Intrahepatic Cholangiocarcinoma Cells Research Articles

Gut microbiota regulates the ALK5/NOX1 axis by altering glutamine metabolism to inhibit ferroptosis of intrahepatic cholangiocarcinoma cells

Intrahepatic cholangiocarcinoma (ICC) is a kind of hepatobiliary tumor that is increasing in incidence and mortality. The gut microbiota plays a role in the onset and progression of cancer, however, the specific mechanism by which the gut microbiota acts on ICC remains unclear. In this study, feces and plasma from healthy controls and ICC patients were collected for 16S rRNA sequencing or metabolomics analysis. Gut microbiota analysis showed that gut microbiota abundance and biodiversity were altered in ICC patients compared with controls. Plasma metabolism analysis showed that the metabolite glutamine content of the ICC patient was significantly higher than that of the controls. KEGG pathway analysis showed that glutamine plays a vital role in ICC. In addition, the use of antibiotics in ICC animals further confirmed that changes in gut microbiota affect changes in glutamine. Further experiments showed that supplementation with glutamine inhibited ferroptosis and downregulated ALK5 and NOX1 expression in HuCCT1 cells. ALK5 overexpression or NOX1 overexpression increased NOX1, p53, PTGS2, ACSL4, LPCAT3, ROS, MDA and Fe2+ and decreased FTH1, SLC7A11 and GSH. Knockdown of NOX1 suppressed FIN56-induced ferroptosis. In vivo, supplementation with glutamine promoted tumor growth. Overexpression of ALK5 repressed tumor growth and induced ferroptosis in nude mice, which could be reversed by the addition of glutamine. Our results suggested that the gut microbiota altered glutamine metabolism to inhibit ferroptosis in ICC by regulating the ALK5/NOX1 axis.

SRC inhibition enables formation of a growth suppressive MAGI1-PP2A complex in isocitrate dehydrogenase-mutant cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

Mitochondria targeted drug delivery system overcoming drug resistance in intrahepatic cholangiocarcinoma by reprogramming lipid metabolism

The challenge of drug resistance in intrahepatic cholangiocarcinoma (ICC) is intricately linked with lipid metabolism reprogramming. The hepatic lipase (HL) and the membrane receptor CD36 are overexpressed in BGJ398-resistant ICC cells, while they are essential for lipid uptake, further enhancing lipid utilization in ICC. Herein, a metal-organic framework-based drug delivery system (OB@D-pMOF/CaP-AC, DDS), has been developed. The specifically designed DDS exhibits a successive targeting property, enabling it to precisely target ICC cells and their mitochondria. By specifically targeting the mitochondria, DDS produces reactive oxygen species (ROS) through its sonodynamic therapy effect, achieving a more potent reduction in ATP levels compared to non-targeted approaches, through the impairment of mitochondrial function. Additionally, the DDS strategically minimizes lipid uptake through the incorporation of the anti-HL drug, Orlistat, and anti-CD36 monoclonal antibody, reducing lipid-derived energy production. This dual-action strategy on both mitochondria and lipids can hinder energy utilization to restore drug sensitivity to BGJ398 in ICC. Moreover, an orthotopic mice model of drug-resistant ICC was developed, which serves as an exacting platform for evaluating the multifunction of designed DDS. Upon in vivo experiments with this model, the DDS demonstrated exceptional capabilities in suppressing tumor growth, reprogramming lipid metabolism and improving immune response, thereby overcoming drug resistance. These findings underscore the mitochondria-targeted DDS as a promising and innovative solution in ICC drug resistance.

