Therapeutic Target For Intrahepatic Cholangiocarcinoma Research Articles

HSF1 is a prognostic determinant and therapeutic target in intrahepatic cholangiocarcinoma

BackgroundIntrahepatic cholangiocarcinoma (iCCA) is a lethal primary liver tumor characterized by clinical aggressiveness, poor prognosis, and scarce therapeutic possibilities. Therefore, new treatments are urgently needed to render this disease curable. Since cumulating evidence supports the oncogenic properties of the Heat Shock Factor 1 (HSF1) transcription factor in various cancer types, we investigated its pathogenetic and therapeutic relevance in iCCA.MethodsLevels of HSF1 were evaluated in a vast collection of iCCA specimens. The effects of HSF1 inactivation on iCCA development in vivo were investigated using three established oncogene-driven iCCA mouse models. In addition, the impact of HSF1 suppression on tumor cells and tumor stroma was assessed in iCCA cell lines, human iCCA cancer-associated fibroblasts (hCAFs), and patient-derived organoids.ResultsHuman preinvasive, invasive, and metastatic iCCAs displayed widespread HSF1 upregulation, which was associated with a dismal prognosis of the patients. In addition, hydrodynamic injection of a dominant-negative form of HSF1 (HSF1dn), which suppresses HSF1 activity, significantly delayed cholangiocarcinogenesis in AKT/NICD, AKT/YAP, and AKT/TAZ mice. In iCCA cell lines, iCCA hCAFs, and patient-derived organoids, administration of the HSF1 inhibitor KRIBB-11 significantly reduced proliferation and induced apoptosis. Cell death was profoundly augmented by concomitant administration of the Bcl-xL/Bcl2/Bcl-w inhibitor ABT-263. Furthermore, KRIBB-11 reduced mitochondrial bioenergetics and glycolysis of iCCA cells.ConclusionsThe present data underscore the critical pathogenetic, prognostic, and therapeutic role of HSF1 in cholangiocarcinogenesis.

N6-methyladenosine-modified circSLCO1B3 promotes intrahepatic cholangiocarcinoma progression via regulating HOXC8 and PD-L1.

Refractoriness to surgical resection and chemotherapy makes intrahepatic cholangiocarcinoma (ICC) a fatal cancer of the digestive system with high mortality and poor prognosis. Important function invests circRNAs with tremendous potential in biomarkers and therapeutic targets. Nevertheless, it is still unknown how circRNAs contribute to the evolution of ICC. CircRNAs in paired ICC and adjacent tissues were screened by circRNAs sequencing. To explore the impact of circRNAs on ICC development, experiments involving gain and loss of function were conducted. Various experimental techniques, including quantitative real-time PCR (qPCR), western blotting, RNA immunoprecipitation (RIP), luciferase reporter assays, RNA pull-down, chromatin immunoprecipitation (ChIP), ubiquitination assays and so on were employed to identify the molecular regulatory role of circRNAs. Herein, we reported a new circRNA, which originates from exon 9 to exon 15 of the SLCO1B3 gene (named circSLCO1B3), orchestrated ICC progression by promoting tumor proliferation, metastasis and immune evasion. We found that the circSLCO1B3 gene was highly overexpressed in ICC tissues and related to lymphatic metastasis, tumor sizes, and tumor differentiation. Mechanically, circSLCO1B3 not only promoted ICC proliferation and metastasis via miR-502-5p/HOXC8/SMAD3 axis, but also eradicated anti-tumor immunity via suppressing ubiquitin-proteasome-dependent degradation of PD-L1 by E3 ubiquitin ligase SPOP. We further found that methyltransferase like 3 (METTL3) mediated the m6A methylation of circSLCO1B3 and stabilizes its expression. Our findings indicate that circSLCO1B3 is a potential prognostic marker and therapeutic target in ICC patients. Taken together, m6A-modified circSLCO1B3 was correlated with poor prognosis in ICC and promoted ICC progression not only by enhancing proliferation and metastasis via potentiating HOXC8 expression, but also by inducing immune evasion via antagonizing PD-L1 degradation. These results suggest that circSLCO1B3 is a potential prognostic marker and therapeutic target for ICC.

