Elsevier

Journal of Proteomics

Volume 124, 21 June 2015, Pages 50-78
Journal of Proteomics

Determination of the protein expression profiles of breast cancer cell lines by quantitative proteomics using iTRAQ labelling and tandem mass spectrometry

https://doi.org/10.1016/j.jprot.2015.04.018Get rights and content

Highlights

  • Overexpressed network involved glycolysis, DNA topology, and splicing, among others

  • The subexpressed protein network mainly affected was oxidative phosphorylation

  • BAG6, DDX39, ANXA8 and COX4 are proposed as putative biomarkers in breast cancer

Abstract

Breast cancer is the principal cancer in women worldwide. Although there are serum tumor markers such as CEA and HER2, they are detected in advanced stages of the disease and used as progression and recurrence markers. Therefore, there is a necessity for the identification of new markers that might lead to an early detection and also provide evidence of an effective treatment. The aim of this work was to determine the differential protein expression profiles of four breast cancer cell lines in comparison to a normal control cell line by iTRAQ labelling and tandem mass spectrometry, in order to identify putative biomarkers of the disease. We identified 1,020 iTRAQ-labelled polypeptides with at least one peptide identified with more than 95% in confidence. Overexpressed polypeptides in all cancer cell lines were 78, whilst the subexpressed were 128. We categorised them with PANTHER program into biological processes, being the metabolic pathways the most affected. We detected six groups of proteins with the STRING program involved in DNA topology, glycolysis, translation initiation, splicing, pentose pathway, and proteasome degradation. The main subexpressed protein network included mitochondrial proteins involved in oxidative phosphorylation. We propose BAG6, DDX39, ANXA8 and COX4 as putative biomarkers in breast cancer.

Biological significance

We report a set of differentially expressed proteins in the MCF7 and T47D (Luminal A), MDA-MB-231 (Claudin low) and SK-BR-3 (HER2+) breast cancer cell lines that have not been previously reported in breast cancer disease. From these proteins, we propose BAG6, DDX39, ANXA8 and COX4 as putative biomarkers in breast cancer. On the other hand, we propose sets of unique polypeptides in each breast cancer cell line that can be useful in the classification of different subtypes of breast cancer.

Introduction

In the last twenty years, great technological advances have changed all biology search fields. These advances include high throughput technologies, such as DNA microarrays, automated DNA sequencing systems, lab-on-a-chip devices, mass spectrometry, the huge amount of information contained in genome and metagenome databases, and their easy accessibility by using personal computers through the internet. All together have raised new forefronts for searching in biology including genomics, proteomics, metabolomics, glycomics, and epigenomics, among many others. Several of these leading search fields have been involved in the discovery of new molecular biomarkers for a number of human disorders including genetic, neurodegenerative, diabetes and cancer.

Breast cancer is the most common type of cancer in women worldwide and the major cause of death by cancer in females. It has been estimated 1.67 million of new cases of breast cancer in women, corresponding to 25% of all cases of cancer diagnosed in 2012 [1]. From the total number of breast cancer deaths in women, about 60% occurred in developing countries. Current estimations for breast cancer indicate that there might be an increase of 55% and 62% in new cases and deaths worldwide by the year 2035, respectively. Moreover, estimations for this cancer in Latin America and the Caribbean indicate that the increase might be as high as 60% in the total number of new cases and approximately up to 89% of deaths by the year 2035 [1]. Therefore, breast cancer is a serious health concern in the world that needs to be diagnosed opportunely.

The diagnosis of this disease is mainly clinical examination followed by screening methods such as digital mammography, ultrasound, magnetic resonance imaging, and biopsy analysis. Some tumor markers are a complementary tool for diagnosis and prognosis. According to the Working Group and Biomarkers Consortium from the National Institutes of Health, biomarkers are molecules that are useful as indicators of normal or pathological conditions in cells, tissues or organisms, or pharmacological responses to therapy [2]. In the case of cancer biomarkers, they are classified as: i) Diagnostic or screening biomarkers for the identification of individuals with a certain type of cancer with high specificity and sensibility; ii) Prognostic biomarkers, to predict the likely course of the disease and its recurrence, once this condition has been established; iii) Predictive biomarkers, which are principally based on mRNA expression profiles and that can be used in predicting disease outcome or the likely response to one or several drugs before starting treatment and, consequently, classifying patients as responders or non-responders to treatment [3], [4]. In the case of breast cancer, some molecules have been used for the determination of recurrence of this disease, including the carcinoembryonic antigen (CEA) and carcinoma antigen 15-3 (CA 15-3) [5]. Her2/Neu/ErbB-2 has also been used. Her2/Neu is a membrane receptor characterized by three domains: the intracellular tyrosine kinase domain, the transmembrane domain and the extracellular domain (ECD). Her2-ECD can be released by shedding and consequently be found in serum. Several groups have used the presence of the Her2-ECD as marker for relapse or prognosis in breast cancer [6], [7], [8], [9]. However, these markers are not enough to diagnose breast cancer. Therefore, there is an urgent necessity for identifying new putative biomarkers.

