Targeting fructose metabolism by glucose transporter 5 regulation in human cholangiocarcinoma.

Alterations in cellular metabolism may contribute to tumor proliferation and survival. Upregulation of the facilitative glucose transporter (GLUT) plays a key role in promoting cancer. GLUT5 mediates modulation of fructose utilization, and its overexpression has been associated with poor prognosis in several cancers. However, its metabolic regulation remains poorly understood. Here, we demonstrated elevated GLUT5 expression in human cholangiocarcinoma (CCA), using RNA sequencing data from samples of human tissues and cell lines, as compared to normal liver tissues or a cholangiocyte cell line. Cells exhibiting high-expression of GLUT5 showed increased rates of cell proliferation and ATP production, particularly in a fructose-supplemented medium. In contrast, GLUT5 silencing attenuated cell proliferation, ATP production, cell migration/invasion, and improved epithelial-mesenchymal transition (EMT) balance. Correspondingly, fructose consumption increased tumor growth in a nude mouse xenograft model, and GLUT5 silencing suppressed growth, supporting the tumor-inhibitory effect of GLUT5 downregulation. Furthermore, in the metabolic pathways of fructolysis-Warburg effect, the expression levels of relative downstream genes, including ketohexokinase (KHK), aldolase B (ALDOB), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 4 (MCT4), as well as hypoxia-inducible factor 1 alpha (HIF1A), were altered in a GLUT5 expression-dependent manner. Taken together, these findings indicate that GLUT5 could be a potential target for CCA therapeutic approach via metabolic regulation.

Promoter hypermethylation of early B cell factor 1 (EBF1) is associated with cholangiocarcinoma progression.

DNA hypermethylation in a promoter region causes gene silencing via epigenetic changes. We have previously reported that early B cell factor 1 (EBF1) was down-regulated in cholangiocarcinoma (CCA) tissues and related to tumor progression. Thus, we hypothesized that the DNA hypermethylation of EBF1 promoter would suppress EBF1 expression in CCA and induce its progression. In this study, the DNA methylation status of EBF1 and mRNA expression levels were analyzed in CCA and normal bile duct (NBD) tissues using a publicly available database of genome-wide association data. The results showed that the DNA methylation of EBF1 promoter region was significantly increased in CCA tissues compared with those of NBD. The degree of methylation was negatively correlated with EBF1 mRNA expression levels. Using methylation-specific PCR technique, the DNA methylation rates of EBF1 promoter region were investigated in CCA tissues (n=72). CCA patients with high methylation rates of EBF1 promoter region in the tumor tissues (54/72) had a poor prognosis. Higher methylation rates of EBF1 promoter region have shown in all CCA cell lines than that of an immortal cholangiocyte cell line (MMNK1). Upon treatment with the DNA methyltransferase inhibitor 5-Aza-dC, increased EBF1 expression levels and reduced DNA methylation rates were observed in CCA cells. Moreover, restoration of EBF1 expression in CCA cells led to inhibition of cell growth, migration and invasion. In addition, RNA sequencing analysis suggested that EBF1 is involved in suppression of numerous pathways in cancer. Taken together, DNA hypermethylation in the EBF1 promoter region suppresses EBF1 expression and induces CCA progression with aggressive clinical outcomes.

Opposing Roles of FoxA1 and FoxA3 in Intrahepatic Cholangiocarcinoma Progression.

Cholangiocarcinoma (CCA), a malignancy of biliary epithelium, is related to liver stem cell deregulation. FoxAs are a group of transcription factors that play critical roles in liver stem cell differentiation. In this study, the expression levels of FoxAs (i.e., FoxA1, FoxA2 and FoxA3) were detected in intrahepatic CCA tissues and the functions of FoxAs were studied in CCA cell lines. FoxA1 and FoxA2 were mainly localized in the nuclei of normal bile duct (NBD) cells and some of the cancer cells. Low expression of FoxA1 in CCA tissues (72%) was significantly correlated with poor prognosis. FoxA3 expression of CCA cells was localized in the nucleus and cytoplasm, whereas it was slightly detected in NBDs. High expression of FoxA3 in cancer tissues (61%) was significantly related to high metastasis status. These findings suggest the opposing roles of FoxA1 and FoxA3 in CCA. Moreover, the FoxA1-over-expressing CCA cell line exhibited a significant reduction in proliferative and invasive activities compared to control cells. Knockdown of FoxA3 in CCA cells resulted in a significant decrease in proliferative and invasive activities compared with control cells. Taken together, in CCA, FoxA1 is down-regulated and has tumor suppressive roles, whereas FoxA3 is up-regulated and has oncogenic roles.

Current omics-based biomarkers for cholangiocarcinoma.

