Intrahepatic Cholangiocarcinoma Cells Research Articles

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.

Comparison of growth features and cancer stem cell prevalence in intrahepatic and extrahepatic cholangiocarcinoma cell lines

Aim of the studyIntra- and extrahepatic cholangiocarcinoma (I-CCA and E-CCA respectively) exhibit different growth features that contribute to different clinical outcomes. Cancer stem cells (CSCs) influence tumor growth and thereby may be responsible for these differences. The aim of this study was to document and compare the growth features of human I-CCA and E-CCA cell lines and determine whether any differences observed could be explained by differences in the prevalence and/or stem cell surface marker (SCSM) expression profiles of CSCs within the tumor cell lines.Material and methodsSix CCA cells lines, three I-CCA and three E-CCA, were studied. Tumor cell growth features including cell proliferation, colony/spheroid formation, migration and invasion were documented. CSC prevalence and SCSM expression profiles were examined by flow cytometry.ResultsI-CCA cells had significantly increased proliferative activity, shorter doubling times and were more invasive than E-CCA cells, while colony/spheroid formation and migration were similar in the two cell populations. There were no significant differences in CSC prevalence rates or SCSM expression profiles.ConclusionsThese findings suggest that I-CCA cells proliferate at a more rapid rate and are more invasive than E-CCA cells but the differences cannot be explained by differences in the prevalence or SCSM expression profiles of CSCs within the tumor cell population.

Driver mutations of intrahepatic cholangiocarcinoma shape clinically relevant genomic clusters with distinct molecular features and therapeutic vulnerabilities.

Purpose: To establish a clinically applicable genomic clustering system, we investigated the interactive landscape of driver mutations in intrahepatic cholangiocarcinoma (ICC).Methods: The genomic data of 1481 ICCs from diverse populations was analyzed to investigate the pair-wise co-occurrences or mutual exclusivities among recurrent driver mutations. Clinicopathological features and outcomes were compared among different clusters. Gene expression and DNA methylation profiling datasets were analyzed to investigate the molecular distinctions among mutational clusters. ICC cell lines with different gene mutation backgrounds were used to evaluate the cluster specific biological behaviors and drug sensitivities.Results: Statistically significant mutation-pairs were identified across 21 combinations of genes. Seven most recurrent driver mutations (TP53, KRAS, SMAD4, IDH1/2, FGFR2-fus and BAP1) showed pair-wise co-occurrences or mutual exclusivities and could aggregate into three genetic clusters: Cluster1: represented by tripartite interaction of KRAS, TP53 and SMAD4 mutations, exhibited large bile duct histological phenotype with high CA19-9 level and dismal prognosis; Cluster2: co-association of IDH/BAP1 or FGFR2-fus/BAP1 mutation, was characterized by small bile duct phenotype, low CA19-9 level and optimal prognosis; Cluster3: mutation-free ICC cases with intermediate clinicopathological features. These clusters showed distinct molecular traits, biological behaviors and responses to therapeutic drugs. Finally, we identified S100P and KRT17 as “cluster-specific”, “lineage-dictating” and “prognosis-related” biomarkers, which in combination with CA19-9 could well stratify Cluster3 ICCs into two biologically and clinically distinct subtypes.Conclusions: This clinically applicable clustering system can be instructive to ICC prognostic stratification, molecular classification, and therapeutic optimization.

IL-6-induced cGGNBP2 encodes a protein to promote cell growth and metastasis in intrahepatic cholangiocarcinoma.

