SILAC-based quantitative proteomic analysis of gastric cancer secretome.
ABSTRACT PURPOSE: Gastric cancer is a commonly occurring cancer in Asia and one of the leading causes of cancer deaths. However, there is no reliable blood-based screening test for this cancer. Identifying proteins secreted from tumor cells could lead to the discovery of clinically useful biomarkers for early detection of gastric cancer. EXPERIMENTAL DESIGN: A SILAC-based quantitative proteomic approach was employed to identify secreted proteins that were differentially expressed between neoplastic and non-neoplastic gastric epithelial cells. Proteins from the secretome were subjected to SDS-PAGE and SCX-based fractionation, followed by mass spectrometric analysis on an LTQ-Orbitrap Velos mass spectrometer. Immunohistochemical labeling was employed to validate a subset of candidates using tissue microarrays. RESULTS: We identified 2,205 proteins in the gastric cancer secretome of which 263 proteins were overexpressed >4-fold in gastric cancer-derived cell lines as compared to non-neoplastic gastric epithelial cells. Three candidate proteins, proprotein convertase subtilisin/kexin type 9 (PCSK9), lectin mannose binding 2 (LMAN2) and PDGFA associated protein 1 (PDAP1), were validated by immunohistochemical labeling. CONCLUSIONS AND CLINICAL RELEVANCE: We report here the largest cancer secretome described to date. The novel biomarkers identified in the current study are excellent candidates for further testing as early detection biomarkers for gastric adenocarcinoma.
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ABSTRACT: Although patients with Glioblastoma multiforme (GBM) have grave prognosis, significant variability in patient outcome is observed. The objective of this study is to identify a molecular signature for GBM prognosis. We subjected 355 mRNA and microRNA expression profiles to elastic net-regulated Cox regression for identification of an integrated RNA signature for GBM prognosis. A prognostic index (PI) was generated for patient stratification. Survival comparison was conducted by Kaplan-Meier method and a general multivariate Cox regression procedure was applied to evaluate the independence of the PI. The abilities and efficiencies of signatures to predict GBM patient outcome was assessed and compared by the area under the curve (AUC) of the receiver-operator characteristic (ROC). An integrated RNA prognostic signature consisted by 4 protective mRNAs, 12 risky mRNAs, and 1 risky microRNA was identified. Decreased survival was associated with being in the high-risk group (hazard ratio = 2.864, P<0.0001). The prognostic value of the integrated signature was validated in five independent GBM expression datasets (n = 201, hazard ratio = 2.453, P<0.0001). The PI outperformed the known clinical factors, mRNA-only, and miRNA-only prognostic signatures for GBM prognosis (area under the ROC curve for the integrated RNA, mRNA-only, and miRNA-only signatures were 0.828, 0.742, and 0.757 at 3 years of overall survival, respectively, P<0.0001 by permutation test). We describe the first, to our knowledge, robust transcriptome-based integrated RNA signature that improves the current GBM prognosis based on clinical variables, mRNA-only, and miRNA-only signatures.PLoS ONE 01/2014; 9(5):e98419. · 3.53 Impact Factor
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ABSTRACT: Cancer progression involves changes in extracellular proteolysis but the contribution of stromal cell secretomes to the cancer degradome remains uncertain. We have now defined the secretome of a specific stromal cell type, the myofibroblast, in gastric cancer, and its modification by proteolysis. SILAC labelling and COFRADIC isolation of methionine containing peptides allowed us to quantify differences in gastric cancer-derived myofibroblasts compared with myofibroblasts from adjacent tissue, revealing increased abundance of several proteases in cancer myofibroblasts including matrix metalloproteinases (MMP) -1 and -3. Moreover N-terminal COFRADIC analysis identified cancer-restricted proteolytic cleavages included liberation of the active forms of MMP-1, -2 and -3 from their inactive precursors. In vivo imaging confirmed increased MMP activity when gastric cancer cells were xenografted in mice together with gastric cancer myofibroblasts. Western blot and enzyme activity assays confirmed increased MMP-1, -2 and -3 activity in cancer myofibroblasts, and cancer cell migration assays indicated stimulation by MMP-1, -2 and -3 in cancer-associated myofibroblast media. Thus cancer-derived myofibroblasts differ from their normal counterparts by increased production and activation of MMP-1 , -2 and -3 and this may contribute to the remodelling of the cancer cell microenvironment.Journal of Proteome Research 05/2013; · 5.06 Impact Factor
- Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 04/2013; · 3.73 Impact Factor