Identification of RNF213 as a potential suppressor of local invasion in intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is a lethal cancer with poor survival especially when it spreads. The histopathology of its rare intraductal papillary neoplasm of the bile duct type (IPNB) characteristically shows cancer cells originating within the confined bile duct space. These cells eventually invade and infiltrate the nearby liver tissues, making it a good model to study the mechanism of local invasion, which is the earliest step of metastasis. To discover potential suppressor genes of local invasion in ICC, we analyzed the somatic mutation profiles and performed clonal evolution analyses of the 11 pairs of macrodissected IPNB tissues with local invasion (LI-IPNB) and IPNB tissues without local invasion from the same patients. We identified a protein-truncating variant (PTV) in an E3 ubiquitin ligase, RNF213 (c.6967C>T; p.Gln2323X; chr17: 78,319,102 [hg19], exon 29), as the most common PTV event in LI-IPNB samples (4/11 patients). Knockdown of RNF213 in HuCCT1 and YSCCC cells showed increased migration and invasion, and reduced vasculogenic mimicry, but maintained normal proliferation. Transcriptomic analysis of the RNF213-knockdown versus control cells was then performed in the HuCCT1, YSCCC, and KKU-100 cells. Gene Ontology (GO) enrichment analysis of the common differentially expressed genes revealed significantly altered cytokine and oxidoreductase-oxidizing metal ions activities, as confirmed by western blotting. Gene Set Enrichment Analysis (GSEA) identified the most enriched pathways being oxidative phosphorylation, fatty acid metabolism, reactive oxygen species, adipogenesis, and angiogenesis. In sum, loss of function of RNF213 is a common genetic alteration in LI-IPNB tissues. RNF213 knockdown leads to increased migration and invasion of ICC cells, potentially through malfunctions of the pathways related inflammation, and energy metabolisms.

YBX1 promotes stemness and cisplatin insensitivity in intrahepatic cholangiocarcinoma via the AKT/β-catenin axis.

Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive malignancy characterized by a poor prognosis and closely linked to tumor stemness. However, the key molecules that regulate ICC stemness remain elusive. Although Y-box binding protein 1 (YBX1) negatively affects prognosis in various cancers by enhancing stemness and chemoresistance, its effect on stemness and cisplatin sensitivity in ICC remains unclear. Three bulk and single-cell RNA-seq datasets were analyzed to investigate YBX1 expression in ICC and its association with stemness. Clinical samples and colony/sphere formation assays validated the role of YBX1 in stemness and sensitivity to cisplatin. AZD5363 and KYA1979K explored the interaction of YBX1 with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) and WNT/β-catenin pathways. YBX1 was significantly upregulated in ICC, correlated with worse overall survival and shorter postoperative recurrence time, and was higher in chemotherapy-non-responsive ICC tissues. The YBX1-high group exhibited significantly elevated stemness scores, and genes linked to YBX1 upregulation were enriched in multiple stemness-related pathways. Moreover, YBX1 expression is significantly correlated with several stemness-related genes (SOX9, OCT4, CD133, CD44 and EPCAM). Additionally, YBX1 overexpression significantly enhanced the colony- and spheroid-forming abilities of ICC cells, accelerated tumor growth in vivo and reduced their sensitivity to cisplatin. Conversely, the downregulation of YBX1 exerted the opposite effect. The transcriptomic analysis highlighted the link between YBX1 and the PI3K/AKT and WNT/β-catenin pathways. Further, AZD5363 and KYA1979K were used to clarify that YBX1 promoted ICC stemness through the regulation of the AKT/β-catenin axis. YBX1 is upregulated in ICC and promotes stemness and cisplatin insensitivity via the AKT/β-catenin axis. Our study describes a novel potential therapeutic target for improving ICC prognosis.

