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Effects of ErbB2 Overexpression on the Proteome and ErbB Ligand-specific Phosphosignaling in Mammary Luminal Epithelial Cells *

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ABSTRACT

Most breast cancers arise from luminal epithelial cells, and 25–30% of these tumors overexpress the ErbB2/HER2 receptor that correlates with disease progression and poor prognosis. The mechanisms of ErbB2 signaling and the effects of its overexpression are not fully understood. Herein, stable isotope labeling by amino acids in cell culture (SILAC), expression profiling, and phosphopeptide enrichment of a relevant, non-transformed, and immortalized human mammary luminal epithelial cell model were used to profile ErbB2-dependent differences in protein expression and phosphorylation events triggered via EGF receptor (EGF treatment) and ErbB3 (HRG1β treatment) in the context of ErbB2 overexpression. Bioinformatics analysis was used to infer changes in cellular processes and signaling events. We demonstrate the complexity of the responses to oncogene expression and growth factor signaling, and we identify protein changes relevant to ErbB2-dependent altered cellular phenotype, in particular cell cycle progression and hyper-proliferation, reduced adhesion, and enhanced motility. Moreover, we define a novel mechanism by which ErbB signaling suppresses basal interferon signaling that would promote the survival and proliferation of mammary luminal epithelial cells. Numerous novel sites of growth factor-regulated phosphorylation were identified that were enhanced by ErbB2 overexpression, and we putatively link these to altered cell behavior and also highlight the importance of performing parallel protein expression profiling alongside phosphoproteomic analysis.

No MeSH data available.


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A, Western blotting showing suppression of IRF9 expression in C3.6 cells that could be induced by IFNβ or IFNγ treatment (24 h) and blocked by co-treatment with EGF or HRGβ1. CDK4 expression served as a loading control. B, IRF9 mRNA levels in IFNγ and IFNγ plus EGF (γ+E) co-treated HMLECs measured by qRT-PCR. C, IRF9 expression and ERK signaling in HMLECs treated with IFNγ and IFNγ plus EGF with or without pretreatment with protein synthesis inhibitor cycloheximide (CHX), MEK inhibitor PD098059 (PD), proteasome inhibitor PS341 (PS), or ErbB receptor kinase inhibitor AG1478 (AG). CDK4 expression served as a loading control. D, IRF9 protein expression is decreased by proteasome inhibitor (PS341) treatment in IFNγ-stimulated C3.6 cells (upper panel) and randomly growing HB4a cells (lower panel).
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Figure 5: A, Western blotting showing suppression of IRF9 expression in C3.6 cells that could be induced by IFNβ or IFNγ treatment (24 h) and blocked by co-treatment with EGF or HRGβ1. CDK4 expression served as a loading control. B, IRF9 mRNA levels in IFNγ and IFNγ plus EGF (γ+E) co-treated HMLECs measured by qRT-PCR. C, IRF9 expression and ERK signaling in HMLECs treated with IFNγ and IFNγ plus EGF with or without pretreatment with protein synthesis inhibitor cycloheximide (CHX), MEK inhibitor PD098059 (PD), proteasome inhibitor PS341 (PS), or ErbB receptor kinase inhibitor AG1478 (AG). CDK4 expression served as a loading control. D, IRF9 protein expression is decreased by proteasome inhibitor (PS341) treatment in IFNγ-stimulated C3.6 cells (upper panel) and randomly growing HB4a cells (lower panel).

Mentions: We wanted to address the possible mechanisms by which ErbB2 may suppress ISG expression. We focused on IRF9/ISGF3G as a key IFNγ-induced gene that associates with the phosphorylated and activated STAT1-STAT2 dimer to form the transcription factor complex ISGF3G that binds to IFN-stimulated response elements in target ISGs and triggers their expression to drive cells into an antiviral state (31). We had also previously observed an inverse correlation between IRF9 and ErbB2 expression in a panel of breast tumor cell lines (17). IRF9 protein expression was confirmed as down-regulated in the ErbB2-overexpressing C3.6 cells, although it was inducible by treatment with either IFNβ (type I) or IFNγ (type II) treatment, indicating that the cells have an intact IFN signaling pathway (Fig. 5A). IRF9 expression was relatively unaffected by EGF or HRG treatments alone, but co-treatment with IFNγ and either growth factor significantly reduced IFNγ-induced expression in the C3.6 cells. This growth factor-mediated abrogation was not apparent at the mRNA level (Fig. 5B), suggesting a post-transcriptional mechanism of regulation. This effect was found to be ErbB receptor-dependent, as cells pretreated with the ErbB kinase inhibitor AG1478 restored the ability of IFNγ to induce IRF9 in the presence of growth factor (Fig. 5C). Pretreatment with the MEK inhibitor PD098059 also partially restored IRF9 expression. Inhibition of protein translation with cycloheximide blocked IRF9 induction as expected, as did pretreatment with the proteasome inhibitor PS341. Decreased IRF9 expression with PS341 treatment was confirmed in both IFNγ-treated C3.6 cells and over a time course in randomly growing HB4a cells (Fig. 5D).


