Sorbitol Dehydrogenase Overexpression and Other Aspects of Dysregulated Protein Expression in Human Precancerous Colorectal Neoplasms: A Quantitative Proteomics Study
2014; Elsevier BV; Volume: 13; Issue: 5 Linguagem: Inglês
10.1074/mcp.m113.035105
ISSN1535-9484
AutoresAnuli C. Uzozie, Paolo Nanni, Teresa Staiano, Jonas Grossmann, Simon Barkow‐Oesterreicher, Jerry W. Shay, Amit K. Tiwari, Federico Buffoli, Endre Laczkó, Giancarlo Marra,
Tópico(s)Bone and Dental Protein Studies
ResumoColorectal adenomas are cancer precursor lesions of the large bowel. A multitude of genomic and epigenomic changes have been documented in these preinvasive lesions, but their impact on the protein effectors of biological function has not been comprehensively explored. Using shotgun quantitative MS, we exhaustively investigated the proteome of 30 colorectal adenomas and paired samples of normal mucosa. Total protein extracts were prepared from these tissues (prospectively collected during colonoscopy) and from normal (HCEC) and cancerous (SW480, SW620, Caco2, HT29, CX1) colon epithelial cell lines. Peptides were labeled with isobaric tags (iTRAQ 8-plex), separated via OFFGEL electrophoresis, and analyzed by means of LC-MS/MS. Nonredundant protein families (4325 in tissues, 2017 in cell lines) were identified and quantified. Principal component analysis of the results clearly distinguished adenomas from normal mucosal samples and cancer cell lines from HCEC cells. Two hundred and twelve proteins displayed significant adenoma-related expression changes (q-value < 0.02, mean fold change versus normal mucosa ±1.4), which correlated (r = 0.74) with similar changes previously identified by our group at the transcriptome level. Fifty-one (∼25%) proteins displayed directionally similar expression changes in colorectal cancer cells (versus HCEC cells) and were therefore attributed to the epithelial component of adenomas. Although benign, adenomas already exhibited cancer-associated proteomic changes: 69 (91%) of the 76 protein up-regulations identified in these lesions have already been reported in cancers. One of the most striking changes involved sorbitol dehydrogenase, a key enzyme in the polyol pathway. Validation studies revealed dramatically increased sorbitol dehydrogenase concentrations and activity in adenomas and cancer cell lines, along with important changes in the expression of other enzymes in the same (AKR1B1) and related (KHK) pathways. Dysregulated polyol metabolism might represent a novel facet of metabolome remodeling associated with tumorigenesis. Colorectal adenomas are cancer precursor lesions of the large bowel. A multitude of genomic and epigenomic changes have been documented in these preinvasive lesions, but their impact on the protein effectors of biological function has not been comprehensively explored. Using shotgun quantitative MS, we exhaustively investigated the proteome of 30 colorectal adenomas and paired samples of normal mucosa. Total protein extracts were prepared from these tissues (prospectively collected during colonoscopy) and from normal (HCEC) and cancerous (SW480, SW620, Caco2, HT29, CX1) colon epithelial cell lines. Peptides were labeled with isobaric tags (iTRAQ 8-plex), separated via OFFGEL electrophoresis, and analyzed by means of LC-MS/MS. Nonredundant protein families (4325 in tissues, 2017 in cell lines) were identified and quantified. Principal component analysis of the results clearly distinguished adenomas from normal mucosal samples and cancer cell lines from HCEC cells. Two hundred and twelve proteins displayed significant adenoma-related expression changes (q-value < 0.02, mean fold change versus normal mucosa ±1.4), which correlated (r = 0.74) with similar changes previously identified by our group at the transcriptome level. Fifty-one (∼25%) proteins displayed directionally similar expression changes in colorectal cancer cells (versus HCEC cells) and were therefore attributed to the epithelial component of adenomas. Although benign, adenomas already exhibited cancer-associated proteomic changes: 69 (91%) of the 76 protein up-regulations identified in these lesions have already been reported in cancers. One of the most striking changes involved sorbitol dehydrogenase, a key enzyme in the polyol pathway. Validation studies revealed dramatically increased sorbitol dehydrogenase concentrations and activity in adenomas and cancer cell lines, along with important changes in the expression of other enzymes in the same (AKR1B1) and related (KHK) pathways. Dysregulated polyol metabolism might represent a novel facet of metabolome remodeling associated with tumorigenesis. Colorectal cancer ranks third among the world's high-incidence cancers and is a leading cause of cancer-related death among older adults (1.Jemal A. Bray F. Center M.M. Ferlay J. Ward E. Forman D. Global cancer statistics.CA Cancer J. Clin. 2011; 61: 69-90Crossref PubMed Scopus (30254) Google Scholar, 2.Siegel R. Naishadham D. Jemal A. Cancer statistics, 2013.CA Cancer J. Clin. 2013; 63: 11-30Crossref PubMed Scopus (11509) Google Scholar). In the United States alone, projections for 2013 include 102,480 new cases and 50,830 deaths (2.Siegel R. Naishadham D. Jemal A. Cancer statistics, 2013.CA Cancer J. 