CircPCNXL2 promotes tumor growth and metastasis by interacting with STRAP to regulate ERK signaling in intrahepatic cholangiocarcinoma

Backgroundcircular RNAs (circRNAs) have been reported to exert important effects in the progression of numerous cancers. However, the functions of circRNAs in intrahepatic cholangiocarcinoma (ICC) are still unclear.MethodscircPCNXL2 (has_circ_0016956) were identified in paired ICC by circRNA microarray. Then, we assessed the biological functions of circPCNXL2 by CCK8, EdU, clone formation, transwell, wound healing assays, and xenograft models. RNA pull-down, mass spectrometry, and RNA immunoprecipitation (RIP) were applied to explore the interaction between cirrcPCNXL2 and serine-threonine kinase receptor-associated protein (STRAP). RNA pull-down, RIP and luciferase reporter assays were used to investigate the sponge functions of circPCNXL2. In the end, we explore the effects of circPCNXL2 and trametinib (a MEK1/2 inhibitor) in vivo.ResultscircPCNXL2 was upregulated in ICC tissues and cell lines, which promoted the proliferation and metastasis of ICC in vitro and in vivo. In terms of the mechanisms, circPCNXL2 could directly bind to STRAP and induce the interaction between STRAP and MEK1/2, resulting in the tumor promotion in ICC by activation of ERK/MAPK pathways. Besides, circPCNXL2 could regulate the expression of SRSF1 by sponging miR-766-3p and subsequently facilitated the growth of ICC. Finally, circPCNXL2 could partially inhibit the anti-tumor activity of trametinib in vivo.ConclusioncircPCNXL2 played a crucial role in the progression of ICC by interacting with STRAP to activate the ERK signaling pathway, as well as by modulating the miR-766-3p/SRSF1 axis. These findings suggest that circPCNXL2 may be a promising biomarker and therapeutic target for ICC.

Multiomics analysis reveals metabolic subtypes and identifies diacylglycerol kinase α (DGKA) as a potential therapeutic target for intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous and lethal hepatobiliary tumor with few therapeutic strategies. The metabolic reprogramming of tumor cells plays an essential role in the development of tumors, while the metabolic molecular classification of iCCA is largely unknown. Here, we performed an integrated multiomics analysis and metabolic classification to depict differences in metabolic characteristics of iCCA patients, hoping to provide a novel perspective to understand and treat iCCA. We performed integrated multiomics analysis in 116 iCCA samples, including whole-exome sequencing, bulk RNA-sequencing and proteome analysis. Based on the non-negative matrix factorization method and the protein abundance of metabolic genes in human genome-scale metabolic models, the metabolic subtype of iCCA was determined. Survival and prognostic gene analyses were used to compare overall survival (OS) differences between metabolic subtypes. Cell proliferation analysis, 5-ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, RNA-sequencing and Western blotting were performed to investigate the molecular mechanisms of diacylglycerol kinase α (DGKA) in iCCA cells. Three metabolic subtypes (S1-S3) with subtype-specific biomarkers of iCCA were identified. These metabolic subtypes presented with distinct prognoses, metabolic features, immune microenvironments, and genetic alterations. The S2 subtype with the worst survival showed the activation of some special metabolic processes, immune-suppressed microenvironment and Kirsten rat sarcoma viral oncogene homolog (KRAS)/AT-rich interactive domain 1A (ARID1A) mutations. Among the S2 subtype-specific upregulated proteins, DGKA was further identified as a potential drug target for iCCA, which promoted cell proliferation by enhancing phosphatidic acid (PA) metabolism and activating mitogen-activated protein kinase (MAPK) signaling. Via multiomics analyses, we identified three metabolic subtypes of iCCA, revealing that the S2 subtype exhibited the poorest survival outcomes. We further identified DGKA as a potential target for the S2 subtype.

Prognostic value of programmed cell death ligand 1 expression in patients with intrahepatic cholangiocarcinoma: a meta-analysis.

