Proteome-wide analysis of protein abundance and turnover remodelling during oncogenic transformation of human breast epithelial cells

Tony Ly, Aki Endo, Alejandro Brenes, Marek Gierlinski, Vackar Afzal, Andrea Pawellek, Angus I. Lamond (Lead / Corresponding author)

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Abstract

Background: Viral oncogenes and mutated proto-oncogenes are potent drivers of cancer malignancy. Downstream of the oncogenic trigger are alterations in protein properties that give rise to cellular transformation and the acquisition of malignant cellular phenotypes. Developments in mass spectrometry enable large-scale, multidimensional characterisation of proteomes. Such techniques could provide an unprecedented, unbiased view of how oncogene activation remodels a human cell proteome.

Methods: Using quantitative MS-based proteomics and cellular assays, we analysed how transformation induced by activating v-Src kinase remodels the proteome and cellular phenotypes of breast epithelial (MCF10A) cells. SILAC MS was used to comprehensively characterise the MCF10A proteome and to measure v-Src-induced changes in protein abundance across seven time-points (1-72 hrs). We used pulse-SILAC MS ( Boisvert et al., 2012), to compare protein synthesis and turnover in control and transformed cells. Follow-on experiments employed a combination of cellular and functional assays to characterise the roles of selected Src-responsive proteins.

Results: Src-induced transformation changed the expression and/or turnover levels of ~3% of proteins, affecting ~1.5% of the total protein molecules in the cell. Transformation increased the average rate of proteome turnover and disrupted protein homeostasis. We identify distinct classes of protein kinetics in response to Src activation. We demonstrate that members of the polycomb repressive complex 1 (PRC1) are important regulators of invasion and migration in MCF10A cells. Many Src-regulated proteins are present in low abundance and some are regulated post-transcriptionally. The signature of Src-responsive proteins is highly predictive of poor patient survival across multiple cancer types. Open access to search and interactively explore all these proteomic data is provided via the EPD database ( www.peptracker.com/epd).

Conclusions: We present the first comprehensive analysis measuring how protein expression and protein turnover is affected by cell transformation, providing a detailed picture at the protein level of the consequences of activation of an oncogene.

Original languageEnglish
Article number51
Pages (from-to)1-48
Number of pages48
JournalWellcome Open Research
Volume3
DOIs
Publication statusPublished - 2 May 2018

Fingerprint

Proteome
Breast
Epithelial Cells
Proteins
Oncogenes
Chemical activation
Proteomics
Polycomb Repressive Complex 1
Assays
Phenotype
Neoplasms
Proto-Oncogenes
src-Family Kinases
Mass spectrometry
Mass Spectrometry
Homeostasis
Cells
Databases

Keywords

  • Biomarker
  • Cancer
  • Epigenetic
  • Polycomb Repressive Complex (PRC)
  • Protein half-life
  • Serine protease inhibitor (Serpin)
  • Src Kinase
  • Time-lapse Proteomics

Cite this

@article{ca6947ad5620405082251e041d49a948,
title = "Proteome-wide analysis of protein abundance and turnover remodelling during oncogenic transformation of human breast epithelial cells",
abstract = "Background: Viral oncogenes and mutated proto-oncogenes are potent drivers of cancer malignancy. Downstream of the oncogenic trigger are alterations in protein properties that give rise to cellular transformation and the acquisition of malignant cellular phenotypes. Developments in mass spectrometry enable large-scale, multidimensional characterisation of proteomes. Such techniques could provide an unprecedented, unbiased view of how oncogene activation remodels a human cell proteome.Methods: Using quantitative MS-based proteomics and cellular assays, we analysed how transformation induced by activating v-Src kinase remodels the proteome and cellular phenotypes of breast epithelial (MCF10A) cells. SILAC MS was used to comprehensively characterise the MCF10A proteome and to measure v-Src-induced changes in protein abundance across seven time-points (1-72 hrs). We used pulse-SILAC MS ( Boisvert et al., 2012), to compare protein synthesis and turnover in control and transformed cells. Follow-on experiments employed a combination of cellular and functional assays to characterise the roles of selected Src-responsive proteins.Results: Src-induced transformation changed the expression and/or turnover levels of ~3{\%} of proteins, affecting ~1.5{\%} of the total protein molecules in the cell. Transformation increased the average rate of proteome turnover and disrupted protein homeostasis. We identify distinct classes of protein kinetics in response to Src activation. We demonstrate that members of the polycomb repressive complex 1 (PRC1) are important regulators of invasion and migration in MCF10A cells. Many Src-regulated proteins are present in low abundance and some are regulated post-transcriptionally. The signature of Src-responsive proteins is highly predictive of poor patient survival across multiple cancer types. Open access to search and interactively explore all these proteomic data is provided via the EPD database ( www.peptracker.com/epd).Conclusions: We present the first comprehensive analysis measuring how protein expression and protein turnover is affected by cell transformation, providing a detailed picture at the protein level of the consequences of activation of an oncogene.",
keywords = "Biomarker, Cancer, Epigenetic, Polycomb Repressive Complex (PRC), Protein half-life, Serine protease inhibitor (Serpin), Src Kinase, Time-lapse Proteomics",
author = "Tony Ly and Aki Endo and Alejandro Brenes and Marek Gierlinski and Vackar Afzal and Andrea Pawellek and Lamond, {Angus I.}",
note = "This work was supported by the Wellcome Trust [206211 to TL, 073980 to AIL, 105024 to AIL, 098503 to AIL, 097045 to MG]. TL is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (206211/Z/17/Z).",
year = "2018",
month = "5",
day = "2",
doi = "10.12688/wellcomeopenres.14392.1",
language = "English",
volume = "3",
pages = "1--48",
journal = "Wellcome Open Research",
issn = "2398-502X",
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}

