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    Research Grants Awarded

    Genetic Analysis Of Jagged1 & Notch1, Genes Implicated In Aggressive Breast Cancer

    Grant Mechanism:
    Investigator Initiated Research

    Scientific Abstract:
    Notch signaling is a potent regulator of normal stem and progenitor cell self-renewal and differentiation. In addition, altered Notch receptor signaling has been implicated in transformation in a number of tissues, including the mouse mammary gland. Recently, the Egan lab has analyzed expression of Notch ligand and receptor genes in human breast cancer. Patients with tumors expressing high-levels of JAG1 ligand and NOTCH1 receptor showed very poor overall survival. JAG1 mRNA expression was also associated with basal breast cancer and with poor outcome in a large cohort of patients with node-negative disease. This effect was subtype-specific. Whereas HER2- tumors, including basal tumors, that expressed high levels of JAG1 mRNA and protein showed reduced disease free survival, this association was not observed in HER2+ tumors. The association between Jagged1 and basal breast cancer was also observed in Brca1 mutant breast cancers, which are virtually all basal-subtype tumors. Interestingly, and in contrast, others have shown that Notch1 gene mutation can cooperate with HER2/Neu to induce mammary tumor formation in mice. Thus, Jagged1 and Notch1 may be important in breast cancer, perhaps in a subtype specific fashion. However, it is not yet clear whether JAG1 and NOTCH1 act as surrogates for poor prognosis in specific forms of breast cancer, or whether they play an active role in malignant transformation of mammary stem and/or progenitor cells as suggested from their ability to control stem cell biology in many tissues. To answer this question, the Egan and Zacksenhaus labs are collaborating. The Zacksenhaus lab has developed techniques and assays to identify and characterize the Cancer Stem Cell or Tumor-Initiating Cells (TIC) from mouse mammary tumors. TICs from mice that model HER2/Neu and basal breast cancer, the two most lethal forms, express different cell surface markers. We hypothesize that Jagged1 and Notch1 regulate mammary TIC survival, self-renewal and differentiation, in much the same way that these proteins regulate survival, self-renewal and differentiation of normal stem/progenitor cells. In addition, Jagged1 and Notch1 may well contribute to breast cancer angiogenesis. Using conditional mutant mice we will test this hypothesis directly, and determine whether Jagged1 and/or Notch1 are required for HER2/Neu or Brca1/p53-basal mammary tumor formation as suggested by our studies in humans. Finally, we will determine how these proteins regulate mammary TIC behavior and test whether they are required to maintain tumor growth and dissemination. We will address these issues through the following specific aims: 1) We will identify Jagged1 & Notch1 expressing and Notch signaling cells in HER2/Neu and Brca1/p53-basal models of breast cancer. We will use Flow cytometry analysis with antibodies against mammary stem/progenitor cell surface markers (CD24, CD49f and Sca1) to identify which tumor cells express Jagged1 and Notch1 in HER2/Neu and Brca1/p53-basal breast cancer models. In addition, we will use a recently developed Notch-signal reporter mouse to define which cells in both tumor models are receiving a Notch signal. 2) We will determine whether Jagged1 expressing, Notch1 expressing or Notch signal receiving cells in HER2/Neu and Brca1/p53-basal breast cancer models are TICs. Using HER2/Neu and Brca1/p53-basal mammary tumor cells sorted for expression of Jagged1, Notch1, or for activation of the recently described ?NAS? Notch-signal reporter, we will test whether any of these populations score for TIC activity. 3) We will define the function of Jagged1 and Notch1 in HER2/Neu and Brca1/p53-basal mammary tumor models. We will use Jagged1 and Notch1 conditional mutant mice to determine what role these genes play in HER2/Neu and Brca1/p53-basal mammary tumors. In each case, we will test for effects of Jagged1 or Notch1 gene mutation on tumor frequency, latency, subtype and angiogenesis, as well as TIC phenotype and alterations in gene expression. 4) We will determine the dependence of HER2/Neu and Brca1/p53-basal mammary tumors on Jagged1 & Notch1. We will use shRNA encoding lentiviral vectors to suppress expression of Jagged1 and Notch1 in TIC enriched cell populations from both mammary tumor models. We will then test for effects of gene knock-down on tumor formation in vivo. In this way we can directly test for the continued dependence of TICs from each model on Jagged1 and/or Notch1. Results from these experiments will reveal what role Jagged1 and Notch1 play in Her2/Neu and Brca1/p53-basal mouse mammary TICs, as well as what role these genes play in tumor induction vs. maintenance of the transformed phenotype. We hypothesize that these proteins play an active role in stimulating TIC self-renewal, in determining the phenotype of TICs and in stimulating tumor angiogenesis. If we are right, in either model, or with even one of the two genes to be functionally assessed, then our data will provide an incentive for the development of novel subtype-specific therapeutics to target one or both of the two most deadly forms of breast cancer.

