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Characterization of Effects of MUC1 Overexpression on Epidermal Growth Factor Receptor Signaling
Background: The main goal of this proposal is to elucidate the relationship between two proteins, EGFR and MUC1 both of which are highly expressed in breast carcinomas. EGFR belongs to the family of epidermal growth factor receptor tyrosine kinases that have been extensively studied for their roles in many types of cancers, specifically, breast cancer. Overexpression and/or increased activation of members of the EGFR family is a hallmark of breast carcinomas and is associated with poor prognosis and aggressive disease. MUC1 is a transmembrane glycoprotein that is aberrantly overexpressed in greater than 90% of invasive breast carcinomas. However, the precise function of MUC1 in breast cancer development remains unresolved. Recent studies on transgenic mice that overexpress human MUC1 (MMTV-MUC1) revealed that they develop metastatic mammary gland carcinoma. Preliminary data in our lab indicate that MUC1 and EGFR biochemically interact in mammary tumors but not in the contralateral normal mammary gland. In addition, overexpression of MUC1 promotes a tumor-specific recruitment of Grb2/SOS to EGFR. Furthermore, MUC1 and EGFR associate with each other in a constitutive manner in both SK-BR3 and ZR-75-30 human breast cancer cell lines. Taken together, the above data suggest that there is a correlation between MUC1 expression and EGFR activation and these interactions could be very significant in promoting mammary carcinoma.
Hypothesis: MUC1 overexpression plays a pivotal role in breast cancer progression by potentiating EGFR dependent signaling cascade and modulating many critical events such as mitogenesis and cell survival.
Specific Aims: (1)Determine the role of EGFR activation in MUC1-induced mammary gland carcinoma using transgenic mouse models (2) Analyze the effects of expression and abrogation of MUC1 on mitogenesis and survival of breast cancer cell lines.
Study Design: To define the functional significance of MUC1 and EGFR interactions, we will utilize tumors and normal mammary glands from transgenic mice that overexpress human MUC1 (MMTV-MUC1). We will directly assess the functional importance of EGFR signaling in MUC1-mediated transformation by crossing the MMTV-MUC1 transgenics onto waved-2 mice (a spontaneous EGFR kinase mutant) and analyze the effects on tumor formation. Analysis of downstream signaling components of Grb2/SOS activation (such as MAP Kinase) in both the MMTV-MUC1 and MMTV-MUC1/waved-2 derived tumors will determine if EGFR is required to activate this signaling pathway in the MMTV-MUC1 model of tumorigenesis. RNAi will be used to ablate MUC1 expression in SK-BR3 and ZR-75-30 breast cancer cell lines that over express MUC1 and assays will be performed to analyze how the presence or absence of MUC1 can modulate cell proliferation and survival. MUC1 dependent EGFR signaling pathway will be further dissected in vitro using pharmacological inhibitors of EGFR kinase activity in the presence and absence of MUC1.
Potential Outcomes and Benefits of Research: Results from the proposed studies will help in deciphering a novel mechanism of regulation of EGFR signaling and also provide insight into the role of MUC1 overexpression in breast cancer. The outcome of these studies could aid in identification of novel therapeutic and diagnostic targets for breast cancer treatment.
Background: Cancer, in general, is defined as a disease in which abnormal cells divide without control. In normal conditions, many mechanisms exist to maintain a fine balance between cell proliferation and apoptosis (cell death). EGFR is a protein present in the membrane of cells that plays many important roles in a normal cell as well as in a cancer cell. Some of the pathways that are regulated by EGFR include cell division, cell survival, etc. In many cancers, especially, breast cancer, EGFR signaling is highly activated resulting in increased cell proliferation and inhibited apoptosis. Increased activation of EGFR in breast cancer is associated with poor prognosis and advanced disease. Hence, it is not surprising that intense efforts are focused on understanding the different mechanisms that regulate EGFR signaling, so that better therapeutic targets can be designed. MUC1 is another protein that is overexpressed in greater than 90% of invasive breast carcinomas. In a normal epithelial cell (this is the main cell type in mammary gland), MUC1 is present at the apical region, whereas, EGFR is present in the basolateral region. Hence, they cannot interact with each other due to spatial restrictions. However, in a cancer cell, this apical-basolateral polarity is lost and both MUC1 and EGFR are distributed over the entire cell surface. This provides a means for these two proteins to associate with each other. Overexpression of human MUC1 in mice resulted in the formation of mammary gland carcinoma. Preliminary data in our lab indicate that MUC1 and EGFR interact specifically in tumor, but not in the normal mammary gland and the tumor specific interactions could possibly be modulating EGFR signaling. Taken together, the above data suggest that these interactions could be very significant in promoting mammary carcinoma.
Hypothesis: As a consequence of altered localization and overexpression, MUC1 aids in modulation of EGFR signaling and contributes to breast cancer development.
Specific Aims: (1) Determine the role of EGFR activation in MUC1-induced mammary gland carcinoma.(2) Analyze the effects of expression and removal of MUC1 on mitogenesis and survival of breast cancer cell lines.
Study Design: To test specific aim one, we will utilize tumors and normal mammary glands from transgenic mice that overexpress human MUC1 (MMTV-MUC1). In order to directly address the functional significance of EGFR signaling in MUC1-induced carcinoma, we will cross MMTV-MUC1 mice to waved-2 mice (have an impaired EGFR function) and analyze the effects on tumor formation. We will also determine the activation of different signaling molecules that are important in cell proliferation and survival and compare the differences in the presence of functional EGFR and impaired EGFR to determine how MUC1 overexpression affects the activation. A technique called RNAi will be used to abolish MUC1 expression in the MUC1 overexpressing breast cancer cell lines (SK-Br-3 and ZR-75-30)and assays will be performed to determine how the presence or absence of MUC1 affects cell proliferation and survival. MUC1 dependent EGFR signaling pathway will be further dissected using pharmacological inhibitors of EGFR function in the presence and absence of MUC1.
Potential Outcomes and Benefits of Research: The proposed studies will provide a better understanding of the role of MUC1 overexpression in breast cancer progression and possibly unravel a novel mechanism of regulation of EGFR. The outcome of these studies could aid in designing tools to therapeutically target tumor specific interactions while leaving the normal mammary gland cells unaffected.