Background and AimsIL‐6–induced tumor progression has been well established through the induction of antiapoptotic and proliferative genes. However, whether other mechanisms such as IL‐6 regulation of circular RNAs (circRNAs) may also contribute to tumor development remains unknown.Approach and ResultsHigh‐throughput RNA sequencing was used to identify the differentially expressed circRNAs on IL‐6 stimulation in intrahepatic cholangiocarcinoma (ICC) cells. CircRNA GGNBP2 (derived from ggnbp2 gene, termed as cGGNBP2) was up‐regulated by IL‐6 treatment in a time and concentration‐dependent manner. The biogenesis of cGGNBP2 was regulated by RNA‐binding protein DEx‐H Box Helicase 9, which was also mediated by IL‐6 exposure. Mass spectrometry and western blotting identified a protein cGGNBP2‐184aa encoded by cGGNBP2. cGGNBP2‐184aa promoted ICC cell proliferation and metastasis in vitro and in vivo. Mechanistically, cGGNBP2‐184aa directly interacted with signal transducers and activators of transduction‐3 (STAT3), phosphorylated STAT3Tyr705, and played a positive regulatory role in modulating IL‐6/STAT3 signaling. IL‐6/cGGNBP2‐184aa/STAT3 formed a positive feedback loop to sustain constitutive activation of IL‐6/STAT3 signaling. Elevated cGGNBP2 expression was correlated with poor prognosis of patients with ICC and was identified as an independent risk factor for patient prognosis.ConclusionsOur study demonstrates that cGGNBP2‐184aa, a protein encoded by IL‐6–induced cGGNBP2, formed a positive feedback loop to facilitate ICC progression and may serve as an auxiliary target for clinical IL‐6/STAT3‐targeting treatments in ICC.

Integrative epigenomic profiling reveal AP-1 is a key regulator in intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor with poor prognosis while its mechanisms of pathogenesis remain elusive. In this study, we performed systemic epigenomic and transcriptomic profiling via MNase-seq, ChIP-seq and RNA-seq in normal cholangiocyte and ICC cell lines. We showed that active histone modifications (H3K4me3, H3K4me1 and H3K27ac) were less enriched on cancer-related genes in ICC cell lines compared to control. The region of different histone modification patterns is enrichment in sites of AP-1 motif. Subsequent analysis showed that ICC had different nucleosome occupancy in differentially expressed genes compared to a normal cell line. Furthermore, we found that AP-1 plays a key role in ICC and regulates ICC-related genes through its AP-1 binding site. This study is the first report showing the global features of histone modification, transcript, and nucleosome profiles in ICC; we also show that the transcription factor AP-1 might be a key target gene in ICC.

CAFs shape myeloid-derived suppressor cells to promote stemness of intrahepatic cholangiocarcinoma through 5-lipoxygenase.

We previously demonstrated that cancer-associated fibroblasts (CAFs) promote tumor growth through recruitment of myeloid-derived suppressor cells (MDSCs). 5-lipoxygenase (5-LO) is highly expressed in myeloid cells and is critical for synthesizing leukotriene B4 (LTB4), which is involved in tumor progression by activating its receptor leukotriene B4 receptor type 2 (BLT2). In this study, we investigated whether and how CAFs regulate MDSC function to enhance cancer stemness, the driving force of the cancer aggressiveness and chemotherapy refractoriness, in highly desmoplastic intrahepatic cholangiocarcinoma (ICC). RNA-sequencing analysis revealed enriched metabolic pathways but decreased inflammatory pathways in cancer MDSCs compared with blood MDSCs from patients with ICC. Co-injection of ICC patient-derived CAFs promoted cancer stemness in an orthotopic ICC model, which was blunted by MDSC depletion. Conditioned media (CM) from CAF-educated MDSCs drastically promoted tumorsphere formation efficiency and stemness marker gene expression in ICC cells. CAF-CM stimulation increased expression and activity of 5-LO in MDSCs, while 5-LO inhibitor impaired the stemness-enhancing capacity of MDSCs in vitro and in vivo. Furthermore, IL-6 and IL-33 primarily expressed by CAFs mediated hyperactivated 5-LO metabolism in MDSCs. We identified the LTB4-BLT2 axis as the critical downstream metabolite signaling of 5-LO in promoting cancer stemness, as treatment with LTB4 was elevated in CAF-educated MDSCs, or blockade of BLT2 (which was preferentially expressed in stem-like ICC cells) significantly reduced stemness-enhancing effects of CAF-educated MDSCs. Finally, BLT2 blockade augmented chemotherapeutic efficacy in ICC patient-derived xenograft models. Our study reveals a role for CAFs in orchestrating the optimal cancer stemness-enhancing microenvironment by educating MDSCs, and suggests the 5-LO/LTB4-BLT2 axis as promising therapeutic targets for ICC chemoresistance by targeting cancer stemness.

