Knockdown of anterior gradient 2 expression extenuates tumor-associated phenotypes of SNU-478 ampulla of Vater cancer cells

Anterior gradient 2 (AGR2) has been implicated in tumor-associated phenotypes such as cell viability, invasion and metastasis in various human cancers. However, the tumor promoting activity of AGR2 has not yet been determined in biliary tract cancers. Thus, we examined the expression of AGR2 and its tumor-promoting activity in biliary tract cancer cells in this study. Expression of AGR2 mRNA and protein was analyzed by real time RT-PCR and western blotting, respectively. MTT assay was employed to measure cell viability and pulsed BrdU incorporation by proliferating cells was monitored by flow cytometry. Soft agar colony formation assay and transwell invasion assay were employed to determine anchorage-independent growth and in vitro invasion of the tumor cells, respectively. In vivo tumor formation was examined by injection of tumor cells into immunocompromised mice subcutaneously. Statistical analysis was performed with 2-tailed unpaired Student's t-test for continuous data and with one-way ANOVA for multiple group comparisons. Bonferroni tests were used for post hoc 2-sample comparisons. AGR2 mRNA was detected in SNU-245, SNU-478, and SNU-1196 cell lines, and its protein expression was confirmed in SNU-478 and SNU-245 cell lines by western blot analysis. Knockdown of AGR2 expression with an AGR2-specific short hairpin RNA (shRNA) in SNU-478, an ampulla of Vater cancer cell line resulted in decreased cell viability and in decreased anchorage-independent growth by 98%. The AGR2 knockdown also increased the sensitivity of the cells to chemotherapeutic drugs, including gemcitabine, 5-fluorouracil and cisplatin. In addition, SNU-478 cells expressing AGR2-shRNA failed to form detectable tumor xenografts in nude mice, whereas control cells formed tumors with an average size of 179 ± 84 mm3 in 3 weeks. Overexpression of AGR2 in SNU-869 cells significantly increased cell viability through enhanced cell proliferation and the number of Matrigel™-invading cells compared with AGR2-negative SNU-869 cells. Our findings implicate that AGR2 expression augments tumor-associated phenotypes by increasing proliferative and invasive capacities of the ampulla of Vater cancer cells.