Intrahepatic Cholangiocarcinoma Cells Research Articles

KRAS mutation in intrahepatic cholangiocarcinoma: Linkage with metastasis-free survival and reduced E-cadherin expression.

Although KRAS mutations are the major driver of intrahepatic cholangiocarcinoma (ICC), their role remains unexplored. This study aimed to elucidate the prognostic effects, association with clinicopathologic characteristics and potent functions of KRAS mutations in ICC. A hundred and seven resected stage I-III ICCs were analysed for KRAS mutation status and its link with clinicopathological features. An independent validation cohort (n=138) was included. In vitro analyses using KRAS-mutant ICC cell lines were performed. KRAS mutation was significantly associated with worse overall survival in stage I-III ICCs, which was validated in an independent cohort. Recurrence-free survival did not significantly differ between cases with and without KRAS mutations, but if limited to recurrence with extrahepatic metastasis, KRAS-mutant cases showed significantly worse distant metastasis-free survival than KRAS-wild cases showed. KRAS mutations were associated with frequent tumour budding with reduced E-cadherin expression. In vitro, KRAS depletion caused marked inhibition of cell growth and migration together with E-cadherin upregulation in KRAS-mutant ICC cells. The RNA-sequencing assay revealed that KRAS depletion caused MYC pathway downregulation and interferon pathway upregulation. Our observations suggest that KRAS mutations are associated with aggressive behaviour of ICC, especially the development of extrahepatic metastasis. Mutant KRAS is likely to change the adhesive status of ICC cells, affect the responsiveness of tumour cells to interferon immune signals, and consequently promote extrahepatic metastasis. KRAS mutation status, which predicts the prognoses of patients with ICC after surgical resection, is expected to help stratify patients better for individual postoperative treatment strategies.

Liver kinase B1 in exosomes inhibits immune checkpoint programmed death ligand 1 and metastatic progression of intrahepatic cholangiocarcinoma.

The increasing morbidity and high mortality of intrahepatic cholangiocarcinoma (ICC) has led to the urgent need for new diagnostics and therapeutics. Liver kinase B1 (LKB1) exerts a tumor suppressor role in multiple malignances, while its regulatory role in exosomes secreted by ICC cells is obscure. In the present study, exosomes were extracted from cell culture supernatants of RBE and HCCC-9810 ICC cells as well as plasma of patients with ICC by ultracentrifugation and the morphology of exosomes was identified by transmission electron microscopy. Notably, compared with that of intracellular LKB1, the protein level of exosomal LKB1 was decreased. Silencing intracellular LKB1 increased the protein levels of programmed death ligand 1 (PD-L1), Slug and phosphorylated-AKT in exosomes, accompanied by decreased expression levels of exosomal LKB1. Exosomes with lower protein levels of LKB1 promoted the expression of the immune checkpoint PD-L1, malignant phenotypes of ICC cells in vitro, and cancer metastasis in vivo. Moreover, the low level of exosomal LKB1 in plasma was tightly associated with the poor prognosis of patients with ICC. Collectively, exosomal LKB1 inhibits the immune checkpoint PD-L1 and metastasis of ICC cells. These findings may provide new methods for the diagnosis and immune therapy of ICC.

Novel Drug Candidate Prediction for Intrahepatic Cholangiocarcinoma via Hub Gene Network Analysis and Connectivity Mapping

