Background. Esophageal squamous cell cancer (ESCC) is associated with a poor prognosis and high recurrence rate. Identification of proteins that are useful for prognostic assessment and therapeutic targets in ESCC is required. Quantitative proteomic analysis provides a powerful approach in screening for alterations in protein levels. An analysis of the differential expression of cell-surface membrane proteins would be useful for identification of proteins associated with cell proliferation, cell migration, drug resistance and therapeutic target for antibody-based therapy.
Purpose of study. To identify cell-surface membrane proteins overexpressed in ESCC compared with normal esophageal epithelial cells, which may represent novel biomarkers of disease and therapeutic targets.
Procedures. We performed a biotinylation-based approach for cell-surface membrane enrichment combined with iTRAQ (isobaric tags for relative and absolute quantitation) technology using nano-LC-MS/MS analysis, to identify cell surface proteins overexpressed in ESCC cell lines compared with control normal esophageal epithelial cell line.
Results. By combining biotinylation of cell-surface membrane proteins and iTRAQ analysis, 1635 proteins were identified, which contained 402 cell surface membrane proteins. We identified overexpression of 12 membrane proteins, whose expression increased more than twofold in at least four of six ESCC cell lines compared with a normal esophageal epithelial cell line (HEEpic). By this proteomic approach, we identified several tumor antigens which included antigens previously associated with ESCC and antigens not previously reported in this disease. Furthermore, we also successfully identified novel antigen associated with proliferation of ESCC. Cell surface expression of this antigen on ESCC was confirmed by FACS analysis and immunohistochemistry. By transfection of siRNA specific for this novel antigen, significant growth suppression was shown by WST8 assay and induction of apoptosis was detected by caspase-3 assay.
Conclusions. By using a high throughput cell-surface membrane proteomic analysis combining biotinylation of cell-surface membrane protein and iTRAQ technology, several ESCC candidate antigens were identified. Further studies on the physiological role of novel tumor antigens overexpressed in ESCC and their application as biomarkers/therapeutic targets are underway.
Note: This abstract was not presented at the meeting.
Citation Format: Emi Harada, Satoshi Serada, Tsuyoshi Takahashi, Minoru Fujimoto, Tetsuji Naka. Quantitative proteomic analysis of cell-surface membrane proteins: Biomarker discovery in esophageal squamous cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5312. doi:10.1158/1538-7445.AM2014-5312