Programmed cell death ligand 1 (PD-L1) is highly expressed in intrahepatic cholangiocarcinoma (ICC) tissues. But there is still a dispute over the prognostic value of PD-L1 in patients with ICC. This study aimed to evaluate the prognostic value of PD-L1 expression in patients with ICC. We performed a meta-analysis based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Guidelines. We searched the literature from PubMed, Embase, Web of Science, and the Cochrane Library up to December 5, 2022. Hazard ratios (HR) and their 95% confidence intervals (95% CI) were calculated to analyze the overall survival (OS), recurrence-free survival (RFS), and time to relapse. The quality of the studies was assessed using the Newcastle-Ottawa scale. Publication bias was assessed using a funnel plot and Egger's test. Ten trials with 1944 cases were included in this meta-analysis. The results showed that the low-PD-L1 group had a statistically significant advantage in OS (HR, 1.57; 95% CI, 1.38-1.79, P <0.00001), RFS (HR, 1.62; 95% CI, 1.34-1.97, P <0.00001), and time to relapse (HR, 1.60; 95% CI, 1.25-2.05, P = 0.0002) compared with the high-PD-L1 group. High programmed cell death (PD1)levels, on the other hand, were correlated with poorer OS (HR, 1.96; 95% CI, 1.43-2.70; P <0.0001) and RFS (HR, 1.87; 95% CI, 1.21-2.91; P = 0.005). Multivariate analysis showed that PD-L1 could act as an independent predictor for OS (HR, 1.48; 95% CI, 1.14-1.91; P = 0.003) and RFS (HR, 1.74; 95% CI, 1.22-2.47; P = 0.002), and PD1 acted as an independent predictor for OS (HR, 1.66; 95% CI, 1.15-2.38; P = 0.006). This meta-analysis demonstrated that high PD-L1/PD1 expression is associated with poor survival in ICC. PD-L1/PD1 may be a valuable prognostic and predictive biomarker and potential therapeutic target in ICC. https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42022380093.

Knockdown of FGFR3 inhibits the proliferation, migration and invasion of intrahepatic cholangiocarcinoma.

The FGF/FGFR signaling axis deregulation of the fibroblast growth factor receptor (FGFR) family is closely related to tumorigenesis, tumor progression and drug resistance to anticancer therapy. And fibroblast growth factor receptor 3 (FGFR3) is one member of this family. In this study, we aimed to investigate the effect of siRNA-induced knockdown of FGFR3 on the biological behaviors of intrahepatic cholangiocarcinoma (ICC). The expression levels of FGFR3 were determined in three intrahepatic cholangiocarcinoma cell lines RBE, HUCCT1 and HCCC9810 cell lines by Western blot. FGFR3 expression in RBE cell line was knocked down by siRNA. Our study found that knockdown of FGFR3 inhibited the migration, invasion and proliferation of ICC cells using Wound healing assay, Transwell migration and invasion assays and Cell proliferation assay. And significantly down-regulated the protein expression levels of MMP2, cyclinD1, and NCadherin, but had no significant effect on MMP9, cyclinD3, vimentin, E-cadherin protein. In addition, we found that ERK/c-Myc presumably is its signaling pathway by bioinformatics analysis and Western blot verification. To sum up, knockdown of FGFR3 inhibited the migration, invasion and proliferation of ICC cells. It demonstrated that FGFR3 probably becomes a therapeutic target for ICC and increases the proportion of potentially curable intrahepatic cholangiocarcinoma patients treated with FGFR inhibitors.

CircRNA MBOAT2 promotes intrahepatic cholangiocarcinoma progression and lipid metabolism reprogramming by stabilizing PTBP1 to facilitate FASN mRNA cytoplasmic export

The carcinogenic role of FASN by regulating lipid metabolism reprogramming has been well-established in multiple tumors. However, whether mechanisms during intrahepatic cholangiocarcinoma (ICC) progression, such as circRNAs, regulate FASN expression remains unknown. Here we demonstrate a lipid metabolism-related circRNA, circMBOAT2 (hsa_circ_0007334 in circBase), frequently upregulated in ICC tissues, and positively correlated with ICC malignant features. CircMBOAT2 knockdown inhibits the growth and metastasis of ICC cells. Mechanistically, circMBOAT2 combines with PTBP1 and protects PTBP1 from ubiquitin/proteasome-dependent degradation, impairing the function of PTBP1 to transfer FASN mRNA from the nucleus to the cytoplasm. Moreover, circMBOAT2 and FASN have the same effect on fatty acid profile, unsaturated fatty acids instead of saturated fatty acids are primarily regulated and associated with malignant behaviors of ICC cells. The levels of lipid peroxidation and ROS were significantly higher when FASN was knocked down and recovered when circMBOAT2 was overexpressed. Our results identified that circMBOAT2 was upregulated in ICC and promoted progression by stabilizing PTBP1 to facilitate FASN mRNA cytoplasmic export, which altered lipid metabolic profile and regulated redox homeostasis in ICC, suggesting that circMBOAT2 may serve as an available therapeutic target for ICC with active lipid metabolism.

Suppression of intrahepatic cholangiocarcinoma cell growth by SKI via upregulation of the CDK inhibitor p21.