Simple SummaryOnly about 15% of intrahepatic cholangiocarcinoma (ICC) patients meet the criteria for resection at the time of diagnosis. For patients with advanced and/or metastatic disease, the development of novel therapeutic strategies is urgently needed. The aim of our study was to identify possible novel therapeutic targets and drugs for ICC by using transcriptomic profiles from the Gene Expression Omnibus databases and The Cancer Genome Atlas. The weighted co-expression gene network was constructed to screen hub genes. Potential drug candidates with promise in the treatment of ICC were identified by analyzing key protein–protein interaction (PPI) networks of the hub genes to identify potential interacting drugs based on the Connectivity Map database.Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy, and there is a need for effective systemic therapies. Gene expression profile-based analyses may allow for efficient screening of potential drug candidates to serve as novel therapeutics for patients with ICC. The RNA expression profile of ICC and normal biliary epithelial cells were downloaded from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Function annotation and enrichment pathway analyses of the differentially expressed genes (DEGs) were finished using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A weighted gene co-expression network (WGCN) was constructed by WGCN analysis (WGCNA). Key genes from the DEGs and co-expression gene modules were analyzed to generate a protein–protein interaction (PPI) network. The association between the top 10 screened hub genes and the overall and disease-free survival of ICC patients was examined. The Connectivity Map (cMap) analysis was performed to identify possible drugs for ICC using hub genes. A total of 151 key genes were selected from the overlapping genes of 1287 GSE-DEGs, 8183 TCGA-DEGs and 1226 genes in the mixed modules. A total of 10 hub genes of interest (CTNNB1, SPP1, COL1A2, COL3A1, SMAD3, SRC, VCAN, PKLR, GART, MRPS5) were found analyzing protein–protein interaction. Using the cMap, candidate drugs screened with potential efficacy for ICC included three tyrosine kinase inhibitors (dasatinib, NVP-BHG712, tivantinib), two cannabinoid receptor agonists (palmitoylethanolamide, arachidonamide), two antibiotics (moxifloxacin, amoxicillin), one estrogen receptor agonist (levonorgestrel), one serine/threonine protein kinase inhibitor (MK-2206) and other small molecules. Key genes from network and PPI analysis allowed us to identify potential drugs for ICC. The identification of novel gene expression profiles and related drug screening may accelerate the identification of potential novel drug therapies for ICC.

EIF5A2 promotes proliferation and invasion of intrahepatic cholangiocarcinoma cells

PurposeIntrahepatic cholangiocarcinoma (ICC) can invade and metastasize. EIF5A2 is involved in the invasive metastatic process of several digestive malignancies. However, its role in ICC is yet to be elucidated. MethodsImmunohistochemistry (IHC) and Western blot (WB) were used to detect the level of EIF5A2 in the tumor specimens of ICC patients and evaluate the correlation between its expression and clinicopathological characteristics. The significance of EIF5A2 in the prognosis of ICC patients was further evaluated by Kaplan–Meier and Cox regression analysis. In addition, CCK-8, EdU, Transwell invasion, and scratch assays were utilized to detect tumor cell proliferation, invasion, and metastasis. Furthermore, the role of EIF5A2 in ICC cells was evaluated after modification of EIF5A2 expression. ResultsThe level of EIF5A2 protein was significantly higher in ICC than in adjacent tissues. This high expression in the tumor samples was significantly associated with malignant phenotypes, such as lymph node metastasis (LNM), microvascular or bile duct invasion, and poor differentiation. ICC patients with high expression of EIF5A2 had short overall survival and a high cumulative recurrence rate. The multifactorial analysis showed that EIF5A2 is an independent prognostic marker. Furthermore, high levels of EIF5A2 may activate the PI3K/AKT/mTOR signaling pathway and upregulate Cyclin D1, Cyclin D3, MMP2, and MMP9 to promote ICC cell proliferation, migration, and invasion. ConclusionThe current study found that EIF5A2 promotes ICC progression and is a prognostic biomarker and candidate therapeutic target for ICC patients.

Novel Nitric Oxide Donor Dinitroazetidine-Coumarin Hybrids as Potent Anti-Intrahepatic Cholangiocarcinoma Agents.

Intrahepatic cholangiocarcinoma (iCC) is a serious liver cancer threatening human health. However, there are a few chemotherapeutic drugs for the treatment of iCC in the clinic. It is extremely urgent to develop new drugs for iCC. In this study, twenty dinitroazetidine and coumarin hybrids were synthesized and evaluated anti-iCC bioactivity as a new type of nitric oxide (NO) donors. Among them, compounds 2–5 and 21 showed a higher antiproliferative activity against RBE cell lines (human intrahepatic cholangiocarcinoma cell lines) and low cytotoxicity in nontumor cells (HOSEpiC and T29). The preliminary study of pharmacology mechanism indicated that compounds 2–5 and 21 could release effective concentration of NO in RBE cell lines, which leaded to inhibit the proliferation of RBE cell lines. The research results revealed that compound 3 inhibited the proliferation of RBE cell lines by inducing apoptosis and arresting cell cycle at G2/M phase. Additionally, compound 3 had acceptable metabolic stability. Therefore, compound 3 was merited to further explore for developing a desirable NO donor lead with anti-iCC activity.

