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    Awarded Grants
    The Role of the Hypoxic Microenvironment in Tumor Formation in the Mammary Gland

    Scientific Abstract:
    SCIENTIFIC ABSTRACT TITLE: The role of the hypoxic microenvironment in tumor formation in the mammary gland. BACKGROUND: The role of hypoxia has been intensively studied in many types of cancer, however its role in breast tumorigenesis is still not well understood. The hypoxic response is regulated by the key hypoxia-inducible transcription factor, (HIF)-1alpha. Under normoxic conditions, HIF-1alpha is rapidly degraded by targeted ubiqutination mediated by its interaction with the von Hippel Lindau tumor suppressor protein (pVHL). In response to hypoxia, HIF-1alpha becomes stabilized, activating a myriad of genes that regulate angiogenesis, glycolysis, proliferation, and apoptosis. Clinical studies suggest that hypoxia may play a role in breast tumorigenesis since up-regulation of HIF-1alpha expression has been shown to correlate with areas of breast tumor growth. For example, up-regulation of HIF-1alpha protein has been demonstrated in high-grade, highly proliferative DCIS lesions. However, it is not clear whether HIF or VHL contribute to tumor progression in the breast, or whether dysregulation is a result of tumorigenesis. OBJECTIVE/HYPOTHESIS: The ultimate goal of these studies is to determine whether deletion of HIF-1alpha or VHL in the mammary epithelium accelerates or retards mammary tumorigenesis and/or impacts metastases. The well-characterized MMTV-polyoma middle T (PyMT) tumor model will be used to induce tumorigenesis in all studies. Based on previous studies from our laboratory, in which we showed that loss of HIF-1alpha retarded growth of fibrosarcomas, we hypothesize that loss of HIF-1alpha will delay PyMT-induced tumorigenesis and that loss of VHL will accelerate PyMT-induced tumorigenesis. The impact of loss of each gene in the context of metastases of mammary tumors will also be investigated. SPECIFIC AIMS: 1) To determine the effect of conditional deletion of HIF-1alpha or VHL in the mammary epithelium on tumor progression in the PyMT mammary tumor model. 2) To characterize and compare the PyMT tumors derived from HIF-1alpha or VHL wild type and null mammary epithelium by histopathology and analysis of HIF-1alpha target genes. 3) To establish mammary epithelial tumor cell lines from these tumors to further elucidate the contribution of HIF-1alpha and VHL to proliferation, migration, and intracellular signaling pathways. STUDY DESIGN: A Cre/lox conditional gene deletion strategy will be used to compare the effects of deletion of HIF-1alpha or VHL in the mammary epithelium on PyMT-induced tumorigenesis. Mice harboring two "floxed alleles" (double-floxed, DF) of either exon 2 of HIF-1alpha or exon 1 of VHL, have bred to MMTV-Cre transgenic mice and are currently maintained in the laboratory. These mice (VHL DF; MMTV-Cre+ and HIF DF; MMTV-Cre+) are currently being bred to the PyMT line to generate mice that harbor the "floxed" alleles and MMTV-PyMT and are either positive or negative for the Cre transgene. Tumor latency, burden, histopathology, microvessel density and regulation of hypoxia-inducible direct target genes such as glucose transporter-1 (Glut-1), vascular endothelial growth factor (VEGF), and phosphoglycerate kinase (PGK) will be evaluated in the following groups of mice: HIF null (HIF DF, Cre+) PyMT+ vs. HIF wild type (HIF DF, Cre negative) PyMT+ and VHL null (VHL DF, Cre+) PyMT+ vs. VHL wild type (VHL DF, Cre negative) PyMT+. Since PyMT mice develop lung metastases in at least 90% of cases, the contribution of HIF-1alpha or VHL to metastases will also be determined. POTENTIAL OUTCOMES AND BENEFITS OF RESEARCH: Since tumor cells are rapidly proliferating, they must overcome and adapt to cellular stresses, including hypoxia. Hypoxia is also relevant to the clinical setting since resistance to chemotherapy and radiation treatment has been associated with tumor hypoxia, especially in the necrotic center of large tumors. The results from our proposed studies will facilitate development of hypoxia-responsive specific therapies for treatment of patients with breast cancer.

    Lay Abstract:
    LAY ABSTRACT TITLE: The role of the hypoxic microenvironment in tumor formation in the mammary gland. The maintenance of normal oxygen levels is critical for homeostasis and for normal tissue function. In response to hypoxia, or low oxygen levels, tissues attempt to restore homeostasis by regulating cellular metabolism and by inducing angiogenesis (formation of new blood vessels). Under hypoxic conditions the transcription factor Hypoxia Inducible Factor-1alpha (HIF-1alpha) is rapidly stabilized, leading to dramatic induction of several genes implicated in angiogenesis, glycolytic metabolism, and apoptosis (programmed cell death). In contrast, under normal oxygen tensions, HIF-1alpha protein is degraded through its interaction with the tumor suppressor protein von Hippel Lindau (VHL). In patients with VHL syndrome, inactivation of the VHL gene results in constitutive expression of HIF-1alpha leading to the development of a variety of highly vascularized tumors. Additionally, up-regulation of HIF-1alpha protein has been demonstrated in a variety of human solid tumors, including high-grade ductal carcinoma in situ breast tumors, yet it is unclear whether this contributes to or results from tumorigenesis. Our lab is interested in elucidating the role of HIF-1alpha and VHL in breast tumorigenesis. Based on previous findings in our laboratory, we predict that loss of HIF-1alpha will delay tumorigenesis whereas loss of VHL will accelerate tumorigenesis. There is no established mammary tumor model in which HIF-1alpha or VHL has been specifically deleted in the mammary epithelial cells. Therefore in order to test our hypothesis, the first specific aim will be to produce a mouse breast cancer model in which either HIF-1alpha or VHL has been deleted. We will use genetically-modified mice lacking either the HIF-1alpha or VHL gene and breed them to the mouse mammary tumor virus (MMTV)-polyoma middle T (PyMT) breast cancer mouse model. Using these models, we will be able to examine the role of the hypoxic response in tumor formation and growth. The second aim will be to characterize and compare the PyMT-derived tumors generated from HIF-1alpha and VHL null and wild type epithelial cells, and to determine whether loss of either gene impairs or accelerates tumor formation. The time to palpable tumor onset, tumor size, and tumor histology will be compared. The third aim is to establish epithelial cell cultures from these tumors to examine how deletion of either gene influences the rate of cell growth and the ability of these cells to migrate, a key requirement for metastases. The overall goal of this project is to utilize these unique mouse models to determine how the regulation of the hypoxic response through HIF-1alpha and VHL impacts rate of tumor formation, tumor growth, and metastases. The results from these studies will have clinical relevance for developing new treatment methods to overcome hypoxia related resistance to chemotherapy and radiation treatment in breast cancer patients, and for therapy specifically aimed at stopping tumor growth and metastasis.