Lymphatic spread from left-sided intrahepatic cholangiocarcinoma: reconsiderations based on the lymphatic drainage from the liver

Intrahepatic cholangiocarcinoma (ICC) is known to have a high frequency of lymph node metastasis. Lymph node dissection (LND) is recommended for accurate staging, but the survival benefit of LND remains unclear. Knowledge of the pathways and direction of lymphatic drainage to the regional lymph nodes is essential when considering LND to improve patient survival. The liver has three lymphatic drainage pathways: portal, sublobular, and subcapsular. Of these, the portal lymphatic pathway, which lies along with the portal tracts, is the primary pathway. The efferent portal lymphatic vessels from the left-sided liver, which continue from the portal lymphatic pathway of the liver, communicate with the lymphatic vessels and lymph nodes along the hepatic artery at the hepatoduodenal ligament. In addition, lymphatic flow may also present along the left embryonic (aberrant) hepatic artery in the lesser omentum, based on our experience. This pathway is the previously reported pathway from the left-sided ICC to the lesser curvature of the stomach. However, through this pathway, ICC cells reach lymph nodes along the root of the left gastric artery but not the perigastric lymph nodes along the lesser curvature because of the opposite direction of lymph flow. Although further analyses using a large number of cases are needed to confirm these observations, these two pathways, along the hepatic artery at the hepatoduodenal ligament and the left embryonic (aberrant) hepatic artery in the lesser omentum should be considered when performing LND in the case of ICC in the left-sided liver.

Modulating PCGF4 Stability Is an Efficient Metastasis-Regulatory Strategy Used by Distinct Subtypes of Cancer-Associated Fibroblasts in Intrahepatic Cholangiocarcinoma

Intrahepatic Cholangiocarcinoma (ICC) is highly malignant neoplasm and prone to metastasis. It is unclear if cancer-associated fibroblasts (CAFs) affect the metastasis of ICC. Here, we have established ICC patient-derived CAF lines and related cancerous cell lines and analyzed the effects of CAFs on the tumor progressive properties of the ICC cancerous cells. Results demonstrated that CAFs can be classified into cancer-restraining or promoting categories (rCAFs or pCAFs) based on distinct tumorigenic effects. The RNA-Seq analyses of ICC cancerous cell lines identified B lymphoma Mo-MLV insertion region 1 (PCGF4, also known BMI1) as a potential metastasis regulator. Strikingly, the changes of PCGF4 levels in ICC cells perfectly mirrored the restraining or promoting effects of CAFs on ICC migration. Our Immunohistochemical analyses on the ICC tissue microarrays indicated that PCGF4 was negatively correlated to overall survival of ICC. We confirmed the promoting effects of PCGF4 on cell migration, drug resistance activity, stemness properties. Mechanistically, rCAFs triggered the proteasome-dependent degradation of PCGF4, while pCAFs enhanced the stability of PCGF4 via activating the IL-6/p-STAT3 pathway. In summary, our data identified roles of CAFs on ICC metastasis and revealed a new mechanism of the CAFs on ICC progression in which PCGF4 acted as the key effector by both categories of CAFs. These findings shed light on developing comprehensive therapeutic strategies for ICC.

Heparanase interacting BCLAF1 to promote the development and drug resistance of ICC through the PERK/eIF2α pathway.

Intrahepatic cholangiocarcinoma (ICC) is a primary epithelial carcinoma known for its aggressive nature, high metastatic potential, frequent recurrence, and poor prognosis. Heparanase (HPSE) is the only known endogenous β-glucuronidase in mammals. In addition to its well-established enzymatic roles, HPSE critically exerts non-catalytic function in tumor biology. This study herein aimed to investigate the non-enzymatic roles of HPSE as well as relevant regulatory mechanisms in ICC. Our results demonstrated that HPSE was highly expressed in ICC and promoted the proliferation of ICC cells, with elevated HPSE levels implicating a poor overall survival of ICC patients. Notably, HPSE interacted with Bcl-2-associated factor 1 (BCLAF1) to upregulate the expression of Bcl-2, which subsequently activated the PERK/eIF2α-mediated endoplasmic reticulum (ER) stress pathway to promote anti-apoptotic effect of ICC. Moreover, our in vivo experiments revealed that concomitant administration of gemcitabine and the Bcl-2 inhibitor navitoclax enhanced the sensitivity of ICC cells with highly expressed HPSE to chemotherapy. In summary, our findings revealed that HPSE promoted the development and drug resistance of ICC via its non-enzymatic function. Bcl-2 may be considered as an effective target with therapeutic potential to overcome ICC chemotherapy resistance induced by HPSE, presenting valuable insights into the development of novel therapeutic strategies against ICC.