The role of Tripartite motif containing 59 (TRIM59) in the proliferation and prognosis of intrahepatic cholangiocarcinoma

Tripartite motif containing 59 (TRIM59) is a crucial gene that is involved in the process of various types of cancer，including breast cancer, lung cancer, colorectal cancer,and so on. Its abnormal expression can affect tumor cell proliferation, metastasis, or apoptosis. In liver cancer, the incidence of intrahepatic cholangiocarcinoma (ICC) is increasing. However, However, it has not been clearly reported on TRIM59 affects the progress of intrahepatic cholangiocarcinoma cells.Firstly, we review the expression of TRIM59 in different cancers and the corresponding normal tissues，and the results preliminarily showed that TRIM59 may be abnormally expressed in many cancers. The author focuses on whether TRIM59 plays a crucial biological role in intrahepatic cholangiocarcinoma. Therefore, we have confirmed through online websites that TRIM59 is highly expressed in intrahepatic cholangiocarcinoma tissues. Furthermore we further found that TRIM59 can be used as an effective prognostic marker for the prognostic guidance of patient survival time. Next, we explore whether the expression level of TRIM59 in intrahepatic cholangiocarcinoma is related to proliferation through the CCK-8 and EDU assay in two ICC cell lines. To further explore how TRIM59 affected the molecular mechanism involved in intrahepatic cholangiocarcinoma cell growth, we found that STAT3 promotes TRIM59 transcription and TRIM59 can affect tumor progression by regulating the PI3K/AKT signaling pathway through luciferase reporter assay and Western blot experiments. In summary, we first found that TRIM59 has great research value in ICC through bioinformatic analysis, then its expression level is closely related to the prognosis through the analysis of clinicopathological indicators of patients with ICC, and the biological mechanism of TRIM59 in ICC provides precise research or therapeutic targets for future cancer treatment. The findings improve our understanding of the potential of TRIM59 in biological functions in ICC and may hold promise as markers for the diagnosis,treatment, and prognosis of ICC. Data availabilityThe raw data of this study are derived from the TCGA database, which are publicly available databases.

SQSTM1/p62 in intrahepatic cholangiocarcinoma promotes tumor progression via epithelial-mesenchymal transition and mitochondrial function maintenance.

SQSTM1/p62 is a selective autophagy receptor that regulates multiple signaling pathways participating in the initiation and progression of tumors. Metastasis is still the main cause for intrahepatic cholangiocarcinoma (ICC)-associated mortality. Hence, this study aimed to explore the mechanism of p62 promoting the progression of ICC. Western blotting and immunohistochemical analyses were conducted to detect the expression level of protein p62 in ICC tissues and its correlation with prognosis. Subsequently, the loss-of-function experiments in vitro and in vivo were performed to define the role of p62 in ICC cell proliferation, invasion, and metastasis. Then, the effect of p62 knockdown on mitochondrial function and mitophagy was evaluated by measuring the oxygen consumption rate, and using immunofluorescence and western blotting analyses. The expression of p62 was significantly upregulated in ICC specimens compared with normal tissues. We further illustrated that p62 expression positively correlated with lymph node metastasis and poor prognosis. The loss-of-function assays revealed that p62 not only promoted ICC cell proliferation, migration, and invasive capacities in vitro, but also induced lung metastasis in the xenograft mouse model. Mechanistically, high expression of p62-induced epithelial-mesenchymal transition (EMT) with the upregulation of Snail, vimentin, N-cadherin, and downregulation of E-cadherin. Moreover, the autophagy-dependent function of p62 might play a vital role in maintaining the mitochondrial function of ICC by mitophagy which might further promote EMT. These data provided new evidence for the mechanism by which abundant p62 expression promoted ICC progression, suggesting a promising therapeutic target for antimetastatic strategies in patients with ICC.