Cholangiocarcinoma (CC) has a poor prognosis and different driver genes depending on the site of onset. Intrahepatic CC is the second-most common liver cancer after hepatocellular carcinoma, and novel therapeutic targets are urgently needed. The present study was conducted to identify novel therapeutic targets by exploring differentially regulated genes in human CC. MicroRNA (miRNA) and mRNA microarrays were performed using tissue and serum samples obtained from 24 surgically resected hepatobiliary tumor cases, including 10 CC cases. We conducted principal component analysis to identify differentially expressed miRNA, leading to the identification of miRNA-3648 as a differentially expressed miRNA. We used an in silico screening approach to identify its target mRNA, the tumor suppressor Sloan Kettering Institute (SKI). SKI protein expression was decreased in human CC cells overexpressing miRNA-3648, endogenous SKI protein expression was decreased in human CC tumor tissues, and endogenous SKI mRNA expression was suppressed in human CC cells characterized by rapid growth. SKI-overexpressing OZ cells (human intrahepatic CC cells) showed upregulation of cyclin-dependent kinase inhibitor p21 mRNA and protein expression and suppressed cell proliferation. Nuclear expression of CDT1 (chromatin licensing and DNA replication factor 1), which is required for the G1/S transition, was suppressed in SKI-overexpressing OZ cells. SKI knockdown resulted in the opposite effects. Transgenic p21-luciferase was activated in SKI-overexpressing OZ cells. These data indicate SKI involvement in p21 transcription and that SKI-p21 signaling causes cell cycle arrest in G1, suppressing intrahepatic CC cell growth. Therefore, SKI may be a potential therapeutic target for intrahepatic CC.

WWP1 upregulation predicts poor prognosis and promotes tumor progression by regulating ubiquitination of NDFIP1 in intrahepatic cholangiocarcinoma

WW domain-containing E3 ubiquitin protein ligase1 (WWP1) is reported to be upregulated in many types of human cancers; however, its expression and function in intrahepatic cholangiocarcinoma (ICC) remain unknown. Here, in this study we investigated the expression pattern, clinical prognosis, tumor biological functions, and molecular mechanisms of WWP1 in ICC. The expression of WWP1 in patient tissues was detected by western blotting, immunohistochemistry (IHC), and immunofluorescence. CCK-8, colony formation, EdU, transwell, and xenograft models were used to explore the role of WWP1 in the proliferation and metastasis of ICC. Co-immunoprecipitation, mass spectrometry, chromatin immunoprecipitation, and immunofluorescence were performed to detect the potential mechanisms. Our study revealed that WWP1 was highly expressed in ICC, and high levels of WWP1 were associated with poor prognosis. Functionally, WWP1 overexpression enhanced the proliferation and metastasis of ICC cells and vice versa. Mechanistically, MYC could be enriched in the promoter region of WWP1 to facilitate its expression. Then, WWP1 targets Nedd4 family interacting protein1 (NDFIP1) and reduces NDFIP1 protein levels via ubiquitination. Downregulation of NDFIP1 in ICC cells rescued the effects of silenced WWP1 expression. WWP1 expression was also negatively correlated with the protein level of NDFIP1 in patient tissues. In conclusion, WWP1 upregulated by MYC promotes the progression of ICC via ubiquitination of NDFIP1, which reveals that WWP1 might be a potential therapeutic target for ICC.

PRDX6 knockout restrains the malignant progression of intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) has a poor prognosis. The bifunctional protein peroxiredoxin 6 (PRDX6), which has both calcium-independent phospholipase A2 (iPLA2) and glutathione peroxidase (GPx) activity, participates in the development of multiple tumors. However, the function and clinical significance of PRDX6 in ICC remain unclear. In this study, we characterized PRDX6 in both human ICC and thioacetamide (TAA)-induced rat ICC. We found PRDX6 was significantly increased in ICC tissues, compared with the peritumoral tissues, and PRDX6 expression level was positively correlated with the malignant phenotype in ICC patients. Furthermore, PRDX6 genetic knockout significantly inhibited the tumor progression in rats. By using RNA sequencing analysis, we found 127 upregulated genes and 321 downregulated genes after PRDX6 knockout. In addition, we noticed a significant repression in the Wnt7a/b cascade, which has been shown to play an important role in the occurrence of ICC. We confirmed that gene expressions in the Wnt7a/b cascade were inhibited in ICC tissues after PRDX6 knockout by using qRT-PCR and immunohistochemistry analysis. Collectively, our findings suggest that PRDX6 may promote ICC by regulating the Wnt7a/b pathway, which could be a novel therapeutic target for ICC.

