Endoplasmic Reticulum Chaperones Are Potential Active Factors in

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Endoplasmic reticulum chaperones are potential active factors in thyroid tumorigenesis Elena Uyy, Viorel I. Suica, Raluca M. Boteanu, Dana Manda, Ancuta E. Baciu, Corin Badiu, and Felicia Antohe J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00567 • Publication Date (Web): 01 Aug 2016 Downloaded from http://pubs.acs.org on August 10, 2016

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Journal of Proteome Research

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Endoplasmic reticulum chaperones are potential active factors in thyroid tumorigenesis Elena Uyy1, Viorel I. Suica1, Raluca M. Boteanu1, Dana Manda2, Ancuta E. Baciu2,3, Corin Badiu2, Felicia Antohe1* 1

Institute of Cellular Biology and Pathology “Nicolae Simionescu”, Bucharest, Romania

2

National Institute of Endocrinology “C.I. Parhon”, Bucharest, Romania

3

University of Bucharest, Faculty of Physics, Magurele, Romania

KEYWORDS: mass spectrometry, thyroid cancer biomarkers, endoplasmic reticulum chaperone


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ABSTRACT: The study aimed to evaluate the proteomic changes in benign follicular adenoma versus malignant follicular variant of papillary thyroid carcinoma. Tumor and non-tumor adjacent samples were analyzed by liquid nano-chromatography mass spectrometry and protein abundance was evaluated by label free quantification. Western blotting and quantitative real time polymerase chain reaction were used to validate and complement the mass spectrometry data. The results demonstrated deregulated expression of four endoplasmic reticulum chaperones (78 kDa glucoseregulated protein, endoplasmin, calnexin, protein disulfide-isomerase A4), of glutathione peroxidase 3 and thyroglobulin, all of them involved in thyroid hormone synthesis pathway. The altered tissue abundance of endoplasmic reticulum chaperones in thyroid cancer was correlated with serum expression levels. The identified proteins significantly discriminate between adenoma and carcinoma in both thyroid tissue and corresponding sera. Data are available via ProteomeXchange with identifier PXD004322.

INTRODUCTION Thyroid hormones biosynthesis is essential for maintaining body homeostasis and is accomplished in follicle, the thyroid gland’s structural and functional unit. Benign follicular thyroid adenoma (FTA) and follicular variant of papillary thyroid carcinoma (FVPTC), a malignant lesion, originate in the follicular cells of the thyroid, grow slowly and present many common clinical diagnostic characteristics. Lately, due to high resolution ultrasound imaging and fine needle aspiration biopsy technique, the thyroid tumors are easily detected. However, the correct diagnosis remains a critical medical issue. These types of neoplasms occur mainly in 40 year old female patients. In 2010, the incidences of thyroid cancer in men and women in the United States of America were 7.63 and 21.15, respectively, per 100,000 individuals, with increasing values in women. By 2018 it is considered that the thyroid neoplasia will become the second most common type of neoplasia in women.1,2


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Previously, several genes (e.g., TSH receptor, BRAF, Ras, RET/PTC, PAX8/PPARγ and p53) have been studied as candidates in the development of thyroid cancer.3-7 However, the mRNA expression has not always correlated with the protein level and the RNA-based studies failed to identify protein variants and post-translational modifications that affect the tumor biology.8 Proteomic approaches have been developed to identify and quantify thousands of proteins relevant for cancer.9 Various thyroid proteins are differentially expressed between healthy individuals, as well as between normal and benign or malignant thyroid neoplastic tissues. Thus, thyroid proteomics can be used to identify the patient's tumor profile with impact upon tumor development, progression and adequate personalized treatment. Studies of the thyrocyte and thyroid cancer cell proteome are only on the verge of emergence compared with the genome and transcriptome ones.8 We investigated the proteomic profile of a group of patients with thyroid neoplasia, taking into account both individual variations and thyroid tumors alterations. In the present study, we applied shotgun nano-flow liquid chromatography (nLC) coupled with high performance tandem mass spectrometry (MS/MS), identifying and quantifying proteins that are significantly altered in the thyroid hormone synthesis pathway in benign follicular thyroid adenoma (FTA) versus follicular variant of papillary thyroid carcinoma (FVPTC). EXPERIMENTAL SECTION Reagents All chemicals used for nLC-MS/MS experiments were of liquid chromatography or mass spectrometry grade. Urea, sodium deoxycholate (DOC), trisma hydrochloride (Tris-HCl), DLdithiothreitol (DTT), iodoacetamide (IAA), N-acetyl-L-cysteine (NAC), ammonium bicarbonate, sodium-dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) reagents and all of the solvents were provided by Sigma-Aldrich (Missouri, USA), unless otherwise specified. Sequencing grade modified trypsin was acquired from Promega (Wisconsin, USA). Protease inhibitor cocktail Complete tablets were purchased from Roche (Mannheim, Germany). The protein quantification kit