YTHDF2 promotes intrahepatic cholangiocarcinoma progression and desensitises cisplatin treatment by increasing CDKN1B mRNA degradation

BackgroundIntrahepatic cholangiocarcinoma (ICC) is an aggressive cancer with exceedingly poor prognosis, and chemoresistance is a huge challenge for treatment. N6‐methyladenosine (m6A) modification plays an important role in the progression and chemoresistance of cancers. We aimed to investigate the oncogenic function and therapeutic significance of the m6A binding protein, YTH domain family 2 (YTHDF2), in ICC progression and cisplatin‐based chemotherapy.MethodsSeveral independent data sets were used to assess the expression of YTHDF2 in ICC, particularly in chemoresistant ICC. Knockdown and overexpression were used to evaluate the effects of YTHDF2 on tumourigenesis and cisplatin response in ICC. Multi‐omics sequencing was performed to identify target genes. RIP, dual luciferase reporter, RNA stability experiment and loss‐of‐function assays were conducted to study the mechanisms underlying the oncogenic function of YTHDF2. Furthermore, patient‐derived xenograft (PDX) model was established to determine the effect of combination treatment of YTHDF2 siRNA and cisplatin in ICC.ResultsOur study showed that YTHDF2 was upregulated in ICC tissues, particularly in chemoresistant ICC tissues, and correlated with poor prognosis. Furthermore, silencing YTHDF2 led to inhibited proliferation, promoted apoptosis and G0/G1 cell cycle arrest. Its downregulation also enhanced DNA damage and sensitised ICC cells to cisplatin. YTHDF2 overexpression exerted the opposite results. Integration analysis using RNA‐seq, MeRIP‐seq and anti‐YTHDF2 RIP‐seq elucidated the role of YTHDF2 in tumourigenesis and cisplatin‐desensitising function by promoting the degradation of cyclin‐dependent kinase inhibitor 1B (CDKN1B) mRNA in an m6A‐dependent manner. Downregulation of CDKN1B increased the YTHDF2 silencing‐induced influence on tumourigenesis and cisplatin response to ICC. In addition, the combination treatment of YTHDF2 siRNA and cisplatin significantly enhanced the anti‐tumour effect of cisplatin in a chemoresistant ICC PDX model.ConclusionsYTHDF2 exhibits tumour oncogenic and cisplatin‐desensitising properties, which may offer insight into the development of novel combination therapeutic strategies for ICC.

Chemokine (C-C motif) ligand 18/membrane-associated 3/forkhead box O1 axis promotes the proliferation, migration, and invasion of intrahepatic cholangiocarcinoma

ABSTRACT Phosphatidylinositol Transfer Protein, Membrane-Associated 3 (PITPNM3) often bind with chemokine (C-C motif) ligand 18 (CCL18) to promote tumor progression. However, the role of PITPNM3 in intrahepatic cholangiocarcinoma (ICC) is unclear. We first searched GEPIA database and detected the PITPNM3 expression using immunohistochemistry and real-time quantitative PCR. The results showed that PITPNM3 is high expression in ICC tissues and cells. Then we investigated the cell function of CLL18 and PITPNM3 through cell clone formation assay and transwell assay. The results indicated that CCL18 treatment promoted the proliferation, migration, and invasion of ICC cells. Silence of PITPNM3 reversed the effect of CCL18 on cell function. Simultaneously, we detected key protein expression of forkhead box O1 (FOXO1) and nuclear factor kappa B (NF-KB) through western blotting and found that CCL18 activated NF-KB pathway while inhibited FOXO1 pathway, the effect of which were attenuated by silence of PITPNM3. Finally, we confirmed which pathway affected the cell function using inhibitor of FOXO1 (AS1842856) and activator of NF-KB (Asatone). The results showed that AS1842856, not Asatone, relieved the inhibitory effect of si-PITPNM3 on the cell function of CCL18. In short, CCL18 treatment activated PITPNM3 to promote the proliferation, migration, and invasion of ICC via FOXO1 signaling pathway. These results provided a new insight for the diagnosis and therapy of ICC.

Cellular heterogeneity and transcriptomic profiles during intrahepatic cholangiocarcinoma initiation and progression.