INTS8 is a therapeutic target for intrahepatic cholangiocarcinoma via the integration of bioinformatics analysis and experimental validation

Intrahepatic cholangiocarcinoma (CHOL) remains a rare malignancy, ranking as the leading lethal primary liver cancer worldwide. However, the biological functions of integrator complex subunit 8 (INTS8) in CHOL remain unknown. Thus, this research aimed to explore the potential role of INTS8 as a novel diagnostic or therapeutic target in CHOL. Differentially expressed genes (DEGs) in two Gene Expression Omnibus (GEO) datasets were obtained by the “RRA” package in R software. The “maftools” package was used to visualize the CHOL mutation data from The Cancer Genome Atlas (TCGA) database. The expression of INTS8 was detected by performing quantitative reverse transcription-PCR (qRT-PCR) and immunohistochemistry in cell lines and human samples. The association between subtypes of tumour-infiltrating immune cells (TIICs) and INTS8 expression in CHOL was determined by using CIBERSORT tools. We evaluated the correlations between INTS8 expression and mismatch repair (MMR) genes and DNA methyltransferases (DNMTs) in pan-cancer analysis. Finally, the pan-cancer prognostic signature of INTS8 was identified by univariate analysis. We obtained the mutation landscapes of an RRA gene set in CHOL. The expression of INTS8 was upregulated in CHOL cell lines and human CHOL samples. Furthermore, INTS8 expression was closely associated with a distinct landscape of TIICs, MMR genes, and DNMTs in CHOL. In addition, the high INTS8 expression group presented significantly poor outcomes, including overall survival (OS), disease-specific survival (DSS) and disease-free interval (DFI) (p < 0.05) in pan-cancer. INTS8 contributes to the tumorigenesis and progression of CHOL. Our study highlights the significant role of INTS8 in CHOL and pan-cancers, providing a valuable molecular target for cancer research.

Circular RNA ACTN4 promotes intrahepatic cholangiocarcinoma progression by recruiting YBX1 to initiate FZD7 transcription

Intrahepatic cholangiocarcinoma (ICC) is a primary liver cancer with high aggressiveness and extremely poor prognosis. The role of circular RNAs (circRNAs) in ICC carcinogenesis and progression remains to be determined. CircRNA microarray was performed to screen significantly upregulated circRNAs in paired ICC and non-tumor tissues. Colony formation, transwell, and xenograft models were used to examine the role of circRNAs in ICC proliferation and metastasis. RNA pulldown, mass spectrometry, chromatin immunoprecipitation, RNA-binding protein immunoprecipitation, chromatin isolation by RNA purification, electrophoretic mobility shift assay, and luciferase reporter assays were used to explore the molecular sponge role of the circRNA (via miRNA binding), and the interaction between circRNA and RNA-binding proteins. Hsa_circ_0050898, which originated from exon 1 to exon 20 of the ACTN4 gene (named circACTN4), was significantly upregulated in ICC. High circACTN4 expression was associated with enhanced tumor proliferation and metastasis invitro and invivo, as well as a worse prognosis following ICC resection. In addition, circACTN4 upregulated Yes-associated protein 1 (YAP1) expression by sponging miR-424-5p. More importantly, circACTN4 also recruited Y-box binding protein 1 (YBX1) to stimulate Frizzled-7 (FZD7) transcription. Furthermore, circACTN4 overexpression in ICC cells enhanced the interaction between YAP1 and β-catenin, which are the core components of the Hippo and Wnt signaling pathways, respectively. CircACTN4 was upregulated in ICC and promoted ICC proliferation and metastasis by acting as a molecular sponge of miR-424-5p, as well as by interacting with YBX1 to transcriptionally activate FZD7. These results suggest that circACTN4 is a potential prognostic marker and therapeutic target for ICC. Intrahepatic cholangiocarcinoma is a primary liver cancer associated with aggressiveness and extremely poor prognosis. It is essential for therapeutic development that we uncover relevant pathogenic pathways. Herein, we showed that a circular RNA (circACTN4) was highly expressed in intrahepatic cholangiocarcinoma and was positively associated with tumor growth and metastasis through key developmental signaling pathways. Thus, circACTN4 could be a prognostic biomarker and therapeutic target for intrahepatic cholangiocarcinoma.