Alpha5 nicotine acetylcholine receptor subunit promotes intrahepatic cholangiocarcinoma metastasis

Neurotransmitter-initiated signaling pathway were reported to play an important role in regulating the malignant phenotype of tumor cells. Cancer cells could exhibit a “neural addiction” property and build up local nerve networks to achieve an enhanced neurotransmitter-initiated signaling through nerve growth factor-mediated axonogenesis. Targeting the dysregulated nervous systems might represent a novel strategy for cancer treatment. However, whether intrahepatic cholangiocarcinoma (ICC) could build its own nerve networks and the role of neurotransmitters in the progression ICC remains largely unknown. Immunofluorescence staining and Enzyme-linked immunosorbent assay suggested that ICC cells and the infiltrated nerves could generate a tumor microenvironment rich in acetylcholine that promotes ICC metastasis by inducing epithelial-mesenchymal transition (EMT). Acetylcholine promoted ICC metastasis through interacting with its receptor, alpha 5 nicotine acetylcholine receptor subunits (CHRNA5). Furthermore, acetylcholine/CHRNA5 axis activated GSK3β/β-catenin signaling pathway partially through the influx of Ca2+-mediated activation of Ca/calmodulin-dependent protein kinases (CAMKII). In addition, acetylcholine signaling activation also expanded nerve infiltration through increasing the expression of Brain-Derived Neurotrophic Factor (BDNF), which formed a feedforward acetylcholine-BDNF axis to promote ICC progression. KN93, a small-molecule inhibitor of CAMKII, significantly inhibited the migration and enhanced the sensitivity to gemcitabine of ICC cells. Above all, Acetylcholine/CHRNA5 axis increased the expression of β-catenin to promote the metastasis and resistance to gemcitabine of ICC via CAMKII/GSK3β signaling, and the CAMKII inhibitor KN93 may be an effective therapeutic strategy for combating ICC metastasis.

FASN-mediated fatty acid biosynthesis remodels immune environment in Clonorchis sinensis infection-related intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer with high lethality. Clonorchis sinensis (C. sinensis) infection is an important risk factor for ICC. Here we investigated the clinical impact and underlying molecular characteristics of C. sinensis-infected ICC. We performed single-cell RNA sequencing, whole exome sequencing, RNA-sequencing, metabolomics and spatial transcriptomics in 251 ICC patients from three medical centers. The alterations of metabolic and immune microenvironment of C. sinensis-infected ICCs were validated through in vitro co-culture system and hydrodynamic injection ICC mouse model. We revealed that C. sinensis infection was significantly associated with ICC patients' overall survival and immunotherapy response. Fatty acid biosynthesis and the expression of FASN, a key enzyme catalyzing long-chain fatty acid synthesis, were significantly enriched in C. sinensis-infected ICCs. ICC cell lines treated with C. sinensis-produced excretory/secretory products (ESPs) displayed an elevation of FASN and free fatty acid. The metabolic alteration of tumor cells was closely correlated with the enrichment of tumor-associated macrophage-like (TAM-like) macrophages and the impairment function of T cells, which led to the immunosuppressive microenvironment formation and tumor progression. Spatial transcriptomics analysis revealed that malignant cells were in closer juxtaposition with TAM-like macrophages in C. sinensis-infected ICCs than non-C. sinensis-infected ICCs. Importantly, FASN inhibitor significantly reversed immunosuppressive microenvironment and enhanced anti-PD-1 efficacy in ICC mouse models treated with ESPs from C. sinensis. We uncover the metabolic signature and immune microenvironment of C. sinensis-infected ICCs and highlight the combination of FASN inhibitors with immunotherapy as a promising strategy for treating C. sinensis-infected ICCs. C. sinensis-infected ICC patients have a poorer prognosis and worse response to immunotherapy than non-C. sinensis-infected ICCs. The underlying molecular characteristics of C. sinensis-infected ICCs remains unclear. Herein, we demonstrate that up-regulation of FASN and free fatty acids in C. sinensis-infected ICCs leads to immunosuppressive microenvironment formation and tumor progression. Thus, administration of FASN inhibitors could significantly reverse immunosuppressive environment and further enhance anti-PD-1 efficacy in combating C. sinensis-infected ICCs.