Intrahepatic cholangiocarcinoma (ICC) is not fully investigated, and how stromal cells contribute to ICC formation is poorly understood. We aimed to uncover ICC origin, cellular heterogeneity, and critical modulators during ICC initiation/progression, and to decipher how fibroblast and endothelial cells in the stromal compartment favor ICC progression. We performed single-cell RNA sequencing (scRNA-seq) using AKT/Notch intracellular domain-induced mouse ICC tissues at early, middle, and late stages. We analyzed the transcriptomic landscape, cellular classification and evolution, and intercellular communication during ICC initiation/progression. We confirmed the findings using quantitative real-time PCR, western blotting, immunohistochemistry or immunofluorescence, and gene knockout/knockdown analysis. We identified stress-responding and proliferating subpopulations in late-stage mouse ICC tissues and validated them using human scRNA-seq data sets. By integrating weighted correlation network analysis and protein-protein interaction through least absolute shrinkage and selection operator regression, we identified zinc finger, MIZ-type containing 1 (Zmiz1) and Y box protein 1 (Ybx1) as core transcription factors required by stress-responding and proliferating ICC cells, respectively. Knockout of either one led to the blockade of ICC initiation/progression. Using two other ICC mouse models (YAP/AKT, KRAS/p19) and human ICC scRNA-seq data sets, we confirmed the orchestrating roles of Zmiz1 and Ybx1 in ICC occurrence and development. In addition, hes family bHLH transcription factor 1, cofilin 1, and inhibitor of DNA binding 1 were identified as driver genes for ICC. Moreover, periportal liver sinusoidal endothelial cells could differentiate into tip endothelial cells to promote ICC development, and this was Dll4-Notch4-Efnb2 signaling-dependent. Stress-responding and ICC proliferating subtypes were identified, and Zmiz1 and Ybx1 were revealed as core transcription factors in these subtypes. Fibroblast-endothelial cell interaction promotes ICC development.

YTHDF1 promotes intrahepatic cholangiocarcinoma progression via regulating EGFR mRNA translation.

Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive disease with the underlying mechanisms poorly understood. YTHDF1, an N6 -methyladenosine (m6 A) reader protein, has important physiological functions in regulation of tumor development. However, the effect of YTHDF1 on ICC progression remains unknown yet. The expression level of YTHDF1 in human ICC tissue was examined in The Cancer Genome Atlas database and our cohort. The role of YTHDF1 was detected using two human ICC cell lines in vitro. An ICC tumorigenesis mouse model was established via hydrodynamic transfection of AKT/YAP plasmids. m6 A sequencing, RNA immunoprecipitation sequencing, and RNA sequencing were carried out to explore the mechanism of YTHDF1 modulating ICC progression. Here, we find that YTHDF1 is upregulated in ICC and associated with shorter survival of ICC patients. Depletion of YTHDF1 inhibits cell proliferation, migration, and invasion, while overexpression of wild-type YTHDF1, but not m6 A reader domain mutant YTHDF1, significantly enhances tumor cell growth and aggressive abilities in vitro. Moreover, overexpression of YTHDF1 promotes the AKT/YAP transfection-induced orthotopic ICC tumorigenesis and progression in vivo. Mechanistically, we identify that YTHDF1 regulates the translation of epidermal growth factor receptor (EGFR) mRNA via binding m6 A sites in the 3'-UTR of EGFR transcript, thus leading to aberrant activities of downstream signal pathways that impact tumor progression. Our data uncover the oncogenic function and m6 A reader-dependent mechanism of YTHDF1 in regulation of ICC progression. Restricting abnormal oncogenic mRNA translation by targeting YTHDF1 may be a novel and promising strategy for ICC treatment.

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.

Tumor-derived exosomes induce immunosuppressive macrophages to foster intrahepatic cholangiocarcinoma progression.