LHPP suppresses tumorigenesis of intrahepatic cholangiocarcinoma by inhibiting the TGFβ/smad signaling pathway.

Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP), a histidine phosphatase, plays an important role in tumor progression and metastasis as a tumor suppressor. Here, we investigate the effect of LHPP in intrahepatic cholangiocarcinoma (ICC). We discovered that LHPP was downregulated in tumor tissues and low levels of LHPP predicted poor survival. LHPP inhibited ICC cell growth, cell invasion and epithelial-mesenchymal transition (EMT) in vitro and in vivo. Mechanically, LHPP deactivated transforming growth factor‑beta (TGFβ) signaling pathway, and low level LHPP upregulated the expression of TGFβ and phosphorylation of smad2/3. Moreover, inhibition of this pathway reversed the biofunction of LHPP. In summary, these findings demonstrated that LHPP suppressed ICC through inhibiting the activation of TGFβ/smad signaling. Our results indicated that LHPP is a potential therapeutic target in ICC.

Integrated Bioinformatics analysis and clinical validation reveals that high expression of mucin 1 in intrahepatic cholangiocarcinoma predicts recurrence after curative resection.

Intrahepatic cholangiocarcinoma (ICC) is a cancer type with high malignancy and a current lack of biomarkers to predict recurrence. In the present study, to identify potential biomarkers, five ICC datasets from the Gene Expression Omnibus database were analyzed to construct initial datasets by using a robust rank aggregation approach. A total of 19 upregulated genes were identified in the initial datasets. The genes identified were then further analysed using data from The Cancer Genome Atlas. Only mucin 1 (MUC1) exhibited significance regarding differential expression and survival prediction. Finally, the expression levels of MUC1 were assessed using reverse transcription-quantitative PCR in 61 pairs of ICC tumor and matched non-cancerous samples. The expression of MUC1 was significantly elevated in ICC tissues compared with that in matched non-cancerous counterparts (P=0.001). Patients with high MUC1 expression levels had significantly shorter overall survival (OS, P=0.009) and recurrence-free survival (RFS, P=0.012). MUC1 was identified as an independent prognostic factor for OS [hazard ratio (HR)=2.364, 95%CI: 1.214-4.485; P=0.023] and RFS (HR=2.552 95%CI: 1.294-5.032; P=0.007) in the multivariate analysis. Using receiver operating characteristic analysis, a co-index including MUC1 had a high accuracy for predicting survival [MUC1 combined with serum levels of CEA and cancer antigen 19-9, and lymph node metastasis, area under curve (AUC)=0.746, 95%CI: 0.620-0.872] and recurrence (MUC1 combined with bile duct invasion and lymph node metastasis, AUC=0.729, 95%CI: 0.605-854). In conclusion, MUC1 is highly expressed in ICC tissue and is a potential prognostic biomarker and therapeutic target for ICC.

Expression of GLS1 in intrahepatic cholangiocarcinoma and its clinical significance.

Kidney‑type glutaminase (GLS1) plays a significant role in tumor metabolism. Our recent studies demonstrated that GLS1 was aberrantly expressed in hepatocellular carcinoma (HCC) and facilitated tumor progression. However, the roles of GLS1 in intrahepatic cholangiocarcinoma (ICC) remain largely unknown. Thus, the aim of this study was to evaluate the expression and clinical significance of GLS1 in ICC. For this purpose, combined data from the Oncomine database with those of immunohistochemistry were used to determine the expression levels of GLS1 in cancerous and non‑cancerous tissues. Second, a wound‑healing assay and Transwell assay were used to observe the effects of the knockdown and overexpression of GLS1 on the invasion and migration of ICC cells. We examined the associations between the expression of GLS1 and epithelial‑mesenchymal transition (EMT)‑related markers by western blot analysis. Finally, we examined the associations between GLS1 levels and clinicopathological factors or patient prognosis. The results revealed that GLS1 was overexpressed in different digestive system tumors, including ICC, and that GLS1 expression in ICC tissue was higher than that in peritumoral tissue. The overexpression of GLS1 in RBE cells induced metastasis and invasion. Moreover, the EMT‑related markers, E‑cadherin and Vimentin, were regulated by GLS1 in ICC cells. By contrast, the knockdown of GLS1 expression in QBC939 cells yielded opposite results. Clinically, a high expression of GLS1 in ICC samples negatively correlated with E‑cadherin expression and positively correlated with Vimentin expression. GLS1 protein expression was associated with tumor differentiation (P=0.001) and lymphatic metastasis (P=0.029). Importantly, patients with a high GLS1 expression had a poorer overall survival (OS) and a shorter time to recurrence than patients with a low GLS1 expression. Multivariate analysis indicated that GLS1 expression was an independent prognostic indicator. On the whole, the findings of this study demonstrated that GLS1 is an independent prognostic biomarker of ICC. GLS1 facilitates ICC progression and may thus prove to be a therapeutic target in ICC.

