Determination of the protein expression profiles of breast cancer cell lines by quantitative proteomics using iTRAQ labelling and tandem mass spectrometry
Graphical abstract
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.
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- 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.
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Current Address: Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, C/ Doctor Moliner 50, 46100 Burjassot, València, España.