ABSTRACT
The upregulation of anterior gradient 2 (AGR2) has been observed in cholangiocarcinoma (CCA) cells, nras-mutant zebrafish, and specimens derived from CCA patients. Our previous study reported AGR2 splicing into AGR2vH to facilitate CCA cell aggressiveness, while this work aims to investigate the molecular mechanisms underlying AGR2vH. First, AGR2vH upregulation was demonstrated in CCA tissues derived from patients. For in vitro studies, established AGR2vH-overexpressing KKU-213A cells were found to exhibit increased proliferation and clonogenicity. In vivo tumorigenicity assessed in a mouse model represented higher tumorigenic potential in AGR2vH-overexpressing cell xenograft mice. Next, LC-MS/MS was analyzed, indicating that AGR2vH may be associated with CCA cell proliferation via Wnt/ß-catenin signaling pathway activation, which was verified by ß-catenin expression and nuclear translocation. The current results provide evidence that AGR2vH upregulation promotes tumorigenicity in CCA cells linked with an alteration of CCA cell proteome.
Subject(s)
ProteomicsABSTRACT
Cholangiocarcinoma (CCA) is a cancer of bile duct, considered to be an incurable and lethal cancer. High mortality rate of CCA patients is underlined by cancer metastasis, an ability of the cancer cells that spread to secondary organs. Recently, we have identified Anterior Gradient-2 (AGR2), from a pair of non-metastatic/metastatic cell lines (KKU-213/KKU-213L5), as a gene that is highly and specifically upregulated in the metastatic cell line. AGR2 encodes for a disulfide isomerase enzyme, ubiquitously detected in mucus-secreting tissues. Overexpression of AGR2 has been reported in several types of human cancer. Role of the overexpressed AGR2 in cancer is still unclear. Here, we found that upregulation of AGR2 in metastatic CCA cells coincides with an aberrant splicing of AGR2 mRNA, and that isoforms of AGR2 RNA, such as AGR2vE, AGR2vF, and AGR2vH are specific to the metastatic cells. We demonstrated that the AGR2vH isoform enables metastatic-associated phenotypes in CCA cells. Depletion of AGR2vH by an isoform-specific interfering RNA in metastatic KKU-213L5 cell results in significant reduction of cancer cell migration and invasion, and a slight decrease of cell adhesion. Overexpression of AGR2vH in non-metastatic KKU-213 cells promotes cancer cell migration, invasion, adhesion, and moderate cell proliferation. Moreover, we found that expression of a metastasis-associated gene, vimentin, positively correlates with expression of AGR2vH. Our results support the notion that aberrant alternative splicing of AGR2 facilitates an accumulation of the oncogenic AGR2vH isoform, in turn, contributes to the pathogenesis and severity of CCA.
