate experimental model systems to test their function. We have preliminary data demonstrating that some of the most frequently altered genes in breast cancer (such as ERBB2 and p53) can be the targets of regulation by miRNAs. We seek to further characterize how miRNAs may regulate these critical breast cancer genes. Additionally we seek to identify new miRNAs that may play a direct role in causing breast cancer. The miRNAs identified as part of this project can be rapidly translated into new breast cancer biomarkers that can be examined in the context of new clinical trials. Specific miRNAs identified from our functional screens may also serve as novel therapeutic targets for the development of small molecule inhibitors. Based on the findings in the proposed work it is anticipated that novel therapeutics may be developed to target aggressive forms of human breast cancer.