The mass spectrometry (MS)-based proteomics has a key role in the identification of biomarkers useful for the diagnosis and prognosis of many diseases including breast cancer. MS-based proteomics involves the separation of either intact proteins or peptides generated by trypsin treatment (or any other) using different techniques, being two-dimensional gel electrophoresis and multidimensional nano-flow HPLC the most used. Finally, peptides are identified by tandem mass spectrometry [10], [11], [12]. In the case of biomarkers, there is a necessity of quantification of the expression levels of polypeptides. Several strategies have been used in the field of breast cancer biomarkers including Stable Isotope Labelling by Amino Acid in Cell Culture (SILAC) [13], [14], [15], [16], [17], [18], 2D-Fluorescence Difference in Gel Electrophoresis (2D-DIGE) [19], [20], [21], and Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) [22], [23], [24], [25], among others.

There are very few reports of quantitative proteomics applied to several breast cancer cell lines using iTRAQ and tandem mass spectrometry. In this paper, we determined the expression profiles of four breast cancer cell lines that are molecularly classified as Luminal A (MCF7 and T47D), Claudin-low (MDA-MB-231) and HER2+ (SK-BR-3) [26], in comparison to a normal cell line (MCF 10A). We identified 1,020 polypeptides labelled with the isobaric tags with at least one identified peptide with a confidence equal or higher than 95%. From this set, 206 proteins showed a change in their expression level in all breast cancer cell lines studied, 78 were overexpressed and 128 subexpressed. Additionally, we identified sets of proteins that were exclusively found in Luminal A, Claudin-low and HER2+ breast cancer cell lines. We discuss the pathways altered in these breast cancer cell lines and propose three panels to be used as putative biomarkers.

Section snippets

Cell lines and cell cultures

MCF7, MDA-MB-231, SK-BR-3 and T47D breast cancer cell lines, and MCF 10A control cell line were obtained from the American Type Culture Collection (Manassas, VA, USA). MCF 10A was cultured in Dulbecco´s Modified Eagle Medium (DMEM)/F-12 supplemented with 10% fetal bovine serum (FBS), 10 ng/mL epidermal growth factor (EGF), 4 μg/mL insulin, 0.4 μg/mL hydrocortisone, 1X L-glutamine, and 1X penicillin-streptomycin solution. MCF7 and MDA-MB-231 cell lines were maintained in DMEM medium, while T47D was

Differential polypeptide expression profiles of breast cancer cell lines

To determine the protein expression profiles of breast cancer cells (MCF7, MDA-MB-231, SK-BR-3 and T47D) in comparison to a normal control cell line (MCF 10A), we used the approach of iTRAQ labelling and tandem mass spectrometry as described in Methods. In the mass spectrometry analysis, most of the ions eluted between 25 and 150 minutes for each sample. We only show the elution patterns for sections 4 to 10 from the IEF strip (Fig. 1A). As examples, we include the MS/MS spectra for LADHFGGK and

Discussion

We found 78 overexpressed proteins and 128 subexpressed polypeptides in common for all breast cancer cell lines. These proteins were analysed with the IPA software in the module of Biomarker filters for the selection of some putative biomarkers in breast cancer. Through IPA, we established three Biomarker filters: Biomarkers I (all tissues, primary cells and all cell lines), Biomarkers II (mammary gland and breast cancer cell lines) and Biomarkers III (all cancer cell lines excluding breast

Conclusions

We found overexpressed proteins implicated in DNA topology, pentose pathway, regulation of translation initiation, proteins involved in degradation by proteasome, splicing and glycolysis in common in all breast cancer cell lines analysed. In relation to the subexpressed polypeptides found in common in all cells studied, they were involved in different mitochondrial processes, being the most affected oxidative phosphorylation. BAG6, DDX39, ANXA8 and COX4 might be putative biomarkers in breast

List of abbreviations

    DMEM

    Dulbecco´s Modified Eagle Medium

    RPMI

    Roswell Park Memorial Institute

    SFB

    fetal bovine serum

    EGF

    epidermal growth factor

    NEAA

    non-essential amino acids

    PBS

    phosphate buffered saline

    GuHCl

    Guanidine hydrochloride

    ABC

    Ammonium bicarbonate

    TEAB

    Triethylammonium bicarbonate

    TCEP

    Tris-(2-carboxyethyl) phosphine

    MMTS

    Methyl methanethiosulfonate

    TFA

    Trifluoroacetic acid

    FA

    Formic acid

    CHAPS

    3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate

Acknowledgements

We thank to Consejo Nacional de Ciencia y Tecnología (Conacyt) and the Instituto de Ciencia y Tecnología del Distrito Federal (ICyTDF) (now transformed in Secretaría de Ciencia Tecnología e Innovación (SECITI) del Distrito Federal), both from Mexico, for the financial support to Dr. Juan Pedro Luna Arias for doing this work. We thank Conacyt for the scholarship to Karla G. Calderón-González during her Ph.D. studies, and the ECOS Mexico-France program for the financial support to Karla G.