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We therefore decided to explore the proteome of a relatively large series of these precancerous lesions (each with a paired sample of normal colon mucosa) using quantitative shotgun MS with the widely used iTRAQ 1The abbreviations used are: iTRAQ, isobaric tags for relative and absolute quantification; HCEC, human colon epithelial cell; FDR, false discovery rate; GO, Gene Ontology; INHAT, inhibitor of acetyltransferases; MS/MS, tandem mass spectrometry; PSM, peptide spectra match; emPAI, exponentially modified protein abundance index; SORD, sorbitol dehydrogenase; TBST, Tris-buffered saline with Tween 20. 1The abbreviations used are: iTRAQ, isobaric tags for relative and absolute quantification; HCEC, human colon epithelial cell; FDR, false discovery rate; GO, Gene Ontology; INHAT, inhibitor of acetyltransferases; MS/MS, tandem mass spectrometry; PSM, peptide spectra match; emPAI, exponentially modified protein abundance index; SORD, sorbitol dehydrogenase; TBST, Tris-buffered saline with Tween 20. peptide labeling technique (21.Besson D. 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Adenoma-related protein expression variations specific to the epithelial compartments of these lesions were identified with a novel approach, which involved comparing the human tissue proteome with that of colon epithelial cell lines. The results of these studies revealed several protein expression changes previously documented only in advanced colorectal cancers. They also disclosed several novel changes with potentially important roles in early-stage large bowel tumorigenesis, including the marked up-regulation of a key enzyme in the polyol pathway. Human colorectal tissues were prospectively collected from patients undergoing colonoscopy in the Istituti Ospitalieri of Cremona, Italy. Approval was obtained from the local ethics committee, and tissues were used in accordance with the Declaration of Helsinki. Each donor provided written informed consent to sample collection, data analysis, and publication of the findings. Progressive numbers were assigned to each patient to protect human confidentiality. The series comprised 30 colorectal adenomas, each with a paired sample of normal mucosa from the same colon segment, >2 cm from the lesion. Tissues were collected endoscopically, promptly frozen in liquid nitrogen, and stored at −80 °C. Five colorectal cancer cell lines (HT29, Caco2, CX1, SW480, and SW620) were obtained from the Zurich Cancer Network's Cell Line Repository. All had been recently purchased from the American Tissue Culture Collection (Teddington, UK) and were certified as mycoplasma infection free. We cultured Caco2 and CX1 cells in Dulbecco's modified Eagle's medium; HT29 cells in McCoy's medium; and SW480 and SW620 cells in RPMI 1640 medium supplemented with 10% fetal bovine serum, l-glutamine, and 1% penicillin-streptomycin (Sigma, St. Louis, MO). The recently established line of immortalized human colon epithelial cells (HCEC) was obtained from J. W. Shay and grown as described elsewhere (23.Roig A.I. Eskiocak U. Hight S.K. Kim S.B. Delgado O. Souza R.F. Spechler S.J. Wright W.E. Shay J.W. Immortalized epithelial cells derived from human colon biopsies express stem cell markers and differentiate in vitro.Gastroenterology. 