    Lay Abstract:
    Recent studies have led to the identification of so called stem cells that are able to divide to make more stem cells and also to ?differentiate? into the many working cells of an organ. These stem cells are absolutely essential during development in order to create the various tissues in our bodies. In addition, they are required for the repair of tissues that must occur throughout life. Because stem cells have the potential to divide many times, they must be tightly controlled. It is now believed that cancers can result from mutations in growth control genes within a stem cell. Breast tumors contain a mixture of cells. Included in this mix is a small population of cells with the potential to expand and spread the tumor to distant organs, as well as the majority of ?differentiated? tumor cells that are essentially harmless. In the past, many cancer drugs and treatments have been assessed on the basis of whether they can shrink the tumor, which involves killing large numbers of tumor cells, often without ultimately curing the patient. It is this recognition that motivates studies to define the small population of Cancer Stem Cells or Tumor Initiating Cells (TICs), which can sustain a primary breast tumor and seed secondary tumors in lymph nodes, lung, brain and bone. If a therapy could be devised to kill or to differentiate breast TICs, it should be curative. The Zacksenhaus lab has recently found that TICs from different forms of mouse breast cancer have unique and characteristic properties. In other words, the TIC from a mouse HER2/Neu type breast cancer is different than a TIC from a mouse model of basal breast cancer. The Egan lab has been studying the Notch receptor and its activators (Jagged proteins) that control normal breast stem cell growth and behavior. The Notch receptors, when inappropriately activated can cause breast cancer in mice, presumably by changing normal breast cells into breast cancer stem cells/TICs. The Egan lab therefore tested for the involvement of Notch and Jagged genes in human breast cancer and found that the genes for Notch1 and Jagged1 are co-expressed in aggressive human breast tumors. Indeed, patients with tumors expressing high levels of Jagged1 and Notch1 showed very poor overall survival. Both labs will now team up to directly test whether Notch1 and Jagged1 regulate Tumor Initiating Cells in mouse models of HER2/Neu and basal breast cancer (the two most malignant breast cancer subtypes in humans). We will mate mice that were designed to model HER2 and basal breast cancers with mice that have lost the Jagged1 or Notch1 genes specifically in the breast. We will then compare breast cancer formation and TIC properties in mice with, and without, Jagged1/Notch1 genes. Based on our previous studies and the known role of Jagged and Notch proteins in regulating stem cells, we expect that removing Jagged1 or Notch1 in one or other of these mouse models will dramatically decrease tumor formation and malignant spread of tumors that do form. Furthermore, we expect that such a result will be associated with suppression of TIC cell division and survival or with increased TIC differentiation. Large biotechnology and pharmaceutical companies are beginning to develop drugs and approaches to stop specific Notch receptors, including Notch1, from working. Results from our study may well provide pre-clinical data to justify targeting Notch1 and/or Jagged1 in patients with very aggressive breast disease, and should help define which patients will benefit from such therapy.