Dataset Information

Proteomic and phosphoproteomic profiling of ErbB2-dependent signalling

ABSTRACT: The mechanisms of ErbB2 signalling and the effects of its overexpression are not fully understood. Herein, SILAC expression profiling and phosphopeptide enrichment of a relevant, non-transformed, immortalized human mammary luminal epithelial cell model were used to profile ErbB2-dependent differences in protein expression and phosphorylation events triggered via EGFR (EGF treatment) and ErbB3 (HRGbeta1 treatment) in the context of ErbB2 overexpression. Bioinformatics analysis was used to infer changes in cellular processes and signalling events. We demonstrate the complexity of the responses to oncogene expression and growth factor signalling and identify protein changes relevant to ErbB2-dependent altered cellular phenotype, in particular cell cycle progression and hyper-proliferation, reduced adhesion and enhanced motility. Numerous novel sites of growth factor-regulated phosphorylation were also identified that were enhanced by ErbB2 overexpression and we putatively link these to altered cell behaviour. Moreover, we have defined a novel mechanism by which ErbB signalling suppresses basal interferon signalling that would promote the survival and proliferation of mammary luminal epithelial cells.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Luminal Epithelial Cell Of Mammary Gland

DISEASE(S): Breast Cancer

SUBMITTER: John Timms

LAB HEAD: John Francis Timms

PROVIDER: PXD004195 | Pride | 2018-10-26

REPOSITORIES: Pride

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Publications

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

Worthington Jenny J Spain Georgia G Timms John F JF

Molecular & cellular proteomics : MCP 20170207 4

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 ...[more]

PMID: 28174229

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Project description:The epithelial growth factor receptor (EGFR) signalling network plays an essential role inproliferation and survival of colorectal cancer cells. For this reason, EGFR is targeted using therapeutic antibodies blocking EGFR signalling (e.g. cetuximab). However, anti-EGFR therapy is rarely curative as subpopulations of tumor cells survive the initial treatment and develop therapy-resistance. To analyse cellular signalling and assess the quantitative changes of the proteome and the phosphoproteome several proteomics approaches have been established. Here, we compared the widely used quantification strategies label-free (LF), SILAC and TMT and systematically evaluated their technical characteristics including coverage, technical variability, complementarity and the ability to assess statistical significant differences. We studied the dynamics of the EGFR signalling network upon treatment withcetuximab after 0 h, 3 h and 24 h, representing the first cellular adaption towards anti-EGFR therapy, with the aim to identify the most powerful approach for monitoring cellular signalling in this setup. We demonstrate that LF had a superior coverage regarding quantification of both, the proteome and phosphoproteome, while SILAC exhibited the highest precision resulting in superior assessment of differentially abundant proteins and class I phosphosites (p-sites). TMT was outperformed by the other two approaches, illustrating its critical dependency on the applied labelling-design. On the protein level, we observed only little regulation upon cetuximab treatment, whereas a great fraction of p-sites was significantly regulated after 3 h and 24 h. The dynamics of significantly regulated p-sites illustrated an initial downregulation of the EGFR and MAPK signalling pathways, which was partially rescued as soon as after 24h. We identified upregulation and signalling via ERBB3 as a possible mechanism bypassing the blockage of EGFR. Moreover, phosphorylation motifs associated to calcium signalling were continuously upregulated, thereby representing compensatory signalling presumably resulting in the observed reactivation of MAPK1/3 (Erk1/2).

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Dataset Information

Proteomic and phosphoproteomic profiling of ErbB2-dependent signalling

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Publications

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

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