Introduction: Cholangiocarcinoma (CCA) is a malignancy of the biliary tract. CCA generally has a low incidence worldwide but incidence is typically high in Southeast Asian countries, particularly in northeastern Thailand, where small liver-fluke (Opisthorchis viverrini) infection is endemic. CCA has a poor prognosis as most CCA patients present with advanced stages. Poor prognosis and worse outcomes are due to the lack of specific and early-stage CCA biomarkers. Areas covered: In this review, we discuss the use of CCA tissues, serum and bile samples as sources of diagnostic and prognostic markers by using -omics approaches, including genomics, epigenomics, transcriptomics and proteomics. The current state of the discovery of molecular candidates and their potential to be used as diagnostic and prognostic biomarkers for CCA are summarized and discussed. Expert opinion: Various potential molecules have been discovered, some of which have been verified as diagnostic biomarkers for CCA. However, most identified molecules require much further evaluation to help us find markers with high specificity, low cost and ease-of-use in routine diagnostic laboratories.

Roles of Zinc Finger Protein 423 in Proliferation and Invasion of Cholangiocarcinoma through Oxidative Stress.

Zinc finger protein 423 (ZNF423) is a transcriptional factor involved in the development and progression of cancers but has not yet been examined in cholangiocarcinoma (CCA), an oxidative stress-driven cancer of biliary epithelium. In this study, we hypothesized that oxidative stress mediated ZNF423 expression regulates its downstream genes resulting in CCA genesis. ZNF423 protein expression patterns and 8-oxodG (an oxidative stress marker) formation in CCA tissues were investigated using immunohistochemical analysis. The results showed that ZNF423 was overexpressed in CCA cells compared to normal bile duct cells adjacent of the tumor. Notably, ZNF423 expression was positively correlated with 8-oxodG formation. Moreover, ZNF423 expression in an immortalized cholangiocyte cell line (MMNK1) was increased by hydrogen peroxide-treatment, suggesting that oxidative stress induces ZNF423 expression. To investigate the roles of ZNF423 in CCA progression, ZNF423 mRNA was silenced using specific siRNA in CCA cell lines, KKU-100 and KKU-213. Silencing of ZNF423 significantly inhibits cell proliferation and invasion of both CCA cell lines. Taking all these results together, the present study denoted that ZNF423 is an oxidative stress-responsive gene with an oncogenic property contributing to the regulation of CCA genesis.

The Importance of CYP19A1 in Estrogen Receptor-Positive Cholangiocarcinoma.

CYP19A1, also called aromatase, is a key enzyme for converting androgens to estrogens of estrogen synthesis. Elevated serum estrogen and high expression levels of estrogen-related proteins are found in cholangiocarcinoma (CCA; bile duct cancer). However, the expression of CYP19A1 in relation to estrogen-related proteins, including estrogen receptors (ERα, ERß, and GPR30) and an estrogen response protein (TFF1), has never been explored in CCA. In this study, we investigated the expressions of CYP19A1 and estrogen-related proteins in CCA tissues (n = 74; 51 males and 23 females) using immunohistochemistry. The results showed that CYP19A1 was overexpressed in CCA cells compared with that in normal bile duct cells in the adjacent tissues. High expression of CYP19A1 was correlated with the metastatic status of the patients. High CYP19A1 expression was also positively correlated with GPR30 expression. Correlation between high CYP19A1 expression in the tumor tissues and shorter survival time was more prominent in male than in female CCA patients. To elucidate further, the effect of CYP19A1 knockdown on a CCA cell line was examined using a specific siRNA. When CYP19A1 gene expression was suppressed, migration and proliferation activities of CCA cells were significantly reduced. Moreover, the cell proliferation of high CYP19A1-expressing KKU-213 cells was more profoundly suppressed by CYP19A1 inhibitors (exemestane and letrozole) than low CYP19A1-expressing KKU-100 cells. Thus, CYP19A1 promotes CCA progression with aggressive clinical outcomes via increased migration and proliferation activities of cancer cells. CYP19A1 can be a potential chemotherapeutic target for CCA, especially in male patients.

Anti-cancer activity of asiatic acid against human cholangiocarcinoma cells through inhibition of proliferation and induction of apoptosis.

Plant-derived anti-cancer agents have been of considerable interest due to their promising effectiveness with low side effects. Asiatic acid, the main constituent of the medicinal plant Centella asiatica (L.) Urban, has a wide range of biological properties such as antioxidant, anti-inflammatory and anti-cancer activities. Cholangiocarcinoma (CCA), which is a malignant tumor of bile duct epithelium, is one of the leading cancers in Southeast Asia, notably the northeast of Thailand where the liver fluke, Opisthorchis viverrini predominates. Many in vitro and in vivo studies have provided evidence supporting that oxidative stress induced by chronic inflammation is involved in CCA genesis with aggressive clinical outcomes. This study was performed to evaluate the cytotoxic effects of asiatic acid on two human CCA cell lines (KKU-156 and KKU-213). Cell viability was determined by a sulforhodamine B (SRB) assay. Morphological changes of the cells were observed by microscopy. Cell apoptosis was detected by flow cytometry using annexin V and propidium iodide (PI) staining. Messenger RNA (mRNA) expression levels of BAX, BCL2 and Survivin/BIRC5 were analyzed by real-time polymerase chain reaction (PCR). It was found that asiatic acid efficiently suppressed CCA cellular viability via induction of apoptosis. In addition, the occurrence of asiatic acid-induced apoptosis was confirmed by microscopic observation of apoptotic vesicles, down-regulation of anti-apoptotic genes (BCL2 and Survivin/BIRC5) and increased early and late apoptotic cells. Our results showed the chemotherapeutic activities of asiatic acid, suggesting the anti-cancer properties of this compound should be clinically assessed and its supplementation may lead to an improvement of survival of CCA patients.

