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

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.


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Study workflow for protein expression (A) and phosphoproteomic (B) profiling. Independent cell cultures were reciprocally labeled with heavy and light lysine/arginine as shown for at least six passages. Cells were serum-starved and then stimulated with EGF or HRGb1 for 10 min (or left unstimulated) as shown. Cells were lysed, and equal amounts of protein were mixed from each condition, and then these were mixed to generate reciprocally labeled biological duplicate pools. These were separated by SDS-PAGE, and 50 gel slices per lane (n = 100) were excised and digested with trypsin, prior to LC-MS/MS. For phosphoproteomic profiling, a common reference pool was generated by pooling equal amounts of protein from light-labeled cultures, and this was used in singlet comparisons for each heavy-labeled condition. The six heavy/light mixtures were digested, separated into five SCX fractions, and each was subjected to SIMAC phosphopeptide enrichment generating three fractions each. Thus, 90 samples were generated and analyzed by LC-MS/MS. Protein identification, phosphosite identification, and SILAC-based quantification were performed for both datasets using MaxQuant software.
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Figure 1: Study workflow for protein expression (A) and phosphoproteomic (B) profiling. Independent cell cultures were reciprocally labeled with heavy and light lysine/arginine as shown for at least six passages. Cells were serum-starved and then stimulated with EGF or HRGb1 for 10 min (or left unstimulated) as shown. Cells were lysed, and equal amounts of protein were mixed from each condition, and then these were mixed to generate reciprocally labeled biological duplicate pools. These were separated by SDS-PAGE, and 50 gel slices per lane (n = 100) were excised and digested with trypsin, prior to LC-MS/MS. For phosphoproteomic profiling, a common reference pool was generated by pooling equal amounts of protein from light-labeled cultures, and this was used in singlet comparisons for each heavy-labeled condition. The six heavy/light mixtures were digested, separated into five SCX fractions, and each was subjected to SIMAC phosphopeptide enrichment generating three fractions each. Thus, 90 samples were generated and analyzed by LC-MS/MS. Protein identification, phosphosite identification, and SILAC-based quantification were performed for both datasets using MaxQuant software.

Mentions: For protein expression profiling, a reciprocal duplicate SILAC-labeling strategy was used to compare two biological replicates each of ErbB2-overexpressing cells and control parental cells by Gel LC-MS/MS (Fig. 1). Fifty gel slices from each of two lanes of the reciprocally labeled and mixed samples were digested and analyzed in 100 LC-MS/MS runs. For phosphopeptide comparisons of six different conditions (±ErbB2, ±EGF, and ±HRGβ1), a common reference sample comprising a pool of equal protein amounts from the six different light-labeled cultures was used in singlet comparisons with each heavy-labeled condition. The six heavy/light mixtures were digested, separated into 15 fractions by strong cation exchange (SCX) chromatography and sequential elution from immobilized metal ion affinity chromatography (SIMAC) phosphopeptide enrichment, and the resulting 90 samples were analyzed by LC-MS/MS (Fig. 1). Data were searched and analyzed using MaxQuant and Perseus software as described below. Proteins were accepted as being significantly up/down-regulated with a significance B value of <0.05.


Effects of ErbB2 Overexpression on the Proteome and ErbB Ligand-specific Phosphosignaling in Mammary Luminal Epithelial Cells *
Study workflow for protein expression (A) and phosphoproteomic (B) profiling. Independent cell cultures were reciprocally labeled with heavy and light lysine/arginine as shown for at least six passages. Cells were serum-starved and then stimulated with EGF or HRGb1 for 10 min (or left unstimulated) as shown. Cells were lysed, and equal amounts of protein were mixed from each condition, and then these were mixed to generate reciprocally labeled biological duplicate pools. These were separated by SDS-PAGE, and 50 gel slices per lane (n = 100) were excised and digested with trypsin, prior to LC-MS/MS. For phosphoproteomic profiling, a common reference pool was generated by pooling equal amounts of protein from light-labeled cultures, and this was used in singlet comparisons for each heavy-labeled condition. The six heavy/light mixtures were digested, separated into five SCX fractions, and each was subjected to SIMAC phosphopeptide enrichment generating three fractions each. Thus, 90 samples were generated and analyzed by LC-MS/MS. Protein identification, phosphosite identification, and SILAC-based quantification were performed for both datasets using MaxQuant software.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383782&req=5

Figure 1: Study workflow for protein expression (A) and phosphoproteomic (B) profiling. Independent cell cultures were reciprocally labeled with heavy and light lysine/arginine as shown for at least six passages. Cells were serum-starved and then stimulated with EGF or HRGb1 for 10 min (or left unstimulated) as shown. Cells were lysed, and equal amounts of protein were mixed from each condition, and then these were mixed to generate reciprocally labeled biological duplicate pools. These were separated by SDS-PAGE, and 50 gel slices per lane (n = 100) were excised and digested with trypsin, prior to LC-MS/MS. For phosphoproteomic profiling, a common reference pool was generated by pooling equal amounts of protein from light-labeled cultures, and this was used in singlet comparisons for each heavy-labeled condition. The six heavy/light mixtures were digested, separated into five SCX fractions, and each was subjected to SIMAC phosphopeptide enrichment generating three fractions each. Thus, 90 samples were generated and analyzed by LC-MS/MS. Protein identification, phosphosite identification, and SILAC-based quantification were performed for both datasets using MaxQuant software.
Mentions: For protein expression profiling, a reciprocal duplicate SILAC-labeling strategy was used to compare two biological replicates each of ErbB2-overexpressing cells and control parental cells by Gel LC-MS/MS (Fig. 1). Fifty gel slices from each of two lanes of the reciprocally labeled and mixed samples were digested and analyzed in 100 LC-MS/MS runs. For phosphopeptide comparisons of six different conditions (±ErbB2, ±EGF, and ±HRGβ1), a common reference sample comprising a pool of equal protein amounts from the six different light-labeled cultures was used in singlet comparisons with each heavy-labeled condition. The six heavy/light mixtures were digested, separated into 15 fractions by strong cation exchange (SCX) chromatography and sequential elution from immobilized metal ion affinity chromatography (SIMAC) phosphopeptide enrichment, and the resulting 90 samples were analyzed by LC-MS/MS (Fig. 1). Data were searched and analyzed using MaxQuant and Perseus software as described below. Proteins were accepted as being significantly up/down-regulated with a significance B value of <0.05.

View Article: PubMed Central - PubMed

ABSTRACT

Most breast cancers arise from luminal epithelial cells, and 25&ndash;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 (HRG1&beta; 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