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    The Significance of the Epidermal Growth Factor Receptor and Cytochrome c Oxidase Subunit II Association in Breast Cancer: Implications in Mediating Cellular Survival

    Scientific Abstract:
    DS: Parsons, S Tracking # DISS0402970 The Significance of the Epidermal Growth Factor Receptor and Cytochrome c Oxidase Subunit II Association In Breast Cancer: Implications in Mediating Cellular Survival The epidermal growth factor receptor (EGFR) and the tyrosine kinase c-Src are co-overexpressed in approximately 30% of breast cancer cells and tissues, suggesting the two proteins functionally interact. EGFR overexpression is associated with poor prognosis, further suggesting that the mechanism of EGFR action in breast cancer cells is important. When EGFR and c-Src were co-overexpressed in the murine fibroblast model C3H10T1/2 and in breast cancer cells, synergistic increases in EGF-induced DNA synthesis, growth in soft agar, and tumor formation in nude mice were seen in comparison to cells singly overexpressing EGFR or c-Src. These synergistic increases were found to be dependent on association of c-Src with EGFR and its phosphorylation of the EGFR tyrosine 845 (Y845) following EGF stimulation. Mutation of Y845 does not alter the intrinsic kinase activity of EGFR or its ability to phosphorylate Shc and to activate MAP kinase. However, mutation of Y845 to phenylalanine (Y845F) does inhibit EGF induced DNA synthesis. These findings suggest that phosph-Y845 (pY845) elicits downstream signals that are independent of Shc and MAPK and are mediated through alternate effectors. In support of our hypothesis that pY845 of the EGFR elicits a tumorigenic signal, we further showed that Stat5b is a mediator of EGF-induced DNA synthesis through pY845 of the EGFR. To obtain other mediators of EGFR pY845 signaling in breast cancer cells, EGFR pY845 binding partners were found by employing the technique of phage display. Cytochrome c oxidase subunit II (CoxII) was identified from a breast cancer tissue library. CoxII is a mitochondrial-encoded protein that functions as an electron acceptor by interacting with cytochrome c in complex IV of the electron transport chain. During oxidative phosphorylation, CoxII directly binds to cytochrome c. The ability of CoxII to regulate cytochrome c release has been postulated as a mechanism by which CoxII can regulate apoptotsis. We further hypothesize that binding of EGFR to CoxII enhances CoxII’s ability to retain cytochrome c, thus promoting cellular survival, evasion of apoptosis, and tumorigenesis. Thus, the association of EGFR and CoxII and its effects on breast cancer cell survival were investigated. Co-immunoprecipitation showed that the EGFR physically associates with CoxII in an EGF- and pY845-dependent manner. Furthermore, in serum free conditions, increased caspase 3 activation and TUNEL staining were apparent in the Y845F mutant EGFR expressing cells as compared to wild-type(wt) EGFR-expressing cells, thus, leading to our hypothesis that EGFR promotes cellular survival or anti-apoptosis through the Y845 phosphorylation site. This study proposes to elucidate in breast cancer cells the mechanism by which association of the wt EGFR with CoxII activates a pro-survival signaling cascade, thereby, potentially leading to unregulated cell proliferation and, ultimately, promoting tumorigenesis. Firstly, investigations into how the EGFR and/or c-Src translocates to the mitochondria and its dependence on EGF will be carried out. The studies will employ confocal and electron microscopy as well as biochemical fractionation of breast cancer cells co-overexpressing c-Src and either wt or Y845F mutant EGFR. Secondly, breast cancer cell lines co-overexpressing c-Src and either EGFR or the EGFR mutant Y845F will be used to determine their involvement in the mitochondrial apoptotic pathway induced by adriamycin, a known breast cancer chemotherapeutic agent. Cytochrome c efflux, mitochondrial inner membrane potential loss, Bcl-2 family member activation order and localization, and caspase activation order and importance to the apoptotic signal will be determined in the various breast cancer cell lines. Thirdly, the effect of EGFR and/or c-Src on CoxII activity will be assessed. EGFR or c-Src phosphorylation of CoxII will be determined through in vitro kinase assays. The effect of tyrosine phosphorylation of CoxII on its activity and on pro-survival signals will be determined in breast cancer cells expressing variants of EGFR and/or c-Src or treated with inhibitors of EGFR or c-Src.