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.

Establishment and Characterization of a New Intrahepatic Cholangiocarcinoma Cell Line, ICC-X2.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant tumor of the biliary tract that is prone to recurrence and metastasis and is characterized by poor sensitivity to chemotherapy and overall prognosis. For these reasons, there is an urgent need to understand its pathological mechanisms and develop effective treatments. To address this challenge, the establishment of suitable preclinical models is critical. Fresh ICC tissue samples were used for primary culture and subculture. The cell line was evaluated by cell proliferation assays, clonal formation assays, karyotype analysis, and short tandem repeat (STR) analysis. Drug resistances against oxaliplatin, paclitaxel, gemcitabine and 5-fluorouracil (5-FU) were evaluated by CCK-8 assay. Subcutaneous injection of 1 × 106 cells to three BALB/c nude mice was conducted for xenograft studies. The hematoxylin and eosin (H&E) staining was used to detect the pathological status of the cell line. The expression of biomarkers CK7, CK19, Ki-67, E-cadherin and vimentin was determined by immunocytochemistry assay. A new ICC cell line named ICC-X2 was successfully established. Like ICC-X3 established using the same patient's metastatic tumor, the cell line has been continuously cultured in vitro for more than a year and has been passaged more than 100 times. ICC-X2 retained the typical biliary epithelial morphology. The population doubling time of ICC-X2 is 48 h. The cells demonstrated an abnormal nearly tetraploid karyotype. The STR analysis confirmed that ICC-X2 was highly consistent with the primary tumor tissue and not cross-contaminated by existing cell lines. ICC-X2 cells positively expressed CK7, CK19, E-cadherin, and vimentin, and the positive expression of Ki-67 in ICC-X2 cells was 40%. The ICC-X2 cells exhibited a strong clonogenic ability. The drug sensitivity test indicated that ICC-X2 was sensitive to oxaliplatin and paclitaxel, but naturally resistant to gemcitabine and 5-FU. ICC-X2 was rapidly able to form transplanted tumors in vivo after subcutaneous inoculation in nude mice. ICC-X2 is an excellent experimental model that can be used for studying the occurrence, development, and metastasis of ICC and investigating the mechanism of tumor drug resistance.

THBS1 and THBS2 Enhance the In Vitro Proliferation, Adhesion, Migration and Invasion of Intrahepatic Cholangiocarcinoma Cells.

In intrahepatic cholangiocarcinoma (iCCA), thrombospondin 1 (THBS1) and 2 (THBS2) are soluble mediators released in the tumor microenvironment (TME) that contribute to the metastatic spreading of iCCA cells via a lymphatic network by the trans-differentiation of vascular endothelial cells to a lymphatic-like phenotype. To study the direct role of THBS1 and THBS2 on the iCCA cells, well-established epithelial (HuCCT-1) and mesenchymal (CCLP1) iCCA cell lines were subjected to recombinant human THBS1 and THBS2 (rhTHBS1, rhTHBS2) for cellular function assays. Cell growth, cell adhesion, migration, and invasion were all enhanced in both CCLP1 and HuCCT-1 cells by the treatment with either rhTHBS1 or rhTHBS2, although they showed some variability in their intensity of speeding up cellular processes. rhTHBS2 was more intense in inducing invasiveness and in committing the HuCCT-1 cells to a mesenchymal-like phenotype and was therefore a stronger enhancer of the malignant behavior of iCCA cells compared to rhTHBS1. Our data extend the role of THBS1 and THBS2, which are not only able to hinder the vascular network and promote tumor-associated lymphangiogenesis but also exacerbate the malignant behavior of the iCCA cells.