TY - JOUR

T1 - Proteome-wide analysis of protein abundance and turnover remodelling during oncogenic transformation of human breast epithelial cells

AU - Ly, Tony

AU - Endo, Aki

AU - Brenes, Alejandro

AU - Gierlinski, Marek

AU - Afzal, Vackar

AU - Pawellek, Andrea

AU - Lamond, Angus I.

N1 - This work was supported by the Wellcome Trust [206211 to TL, 073980 to AIL, 105024 to AIL, 098503 to AIL, 097045 to MG]. TL is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (206211/Z/17/Z).

PY - 2018/5/2

Y1 - 2018/5/2

N2 - Background: Viral oncogenes and mutated proto-oncogenes are potent drivers of cancer malignancy. Downstream of the oncogenic trigger are alterations in protein properties that give rise to cellular transformation and the acquisition of malignant cellular phenotypes. Developments in mass spectrometry enable large-scale, multidimensional characterisation of proteomes. Such techniques could provide an unprecedented, unbiased view of how oncogene activation remodels a human cell proteome.Methods: Using quantitative MS-based proteomics and cellular assays, we analysed how transformation induced by activating v-Src kinase remodels the proteome and cellular phenotypes of breast epithelial (MCF10A) cells. SILAC MS was used to comprehensively characterise the MCF10A proteome and to measure v-Src-induced changes in protein abundance across seven time-points (1-72 hrs). We used pulse-SILAC MS ( Boisvert et al., 2012), to compare protein synthesis and turnover in control and transformed cells. Follow-on experiments employed a combination of cellular and functional assays to characterise the roles of selected Src-responsive proteins.Results: Src-induced transformation changed the expression and/or turnover levels of ~3% of proteins, affecting ~1.5% of the total protein molecules in the cell. Transformation increased the average rate of proteome turnover and disrupted protein homeostasis. We identify distinct classes of protein kinetics in response to Src activation. We demonstrate that members of the polycomb repressive complex 1 (PRC1) are important regulators of invasion and migration in MCF10A cells. Many Src-regulated proteins are present in low abundance and some are regulated post-transcriptionally. The signature of Src-responsive proteins is highly predictive of poor patient survival across multiple cancer types. Open access to search and interactively explore all these proteomic data is provided via the EPD database ( www.peptracker.com/epd).Conclusions: We present the first comprehensive analysis measuring how protein expression and protein turnover is affected by cell transformation, providing a detailed picture at the protein level of the consequences of activation of an oncogene.

AB - Background: Viral oncogenes and mutated proto-oncogenes are potent drivers of cancer malignancy. Downstream of the oncogenic trigger are alterations in protein properties that give rise to cellular transformation and the acquisition of malignant cellular phenotypes. Developments in mass spectrometry enable large-scale, multidimensional characterisation of proteomes. Such techniques could provide an unprecedented, unbiased view of how oncogene activation remodels a human cell proteome.Methods: Using quantitative MS-based proteomics and cellular assays, we analysed how transformation induced by activating v-Src kinase remodels the proteome and cellular phenotypes of breast epithelial (MCF10A) cells. SILAC MS was used to comprehensively characterise the MCF10A proteome and to measure v-Src-induced changes in protein abundance across seven time-points (1-72 hrs). We used pulse-SILAC MS ( Boisvert et al., 2012), to compare protein synthesis and turnover in control and transformed cells. Follow-on experiments employed a combination of cellular and functional assays to characterise the roles of selected Src-responsive proteins.Results: Src-induced transformation changed the expression and/or turnover levels of ~3% of proteins, affecting ~1.5% of the total protein molecules in the cell. Transformation increased the average rate of proteome turnover and disrupted protein homeostasis. We identify distinct classes of protein kinetics in response to Src activation. We demonstrate that members of the polycomb repressive complex 1 (PRC1) are important regulators of invasion and migration in MCF10A cells. Many Src-regulated proteins are present in low abundance and some are regulated post-transcriptionally. The signature of Src-responsive proteins is highly predictive of poor patient survival across multiple cancer types. Open access to search and interactively explore all these proteomic data is provided via the EPD database ( www.peptracker.com/epd).Conclusions: We present the first comprehensive analysis measuring how protein expression and protein turnover is affected by cell transformation, providing a detailed picture at the protein level of the consequences of activation of an oncogene.

KW - Biomarker

KW - Cancer

KW - Epigenetic

KW - Polycomb Repressive Complex (PRC)

KW - Protein half-life

KW - Serine protease inhibitor (Serpin)

KW - Src Kinase

KW - Time-lapse Proteomics

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U2 - 10.12688/wellcomeopenres.14392.1

DO - 10.12688/wellcomeopenres.14392.1

M3 - Article

C2 - 29904729

VL - 3

SP - 1

EP - 48

JO - Wellcome Open Research

JF - Wellcome Open Research

SN - 2398-502X

M1 - 51

ER -