Abstract
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.
Highlights
From the ‡Institute of Molecular Cancer Research, University of Zurich, Zurich 8057, Switzerland; §Functional Genomics Center of the University of Zurich and ETH, Zurich 8057, Switzerland; ¶Gastroenterology and Endoscopy Unit, Hospital of Cremona, Cremona 26100, Italy; ʈUniversity of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75211
Proteomic Analysis of Human Colorectal Tissues and Colon Cell Lines—We used a quantitative-MS-based discovery strategy to explore the proteome of human colorectal tissues and colon cell lines
When principal component analysis was performed on the expression intensity values of the 1496 nonredundant proteins expressed and quantified in all tissues and cell lines (i.e. those representing the intersection of the tissue (n ϭ 10,452) and cell line (n ϭ 5056) protein sets reported in Table II), colon cancer cell lines clustered with adenomas, whereas human colon epithelial cells (HCEC) cells were closer to the normal mucosa samples (Fig. 3C)
Summary
Human Tissue Samples and Cell Lines—Human colorectal tissues were prospectively collected from patients undergoing colonoscopy in the Istituti Ospitalieri of Cremona, Italy. The 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. The absolute concentrations of D-sorbitol, ␣ and  D-fructose, and ␣ and  D-glucose were calculated on the basis of the ratio of the intensity of specific fragments originating from the unlabeled compound to that of the added labeled analog (internal standard). These concentrations were used to estimate intracellular levels per milligram of tissue (adenoma versus normal mucosa). The relative concentration of lactate was estimated from the ratio of the intensity of specific fragments originating from the unlabeled compound to that of the added 13C1-sorbitol (internal standard)
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