2010; 138: 1012-1021Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). For MS studies, frozen tissue samples were quickly weighed and homogenized on ice (1 min of grinding, 1 min on ice, 1 min of grinding) in a Wheaton glass borosilicate grinder containing a solution of 100 mm triethylammonium bicarbonate (Sigma, St Louis, MO), 1X Complete EDTA-free Protease Inhibitor Mixture (Roche, Mannheim, Germany), 1 m urea, 5 mm β-glycerophosphate disodium salt hydrate, 1 mm sodium orthovanadate, and 5 mm sodium fluoride (Sigma). The efficiency of cell lysis was microscopically confirmed. The homogenates were then sonicated with a Bioruptor (Diagenode, Denville, NJ) (high power, five 10-s/10-s on/off cycles) and centrifuged (16,000g for 5 min at 4 °C). The supernatant containing the proteins was collected and stored at −80 °C. Cells (grown to >80% confluence in 15-cm2 dishes) were washed in PBS, covered with 250 μl of the buffer used for tissue sample homogenization (see above), detached from the dish with a cell scraper, and homogenized (25 passages through a 25-gauge needle). The efficiency of cell lysis was microscopically confirmed. Sonication and centrifugation were repeated as described above, and the protein concentration was determined via Bradford assay. Prior to MS analysis, a 5-μg sample of each protein extract was subjected to one-dimensional gel electrophoresis on a 12% bisacrylamide gel to assess protein integrity and extraction protocol reproducibility. The entire proteomic workflow, from tissue/cell processing to statistical analysis, is summarized in Fig. 1 and described in detail in the next five subsections. For sorbitol dehydrogenase (SORD) assays (see below), >80% confluent cells were washed in PBS and covered with a solution consisting of 100 mm triethanolamine (Sigma) and 1X Complete EDTA-free Protease Inhibitor Mixture (Roche). (A simple buffer was used to reduce the risk of introducing anti-enzymatic substances into our extract.) Cells were then scraped and homogenized with 25 passages through a 25-gauge needle. Tissue samples were weighed and homogenized in a Wheaton glass borosilicate grinder containing the buffer described above. After centrifugation (16,000 g, 4 °C, 5 min), the supernatant was aliquoted and stored at −80 °C. Protein concentration was measured via Bradford assay. iTRAQ 8-plex experiments were performed to analyze tissue extracts (10 experiments) and cell-line extracts (1 experiment) (Fig. 1). Labeling efficiency and relative quantitation accuracy were assessed with the aid of two reference protein extract mixtures: one for tissue samples (pooled extracts from three normal tissues and three adenomas) and one for cell lines (pooled aliquots of each of the six cell line extracts). Fifty micrograms of protein per sample were used for each iTRAQ channel. Tryptic digestion (10% w/w, sequencing-grade modified trypsin, Promega, Madison, WI) and iTRAQ 8-plex labeling (AB Sciex, Framingham, MA) were performed according to the manufacturers' instructions (2.5-h incubation of samples with iTRAQ labels). For tissue experiments, two iTRAQ labels, 113 and 114, were chosen for the reference mixture, and labels 115/116, 117/118, and 119/121 were used for the three pairs of normal/adenomatous tissues included in each experiment. For the cell line experiment, labels 113 and 114 were used for the reference mixture, and labels 115–121 represented HCEC, HT29, Caco2, CX1, SW480, and SW620 cells, respectively (Fig. 1). After iTRAQ labeling, the samples (for each experiment) were combined, desalted on 500-mg SepPak C18 columns (Millipore, Billerica, MA), dried in a SpeedVac concentrator (Thermo Scientific), and subjected to peptide fractionation. Peptide fractionation was performed according to the manufacturer's protocols with an Agilent 3100 OFFGEL fractionator and 12-well OFFGEL kit (both from Agilent Technologies, Santa Clara, CA). Briefly, samples were resolubilized in 1.8 ml of 1X OFFGEL peptide stock solution containing carrier ampholytes (pH range 3–10), loaded into the wells (150 μl per well), and focused until 20 kV/h was reached with a maximum current of 50 μA. For each experiment, 12 fractions were collected. A 15-μl aliquot of each fraction was acidified with 1.5 μl of a 50% acetonitrile/1% trifluoroacetic acid solution, desalted using ZipTip C18 (Millipore, Billerica, MA), dried, resolubilized in 15 μl of a 0.1% formic acid/3% acetonitrile solution, and analyzed with MS. Peptide samples (4 μl) were analyzed on an LTQ-Orbitrap Velos mass spectrometer (Thermo Fischer Scientific, Bremen, Germany) coupled to a nano-HPLC system (Eksigent Technologies, Dublin, CA). The solvent compositions were 0.2% formic acid and 1% acetonitrile for channel A and 0.2% formic acid and 80% acetonitrile for channel B. Peptides were loaded onto an in-house-made tip column (75 μm × 80 mm) packed with reverse-phase C18 material (AQ, 3 μm, 200 Å, Bischoff GmbH, Leonberg, Germany) and eluted (flow rate, 250 nL/min; solvent B gradient: from 3% to 30% in 62 min, from 30% to 45% in 70 min, and from 45% to 97% in 75 min). Full-scan MS spectra (300–1700 m/z) were acquired at a resolution