Liensinine inhibits progression of intrahepatic cholangiocarcinoma by regulating TGF-β1 /P-smad3 signaling through HIF-1a.

Intrahepatic cholangiocarcinoma (ICC) is a high-grade malignant digestive system tumor with an insidious onset and unfavorable prognosis. Liensinine, a small molecule derived from plants, has been proven to have significant tumor suppressor activity in other cancers. However, there are no reports on whether liensinine can inhibit the proliferation or metastasis of ICC. This study aimed to explore the tumor-suppressive activity of liensinine in ICC and its underlying mechanisms. The phenotypic changes in ICC cells were monitored in vitro using cell function tests. Western blot and immunofluorescence analyses verified the efficacy of liensinine. Tumor-bearing nude mice were used to explore the effect of liensinine on tumors and its toxicity and side effects in vivo. Liensinine suppressed ICC cell proliferation and arrested the cell cycle at the G1 phase. The epithelial-mesenchymal transition (EMT) of ICC cells was also inhibited, thereby restraining their invasion and migration of tumor cells. In addition, this study found that the potential mechanism of liensinine inhibiting EMT may be via suppression of the TGF-β1/P-smad3 signaling pathway through hypoxia-inducible factor 1 alpha (HIF-1a). In vivo experiments showed that liensinine inhibited the growth of Hucc-T1 transplanted tumors in nude mice. Liensinine restrained the proliferation of ICC cells and suppressed EMT in ICC via the HIF-1a-mediated TGF-β1/P-smad3 signaling pathway.

Identification of FOXP1 as a favorable prognostic biomarker and tumor suppressor in intrahepatic cholangiocarcinoma

BackgroundForkhead-box protein P1 (FOXP1) has been proposed to have both oncogenic and tumor-suppressive properties, depending on tumor heterogeneity. However, the role of FOXP1 in intrahepatic cholangiocarcinoma (ICC) has not been previously reported.MethodsImmunohistochemistry was performed to detect FOXP1 expression in ICC and normal liver tissues. The relationship between FOXP1 levels and the clinicopathological characteristics of patients with ICC was evaluated. Finally, in vitro and in vivo experiments were conducted to examine the regulatory role of FOXP1 in ICC cells.ResultsFOXP1 was significantly downregulated in the ICC compared to their peritumoral tissues (p < 0.01). The positive rates of FOXP1 were significantly lower in patients with poor differentiation, lymph node metastasis, invasion into surrounding organs, and advanced stages (p < 0.05). Notably, patients with FOXP1 positivity had better outcomes (overall survival) than those with FOXP1 negativity (p < 0.05), as revealed by Kaplan–Meier survival analysis. Moreover, Cox multivariate analysis showed that negative FOXP1 expression, advanced TNM stages, invasion, and lymph node metastasis were independent prognostic risk factors in patients with ICC. Lastly, overexpression of FOXP1 inhibited the proliferation, migration, and invasion of ICC cells and promoted apoptosis, whereas knockdown of FOXP1 had the opposite role.ConclusionOur findings suggest that FOXP1 may serve as a novel outcome predictor for ICC as well as a tumor suppressor that may contribute to cancer treatment.

Regulation of lipid metabolism by APOE4 in intrahepatic cholangiocarcinoma via the enhancement of ABCA1 membrane expression.