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was The Precision Red Advanced Protein Assay Reagent 2 (Cytoskeleton, Colorado, USA). Antibodies and their sources were as follows: rabbit monoclonal anti-GRP94 (endoplasmin) clone EPR3988, rabbit polyclonal anti-GRP78 (78 kDa glucose-regulated protein or BiP) from Abcam (Cambridge, UK) and anti-rabbit IgG peroxidase conjugates from Sigma-Aldrich. The enhanced chemiluminescence ECL Western Blotting Substrate detection reagent kit was supplied by Thermo Scientific (Illinois, USA). C18 Sep Pak solid phase extraction columns were acquired from Waters Corporation (Massachusetts, USA). Thyroid Tissue Samples The tumor and adjacent non-tumor thyroid tissue fragments from the resected thyroid glands were collected and analyzed from 18 female (17-66 years old) from a cohort of 76 patients hospitalized from January to June 2013, in the National Institute of Endocrinology CI Parhon, Bucharest, Romania. All patients gave their written informed consent and the study was approved by the Ethics Committee in agreement with the Helsinki declaration and all the current Romanian and institutional regulations. Based on clinical evaluation and histological examination the patients were divided into two groups, one with benign follicular thyroid adenoma (FTA, n=9, average age 52±9 years) and the other with follicular variant of papillary thyroid carcinoma (FVPTC, n=9, average age 45±16 years). Table 1 lists the tumor type and classification according to the international standard guidelines10 for all patients enrolled in this study. Control thyroid tissue (CFTA and CFVPTC) was collected by the hospital pathologist and defined as adjacent to the site of lesion with no histological markers of abnormal pathology. The harvested samples were frozen in phosphate buffered saline (PBS) containing 10 µg/ml protease inhibitors mixture. Protein homogenates were prepared on ice from 30 mg of thyroid tissue fragments suspended in 0.3 ml highly denaturant buffer containing 8M urea, 1% DOC and 100 mM Tris-HCl (pH 7.5), with the use of a rotor-stator mechanical homogenizer (5 minutes at high speed). The solubilization was conducted through powerful vortexing for 20 min on ice, followed by centrifugation (11,000 xg,


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10 min, 4˚C). Protein quantification was performed and the supernatant protein fraction was analyzed by biochemical techniques, mass spectrometry and Western blotting (WB) methods. 1D Electrophoresis Equal amounts of protein (40 µg) were loaded and separated by 1D electrophoresis on 10 % SDS polyacrylamide gels (SDS-PAGE). Each gel was loaded with 10 µl of molecular weight Prestained SDS-PAGE Low Range Standards marker (Bio-Rad Laboratories, CA, USA). Dilutions of the samples were mixed in 1:1 proportion with sample buffer (2 % SDS, 0.25 M Tris, 2 mM EDTA, 10 % glycerol, 1 % 2-Mercaptoethanol, 0.5 % Bromophenol blue). The protein denaturation was performed under reducing conditions at 98º C for 3 minutes. All electrophoresis gels were run at 120 V/gel. The proteins from sera samples were identically processed except for the loading volumes (2 µl serum /lane). Immunoblotting assay The SDS-PAGE separated proteins were transferred onto nitrocellulose membrane and analyzed by Western blotting (WB) assay. After checking the proteins' uniform electro-transfer by Ponceau S staining, the membranes were vigorously washed and blocked with 2 % bovine serum albumin (BSA) in TRIS-buffered saline (TBS) containing 0.05 % Tween 20, pH 7.6. The blots were exposed for 2 h to the primary BiP or GRP94 antibodies in TBS with 1 % BSA followed by the appropriate IgG coupled with horse radish peroxidase (IgG–HRP) secondary antibodies for 1 h. The negative controls were performed in similar conditions with non-immune rabbit IgG instead of the first antibody. The reaction product was detected with the ECL Western Blotting Substrate kit and quantified by densitometry with Scion Image analysis freeware. Sera and thyroid tissue sample preparation for mass spectrometric analysis The serum samples (70 mg protein), harvested before surgery, were processed for low abundance protein enrichment using the combinatorial hexapeptide ligand libraries within the ProteoMiner Large-Capacity Kit (Bio-Rad Laboratories, Hercules, CA, USA). The one-step elution


