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. 2014 May;13(5):1198-218.
doi: 10.1074/mcp.M113.035105. Epub 2014 Feb 24.

Sorbitol dehydrogenase overexpression and other aspects of dysregulated protein expression in human precancerous colorectal neoplasms: a quantitative proteomics study

Anuli Uzozie  1 , Paolo Nanni, Teresa Staiano, Jonas Grossmann, Simon Barkow-Oesterreicher, Jerry W Shay, Amit Tiwari, Federico Buffoli, Endre Laczko, Giancarlo Marra
Affiliations

Sorbitol dehydrogenase overexpression and other aspects of dysregulated protein expression in human precancerous colorectal neoplasms: a quantitative proteomics study

Anuli Uzozie et al. Mol Cell Proteomics. 2014 May.

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.

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Figures

Fig. 1.
Fig. 1.
Project design and iTRAQ 8-plex labeling scheme. Sample preparation for shotgun MS/MS and important steps in the analysis of proteomic data for the detection of dysregulated proteins in adenomas and colon cancer cell lines. For each experiment on tissue samples, iTRAQ tags were assigned to a duplicate reference (two identical pools of normal and adenoma samples: 113 and 114, respectively), normal tissues (115, 117, 119) and corresponding adenomas (116, 118, 121). The same pattern was repeated in all 10 experiments. In cell line experiments, two identical pools, each comprising all six cell lines, were used as references (113 and 114), and each of the remaining six tags was used to label a single cell line. The data analysis flow chart depicted in this figure is described in “Experimental Procedures.”
Fig. 2.
Fig. 2.
Protein coverage with iTRAQ shotgun analysis in colorectal tissues. A, analysis of Mascot emPAI values (used as a proxy for emPAI values) revealed a dynamic range of protein abundance in tissues that spanned 4 orders of magnitude (y-axis) and corresponded with known abundance estimates for various proteins in these tissues. The high/moderate-abundance proteins (e.g. ACTB, FABP5, CHGA) and low-abundance protein (e.g. POLR3A) relatively quantified in our samples are highlighted relative to their mean Mascot emPAI value. B, distribution of reported abundance ranges for the proteins with at least one unique peptide identified in tissues, and the high-molecular-weight proteins with the greatest number of unique peptides identified. Subcellular localizations of the proteins identified in colorectal tissues and cell lines (C) and biological processes in which these proteins are involved (D). This analysis was performed using Scaffold and Gene Ontology annotations (see “Experimental Procedures”).
Fig. 3.
Fig. 3.
Principal component analysis of protein expression. Three-dimensional principal component analysis score plot of log2 protein expression intensity values for (A) tissues (normal mucosa, black; adenomas, red), (B) cell lines (HCEC, cyan; colon cancer cell lines, green), and (C) both. The first three principal components (PCs) account for 40%, 82%, and 36% of the total variance in the tissue, cell line, and tissue + cell line sets, respectively. PC1, the main direction of spread in the three groupings, reflects intergroup variance based on tissue or cell line type (i.e. normal/immortalized versus tumorous). Cell lines derived from the same patient: *SW480 and SW620 cells; ^HT29 and CX1 cells.
Fig. 4.
Fig. 4.
Analysis of the 212 proteins displaying significant tumor-related dysregulation. A, hierarchical clustering of iTRAQ abundance ratios (normal versus 114, adenoma versus 114) for the 212 proteins displaying significant adenoma-related dysregulation grouped tissue samples into two discrete clusters: adenoma (A) and normal (N). B, Pearson's correlation test comparing average fold changes (at least ±0.5 log2) for the 212 proteins (red, up-regulated; blue, down-regulated) in the tissue series with average log2 fold changes for the corresponding mRNAs measured in another set of adenoma/normal mucosal samples.
Fig. 5.
Fig. 5.
Significantly up-regulated SORD expression and activity in colorectal cell lines and adenomas. A, tumor-related up-regulation of sorbitol dehydrogenase (SORD) in colon cancer cell lines was confirmed with Western blotting. The SORD dysregulation trend was identical to that observed with iTRAQ-based MS/MS, although when immunoblot results were quantified (B), the log2 fold changes were more than five times greater than those documented in the iTRAQ study. C, SORD protein expression (iTRAQ analysis) in 21 normal mucosa–adenoma tissue pairs. D, SORD mRNA expression in 42 other normal mucosa–adenoma pairs from a previous study by our group (26). Error bars indicate the means and 95% confidence intervals. E, Western blots showing tumor-related up-regulation of SORD expression in four randomly selected adenoma (A)/normal mucosa (N) tissue pairs of the 21 shown in panel C (see Table I for sample descriptions). F, SORD activity also displayed tumor-related up-regulation in cell lines (HT29 and SW480 versus HCEC cells) and tissues (adenomas versus normal mucosa). Columns show mean enzyme activity measured in at least two replicates; error bars indicate standard deviations from means. The Western blot beneath the graph shows SORD levels measured in the extracts used for the enzyme activity assays.
Fig. 6.
Fig. 6.
Anti-SORD immunostaining of colorectal cell lines and tissues. Consistent with proteomic data, SORD expression was (A) negligible or absent in HCECs but (B) clearly expressed in the cytoplasm of HT29 cells. C, in normal colorectal mucosa, SORD expression was limited to the lower portion of the epithelial crypts, where stem cells and highly proliferating cells are located. Higher magnification views show staining at (D) the base versus (E) the mouth of colonic crypts. F, G, its expression was markedly increased in adenomatous glands (red arrowheads) relative to normal crypts (green arrowheads). Panels H and I show abundant expression of SORD in a large adenoma and in a cancer, respectively.
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