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    Role of Gab2 in Breast Cancer

    Scientific Abstract:
    Tracking ID Number: PDF 0402217. Mohamed Bentires-Alj, Ph.D., Role of Gab2 in Breast Cancer. Background: Gab2 is a scaffolding adapter related to Gab1 and Dos, and was initially identified by the Neel lab as a major binding protein for the tyrosine phosphatase Shp-2. Gab2 plays a role in several normal cytokine and receptor tyrosine kinase signaling pathways. Gab2 also is involved in some types of neoplasia. For example, the sponsor’s lab showed that Gab2 is tyrosyl phosphorylated in Bcr-Abl-transformed cells, and essential for Bcr-Abl-evoked transformation and leukemogenesis. Moreover, Gab2 is over-expressed in ~30% of human breast tumors and cell lines. The consequences of this over-expression had remained unclear. However, I have obtained substantial preliminary data implicating Gab2 over-expression in breast carcinogenesis and/or metastasis. Objective: The goal of my research is to determine the effects of Gab2 over-expression and deficiency on breast carcinogenesis, using an ex vivo three dimensional (3D) culture system and transgenic mouse breast cancer models. Specific Aims: Aim 1 tests the effect of wild type Gab2 over-expression on MCF10A “normal” breast epithelial cells. Aim 2 uses Gab2 transgenic and knockout mice to clarify the role of this scaffold in breast carcinogenesis and metastasis in vivo. Study design: In 3D cultures, MCF10A cells adopt several features of normal breast epithelium, including acinar development and lumen generation. Co-expression of proliferation-inducing and apoptosis-suppressing genes in this system leads to non-invasive lumen filling that resembles ductal carcinoma in situ (DCIS). My experiments show that Gab2 over-expression increases MCF10A cell proliferation, but this is balanced by enhanced lumenal apoptosis. These effects suggest that Gab2 over-expression contributes to, but is not sufficient for, breast cancer initiation. In Aim 1, the effects of Gab2 over-expression on proliferation and apoptosis will be quantified. The mechanism underlying these effects will be studied by expressing Gab2 mutants that do not bind SHP-2, p85 or both, and assessing proliferation, apoptosis and lumen formation at different time intervals. The balance of proliferation and apoptosis in Gab2 over-expressors suggests that Gab2 is not sufficient for carcinogenesis. Other oncogenes, e.g., those that prevent apoptosis, may collaborate with Gab2 in tumorigenesis; I will test this hypothesis by co-expressing Gab2 with Bcl2. Also, because my transgenic studies suggest a separate role for Gab2 in metastasis, I will ask whether Gab2 over-expression can collaborate with ErbB2 (which causes lumenal filling but not invasion) to cause the invasive phenotype in 3D cultures. Aim 2 explores the effect of Gab2 in mouse models. I have generated transgenic mice over-expressing Gab2 in the mammary gland. Virgin, pregnant, lactating and involuting mammary tissue from these mice will be assessed for histology, proliferation and apoptosis, and cohorts of Gab2 over-expressors will be followed to see if they develop mammary tumors. I also have found that when mice expressing polyoma virus middle T antigen in mammary gland (MMTV-PymT) are placed on a Gab2-knockout background, primary tumors develop as in WT mice, but pulmonary metastasis is markedly suppressed. I will determine whether this effect is due to epithelial cells or stromal actions of Gab2 by means of transplantation studies. If an epithelial action is defined, Gab2 mutants will be used to delineate the relevant signaling pathway(s). Finally, I have also found that MMTV-Neu-evoked primary tumor generation is dramatically impaired in Gab2-/- mice. As for the MMTV-PymT mice, I will use transplantation studies to determine whether these effects are epithelial or stromal-dependent and if the former pertains, use Gab2 mutants to assess the relevant pathways involved. Potential Outcomes and Benefits of the Research: The proposed experiments should help clarify how Gab2 over-expression contributes to human breast carcinogenesis, and may identify novel therapeutic targets that are components of Gab2-dependent pathways.

    Lay Abstract:
    Tracking ID Number: PDF 0402217. Mohamed Bentires-Alj, Ph.D., Role of Gab2 in Breast Cancer. Breast cancer ranks second among cancer deaths in women. In 2003 alone, an estimated 267,000 new cases of breast cancer are expected to occur among US women and 39,800 lives will be lost to this disease. Improved understanding of how breast cancer arises and the factors that determine breast tumor biology, and in particular, tumor metastasis, are urgently needed to improve treatment options. A hallmark of cancer is the loss of normal cell growth control. Cancer cells bypass normal proliferation control mechanisms and typically escape programmed cell death. Cancer cells are able to do this because they have numerous mutations that either activate proliferation-promoting genes or inactivate proliferation/cell death-inhibiting genes. Although defining the genetic abnormalities in breast cancers is certainly important, new, more specific therapeutic approaches are likely to result only from a more thorough understanding of how these genetic abnormalities cause differences in the signaling pathways of breast cancer cells. The transduction of many extracellular signals into a cellular response is mediated by a family of proteins termed “scaffolding adapters “(scaffolds), which assemble protein complexes by increasing the local concentration of cascade components. This proposal focuses on the role of a particular scaffolding protein, Gab2, in breast cancer. The sponsor’s laboratory has shown that Gab2 is essential for leukemia caused by Bcr-Abl, the mutant cellular protein responsible for chronic myeologenous leukemia (CML). His laboratory also found that Gab2 is over-expressed in ~30% of human breast cancers. The consequences of Gab2 over-expression in breast carcinogenesis had been unknown, but my preliminary data indicate that Gab2 has important cancer-promoting functions in both in breast cancer initiation and metastasis. Gab2 induces the proliferation of a normal breast cell line and is indispensable for ErbB2/Her2 tumorigenesis in a mouse model of breast cancer. This model is particularly relevant to human breast carcinogenesis, because ~25-30% of human breast tumors have ErbB2/Her2 amplification, and the product of this gene is the target of the novel anticancer agent, Herceptin (Trastuzumab). Gab2 also is required for lung metastasis in another mouse model of breast cancer, driven by the oncogene of the DNA tumor virus polyoma (middle T). Taken together, my data strongly suggest a causal link between Gab2 over-expression and breast cancer pathology, and argue for studies aimed at understanding the mechanism of Gab2 action in breast cancer. My project will use a combination of cell biological, biochemical and mouse genetic approaches to study how Gab2 contributes to breast cancer initiation and metastasis. In Aim1, I will further define the effects of Gab2 over-expression on an immortal non-cancerous human breast cell line, MCF10A. In Aim2, I will analyze the effects of Gab2 over-expression in the mouse mammary gland. I will test whether, as in MCF10A cells, Gab2 can drive proliferation, but also promote apoptosis and/or whether over-expression leads to tumorigenesis. Second, I will further define how Gab2 deficiency prevents primary tumor generation in ErbB2/Her2 transgenic mice, and how Gab2 deficiency prevents lung metastasis in mice expressing the polyoma middle T oncogene. Finally, in studies in both Aims, I will use Gab2 mutants to delineate which Gab2-dependent pathways are required for its effects in MCF10A cells and in mice. Identifying these downstream pathways may suggest new therapeutic targets in breast cancer.