Macrophages are prominent components of solid tumors and exhibit distinct functions in different tumor microenvironments. Exosomes are emerging as necessary mediators of the cross-talk between tumor cells and the microenvironment. However, the underlying mechanisms of exosomes involving into crosstalk between tumor cells and macrophages during disease progression of intrahepatic cholangiocarcinoma (ICC) have not been yet fully realized. We found that the macrophages of ICC tumor tissues up-regulated the expression levels of immunosuppressive molecule programmed death-ligand 1 (PD-L1). Increased PD-L1+ macrophages in tumor tissues effectively suppressed T-cell immunity and correlated with poor survival rates in patients with ICC. High-throughput RNA-sequencing analysis that was performed to identify differential levels of microRNAs (miRNAs) between exosomes derived from ICC cells and primary human intrahepatic biliary epithelial cells revealed that miR-183-5p was increased in ICC cell-derived exosomes. Exosomal miR-183-5p inhibited phosphatase and tensin homolog (PTEN) expression, to subsequently affect the elevations on both phosphorylated AKT and PD-L1 expression in macrophages. Furthermore, macrophages that treated with ICC cell-derived exosomes significantly suppressed T-cell immunity in vitro and contributed to the growth and progression of ICC in vivo, which were reversible through blockages on PD-L1 of these macrophages. Finally, clinical data showed that up-regulated levels of plasma exosomal miR-183-5p correlated with poor prognosis of patients with ICC after curative resection. Tumor-derived exosomal miR-183-5p up-regulates PD-L1-expressing macrophages to foster immune suppression and disease progression in ICC through the miR-183-5p/PTEN/AKT/PD-L1 pathway. Exosomal miR-183-5p is a potential predictive biomarker for ICC progression and a potential target for development of therapeutic strategies against immune tolerance feature of ICC.

Steroidal saponins from Trillium tschonoskii rhizome repress cancer stemness and proliferation of intrahepatic cholangiocarcinoma

A phytochemical study was carried out on the extract of Trillium tschonoskii rhizomes, resulting in the isolation of thirty-six steroidal glycosides (1–36). Their structures were established mainly by spectroscopic analyses as well as necessary chemical evidence, of which 1–25 were identified as new analogues. Herein, all the isolated analogues were screened for the cytotoxicity against intrahepatic cholangiocarcinoma (ICC) cell lines of HuCCT1 and RBE through tumor colony formation and CCK-8 survival analysis, and the results demonstrated that three compounds 9, 12, and 26 significantly repressed tumor colony and sphere formation in both cell lines, respectively. Furthermore, the three analogues possessed a remarkable inhibitory role of organoid formation established from hydrodynamic induced mouse primary intrahepatic cholangiocarcinoma. Moreover, the functional assays of flow cytometry analysis, cancer stemness related gene expression, and western blotting assays all indicated that compound 26 could significantly repress cancer stem markers. Taken together, these results demonstrate that steroidal glycosides derived from T. tschonoskii rhizomes could be potentially implicated in human ICC therapy.

TNF receptor-related factor 3 inactivation promotes the development of intrahepatic cholangiocarcinoma through NF-κB-inducing kinase-mediated hepatocyte transdifferentiation.

Intrahepatic cholangiocarcinoma (ICC) is a deadly but poorly understood disease, and its treatment options are very limited. The aim of this study was to identify the molecular drivers of ICC and search for therapeutic targets. We performed a Sleeping Beauty transposon-based in vivo insertional mutagenesis screen in liver-specific Pten -deficient mice and identified TNF receptor-related factor 3 ( Traf3 ) as the most significantly mutated gene in murine ICCs in a loss-of-function manner. Liver-specific Traf3 deletion caused marked cholangiocyte overgrowth and spontaneous development of ICC in Pten knockout and KrasG12D mutant mice. Hepatocyte-specific, but not cholangiocyte-specific, Traf3 -deficient and Pten -deficient mice recapitulated these phenotypes. Lineage tracing and single-cell RNA sequencing suggested that these ICCs were derived from hepatocytes through transdifferentiation. TRAF3 and PTEN inhibition induced a transdifferentiation-like phenotype of hepatocyte-lineage cells into proliferative cholangiocytes through NF-κB-inducing kinase (NIK) up-regulation in vitro. Intrahepatic NIK levels were elevated in liver-specific Traf3 -deficient and Pten -deficient mice, and NIK inhibition alleviated cholangiocyte overgrowth. In human ICCs, we identified an inverse correlation between TRAF3 and NIK expression, with low TRAF3 or high NIK expression associated with poor prognosis. Finally, we showed that NIK inhibition by a small molecule inhibitor or gene silencing suppressed the growth of multiple human ICC cells in vitro and ICC xenografts in vivo. TRAF3 inactivation promotes ICC development through NIK-mediated hepatocyte transdifferentiation. The oncogenic TRAF3-NIK axis may be a potential therapeutic target for ICC.