References (92)

  • H. Lu et al.

    The identification of potential factors associated with the development of type 2 diabetes: a quantitative proteomics approach

    Mol Cell Proteomics

    (2008)
  • R.M. Neve et al.

    A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes

    Cancer Cell

    (2006)
  • D.R. Littler et al.

    The enigma of the CLIC proteins: Ion channels, redox proteins, enzymes, scaffolding proteins?

    FEBS Lett

    (2010)
  • K.S. Wilson et al.

    Differential gene expression patterns in HER2/neu-positive and -negative breast cancer cell lines and tissues

    Am J Pathol

    (2002)
  • P. Flavin et al.

    RuvBl2 cooperates with Ets2 to transcriptionally regulate hTERT in colon cancer

    FEBS Lett

    (2011)
  • K. Matsumoto et al.

    Gene regulation by Y-box proteins: coupling control of transcription and translation

    Trends Cell Biol

    (1998)
  • GLOBOCAN

    Estimated cancer incidence, mortality and prevalence worldwide in 2012

    (2012)
  • Biomarkers Definitions Working G

    Biomarkers and surrogate endpoints: preferred definitions and conceptual framework

    Clin Pharmacol Ther

    (2001)
  • M. Hamdan
  • L.J. van 't Veer et al.

    Gene expression profiling predicts clinical outcome of breast cancer

    Nature

    (2002)
  • M.J. Duffy

    Serum tumor markers in breast cancer: are they of clinical value?

    Clin Chem

    (2006)
  • W.P. Carney et al.

    Circulating HER2 extracellular domain: A specific and quantitative biomarker of prognostic value in all breast cancer patients?

    Biomark Cancer

    (2013)
  • J.H. Ha et al.

    Serial serum HER2 measurements for the detection of breast cancer recurrence in HER2-positive patients

    J Breast Cancer

    (2014)
  • L. Lam et al.

    Interference-Free HER2 ECD as a Serum Biomarker in Breast Cancer

    J Mol Biomark Diagn

    (2014)
  • H. Steen et al.

    The ABC's (and XYZ's) of peptide sequencing

    Nat Rev Mol Cell Biol

    (2004)
  • T.C. Walther et al.

    Mass spectrometry-based proteomics in cell biology

    J Cell Biol

    (2010)
  • A. Goncalves et al.

    Clinical application of proteomics in breast cancer: state of the art and perspectives

    Med Princ Pract

    (2011)
  • X. Liang et al.

    Quantification of membrane and membrane-bound proteins in normal and malignant breast cancer cells isolated from the same patient with primary breast carcinoma

    J Proteome Res

    (2006)
  • R. Lund et al.

    Efficient isolation and quantitative proteomic analysis of cancer cell plasma membrane proteins for identification of metastasis-associated cell surface markers

    J Proteome Res

    (2009)
  • X. Xu et al.

    Quantitative proteomics study of breast cancer cell lines isolated from a single patient: discovery of TIMM17A as a marker for breast cancer

    Proteomics

    (2010)
  • T. Geiger et al.

    Proteomic portrait of human breast cancer progression identifies novel prognostic markers

    Cancer Res

    (2012)
  • T.C. Lai et al.

    Secretomic and proteomic analysis of potential breast cancer markers by two-dimensional differential gel electrophoresis

    J Proteome Res

    (2010)
  • D.M. Schulz et al.

    Identification of differentially expressed proteins in triple-negative breast carcinomas using DIGE and mass spectrometry

    J Proteome Res

    (2009)
  • H.L. Huang et al.

    Biomarker discovery in breast cancer serum using 2-D differential gel electrophoresis/ MALDI-TOF/TOF and data validation by routine clinical assays

    Electrophoresis

    (2006)
  • J. Ho et al.

    Novel breast cancer metastasis-associated proteins

    J Proteome Res

    (2009)
  • P. Bouchal et al.

    Biomarker discovery in low-grade breast cancer using isobaric stable isotope tags and two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) based quantitative proteomic analysis

    J Proteome Res

    (2009)
  • S.U. Shaheed et al.

    Identification of stage-specific breast markers using quantitative proteomics

    J Proteome Res

    (2013)
  • D.L. Holliday et al.