H2A.Z regulates tumorigenesis, metastasis and sensitivity to cisplatin in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a fatal, malignant tumor of the liver; effective diagnostic biomarkers and therapeutic targets for ICC have not been identified yet. High expression of H2A histone family member Z (H2A.Z) is a high-risk factor for poor prognosis in patients with breast cancer and primary hepatocellular cancer. However, the significance of H2A.Z and its expression in ICC remains unknown. The present study demonstrated that H2A.Z is overexpressed in ICC and expression of H2A.Z correlated with poor prognosis in patients with ICC. H2A.Z regulated cell proliferation in vitro and in vivo via H2A.Z/S-phase kinase-associated protein 2/p27/p21 signaling. Inhibition of H2A.Z reduced cell proliferation and induced apoptosis in ICC. In addition, downregulation of H2AZ reduced tumor metastasis by repressing epithelial-mesenchymal transition and enhanced the antitumor effects of cisplatin in the treatment of ICC. Overall, H2A.Z promoted cell proliferation and epithelial-mesenchymal transition in ICC, suggesting that H2A.Z may be a novel biomarker and therapeutic target for ICC.

Osteopontin promotes metastasis of intrahepatic cholangiocarcinoma through recruiting MAPK1 and mediating Ser675 phosphorylation of \u03b2-Catenin

The incidence and mortality of intrahepatic cholangiocarcinoma (ICC) are increasing worldwide in recent decades. Osteopontin (OPN) plays an important role in cancer metastasis, but its functional mechanism in ICC is not clear yet. In this study, we found that OPN level was elevated both in plasma and tumor tissues of ICC patients, which was closely related to a shorter overall survival (OS) and high probability of tumor relapse after curative resection. The gain- and loss-of-function studies determined that OPN could promote ICC growth and metastasis. OPN selectively interacted with β-Catenin and knockdown of β-Catenin abrogated the effects induced by OPN. OPN recruited MAPK1 and activated MEK–MAPK1 pathway to mediate the S675 phosphorylation of β-Catenin and nucleus accumulation, which induced the activation of Wnt signaling. Moreover, a significant correlation between OPN and β-Catenin was found in ICC tissues. OPN, β-Catenin, and their combination were independent prognostic indicator for ICC patients. In conclusion, OPN promotes ICC progression through recruiting MAPK1 and activating the Wnt/β-Catenin pathway and can serve as a novel prognostic marker and therapeutic target for ICC.

Upregulation of long non‑coding RNA CCAT2 indicates a poor prognosis and promotes proliferation and metastasis in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (IHCC) is an aggressive cancer with a poor survival rate and is the second most common type of primary cancer of the hepatobiliary system. At present, the molecular mechanisms of IHCC initiation and progression remain unclear. Recent evidence has indicated that long non‑coding RNAs (lncRNAs) serve a crucial role in cancer development; however, the functional role of lncRNAs in IHCC has not been investigated in detail. In the present study, a marked overexpression of lncRNA colon cancer‑associated transcript2 (CCAT2) was observed in IHCC cell lines and clinical specimens. Statistical analysis of IHCC clinicopathological characteristics and CCAT2 expression data revealed that high CCAT2 expression levels correlated with microvascular invasion, differentiation grade, tumor (T), lymph node (N), metastasis (M) and overall TNM stages of IHCC (P<0.05). Kaplan‑Meier analysis demonstrated that CCAT2 upregulation was associated with poor overall survival and progression‑free survival in IHCC. Furthermore, high CCAT2 expression was identified as an independent risk factor of IHCC poor prognosis in both univariate and multivariate Cox regression analyses. The role of CCAT2 in promoting IHCC cell proliferation, motility and invasion was further confirmed with invitro assays. Therefore, CCAT2 may promote IHCC progression and metastasis, and may be a promising prognostic biomarker and therapeutic target in IHCC.

