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    A Novel Strategy for the Identification of Tumor Suppressors in Human Breast Cancer

    Scientific Abstract:
    A Novel Strategy for the Identification of Tumor Suppressors in Human Breast Cancer Background: Tumorigenesis is a process requiring the cooperation of multiple genetic alterations. Although the dysregulation of several pathways has been demonstrated in human breast cancer, it is clear that many crucial tumor suppressors responsible for restraining malignant transformation of the breast epithelium remain unidentified. Indeed, the recessive nature of tumor suppressors has made these genes particularly elusive, thus emphasizing the need to develop new strategies for their identification. Recently, cell culture models of breast cell transformation have been created by introduction of defined genetic elements into explanted human mammary epithelial cells (HMEC). These models have been used to describe the signaling requirements for in vitro and in vivo transformation of human breast cells. Given their relative flexibility, such models also provide a potentially useful platform for the identification of novel pathways that contribute to the transformation process through the use of forward genetic screens. RNA interference is a powerful tool for manipulating gene expression. Recent advances in RNAi technology have made possible the stable suppression of specific genes in mammalian cells, allowing for loss-of-function studies in cell culture systems. Objective: The primary objectives of this proposal are to identify candidate tumor suppressors by applying a new RNAi technology to an in vitro model of human breast cell transformation and to subsequently characterize the role(s) of these candidates in mammary tumorigenesis. Specific Aims: To elucidate novel tumor suppressor pathways with significance to breast cancer, we aim to 1) identify negative regulators of transformation by applying an RNAi-based loss-of-function screen to a previously described model of HMEC transformation; 2) examine the effects of normal and aberrant candidate function in the transformation process; 3) determine the status of these candidates in clinical samples of human breast cancer; 4) establish mouse knock-out models to explore the physiologic consequences of a candidate’s loss-of-function in vivo. Study Design: In collaboration with Dr. Greg Hannon, our laboratory has constructed an amphotropic retroviral library expressing ~35,000 short-hairpin RNAs (shRNAs) corresponding to ~12,000 human genes, thus providing a unique opportunity to perform loss-of-function-based forward genetic screens in human cells. HMEC derivatives with previously characterized genetic alterations will be transduced with this library and subsequently selected for anchorage-independent growth, an in vitro indicator of cellular transformation. The contribution of isolated shRNAs to transformation will be measured in in vivo tumorigenicity assays, and selected candidates will be further evaluated for their effects on the individual phenotypic hallmarks of malignant transformation. We will examine the molecular relationship of candidate genes to pathways already implicated in mammary tumorigenesis and explore these genes for novel functions using biochemical and cell biological methods. Clinical samples of human breast cancer will be analyzed for the sequence integrity and expression levels of promising tumor suppressor candidates. To complement these studies, we will also generate mouse knock-out models and characterize these models with respect to breast carcinogenesis. Potential Outcomes and Benefits of the Research: Together, these studies will identify novel tumor suppressors and provide insight into the essential pathways whose dysregulation contributes to the genesis of breast cancer. Importantly, by altering the complement of alleles in this model, the strategy can be easily modified to identify alternative classes of tumor suppressors. Furthermore, these studies have the potential to provide new targets for therapeutic intervention and increasingly relevant models for drug discovery.

    Lay Abstract:
    A Novel Strategy for the Identification of Tumor Suppressors in Human Breast Cancer The conversion of normal breast cells into cancer cells is restrained by a class of genes appropriately named tumor suppressors. When these genes become dysfunctional, cells acquire many of the changes necessary to form a cancer including the ability to proliferate under suboptimal conditions, a reduced sensitivity to signals that initiate cell death, an increased life span, and the ability to inappropriately migrate to other tissues. Cancers often acquire extensive genetic changes, with alterations occurring in genes governing cancer progression as well as genes that are unrelated to this process. As such, the number of tumor suppressors that exist in the human genome is difficult to predict. However, it is clear that many tumor suppressors and their functions remain undefined. Importantly, the specific tumor suppressors (or combinations of tumor suppressors) that become dysfunctional may vary significantly among cancers from different tissues. For this reason, it is important to identify and examine tumor suppressors that regulate breast cancer development in models that reflect this tissue specificity. Recently, a cell culture based model has been developed to examine the minimal genetic requirements for transformation of a healthy breast cell into a breast cancer. The primary objective of this proposal is to identify novel genes that regulate breast cancer formation in this in vitro model and to test these genes as candidate tumor suppressors. In collaboration with Dr. Greg Hannon, we have developed a new technology that reduces the expression of individual genes on a large-scale (thousands of genes) basis. We will apply this technology to the in vitro breast cancer model in a high throughput manner to identify genes whose loss of function promotes the transformation of breast cells into cancer derivatives. The contribution of these genes to the individual hallmarks of cancer (listed above) will be determined. Importantly, the functional status of these candidates will be examined in human breast cancer samples. Furthermore, mouse models will be used to examine the physiologic role of selected candidates in breast cancer pathogenesis. Collectively, these studies will define new factors that regulate the progression of breast cancer. In addition, this work has the potential to uncover new pathways that can be targeted in breast cancer therapies as well as provide more relevant models for drug discovery.