Upregulation of AGR2vH facilitates cholangiocarcinoma cell survival under endoplasmic reticulum stress via the activation of the unfolded protein response pathway

Abstract

Cholangiocarcinoma (CCA) is an epithelial cell malignancy arising within the biliary tree in the liver. CCA is usually diagnosed at an advanced stage, subsequent to developing with metastasis. Recently, anterior gradient-2 (AGR2) was characterized as one of the most highly upregulated genes among all metastasis-associated genes in highly metastatic CCA cell lines. Previous reports have demonstrated that AGR2 is required for triggering the unfolded protein response (UPR) pathway to support cancer cell survival, particularly under endoplasmic reticulum (ER) stress conditions. A previous study identified an AGR2 short isoform generated by aberrant splicing, AGR2vH, which contributed to the metastatic phenotype of CCA cells. The aim of the present study was to determine the function of AGR2vH in UPR pathway activation to support cancer cell survivability and apoptosis evasion. Subsequent to experimentally inducing ER stress in AGR2vH-overexpressing CCA cells using tunicamycin, the UPR pathway was activated by the upregulation of UPR marker genes (activating transcription factor 6, eukaryotic initiation factor 2a and spliced X-box binding protein 1), UPR proteins [binding immunoglobulin protein/glucose-regulated protein (GRP)78 kDa and phosphorylated eukaryotic translation initiation factor 2a] and UPR downstream targets (GRP94). In addition, the results were verified by AGR2vH knockdown using specific small interfering RNAs. Under ER stress conditions, the overexpression of AGR2vH reduced the number of apoptotic cells by decreasing caspase-3/7 activity and downregulating C/EBP homologous protein mRNA and B-cell lymphoma-2 (Bcl-2)-associated X protein expression, whereas the Bcl-2 protein was upregulated, resulting in a higher number of viable cells. The results of the present study support the previous data that indicate that an oncogenic AGR2vH isoform may not only promote metastasis-associated phenotypes, but also CCA cell survival and apoptosis evasion, thereby favoring cancer progression.