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was performed using 6 M guanidine-HCl, pH 6, which dissociates all types of interactions, as previously specified.11 The resulting protein samples were concentrated up to a volume of ~130 µL for treatment with Zeba Spin desalting columns (Thermo Scientific) with a molecular weight cut-off of 7 kDa. We performed molecular weight fractionation with liquid phase recovery using the GELFREE 8100 fractionation system (Expedeon Inc., California, USA) and 10 % Tris-Acetate cartridge kit, which separated and collected 12 fractions of proteins ranging from 3.5 to 100 kDa, with optimized resolution at 15-100 kDa, using the vendor recommendations. Briefly, 350 µg of the salt-free samples were combined with a reducing agent (20 mM DTT) and kit buffer, heat-denatured at 50°C for 10 min and selective channels were run using negative polarity for a total of 125 min. All fractions of sera proteins and the tissue thyroid samples (after solubilization) were processed as previously mentioned.12 Briefly, the samples were purified by acetone precipitation using four times the sample volume of cold (-20˚ C) acetone, after which the samples were incubated for 60 min at -20˚ C. After 20 min centrifugation (at 20,000 xg), the protein pellet was suspended in a freshly prepared denaturant buffer containing 8 M urea, 0.1 M Tris-HCl, 0.1 mM EDTA and 20 mM DTT, pH 8.8. The mixture was maintained for 60 min under agitation at room temperature to reduce the cysteine residues. Alkylation was conducted using 80 mM IAA in 0.1 M Tris-HCl and 0.1 mM EDTA buffer, for 90 min, in the dark, under agitation, at room temperature. Quenching was performed using 80 mM NAC in 0.1 M Tris-HCl and 0.1 mM EDTA buffer, for 30 min, in the dark, under agitation, at room temperature. Before the digestion process, the sample buffer was diluted up to 1 M urea using 50 mM ammonium bicarbonate (pH 8.8) and DOC (1 % final concentration). Proteolysis was performed overnight at 37˚ C with sequencing grade modified trypsin, under agitation, using a 1:20 enzyme to substrate quantitative ratio. After 14 h, the resulted peptide mixtures were acidified with formic acid to pH 2-3 for trypsin inhibition and DOC precipitation. DOC was discarded following a 20 min, 20,000 x g centrifugation. The desalting step was conducted using Sep Pek C18 columns. The purified peptides were eluted using 0.1 % formic acid in 50 %


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acetonitrile in water. The peptides were dried using the Concentrator plus system from Eppendorf (Hamburg, Germany) and stored at -80˚ C until nLC-MS/MS analysis. Prior to the nLC separation, the peptides were suspended in 0.1 % formic acid, 5 % acetonitrile solution to 0.5 µg/µl final concentration, using an ultra-sonication bath. nLC-MS/MS analysis, label free quantification and data mining Analitical procedures Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) experiments were performed using the EASY –nLC II nano system coupled to the LTQ Orbitrap Velos Pro mass spectrometer (Thermo Scientific). The instrument operating software was Xcalibur 2.2 SP1 build 48 and LTQ Orbitrap Velos MS 2.7.01103 SP1. Three replicates from each sample were successively loaded into the trap and analytical columns. Peptides were eluted using a 90 min. 2–35 % solvent B (acetonitrile with 0.1 % formic acid) over A (water with 0.1 % formic acid) separation gradient (125 min total chromatographic method and MS acquisition time) at a flow rate of 300 nL/min. Dynamic nano-electrospray source was utilized with stainless steel nano-bore emitters (40 mm length, OD 1/32”). The mass spectrometer was operated in a top 6 data-dependent configuration (for tissue sample) and in a top 12 data dependent configuration (for serum samples) at 60k resolving power for a full scan, with monoisotopic precursor selection enabled and mass correction using lock mass method, across the 50-2000 m/z domain (for tissue sample) and 350-1700 m/z domain (for sera samples). The analyses were carried out with collision induced dissociation (CID) fragmentation mode (with the m/z width of precursor window set to 2 and normalized collision energy of 35). Bioinformatics and statistical approaches Protein identification was performed using Proteome Discoverer 1.4 software (Thermo Scientific). The search engine was Mascot 2.4.1 (Matrix Science, London, UK) and the taxonomy was set on Homo sapiens organism in UniProtKB/SwissProt fasta database, build 04.2013. A maximum of 2 missed cleavage sites was allowed. The mass tolerance for the precursor ion was set


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on 10 ppm and for the fragment on 0.8 Da. Oxidation of methionine and deamidation of asparagine and glutamine were enabled as dynamic modifications while carbamidometylation of cysteine was set as fixed modification. The search workflow contained also a Percolator validation node13,14 using a decoy database search with a strict False Discovery Rate (FDR) target lower than 5 %. Proteome Discoverer Daemon 1.4 was utilized for performing raw files combination as well as batch searches based on 3 technical replicates for each biological condition. The label free quantification on the precursor level was performed with SIEVE 2.1 software (Thermo Scientific) as previously described.15 This software uses mass spectra alignment over retention time for different experimental conditions to detect features - frames (well defined rectangular regions in the m/z versus retention time plane) that change in the different biological conditions. The total ion current (TIC) normalization16 of all frames from each mass spectrometry experiment was performed to convert the spectra within the same intensity range. The normalized frames or peptide precursor intensities were filtered by coefficient of variation (

Endoplasmic Reticulum Chaperones Are Potential Active Factors in

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