CircNFIB inhibits tumor growth and metastasis through suppressing MEK1/ERK signaling in intrahepatic cholangiocarcinoma

BackgroundConsiderable evidence shows that circular RNAs (circRNAs) play an important role in tumor development. However, their function in intrahepatic cholangiocarcinoma (ICC) metastasis and the underlying mechanisms are incompletely understood.MethodscircNFIB (hsa_circ_0086376, termed as cNFIB hereafter) was identified in human ICC tissues through circRNAs sequencing. The biological role of cNFIB was determined in vitro and in vivo by gain or loss of functional experiments. Fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP) and RNA pull-down assays were conducted to analyze the interaction of cNFIB with dual specificity mitogen-activated protein kinase kinase1 (MEK1). Duolink in situ proximity ligation assay (PLA) and coimmunoprecipitation (co-IP) assay were used to investigate the effects of cNFIB on the interaction between MEK1 and mitogen-activated protein kinase 2 (ERK2). Finally, a series of in vitro and in vivo experiments were performed to explore the influences of cNFIB on the anti-tumor activity of trametinib (a MEK inhibitor).ResultscNFIB was significantly down-regulated in human ICC tissues with postoperative metastases. The loss of cNFIB was highly associated with aggressive characteristics and predicted unfavorable prognosis in ICC patients. Functional studies revealed that cNFIB inhibited the proliferation and metastasis of ICC cells in vitro and in vivo. Mechanistically, cNFIB competitively interacted with MEK1, which induced the dissociation between MEK1 and ERK2, thereby resulting in the suppression of ERK signaling and tumor metastasis. Moreover, we found that ICC cells with high levels of cNFIB held the potential to delay the trametinib resistance. Consistently, in vivo and in vitro studies demonstrated that cotreatment with trametinib and lentivirus vector encoding cNFIB showed greater inhibitory effect than isolated trametinib treatment.ConclusionsOur findings identified that cNFIB played a key role in ICC growth and metastasis by regulating MEK1/ERK signaling. Given the efficacy of cNFIB modulation on ICC suppression and trametinib sensitivity, cNFIB appears to be a potential therapeutic molecule for ICC treatment.

Screening a redox library identifies the anti-tumor drug Hinokitiol for treating intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a highly malignant and heterogeneous cancer with a poor prognosis. At present, there is no optimal treatment except for surgical resection, and recurrence after resection will lead to death due to multidrug resistance. Changes in the redox signal have been found to be closely related to the growth and drug resistance of tumor cells. Therefore, the purpose of this study was to screen small molecule compounds from the redox library to find a drug for anti-ICC and to explore its downstream mechanism. Tumor clone and sphere formation of ICC cell lines, as well as mouse ICC organoid proliferation assays were utilized to screen the candidate drug in the Redox library. Western blotting, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), as well as cell apoptosis and cell cycle flow cytometry assays were used to explore the mechanism. We found that Hinokitiol was a candidate drug through inhibition of tumor clone and sphere formation, and the expression of cancer stem cell (CSC)-related genes. Furthermore, Hinokitiol significantly inhibited the proliferation of ICC cells by downregulating the ERK and P38 pathways. In addition, the combination of Hinokitiol and Palbociclib showed a significant inhibitory effect on human ICC cells and mouse ICC organoids. Hinokitiol may have the potential to be developed as a clinical therapeutic drug for ICC treatment.

Hsa_circ_0019054 up-regulates HIF1A through sequestering miR-340-5p to promote the tumorigenesis of intrahepatic cholangiocarcinoma.

