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    Awarded Grants
    Endocytosis and Signaling of the Epidermal Growth Factor: Role of Abl Tyrosine Kinases and Abi Adaptors

    Scientific Abstract:
    SCIENTIFIC ABSTRACT Title: Endocytosis and signaling of the Epidermal Growth Factor: Role of Abl tyrosine kinases and Abi adaptors. Background: Overexpression of HER2 (Human Epidermal Growth Factor Receptor 2) or the Epidermal Growth Factor Receptor (EGFR) is associated with enhanced proliferation and malignancy in breast cancer. EGFR is activated upon ligand-binding, ubiquitinated by the Cbl E3 ligase, internalized and targeted to lysosomes for degradation. Failure to remove activated EGFR from the surface and increased EGF responsiveness is associated with malignant breast cancers. The EGFR signal is inactivated mostly through endocytosis and degradation of the receptor-ligand complex. The Cbl adaptor protein is an important regulator of EGFR/HER2 downregulation after growth factor binding. We have recently shown that constitutively active forms of the Abl nonreceptor tyrosine kinase block internalization of the EGFR normally induced by binding to the EGF ligand. We previously showed that the normal c-Abl tyrosine kinase is activated downstream of the ligand-stimulated EGFR. Recently, we have also demonstrated that EGF stimulation results in the formation of complex between Cbl and the Abl-interactor 1 (Abi1) adaptor protein, and that this complex is disrupted by expression of the activated Abl tyrosine kinases. Both, Abi1 and Cbl undergo enhanced tyrosine phosphorylation following expression of the activated Abl kinase. In addition to Cbl and Abi1, the EGFR itself is also a target of Abl-induced tyrosine phosphorylation. Significantly, c-Abl, Abi and Cbl are mutated in human cancers, and deregulation of their activities leads to proliferation and metastasis. Hypothesis/Objectives: The goal of this proposal is to define the role of c-Abl and Abi proteins in endocytosis and signaling of the EGFR. We will analyze the role of the Cbl/Abi1 complex in EGFR endocytosis. We hypothesize that Abi1 may target Cbl to sites where Cbl can carry out its function in endocytosis, and that c-Abl mediated disruption of the Cbl/Abi1 complex may affect subsequent association of Cbl with the activated EGFR. We will examine whether c-Abl regulates EGFR function by affecting its phosphorylation and or ubiquitination levels. We hypothesize that inhibiting Abl kinase activity in breast cancer cells may affect their proliferation and invasiveness. Specific Aims: (1) To analyze whether Abl-mediated phosphorylation of Cbl and EGFR affects their association upon EGF stimulation and to analyze the effect of activated Abl on EGFR phosphorylation and ubiquitination upon EGF stimulation in breast cancer cells. (2)- To analyze whether the Cbl/Abi1 complex is present in breast cancer cells and to analyze the effect of complex disruption in proliferation and malignancy of breast cancer cells. (3) To analyze the mechanism by which kinase-active Abl disrupts the Cbl/Abi1 complex and whether this is dependent on the phosphorylation of Cbl and/or Abi1. (4) To analyze whether inhibiting Abl kinase activity in breast cancer cells could affect the proliferation and migration properties of these cells. Study design: We will use standard molecular biology techniques such as western blot and PCR to determine c-Abl and Abi1 levels in breast cancer cells with different degrees of malignancy and invasiveness. We will determine the levels of Abl activation, Cbl, Abi1 and EGFR phosphorylation in these cell lines. We will use different Abi1 and Cbl mutants to map the Cbl/Abi1 interaction. We will use mass spectrometry techniques in order to determine the specific Abl-induced phosphorylation sites on the EGFR, Abi1 and Cbl and site directed mutagenesis mutate these sites. Fluorescence and confocal microscopy will be utilized to examine complex formation in cells. Downregulation of protein expression will be achieved through use of RNA interference techniques. We will inhibit tyrosine phosphorylation through use of specific inhibitors of the Abl and the EGFR available commercially. Cell proliferation and cell migration assays will be performed as previously described. Relevance: EGF activates cell proliferation and migration of breast cancer cells, which is critical for invasion. The Abl kinase, Abi and Cbl are mutated in human cancers, and deregulation of their activities leads to proliferation and metastasis. The activated Abl kinase phosphorylates the EGFR, Cbl and Abi1, and blocks EGFR endocytosis, suggesting a role for Abl in EGFR signaling. Insights learned from this project, on the mechanisms of EGFR internalization, regulation of Cbl function and migration of breast cancer cells will enhance current knowledge of the biology of breast cancer and the progression towards its cure.

    Lay Abstract:
    LAY ABSTRACT Title: Endocytosis and signaling of the Epidermal Growth Factor: Role of Abl tyrosine kinases and Abi adaptors. Breast cancer is one of the most common malignancies in women. Overexpression of HER2 (Human Epidermal Growth Factor Receptor 2) is associated with enhanced proliferation, malignancy and poor prognosis in breast cancer. HER2 is activated upon ligand-binding. Upon activation, HER2 and other members of the Epidermal Growth Factor Receptor (EGFR) family auto-phosphorylate, and induce activation of several signal transduction cascades. The EGFR signal is inactivated, primarily through endocytosis or internalization of the receptor-ligand complex. The Cbl adaptor protein has emerged as an important regulator of EGFR/HER2 downregulation after growth factor binding, through the process of endocytosis. The antitumor efficacy of HER2-specific antibodies such as Herceptin, currently used in the clinic, relates to their ability to direct HER2 to a Cbl- dependent endocytosis and degradation pathway. To induce the downregulation of EGFR and HER2, Cbl must associate with several proteins. Two proteins that interact with Cbl are the Abl tyrosine kinase and the Abi adaptor protein. Moreover, Cbl is tyrosine phosphorylated by the c-Abl kinase. Both Abl and Abi1 interact with proteins implicated in endocytosis, and both proteins are modified by phosphorylation events following stimulation of the EGFR. We hypothesize that Abl and Abi1 modulate the activity of Cbl towards the downregulation of EGFR. Also, Abl-dependent phosphorylation of the EGFR and Cbl may affect their function and association upon EGF stimulation. The Abl kinases, Abi1 and Cbl are mutated in human cancers, and deregulation of their activities leads to proliferation and/or metastasis. The goal of this proposal is to define the role of the Abl kinase and Abi1 in endocytosis and signaling of the EGFR. We will analyze the effect of Abl on EGFR phosphorylation and ubiquitination upon EGF stimulation. Also, we will analyze whether Abl-mediated phosphorylation of Cbl and EGFR affects their association upon ligand binding. To analyze whether inhibiting endogenous Abl in breast cancer cells affects their invasiveness, we will carry out migration assays upon EGF stimulation in the absence or presence of c-Abl specific inhibitors such as STI-571, Gleevec. We will test if Abi mutants or loss of Abi expression can affect proliferation and survival of cells upon EGFR activation. The insights learned from this project could be used to design new strategies for the clinical use of Abl specific inhibitors, such as STI-571, as well as development of Abi1 inhibitors in breast cancer. EGF activates cell migration of breast cancer cells, which is critical for invasion. Breast cancer cells vary in their responsiveness to EGF, and this correlates with a higher degree of malignancy and invasiveness. Because Abl, Abi and Cbl are downstream targets of the EGFR, and are known to be mutated in human cancers, deregulation of their activities leads to proliferation and metastasis. Insights learned from this project on the mechanisms of EGFR internalization, regulation of Cbl function and migration in breast cancer cells will contribute to the knowledge of the biology of breast cancer and the progression towards its cure.