Intrahepatic cholangiocarcinoma (ICC) is a malignancy with a dismal prognosis, thus the discovery of promising diagnostic markers and treatment targets is still required. In this study, 1,852 differentially expressed genes (DEGs) were identified in the GSE45001 dataset for weighted gene co-expression network analysis (WGCNA), and the turquoise module was confirmed as the key module. Next, the subnetworks of the 1,009 genes in the turquoise module analyzed by MCODE, MCC, and BottleNeck algorithms identified nine overlapping genes (CAT, APOA1, APOC2, HSD17B4, EHHADH, APOA2, APOE4, ACOX1, AGXT), significantly associated with lipid metabolism pathways, such as peroxisome and cholesterol metabolism. Among them, APOE4 exhibited a potential tumor-suppressive role in ICC and high diagnostic value for ICC in both GSE45001 and GSE32879 datasets. In vitro experiments demonstrated Apolipoprotein E4 (APOE4) overexpression suppressed ICC cell proliferation, migration, and invasion, knockdown was the opposite trend. And in ICC modulated lipid metabolism, notably decreasing levels of TG, LDL-C, and HDL-C, while concurrently increasing the expressions of TC. Further, APOE4 also downregulated lipid metabolism-related genes, suggesting a key regulatory role in maintaining cellular homeostasis, and regulating the expression of the membrane protein ATP-binding cassette transporter A1 (ABCA1). These findings highlighted the coordinated regulation of lipid metabolism by APOE4 and ABCA1 in ICC progression, providing new insights into ICC mechanisms and potential therapeutic strategies.

Csi-miR-96-5p delivered by Clonorchis sinensis extracellular vesicles promotes intrahepatic cholangiocarcinoma proliferation and migration via the ferroptosis-related PTEN/SLC7A11/GPX4 axis

BackgroundClonorchis sinensis (CS) is classified as a group 1 carcinogen and can cause intrahepatic cholangiocarcinoma (ICC). CS extracellular vesicles (CsEVs) play important roles in mediating communication between parasitic helminths and humans. Ferroptosis is a novel cell death mechanism that is mainly induced by lipid peroxidation and iron overload. However, the role of CsEVs in the regulation of ferroptosis in ICC remains unclear. This study aimed to explore the role of CS-secreted miR-96-5p (csi-miR-96-5p) delivered by CsEVs in ICC progression and ferroptosis.MethodsTissue samples were collected from ICC patients with CS infection (CS-ICC) or without CS infection (NC-ICC). The levels of csi-miR-96-5p and PTEN gene were determined by quantitative polymerase chain reaction (qPCR) and western blotting, and survival analysis was performed. CsEVs were isolated and identified by ultracentrifugation and transmission electron microscopy. Lentiviruses were used to establish stable cell lines with csi-miR-96-5p mimic expression, PTEN overexpression (PTEN-EXO) and PTEN CRISPR/Cas9-based knockout (PTEN-KO) and their respective negative controls. Cell proliferation was assessed by performing Cell Counting Kit-8 assays in vitro and in a tumor xenograft model in vivo, and cell migration was assessed by performing Transwell assays. Erastin is used to induce ferroptosis. Ferroptosis levels were evaluated using biomarkers.ResultsHigh csi-miR-96-5p and low PTEN expression was observed in CS-ICC tissues and was associated with poor overall survival. csi-miR-96-5p was highly enriched in CsEVs and was taken up by ICC cells. csi-miR-96-5p mimics or PTEN-KO significantly promoted the growth and migration of ICC cells in vitro and in vivo, whereas PTEN-EXO exerted the opposite effect. Mechanistically, csi-miR-96-5p mimics or PTEN-KO inhibited erastin-induced ferroptosis, including reducing the accumulation of Fe2+, lipid reactive oxygen species, and malondialdehyde, increasing the GSH/GSSG ratio and levels of SLC7A11 and GPX4, whereas PTEN-EXOs exerted the opposite effect.Conclusionscsi-miR-96-5p delivered by CsEVs reduced ferroptosis by regulating the expression of the PTEN/SLC7A11/GPX4 axis, thereby promoting ICC proliferation and migration. For the first time to our knowledge, we found that CS miRNAs could promote tumor development through ferroptosis.Graphical

Ferroptosis-related gene glutathione peroxidase 4 promotes reprogramming of glucose metabolism via Akt-mTOR axis in intrahepatic cholangiocarcinoma.