    Choosing the right cell line for breast cancer research

    Breast Cancer Res

    (2011)
  • H. Mi et al.

    PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees

    Nucleic Acids Res

    (2013)
  • K.G. Calderón-González et al.

    Data set of the protein expression profiles of Luminal A, Claudin-low and overexpressing HER2 + breast cancer cell lines by iTRAQ labelling and tandem mass spectrometry

    Data in Brief

    (2015)
  • T. Stein et al.

    Annexin A8 is up-regulated during mouse mammary gland involution and predicts poor survival in breast cancer

    Clin Cancer Res

    (2005)
  • F. Desmots et al.

    The reaper-binding protein scythe modulates apoptosis and proliferation during mammalian development

    Mol Cell Biol

    (2005)
  • T. Tsukahara et al.

    Scythe/BAT3 regulates apoptotic cell death induced by papillomavirus binding factor in human osteosarcoma

    Cancer Sci

    (2009)
  • S.T. Yong et al.

    A novel, non-apoptotic role for Scythe/BAT3: a functional switch between the pro- and anti-proliferative roles of p21 during the cell cycle

    PLoS One

    (2012)
  • T. Sasaki et al.

    HLA-B-associated transcript 3 (Bat3)/Scythe is essential for p300-mediated acetylation of p53

    Genes Dev

    (2007)
  • V.R. Simhadri et al.

    Dendritic cells release HLA-B-associated transcript-3 positive exosomes to regulate natural killer function

    PLoS One

    (2008)
  • Cited by (30)

    • Applications of proteomics in cancer diagnosis

      2023, Proteomics: A Promising Approach for Cancer Research
    • Proteomic identification of tumor- and metastasis-associated galectin-1 in claudin-low breast cancer

      2021, Biochimica et Biophysica Acta - General Subjects
      Citation Excerpt :

      Published studies of Gal-1 function in claudin-low breast cancers, however, are limited to several in vitro studies of tumor cell migration, invasion and drug resistance using the human cancer cell line MDA-MB-231 [52,53]. Our nano-LC-MS/MS findings, which identified high Gal-1 expression in murine claudin-low T11 tumors, are consistent with two previous studies that also used mass spectrometry-based discovery approaches and reported overexpression of Gal-1 in the human claudin-low cell line, MDA-MB-231, and in a highly metastatic MDA-MB-231 variant [51,54]. However, in contrast to our study, these investigations used cell cultures, not primary or metastatic tumors and Gal-1 identity and overexpression were not confirmed by additional methods.

    • URH49 exports mRNA by remodeling complex formation and mediating the NXF1-dependent pathway

      2020, Biochimica et Biophysica Acta - Gene Regulatory Mechanisms
      Citation Excerpt :

      Thus, we speculated that URH49 may play a fundamental role during the assembling of the Apo-AREX complex, while CIP29 acts as an auxiliary factor. Elevated expression level of URH49 as well as CIP29 has been observed in several cancer types, identifying URH49 as a cancer cell biomarker [54–58]. In addition, URH49 and CIP29 have also been demonstrated to contribute to the progression of several cancers [54,59].

    • Irradiation of rainbow trout at early life stages results in a proteomic legacy in adult gills. Part A; proteomic responses in the irradiated fish and in non-irradiated bystander fish

      2018, Environmental Research
      Citation Excerpt :

      Although the downregulation of alpha-1-antiprotease-like protein / alpha-1 antitrypsin therefore suggests an anti-tumorigenic response, more recently it has been confirmed that alpha-1-antitrypsin can be up- or down-regulated in cancer (Humphries et al., 2014). Similarly while, glycine rich protein was, like in the present investigation, overexpressed in cancer cell lines (Calderón-González et al., 2015), it is downregulated (i.e. the opposite response) in a mouse model susceptible to colon cancer (Ðermadi Bebek et al., 2014) and during cancer progression and metastasis in breast cancer cells (Zeng et al., 2014). Interestingly these changes in dual function proteins appeared to be a unique characteristic of two year old trout which had been irradiated in the early life stages following hatching.

    • Use of nanostructured materials in drug delivery

      2018, Nanobiomaterials: Nanostructured Materials for Biomedical Applications
    View all citing articles on Scopus
    1

    Current Address: Secció de Proteómica, Servei Central de Suport a la Investigació Experimental (SCSIE), Universitat de València, C/Doctor Moliner 50, 46100 Burjassot, València, España.

    2

    Current Address: Centro Nacional de Investigaciones Oncológicas (CNIO), Programa de Genética del Cáncer Humano, Grupo de Genética Humana, Melchor Fernández Almagro 3, 28029 Madrid, España.

    3

    Current Address: Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, C/ Doctor Moliner 50, 46100 Burjassot, València, España.

    View full text