CircRTN4IP1 regulates the malignant progression of intrahepatic cholangiocarcinoma by sponging miR-541-5p to induce HIF1A production

BackgroundRecent studies indicate that circular RNA (circRNA) serves important roles in the development of intrahepatic cholangiocarcinoma (ICC). However, the role of circRNA reticulon 4 interacting protein 1 (circRTN4IP1) in ICC progression remains unknown. MethodsExpression of circRTN4IP1, microRNA-541-5p (miR-541-5p), hypoxia inducible factor 1 subunit alpha (HIF1A) and other indicated protein markers was detected by quantitative real-time polymerase chain reaction or Western blot. The functional effects of circRTN4IP1 knockdown in ICC cells were analyzed by cell counting kit-8, cell colony formation, flow cytometry analysis, Western blot, glucose and lactate kit assays. The positive expression rate of HIF1A was detected by immunohistochemistry assay. The interaction between miR-541-5p and circRTN4IP1 or HIF1A was identified by dual-luciferase reporter, RNA immunoprecipitation or RNA pull-down assays. Xenograft mouse model assay was performed to determine the effect of circRTN4IP1 depletion on tumor formation. ResultsIn contrast, ICC tissues and cells showed high expression of circRTN4IP1 and HIF1A, but low expression of miR-541-5p. Knockdown of circRTN4IP1 led to repression of cell proliferation and glucose metabolism, but promotion of cell apoptosis; however, circRTN4IP1 overexpression had opposite effects. In mechanism, circRTN4IP1 acted as a sponge for miR-541-5p, which was found to target HIF1A. MiR-541-5p inhibitors could remit circRTN4IP1 knockdown-mediated action. Also, HIF1A participated in the regulation of miR-541-5p in ICC progression. In support, circRTN4IP1 depletion impeded tumor formation in vivo. ConclusionCircRTN4IP1 knockdown inhibited ICC cell malignancy by miR-541-5p/HIF1A axis, providing us with a reliable target for the therapy of ICC.

Loss of FBP1 by aPKC-ι/Snail Pathway-Mediated Repression Promotes Invasion and Aerobic Glycolysis of Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is one of the most commonly diagnosed malignancies worldwide, and the second most common primary liver tumor. The lack of effective diagnostic and treatment methods results in poor patient prognosis and high mortality rate. Atypical protein kinase C-ι (aPKC-ι) is highly expressed in primary and metastatic ICC tissues, and regulates epithelial mesenchymal transition (EMT) through the aPKC-ι/P-Sp1/Snail signaling pathway. Recent studies have correlated aberrant glucose metabolism with EMT. Given the vital role of FBP1 in regulating glucose metabolism in cancer cells, we hypothesized that aPKC-ι downregulates FBP1 in ICC cells through the Snai1 pathway, and enhances glycolysis and metastasis. We confirmed the ability of aPKC-ι promotes glycolysis, invasion and metastasis of cancer cells, and further demonstrated that FBP1 inhibits the malignant properties of ICC cells by antagonizing aPKC-ι. Our findings provide novel insights into the molecular mechanisms of ICC progression and metastasis, as well as a theoretical basis for exploring new treatment strategies.

Genomic evolution and the impact of SLIT2 mutation in relapsed intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is aggressive and has high rates of relapse, conferring poor long-term survival after curative resection. Little is known about the genomic evolution that occurs during ICC relapse. We conducted whole-exome sequencing of 30 paired primary and relapsed tumors from 10 patients with ICC who received curative resection. We sought to identify frequently altered genes, infer tumor subclonal architectures, and track genomic evolution from primary to relapsed tumors. We examined functional effects and the mechanism of action of SLIT2, a gene specifically mutated in relapsed tumors, on tumor growth and metastasis and the tumor microenvironment. Our results indicated that relapsed ICCs were genetically derived from intrahepatic dissemination of primary tumors. However, they acquired additional mutations while maintaining most drivers, such as TP53 and IDH1. Multiregion sequencing suggested polyclonal seeding of ICC dissemination. Four of 10 relapsed ICCs acquired SLIT2 mutations that were not present in the corresponding primary tumors. Validation in an expanded sample revealed SLIT2 mutations in 2.3% (1/44) of primary ICCs and 29.5% (13/44) of relapsed ICCs. Biofunctional investigations revealed that inactivating mutation of SLIT2 resulted in activation of PI3K-Akt signaling in ICC cells, directly enhanced neutrophil chemotaxis, mediated tumor-associated neutrophil infiltration, and contributed to ICC growth and metastasis. We characterized genomic evolution during ICC relapse and identified SLIT2 as a driver of tumor dissemination and tumor-associated neutrophil infiltration.