In humans, Anterior gradient (AG) proteins are mainly distributed in endoderm-derived organs, such as the lungs, stomach, small intestine, colon, and prostate (27, 33). AGR2 was initially found in human breast cancer specimens (27), contrarily, decreased AGR2 expression was found in breast cancer (27, 34), esophagus (35), stomach (36), lung (37), pancreas(38), ovarian (39) and prostate cancer. (40). AGR2 is a member of the disulfide isomerase family of endoplasmic reticulum (ER) proteins, it catalyzes protein folding and thiol-disulfide interchange reactions (41).
DiMaioet al. retrospectively examined 116 specimens of esophageal carcinoma; they demonstrated that the presence of diffused AGR2 expression is highly sensitive to esophageal adenocarcinoma. However, focal expression of AGR2 was found in only approximately one-third (36.59%) of ESCC specimens (42). In this study, we firstly attempted to identify biomarker to predict nCRT responses in esophageal cancer patients (n = 32) by next-generation sequencing (NGS). We found 464 genes were differentially expressed in ESCC that were associated with nCRT response. We then assessed gene expression in up-regulated genes between Complete response and Non-complete response group, and further selected four genes that are associated with cell proliferation and cell migration, namely AGR2 (27), PPP1R15A (28), GADD45B (29), and LRG1 (30). Based on the gene fold change differences, we scrutinized AGR2 with 2.8 fold difference in the patients with Non-complete response than treatment group. Thus we decided to use AGR2 to do further investigation. AGR2 expression was significantly correlated with metastasis and poor prognosis in breast cancer and biomarker in prostate cancer (43–45).
Previous study showed that, AGR2 induces cancer cell proliferation, invasion and survival, chemotherapy resistance, metastasis and tumor growth. Therefore in this present study at first we knock-downed AGR2 expression in esophageal cancer cell lines (CE146T/VGH, TE2, and CE48T/VGH cells) and then analyzed for AGR2 expression using Western blotting. AGR2 protein expression was greatly decreased in all the three si-AGR2 transfected cancer cells. To further correlate the role of AGR2 with nCRT response, we knockdown AGR2 expression and treated cells with cisplatin and 5-FU and analysed for its cell viability. Cancer cells -CE48T/VGH and CE146T/VGH treated with low concentration of 2.5µM cisplatin and 3µM 5-FU showed no any change in the cell viability percentage compared with TE2 cell lines. This may be due to nature of cell type, contrarily when the treatment dose was increased all the three cells showed significant decrease in cell viability compared to control group. This data clearly explains us the significant role of AGR2 down-regulation in cisplatin and 5-FU treatment in esophageal cancer cells.
Previous study showed that AGR2 expression and cell cycle proteins showed as good prognosis marker in epithelial ovarian cancer (46), in this study in AGR2-knockdown cells, cisplatin and 5-FU treatment increased p21 protein level compared to control group. These results suggest that the cytotoxicity sensitivity to cisplatin and 5-FU in esophageal cancer was associated with AGR2 expression and might be through the p21 pathway. This was a retrospective study; further prospective studies are required to verify the correlation and precise pathway. Although many studies have confirmed that endoscopic biopsy results in a high concordance between biopsy and resected specimens (18, 47, 48), the bias of gastroscopic biopsy remains a concern.