SILAC-based quantitative proteomic approach to identify potential biomarkers from the esophageal squamous cell carcinoma secretome

Abstract

The identification of secreted proteins that are differentially expressed between non-neoplastic and esophageal squamous cell carcinoma (ESCC) cells can provide potential biomarkers of ESCC. We used a SILAC-based quantitative proteomic approach to compare the secretome of ESCC cells with that of non-neoplastic esophageal squamous epithelial cells. Proteins were resolved by SDS-PAGE, and tandem mass spectrometry analysis (LC-MS/MS) of in-gel trypsin-digested peptides was carried out on a high-accuracy qTOF mass spectrometer. In total, we identified 441 proteins in the combined secretomes, including 120 proteins with > 2-fold upregulation in the ESCC secretome vs. that of non-neoplastic esophageal squamous epithelial cells. In this study, several potential protein biomarkers previously known to be increased in ESCC including matrix metalloproteinase 1, transferrin receptor, and transforming growth factor beta-induced 68 kDa were identified as overexpressed in the ESCC-derived secretome. In addition, we identified several novel proteins that have not been previously reported to be associated with ESCC. Among the novel candidate proteins identified, protein disulfide isomerase family a member 3 (PDIA3), GDP dissociation inhibitor 2 (GDI2), and lectin galactoside binding soluble 3 binding protein (LGALS3BP) were further validated by immunoblot analysis and immunohistochemical labeling using tissue microarrays. This tissue microarray analysis showed overexpression of protein disulfide isomerase family a member 3, GDP dissociation inhibitor 2, and lectin galactoside binding soluble 3 binding protein in 93%, 93% and 87% of 137 ESCC cases, respectively. Hence, we conclude that these potential biomarkers are excellent candidates for further evaluation to test their role and efficacy in the early detection of ESCC.

Figure 1

The work flow for discovery and initial validation of biomarkers for esophageal squamous cell carcinoma. For SILAC labeling, Het-1A cells were grown in ‘heavy’ medium and the TE-series ESCC cells were grown in ‘light’ medium as indicated. The secretome was normalized, pooled and resolved by SDS-PAGE. Gel bands were excised and in-gel trypsin digested followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on a qTOF mass spectrometer. The data was searched using Mascot and Spectrum Mill search engines. Some of the overexpressed proteins that were not previously described (e.g., PDIA3) were validated using western blot and IHC labeling using tissue microarrays.

Figure 2

Protein profiled using the SILAC strategy. (A) Venn diagram showing proteins identified by Mascot and Spectrum Mill search algorithms. (B) Distribution of proteins identified by Spectrum Mill plotted against the log₂ ratios as indicated. Proteins for which peptides were only observed in cancer cell lines are indicated in red while those for which peptides were identified only in the normal cell line are shown in green. (C) Distribution of proteins identified by Mascot plotted against the log2 ratios as indicated. Proteins for which peptides were only observed in cancer cell lines are indicated in red while those for which peptides were identified only in the normal cell line are shown in green.

Figure 3

MS and MS/MS spectra of selected differentially expressed proteins. MS and MS/MS spectra of peptide from representative differentially expressed proteins identified in this study. (A) Matrix metalloproteinase 1 (MMP1); (B) TGFbeta induced, 68 KD (TGFBI); (C) GDP dissociation inhibitor 2 (GDI2); (D) Protein disulfide isomerase A3 (PDIA3); (E) Nicotinamide phosphoribosyltransferase (NAMPT) and (F) Lectin, galactoside-binding, soluble, 3 binding protein (LGALS3BP).

Figure 4

Western blot validation for selected proteins identified in the ESCC secretome. Pooled conditioned media from different ESCC cell lines and normal cell line was tested for expression of the indicated proteins using commercially available antibodies.

Figure 5

Validation of TGFBI using immunohistochemical labeling. Expression of TGFBI in representative normal esophageal squamous mucosa (A). Expression of TGFBI in ESCC is observed in both stromal and epithelial cell compartments (B).

Figure 6

Validation of PDIA3 using immunohistochemical labeling. Expression of PDIA3 in representative normal esophageal squamous mucosa (A). Expression of PDIA3 in ESCC is observed in both stromal and epithelial cell compartments (B).

Figure 7

Validation of LGALS3BP using immunohistochemical labeling. Expression of LGALS3BP in representative normal esophageal squamous mucosa (A). Expression of LGALS3BP in ESCC is observed in both stromal and epithelial compartments (B).

Figure 8

Validation of GDI2 using immunohistochemical labeling. Expression of GDI2 in representative normal esophageal squamous mucosa (A). Expression of GDI2 in ESCC is observed in both stromal and epithelial compartments (B).