Overexpression of long non‑coding RNA CRNDE facilitates epithelial‑mesenchymal transition and correlates with poor prognosis in intrahepatic cholangiocarcinoma

The clinical significance and essential role of long non-coding RNA colorectal neoplasia differentially expressed (lncRNA CRNDE) have been well illuminated in various cancers. However, the function of CRNDE in intrahepatic cholangiocarcinoma (IHCC) has not been reported at present. The aim of the present study was to investigate the role of CRNDE in IHCC. Firstly, the relative expression of CRNDE was observed to be upregulated in IHCC cell lines and tissues. And high CRNDE expression was statistically associated with IHCC differentiation grade, lymph node metastasis, tumor-nodes-metastasis (TNM) stage and size. Survival analysis identified that high CRNDE expression is a predictor of worse overall survival (OS) and progression-free survival (PFS) in patients with IHCC. Moreover, high CRNDE expression was identified as an independent risk factor of IHCC poor OS and PFS. Further studies of in vitro assays suggested that CRNDE silencing could suppress the proliferation of HuCCT1 cells following CCK-8 and colony formation assays, while CRNDE ectopic expression in HCCC9810 cells promoted proliferation. Moreover, the migration and invasion of HuCCT1 cells were greatly repressed with CRNDE deficiency following Transwell and Matrigel assays. Accordingly, the motility of HCCC9810 cells was notably accelerated with CRNDE overexpression. Mechanistically, CRNDE was revealed to facilitate the epithelial-mesenchymal transition (EMT) of IHCC cells. In conclusion, these observations indicated that CRNDE could promote the clinical progression and metastasis of IHCC by facilitating EMT. CRNDE may be a novel prognostic marker and therapeutic target in IHCC.

Abstract 5768: Intrahepatic cholangiocarcinoma cells can be converted into functional hepatocytes by inhibition of Wnt signaling pathway

Abstract Background and Aim: Intrahepatic cholangiocarcinoma (IHCC) is the second most prevalent malignancy in the liver, and is one of the most aggressive malignancies characterized by a poor prognosis. It is believed that IHCC is an epithelial cell malignancy arising from cholangiocytes composing the intrahepatic bile ducts. However, patients with viral hepatitis develop IHCC, suggesting that IHCC is derived from transformed hepatocytes. Indeed, recent studies have revealed that IHCC can arise from Notch-mediated conversion of hepatocytes in mice. The new 3D culture system called organoid culture has been developed, which allows long-term expansion of Lgr5-positive stem cells. To clarify whether IHCC cells harbor the potency of conversion into functional hepatocytes, we established organoids derived from human IHCC and cultured them in different culture conditions. Methods: We established organoids derived from human IHCC and cultured these organoids with expansion medium (EM) containing R-spondin 1. Conversion of IHCC cells into hepatocytes was conducted by organoid culture with differentiation medium (DM) containing BMP7, DAPT, FGF19 and dexamethasone. Results: Gene set enrichment analysis (GSEA) showed that genes associated with hepatocyte differentiation signature were significantly enriched in IHCC organoids cultured with DM compared with those cultured with EM. IHCC organoids cultured with DM induced up-regulation of hepatocyte markers albumin, HNF4a and CYP3A4, and acquired mature hepatocyte functions including albumin secretion, bile acid production and increased CYP3A4 activity. Removal of R-spondin 1, a ligand of Wnt signaling pathway, from EM enhanced hepatocyte differentiation. IHCC organoids cultured with DM significantly reduced the malignant potential in vitro and in vivo compared with those cultured with EM. Further studies using thioacetamide (TAA)-induced IHCC model mice showed that TAA-induced inflammation in the liver promoted recruited macrophages to secrete Wnt3a. Conclusions: These findings indicated that IHCC cells can be converted into functional hepatocytes by inhibition of Wnt signaling pathway. Activation of Wnt signaling pathway by recruited macrophages may play a critical role in the malignant transformation of hepatocytes into IHCC, which could be an important therapeutic target for IHCC. Citation Format: Toshiaki Nakaoka, Yoshimasa Saito, Toshihide Muramatsu, Hidenori Ojima, Yae Kanai, Yuko Sugiyama, Masaki Kimura, Takanori Kanai, Toshiro Sato, Hidetsugu Saito. Intrahepatic cholangiocarcinoma cells can be converted into functional hepatocytes by inhibition of Wnt signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5768. doi:10.1158/1538-7445.AM2017-5768