    Lay Abstract:
    DS: Parsons, S Tracking # DISS0402970 The Significance of the Epidermal Growth Factor Receptor and Cytochrome c Oxidase Subunit II Association In Breast Cancer: Implications in Mediating Cellular Survival To maintain normal tissue homeostasis, a balance of cell division and cell death occurs. As the number of cells increases, by division and proliferation, an approximately equal number undergo programmed cellular death, i.e. apoptosis. The intracellular signaling cascades that initiate and carry out apoptosis are well studied, but research continues to clarify previously identified pathways and define new pathways. Evasion of apoptosis can lead to the unregulated and excessive cell proliferation that is a hallmark of cancer. Consequently, understanding any mechanism cancer cells utilize to evade apoptosis will be extremely important in developing future cancer therapies. The epidermal growth factor receptor (EGFR) is a transmembrane receptor that resides on the plasma membrane where it can convert extracellular stimuli into intracellular information by initiating intracellular signaling cascades. Using this mechanism, the EGFR has been shown to activate proliferative and pro-survival (anti-apoptotic) signaling cascades. These cascades can be initiated through the association of molecules with phosphorylated amino acids located on the cytoplasmic tail of the receptor. Of particular interest to this proposal, however, is the amino acid tyrosine 845 (Y845), which resides in the catalytic domain of the molecule. Phosphorylation of Y845 has been shown to occur in cells overexpressing the tyrosine kinase c-Src and the EGFR and this phosphorylation is regulated by c-Src kinase activity. Previous studies in our laboratory strongly suggested that this phosphorylated amino acid may have downstream effectors that are important to cellular survival and tumorigenesis. Investigation of the downstream effects of Y845 are particularly pertinent to breast cancer because EGFR and c-Src are overexpressed in approximately 30% of breast cancers most of which represent late stage cancers for which few effective therapies are available. Thus, if Y845 is mediating a pro-survival signaling cascade, then research investigating this pathway and its effectors may lead to more efficient and effective cancer therapies for many breast cancer patients. The mitochondria are commonly referred to as the power producers of the cell. They house the machinery necessary for the production of adenosine triphosphate (ATP), the main energy source for the cell. Previous studies in our laboratory have demonstrated that the EGFR can localize to the mitochondria and that phosphorylated Y845 of the EGFR can associate with a mitochondrial survival protein, cytochrome c oxidase subunit II (CoxII). This survival protein also is a part of the machinery needed to produce cellular energy. We have found that very little EGFR and CoxII associate when Y845 is mutated. Furthermore, the mutated EGFR causes an increase in apoptosis. From these results we hypothesize that the normal EGFR is mediating a pro-survival signaling cascade through the association of phospho-Y845 with CoxII and due to the high prevalence of EGFR and c-Src overexpression in breast cancer cells, this pro-survival signal may ultimately lead to evasion of programmed cellular death and thus promote cancer development. This study proposes to elucidate in breast cancer cells the mechanisms by which the EGFR translocates to the mitochondria where it can associate with CoxII and subsequently activate pro-survival signals. Firstly, we will determine how the EGFR and c-Src localize to the mitochondria where the EGFR can associate with CoxII. Secondly, we will test the role of Y845 of the EGFR in promoting specific intracellular survival signals. Thirdly, we will characterize the effects of EGFR and/or c-Src association with CoxII by assessing cytochrome c oxidase function as part of the machinery involved in making cellular energy. Importantly, the results of this study may identify molecular targets in the pro-survival pathway that could be the focus of new drug development. Such drugs could synergize with known breast cancer chemotherapeutics to ultimately enhance their efficiency and effectiveness.