This line represents an additive relationship between two drugs

This line represents an additive relationship between two drugs. Src family kinase (SFK) inhibitors; however, the combination of EGFR and SFK inhibitors synergistically decreases cell viability. We found that this decrease in cell viability observed with EGFR and SFK inhibitor co-treatment correlates with loss of Akt phosphorylation. In addition, we found that in breast malignancy cell lines with EGFR and c-Src colocalized to lipid rafts, phospho-inositide-3-kinase (PI3K) was also associated with lipid rafts. Collectively, the data herein suggest that lipid rafts provide a platform for the connection of EGFR, c-Src and PI3K, leading to activation of cellular survival signaling in breast cancer cells. strong class=”kwd-title” Key phrases: epidermal growth element receptor, c-Src, breast malignancy, cholesterol, Akt, lipid rafts, crosstalk Intro Epidermal growth element receptor (EGFR) is definitely a mitogenic receptor tyrosine kinase indicated within the Rabbit Polyclonal to Gab2 (phospho-Tyr452) membrane as asymmetric dimers that upon ligand binding undergo a conformational modify to induce autophosphorylation.1 Autophosphorylation of tyrosine residues provides docking sites for mediators of downstream signaling including phospho-inositide-3-kinase (PI3K), Shc, Grb2, phospholipase C (PLC), and signal transducers and activators of transcription (STATs).2 Recruitment and subsequent phosphorylation of these proteins results in activation of downstream signaling leading to cellular proliferation, migration, differentiation and survival (reviewed in ref. 2). While the most well known localization of EGFR is at the plasma membrane, evidence demonstrates that EGFR is definitely active within additional cellular membrane bound organelles, including endosomes, the nucleus and mitochondria.3C5 In addition, EGFR localizes to discrete membrane microdomains, referred to as lipid rafts.6 Within these lipid rafts, EGFR signaling has been shown to be stimulated as well as inhibited, depending on the cellular context.7C10 High concentrations of signaling molecules can be found within lipid rafts.11C13 Such molecules include a variety of GPI-anchored proteins, lipid modified proteins and transmembrane proteins.14,15 Lipid rafts have both positive and negative effects on cellular signaling. Specifically, rafts may act as platforms to localize components of a signaling pathway in close proximity, or may facilitate crosstalk between signaling pathways by keeping components of both pathways near each other. However, lipid rafts may also act as sequestering areas in the membrane to prevent association of pathway parts, leading to downregulation of signaling events (examined in ref. 16). As mentioned above, EGFR is definitely capable of lipid raft localization, which can regulate EGFR-dependent signaling pathways. Another protein family known be present within lipid rafts is the Src family of tyrosine kinases.17,18 Lipid modification, as well as basic Ilorasertib residues within the unique domain of the protein, facilitates Ilorasertib association of c-Src, the prototypical member of this family, to lipid rafts.18 Such localization has been explained in neuronal, hematopoietic, and cervical and lung cancer cell lines.17C21 Like EGFR, lipid rafts also mediate c-Src signaling, including c-Src-dependent activation of PI3K/Akt.17 c-Src interacts with a number of receptor tyrosine kinases including EGFR.22 Activation of EGFR raises c-Src catalytic activity.23 c-Src, in turn, phosphorylates novel sites on EGFR, which promotes signaling downstream of the receptor.24C26 Phosphorylation of EGFR by c-Src has been shown to regulate EGF-induced mitogenesis.27,28 Specifically, phosphorylation of EGFR by c-Src mediates the binding of PI3K to EGFR leading to Akt phosphorylation and cellular survival signaling.26 The interaction between these two proteins enhances transformation of mouse fibroblasts and human being mammary epithelial cell lines, and tumor formation in nude mice.27,29 We have previously demonstrated that EGFR is localized to plasma membrane lipid rafts in breast cancer cells that are resistant to EGFR tyrosine kinase inhibitors.30 Interestingly, inhibiting lipid raft structure with cholesterol biosynthesis inhibitors sensitizes these resistant cells to EGFR inhibitors.30 Mechanistically, we found that inhibiting lipid raft structure decreased signaling through the Akt pathway, as previously reported by others.30,31 Here, we demonstrate co-localization and co-association between c-Src and EGFR within plasma membrane lipid rafts of one such breast cancer cell collection, SUM159. While inhibition of either of these proteins alone was insufficient to reduce the viability of breast cancer cells, co-inhibition of c-Src and EGFR kinase activities resulted in a synergistic decrease in cell viability. Additionally, when EGFR and c-Src colocalized within lipid rafts, c-Src kinase inhibition abrogated EGFR-kinase self-employed Akt phosphorylation. Results c-Src localizes to lipid rafts in SUM159 cells. Previously, we have demonstrated that EGFR localizes to lipid rafts in four EGFR TKI resistant breast malignancy cell lines.30 This localization of EGFR to lipid rafts was required for Akt phosphorylation and EGFR disassociation with lipid rafts subsequently sensitized these cells EGFR kinase inhibition.30 However, the signaling Ilorasertib mechanism by.

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