setting of 30,000 at 400 m/z after accumulation to a target value of 1 × 106. For the eight most intense signals per cycle above a threshold of 1000, both collision-induced dissociation and higher-energy collisional dissociation spectra were acquired in a data-dependent manner (Fig. 1). Collision-induced dissociation scans were recorded in the ion trap (settings: normalized collision energy, 35; maximum injection time, 50 ms; automatic gain control, 1 × 104 ions). For the higher-energy collisional dissociation scans, spectra were recorded at a resolution setting of 7500 at 400 m/z (normalized collision energy, 52; maximum injection time, 125 ms; automatic gain control, 5 × 104 ions). Charge state screening was enabled, and singly charged states were rejected. Precursor masses previously selected for MS/MS were excluded from further selection for 60 s, and the exclusion window was set at 10 ppm. The maximum number of entries in the exclusion list was set at 500. All samples were analyzed in duplicate, and precursors selected in the first run were excluded from fragmentation in the second run. The exclusion list was set on a time window of 4 min and a mass width of 10 ppm. Spectra were acquired using internal lock mass calibration on m/z 429.088735 and 445.120025. As depicted in Fig. 1, Mascot Distiller 2.4.3.3 (Matrix Science, Boston, MA) was used to generate Mascot generic format peak lists. De-isotoping and peak picking were not performed between 112.5 and 121.5 m/z (the range containing iTRAQ reporter ions), and the higher-energy collisional dissociation and collision-induced dissociation spectra were merged by summing. For each of the 11 experiments, the corresponding 24 Mascot generic format peak lists were concatenated and searched, with the aid of Mascot Server 2.3.02 (Matrix Science), against a forward UniProtKB/Swiss-Prot database for human proteins concatenated to a reversed decoyed FASTA database. The concatenated database contained a total of 147,438 proteins with accessions in Gene Ontology–compatible format and 260 common MS contaminants (NCBI taxonomy I.D. 9606, released December 13, 2011). Methylthio (C), iTRAQ 8-plex labeling at the N terminus and lysine were set as fixed modifications, and variable modifications consisted of methionine oxidation and iTRAQ 8-plex labeling of tyrosine. We used the iTRAQ 8-plex-vs114 (Applied Biosystems Zug, Switzerland) quantitation method. The isotope and impurity correction factors used for each iTRAQ label were those provided by the manufacturer. Precursor and fragment tolerances were set at 10 ppm and 0.8 Da, respectively. The enzyme specificity was set to trypsin with an allowance of up to one missed cleavage. Using Mascot internal export scripts, we transformed Mascot DAT files into XML files and parsed them with in-house scripts so that peptide sequences, scores, and intensities of the individual reporter ion channels were reported. Confidently identified and quantified peptides were selected with the following filters: rank 1 (best spectra assignment); ion score, > 15; and presence of iTRAQ intensity values for reporter channels 113 and 114. (These steps are described in the boxes of the lower half of Fig. 1.) Peptide reporter channel intensities were summed for each protein individually using R-scripts. Ratios were built from summed channels (113/114 to 121/114) for all proteins identified in each iTRAQ experiment. False discovery rates (FDRs) (24.Kall L. Storey J.D. MacCoss M.J. Noble W.S. Posterior error probabilities and false discovery rates: two sides of the same coin.J. Proteome Res. 2008; 7: 40-44Crossref PubMed Scopus (210) Google Scholar) were determined at the spectrum, peptide, and protein levels. The results of individual experiments were then merged into one matrix, which was used for statistical analysis in R and Perseus (Version 1.2.7.4). All proteins identified with the same peptide(s) were grouped into families, each of which was identified by a unique protein family number. Ratios of the intensity of each ion channel to that of 114 were converted to base 2 logarithmic values and normalized respectively on the median (which was set at 0), resulting in ratios that followed a Gaussian distribution. Proteins identified on the basis of the same peptide(s) shared the same family number and were represented once in our statistical analysis. The paired t test was used to compare the expression of a given protein in each adenoma and that found in the corresponding sample of normal mucosa. To correct for multiple comparisons, the FDR was controlled