The role of the ferroptosis-related gene glutathione peroxidase 4 (GPX4) in oncology has been extensively investigated. However, the clinical implications of GPX4 in patients with intrahepatic cholangiocarcinoma (ICC) remain unknown. This study aimed to evaluate the prognostic impact of GPX4 and its underlying molecular mechanisms in patients with ICC. Fifty-seven patients who underwent surgical resection for ICC between 2010 and 2017 were retrospectively analyzed. Based on the immunohistochemistry, patients were divided into GPX4 high (n = 15) and low (n = 42) groups, and clinical outcomes were assessed. Furthermore, the roles of GPX4 in cell proliferation, migration and gene expression were analyzed in ICC cell lines in vitro and in vivo. The results from clinical study showed that GPX4 high group showed significant associations with high SUVmax on 18F-fluorodeoxyglucose-positron emission tomography (≥8.0, P = 0.017), multiple tumors (P = 0.004), and showed glucose transporter 1 (GLUT1) high expression with a trend toward significance (P = 0.053). Overall and recurrence-free survival in the GPX4 high expression group were significantly worse than those in the GPX4 low expression group (P = 0.038 and P < 0.001, respectively). In the experimental study, inhibition of GPX4 attenuated cell proliferation and migration in ICC cell lines. Inhibition of GPX4 also decreased the expression of glucose metabolism-related genes, such as GLUT1 or HIF1α. Mechanistically, these molecular changes are regulated in Akt-mechanistic targets of rapamycin axis. In conclusion, this study suggested the pivotal value of GPX4 serving as a prognostic marker for patients with ICC. Furthermore, GPX4 can mediate glucose metabolism of ICC.

Retracted] Downregulation of ROS‑FIG inhibits cell proliferation, colony‑formation, cell cycle progression, migration and invasion, while inducing apoptosis in intrahepatic cholangiocarcinoma cells.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that the western blotting data shown in Fig.9 were strikingly similar to data appearing in different form in other articles written by different authors at different research institutes that had either already been published elsewhere prior to the submission of this paper to International Journal of Molecular Medicine, or were under consideration for publication at around the same time. In view of the fact that certain of these data had already apparently been published previously, the Editor of International Journal of Molecular Medicine has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Molecular Medicine 34: 661‑668, 2014; 10.3892/ijmm.2014.1823].

ΔNp63α facilitates proliferation and migration, and modulates the chromatin landscape in intrahepatic cholangiocarcinoma cells

p63 plays a crucial role in epithelia-originating tumours; however, its role in intrahepatic cholangiocarcinoma (iCCA) has not been completely explored. Our study revealed the oncogenic properties of p63 in iCCA and identified the major expressed isoform as ΔNp63α. We collected iCCA clinical data from The Cancer Genome Atlas database and analyzed p63 expression in iCCA tissue samples. We further established genetically modified iCCA cell lines in which p63 was overexpressed or knocked down to study the protein function/function of p63 in iCCA. We found that cells overexpressing p63, but not p63 knockdown counterparts, displayed increased proliferation, migration, and invasion. Transcriptome analysis showed that p63 altered the iCCA transcriptome, particularly by affecting cell adhesion-related genes. Moreover, chromatin accessibility decreased at p63 target sites when p63 binding was lost and increased when p63 binding was gained. The majority of the p63 bound sites were located in the distal intergenic regions and showed strong enhancer marks; however, active histone modifications around the Transcription Start Site changed as p63 expression changed. We also detected an interaction between p63 and the chromatin structural protein YY1. Taken together, our results suggest an oncogenic role for p63 in iCCA.