Prolonged oxidative stress down-regulates Early B cell factor 1 with inhibition of its tumor suppressive function against cholangiocarcinoma genesis.

Early B cell factor 1 (EBF1) is a transcription factor involved in the differentiation of several stem cell lineages and it is a negative regulator of estrogen receptors. EBF1 is down-regulated in many tumors, and is believed to play suppressive roles in cancer promotion and progression. However, the functional roles of EBF1 in carcinogenesis are unclear. Liver fluke-infection-associated cholangiocarcinoma (CCA) is an oxidative stress-driven cancer of bile duct epithelium. In this study, we investigated EBF1 expression in tissues from CCA patients, CCA cell lines (KKU-213, KKU-214 and KKU-156), cholangiocyte (MMNK1) and its oxidative stress-resistant (ox-MMNK1-L) cell lines. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was used as an oxidative stress marker. Our results revealed that EBF1 expression was suppressed in cancer cells compared with the individual normal bile duct cells at tumor adjacent areas of CCA tissues. CCA patients with low EBF1 expression and high formation of 8-oxodG were shown to correlate with poor survival. Moreover, EBF1 was suppressed in the oxidative stress-resistant cell line and all of CCA cell lines compared to the cholangiocyte cell line. This suggests that prolonged oxidative stress suppressed EBF1 expression and the reduced EBF1 level may facilitate CCA genesis. To elucidate the significance of EBF1 suppression in CCA genesis, EBF1 expression of the MMNK1 cell line was down-regulated by siRNA technique, and its effects on stem cell properties (CD133 and Oct3/4 expressions), tumorigenic properties (cell proliferation, wound healing and cell migration), estrogen responsive gene (TFF1), estrogen-stimulated wound healing, and cell migration were examined. The results showed that CD133, Oct3/4 and TFF1 expression levels, wound healing, and cell migration of EBF1 knockdown-MMNK1 cells were significantly increased. Also, cell migration of EBF1-knockdown cells was significantly enhanced after 17ß-estradiol treatment. Our findings suggest that EBF1 down-regulation via oxidative stress induces stem cell properties, tumorigenic properties and estrogen responses of cholangiocytes leading to CCA genesis with aggressive clinical outcomes.

Development and characterization of a hydrogen peroxide-resistant cholangiocyte cell line: A novel model of oxidative stress-related cholangiocarcinoma genesis.

Oxidative stress is a cause of inflammation-related diseases, including cancers. Cholangiocarcinoma is a liver cancer with bile duct epithelial cell phenotypes. Our previous studies in animal and human models indicated that oxidative stress is a major cause of cholangiocarcinoma development. Hydrogen peroxide (H2O2) can generate hydroxyl radicals, which damage lipids, proteins, and nucleic acids, leading to cell death. However, some cells can survive by adapting to oxidative stress conditions, and selective clonal expansion of these resistant cells would be involved in oxidative stress-related carcinogenesis. The present study aimed to establish H2O2-resistant cell line from an immortal cholangiocyte cell line (MMNK1) by chronic treatment with low-concentration H2O2 (25 µM). After 72 days of induction, H2O2-resistant cell lines (ox-MMNK1-L) were obtained. The ox-MMNK1-L cell line showed H2O2-resistant properties, increasing the expression of the anti-oxidant genes catalase (CAT), superoxide dismutase-1 (SOD1), superoxide dismutase-2 (SOD2), and superoxide dismutase-3 (SOD3) and the enzyme activities of CAT and intracellular SODs. Furthermore, the resistant cells showed increased expression levels of an epigenetics-related gene, DNA methyltransferase-1 (DNMT1), when compared to the parental cells. Interestingly, the ox-MMNK1-L cell line had a significantly higher cell proliferation rate than the MMNK1 normal cell line. Moreover, ox-MMNK1-L cells showed pseudopodia formation and the loss of cell-to-cell adhesion (multi-layers) under additional oxidative stress (100 µM H2O2). These findings suggest that H2O2-resistant cells can be used as a model of oxidative stress-related cholangiocarcinoma genesis through molecular changes such as alteration of gene expression and epigenetic changes.