Circular RNAs (circRNAs) have been uncovered to play an important regulatory function in the tumorigenesis of intrahepatic cholangiocarcinoma (ICC). Hsa_circ_0,019,054 was found to be increased in ICC. Here, we aimed to explore the action and mechanism of hsa_circ_0,019,054 in ICC carcinogenesis. Quantitative real-time PCR (qRT-PCR) and western blotting were used to detect the levels of genes and proteins. The functional experiments were performed using in vitro 5-ethynyl-2'-deoxyuridine (EdU) assay, cell counting Kit-8 (CCK-8) assay, flow cytometry, and in vivo murine xenograft model. The glycolysis was analyzed by detecting glucose uptake and lactate level. The binding between miR-340-5p and hsa_circ_0,019,054 or HIF1A (Hypoxia-inducible factor 1-alpha) was validated using pull-down, dual-luciferase reporter and RNA immunoprecipitation assays. Hsa_circ_0,019,054 expression was higher in ICC tissues and cells. Functionally, hsa_circ_0,019,054 silencing could suppress ICC cell proliferation and glycolysis active, as well as induce apoptosis. Mechanistically, hsa_circ_0,019,054 was demonstrated to act as a sponge for miR-340-5p, which directly targeted HIF1A. Hsa_circ_0,019,054/miR-340-5p/HIF1A formed a feedback loop. HIF1A was up-regulated, while miR-340-5p was decreased in ICC tissues and cells. MiR-340-5p re-expression attenuated ICC cell growth. Besides that, rescue experiments suggested that HIF1A overexpression or miR-340-5p knockdown reversed the anti-proliferation and glycolysis arrest effects mediated by hsa_circ_0,019,054 silencing. Importantly, hsa_circ_0,019,054 silencing also impeded the growth of ICC in nude mice. Hsa_circ_0,019,054 deficiency could attenuate the proliferation and glycolysis of ICC cells via miR-340-5p/HIF1A axis.

ACOT12-mediated acetyl-CoA hydrolysis suppresses intrahepatic cholangiocarcinoma metastasis by inhibiting epithelial-mesenchymal transition.

Intrahepatic cholangiocarcinoma (ICC) is the most common malignant bile duct tumor in the liver and the second most common primary liver cancer with increasing morbidity and poor prognosis. Metabolic aberration plays key roles in cancer progression. As a key metabolic intermediate, acetyl-CoA accumulation shows close association with cancer metastasis. However, the role of acetyl-CoA metabolic aberration in ICC is still undetermined. Here, by investigating tissue samples from ICC patients and ICC cell lines, we found that acyl-CoA thioesterase 12 (ACOT12) expression is significantly down-regulated in ICC tissues, and is associated with poor prognosis of ICC. In vitro and in vivo studies demonstrated that ACOT12 suppressed ICC cells metastasis. Further mechanistic studies revealed that down-regulation of ACOT12 promoted ICC metastasis by inducing Slug expression and epithelial-mesenchymal transition (EMT). Our findings link ACOT12-regulated-acetyl-coA metabolic aberration with ICC metastasis and imply that ACOT12 could be a prognostic marker and a potential therapeutic target for ICC metastasis.

B7-H3 Targeted CAR T Cell Immunotherapy for Primary and Metastatic/Multi-focal Intrahepatic Cholangiocarcinoma

Introduction: The lack of effective therapy for intrahepatic cholangiocarcinoma (ICC) has prompted us to develop a novel immunotherapy using T cells engineered with a chimeric antigen receptor (CAR T cells). The aim of this preliminary study is to demonstrate that B7-H3 CAR T cells are safe and effective in eradicating ICC cells orthotopically grafted in NSG (NOD scid gamma) mice and in prolonging their survival. Method: Primary tumor was established in 15 mice by orthotopic grafting of human ICC cells ICC3-GFP-Luc. Then mice were randomized into 3 groups: treatment group injected through the tail vein with B7-H3 CAR T cells (n=5), intratumorally injected (n=5) and control group (n=5), left untreated. The multi-focal/metastatic mice model was established by tail vein injection of the same cell line in 8 mice. Then mice were randomized into 2 groups: treatment group, injected through the tail vein with B7-H3 CAR T cells (n=4) and control group (n=4), left untreated. Tumor size and development of metastasis were monitored weekly by bioluminescent imaging (BLI). Results: While tail vein injected B7-H3 CAR T cells eradicated primary tumor in all the mice, the intratumorally administered B7-H3 CART cells only reduce the growth of the tumor. As far as mice with multifocal/metastatic disease is concerned, B7-H3 CART cells completely eradicate the tumor in all the mice and prolong their survival. Conclusions: B7-H3 CAR T cell therapy is effective in eradicating both primary and multi- focal disease and ICC metastases established in NSG mice and in prolonging their survival.