with the Benjamini–Hochberg procedure. The average protein-expression fold change in adenomas, relative to the normal mucosa, was then calculated. For this, median normalized ratios for all proteins in each paired adenoma–normal mucosa sample were deconvoluted of the reference standard effects (114) to compute the adenoma versus normal mucosa ratio per protein (deconvoluted fold change, (116/114)/(115/114) = (116/115)) and the mean fold change per protein in all tissue pairs. The Mascot emPAI values for all proteins were included in XML exports for each experiment. Thereafter, the mean Mascot emPAI value was calculated for all proteins. Gene Ontology (GO) annotations and GO terms for proteins in the UniProt/Swiss-Prot database were sourced from the European Bioinformatics Institute. The Scaffold program (Version 3) was used to identify the cellular localizations and biological processes most represented in lists of proteins quantified in tissues and cell lines. The topGO Bioconductor software package in R was used to identify and screen for GO biological process categories displaying enrichment for proteins that were differentially regulated in adenomas (versus normal mucosa) (25.Alexa A. Rahnenfuhrer J. Lengauer T. Improved scoring of functional groups from gene expression data by decorrelating GO graph structure.Bioinformatics. 2006; 22: 1600-1607Crossref PubMed Scopus (1305) Google Scholar). First, we prepared a "universe" comprising all the proteins quantified in our study, each matched to GO terms and annotations. This served as the "background." The "foreground" consisted of the list of significantly dysregulated proteins. The most significant GO terms were scored with the Eliminating Genes (elim) method (25.Alexa A. Rahnenfuhrer J. Lengauer T. Improved scoring of functional groups from gene expression data by decorrelating GO graph structure.Bioinformatics. 2006; 22: 1600-1607Crossref PubMed Scopus (1305) Google Scholar). Proteins were separated on a 10% SDS-polyacrylamide gel and transferred to a hydrophobic polyvinylidene difluoride membrane (GE Healthcare, Amersham Biosciences Hybond-P PVDF membrane, Pittsburgh, PA) according to standard protocols (26.Cattaneo E. Laczko E. Buffoli F. Zorzi F. Bianco M.A. Menigatti M. Bartosova Z. Haider R. Helmchen B. Sabates-Bellver J. Tiwari A. Jiricny J. Marra G. Preinvasive colorectal lesion transcriptomes correlate with endoscopic morphology (polypoid vs. nonpolypoid).EMBO Mol. Med. 2011; 3: 334-347Crossref PubMed Scopus (31) Google Scholar). After 1 h of blocking with 5% nonfat dry milk in TBS with 1% Tween 20 (milk–TBST), membranes were incubated overnight with the primary antibody (anti-SORD (HPA040260, Sigma); anti-aldose reductase, AKR1B1 (GTX113381, GeneTex, Irvine, CA); anti-ketohexokinase, KHK (GTX109591, GeneTex)) diluted 1:1000 in milk–TBST and washed once with milk–TBST (20 min) and twice with TBST (20 min). After 1 h of incubation in horseradish-peroxidase-conjugated secondary antibody (anti-rabbit IgG, GE Healthcare) diluted 1:5000 in milk–TBST, membranes were washed once with milk (20 min) and twice with TBST (20 min). Enhanced chemiluminescence (Amersham Biosciences, catalog no. RPN2106) was used to detect immunoreactive proteins. HT29 and HCEC cells were seeded (3 × 105 per well) on 22-mm × 22-mm cover slips in six-well plates and grown under standard conditions until cells reached 70% to 80% confluence. Cells were then washed twice with PBS and fixed in ethanol:methanol solution (50:50) for 10 min at room temperature. Fixed cells were permeabilized (10 min with 0.25% Triton X-100), blocked (30 min in 10% goat serum (X0907, Dako, Glostrup, Denmark)), and incubated with primary antibody (rabbit polyclonal anti-SORD, HPA040260, Sigma, 1:100) for 18 h at 4 °C. After three washes with PBS, the cells were incubated for 1 h with secondary antibody conjugated to polymer-HRP anti-rabbit (Dako, EnVision+ System-HRP, catalog no. K4010). They were then washed three times in PBS and incubated for 15 min in the substrate-chromogen 3,3-diaminobenzidine tetrahydrochloride (Dako, EnVision+ System-HRP, catalog no. K4010). Cells were washed quickly with PBS and mounted on slides (EUKITT, O. Kindler, GmbH, Freiburg, Germany) for light microscopy (Leica Microsystems GmbH, Wetzlar, Germany). Images were examined and recorded with Leica Application Suite (V3.3.0) software. SORD immunohistochemistry was performed as previously described (27.Truninger K. Menigatti M. Luz J. Russell A. Haider R. Gebbers J.O. Bannwart F. Yur
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