Bcl-2 Family Members Bcl-xL and Bax Cooperatively Contribute to Bortezomib Resistance in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin lymphoma with poor prognosis, due to the inevitable development of drug resistance. Despite being the first-in-class proteasome inhibitor for relapsed/refractory MCL, resistance to bortezomib (BTZ) in MCL patients remains a major hurdle of effective therapy, and relapse following BTZ is frequent. Understanding the mechanisms underlying BTZ resistance is, therefore, important for improving the clinical outcome and developing novel therapeutic strategies. Here, we established de novo BTZ-resistant human MCL-derived cells with the highest resistance index of 300-fold compared to parental cells. We provided compelling evidence that both Bcl-xL and Bax are key mediators in determining BTZ sensitivity in MCL cells. Overexpression of antiapoptotic Bcl-xL and depletion of proapoptotic Bax cooperatively protected MCL cells against BTZ-induced apoptosis, causing acquired BTZ resistance, likely by tilting the balance of Bcl-2 family proteins toward antiapoptotic signaling. Bioinformatics analyses suggested that high BCL2L1 (encoded Bcl-xL) and low BAX were, in part, associated with poor prognosis of MCL patients, e.g., when combined with low OGT, which regulates cellular O-GlcNAcylation. Our findings support recent strategies in small molecule drug discovery co-targeting antiapoptotic Bcl-2 family proteins using BH3 mimetics and Bax using Bax activators to overcome cancer drug resistance.

Overexpression of AGR2vH, an oncogenic AGR2 spliced transcript, potentiates tumorigenicity and proteomic alterations in cholangiocarcinoma cell.

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.

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

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 gradient­2 (AGR2) was characterized as one of the most highly upregulated genes among all metastasis­associated 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 AGR2vH­overexpressing 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 X­box binding protein 1), UPR proteins [binding immunoglobulin protein/glucose­regulated 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 caspase­3/7 activity and downregulating C/EBP homologous protein mRNA and B­cell lymphoma­2 (Bcl­2)­associated X protein expression, whereas the Bcl­2 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 metastasis­associated phenotypes, but also CCA cell survival and apoptosis evasion, thereby favoring cancer progression.

Aberrant mRNA splicing generates oncogenic RNA isoforms and contributes to the development and progression of cholangiocarcinoma.

Cholangiocarcinoma is a lethal biliary cancer, with an unclear molecular pathogenesis. Alternative splicing is a post-transcriptional modification that generates mature mRNAs, which are subsequently translated into proteins. Aberrant alternative splicing has been reported to serve a role in tumor initiation, maintenance and metastasis in several types of human cancer, including cholangiocarcinoma. In this review, the aberrant splicing of genes and the functional contributions of the spliced genes, in the carcinogenesis, progression and aggressiveness of cholangiocarcinoma are summarized. In addition, factors that influence this aberrant splicing that may be relevant as therapeutic targets or prognosis markers for cholangiocarcinoma are discussed.

An aberrantly spliced isoform of anterior gradient-2, AGR2vH promotes migration and invasion of cholangiocarcinoma cell.

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.