Effects of ErbB2 Overexpression on the Proteome and ErbB Ligand-specific Phosphosignaling in Mammary Luminal Epithelial Cells *
A, Western blotting showing suppression of IRF9 expression in C3.6 cells that could be induced by IFNβ or IFNγ treatment (24 h) and blocked by co-treatment with EGF or HRGβ1. CDK4 expression served as a loading control. B, IRF9 mRNA levels in IFNγ and IFNγ plus EGF (γ+E) co-treated HMLECs measured by qRT-PCR. C, IRF9 expression and ERK signaling in HMLECs treated with IFNγ and IFNγ plus EGF with or without pretreatment with protein synthesis inhibitor cycloheximide (CHX), MEK inhibitor PD098059 (PD), proteasome inhibitor PS341 (PS), or ErbB receptor kinase inhibitor AG1478 (AG). CDK4 expression served as a loading control. D, IRF9 protein expression is decreased by proteasome inhibitor (PS341) treatment in IFNγ-stimulated C3.6 cells (upper panel) and randomly growing HB4a cells (lower panel).
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Figure 5: A, Western blotting showing suppression of IRF9 expression in C3.6 cells that could be induced by IFNβ or IFNγ treatment (24 h) and blocked by co-treatment with EGF or HRGβ1. CDK4 expression served as a loading control. B, IRF9 mRNA levels in IFNγ and IFNγ plus EGF (γ+E) co-treated HMLECs measured by qRT-PCR. C, IRF9 expression and ERK signaling in HMLECs treated with IFNγ and IFNγ plus EGF with or without pretreatment with protein synthesis inhibitor cycloheximide (CHX), MEK inhibitor PD098059 (PD), proteasome inhibitor PS341 (PS), or ErbB receptor kinase inhibitor AG1478 (AG). CDK4 expression served as a loading control. D, IRF9 protein expression is decreased by proteasome inhibitor (PS341) treatment in IFNγ-stimulated C3.6 cells (upper panel) and randomly growing HB4a cells (lower panel).
Mentions: We wanted to address the possible mechanisms by which ErbB2 may suppress ISG expression. We focused on IRF9/ISGF3G as a key IFNγ-induced gene that associates with the phosphorylated and activated STAT1-STAT2 dimer to form the transcription factor complex ISGF3G that binds to IFN-stimulated response elements in target ISGs and triggers their expression to drive cells into an antiviral state (31). We had also previously observed an inverse correlation between IRF9 and ErbB2 expression in a panel of breast tumor cell lines (17). IRF9 protein expression was confirmed as down-regulated in the ErbB2-overexpressing C3.6 cells, although it was inducible by treatment with either IFNβ (type I) or IFNγ (type II) treatment, indicating that the cells have an intact IFN signaling pathway (Fig. 5A). IRF9 expression was relatively unaffected by EGF or HRG treatments alone, but co-treatment with IFNγ and either growth factor significantly reduced IFNγ-induced expression in the C3.6 cells. This growth factor-mediated abrogation was not apparent at the mRNA level (Fig. 5B), suggesting a post-transcriptional mechanism of regulation. This effect was found to be ErbB receptor-dependent, as cells pretreated with the ErbB kinase inhibitor AG1478 restored the ability of IFNγ to induce IRF9 in the presence of growth factor (Fig. 5C). Pretreatment with the MEK inhibitor PD098059 also partially restored IRF9 expression. Inhibition of protein translation with cycloheximide blocked IRF9 induction as expected, as did pretreatment with the proteasome inhibitor PS341. Decreased IRF9 expression with PS341 treatment was confirmed in both IFNγ-treated C3.6 cells and over a time course in randomly growing HB4a cells (Fig. 5D).

View Article: PubMed Central - PubMed

ABSTRACT

Most breast cancers arise from luminal epithelial cells, and 25–30% of these tumors overexpress the ErbB2/HER2 receptor that correlates with disease progression and poor prognosis. The mechanisms of ErbB2 signaling and the effects of its overexpression are not fully understood. Herein, stable isotope labeling by amino acids in cell culture (SILAC), expression profiling, and phosphopeptide enrichment of a relevant, non-transformed, and immortalized human mammary luminal epithelial cell model were used to profile ErbB2-dependent differences in protein expression and phosphorylation events triggered via EGF receptor (EGF treatment) and ErbB3 (HRG1β treatment) in the context of ErbB2 overexpression. Bioinformatics analysis was used to infer changes in cellular processes and signaling events. We demonstrate the complexity of the responses to oncogene expression and growth factor signaling, and we identify protein changes relevant to ErbB2-dependent altered cellular phenotype, in particular cell cycle progression and hyper-proliferation, reduced adhesion, and enhanced motility. Moreover, we define a novel mechanism by which ErbB signaling suppresses basal interferon signaling that would promote the survival and proliferation of mammary luminal epithelial cells. Numerous novel sites of growth factor-regulated phosphorylation were identified that were enhanced by ErbB2 overexpression, and we putatively link these to altered cell behavior and also highlight the importance of performing parallel protein expression profiling alongside phosphoproteomic analysis.

No MeSH data available.


Related in: MedlinePlus