Advances in the treatment of intrahepatic cholangiocarcinoma: An overview of the current and future therapeutic landscape for clinicians.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver tumor and remains a fatal malignancy in the majority of patients. Approximately 20%-30% of patients are eligible for resection, which is considered the only potentially curative treatment; and, after resection, a median survival of 53 months has been reported when sequenced with adjuvant capecitabine. For the 70%-80% of patients who present with locally unresectable or distant metastatic disease, systemic therapy may delay progression, but survival remains limited to approximately 1 year. For the past decade, doublet chemotherapy with gemcitabine and cisplatin has been considered the most effective first-line regimen, but results from the recent use of triplet regimens and even immunotherapy may shift the paradigm. More effective treatment strategies, including those that combine systemic therapy with locoregional therapies like radioembolization or hepatic artery infusion, have also been developed. Molecular therapies, including those that target fibroblast growth factor receptor and isocitrate dehydrogenase, have recently received US Food and Drug Administration approval for a defined role as second-line treatment for up to 40% of patients harboring these actionable genomic alterations, and whether they should be considered in the first-line setting is under investigation. Furthermore, as the oncology field seeks to expand indications for immunotherapy, recent data demonstrated that combining durvalumab with standard cytotoxic therapy improved survival in patients with ICC. This review focuses on the current and future strategies for ICC treatment, including a summary of the primary literature for each treatment modality and an algorithm that can be used to drive a personalized and multidisciplinary approach for patients with this challenging malignancy.

Interventional gastroenterology in oncology.

Cancer is one of the foremost health problems worldwide and is among the leading causes of death in the United States. Gastrointestinal tract cancers account for almost one third of the cancer-related mortality globally, making it one of the deadliest groups of cancers. Early diagnosis and prompt management are key to preventing cancer-related morbidity and mortality. With advancements in technology and endoscopic techniques, endoscopy has become the core in diagnosis and management of gastrointestinal tract cancers. In this extensive review, the authors discuss the role endoscopy plays in early detection, diagnosis, and management of esophageal, gastric, colorectal, pancreatic, ampullary, biliary tract, and small intestinal cancers.

Discovery of lirafugratinib (RLY-4008), a highly selective irreversible small-molecule inhibitor of FGFR2.

Fibroblast growth factor receptor (FGFR) kinase inhibitors have been shown to be effective in the treatment of intrahepatic cholangiocarcinoma and other advanced solid tumors harboring FGFR2 alterations, but the toxicity of these drugs frequently leads to dose reduction or interruption of treatment such that maximum efficacy cannot be achieved. The most common adverse effects are hyperphosphatemia caused by FGFR1 inhibition and diarrhea due to FGFR4 inhibition, as current therapies are not selective among the FGFRs. Designing selective inhibitors has proved difficult with conventional approaches because the orthosteric sites of FGFR family members are observed to be highly similar in X-ray structures. In this study, aided by analysis of protein dynamics, we designed a selective, covalent FGFR2 inhibitor. In a key initial step, analysis of long-timescale molecular dynamics simulations of the FGFR1 and FGFR2 kinase domains allowed us to identify differential motion in their P-loops, which are located adjacent to the orthosteric site. Using this insight, we were able to design orthosteric binders that selectively and covalently engage the P-loop of FGFR2. Our drug discovery efforts culminated in the development of lirafugratinib (RLY-4008), a covalent inhibitor of FGFR2 that shows substantial selectivity over FGFR1 (~250-fold) and FGFR4 (~5,000-fold) in vitro, causes tumor regression in multiple FGFR2-altered human xenograft models, and was recently demonstrated to be efficacious in the clinic at doses that do not induce clinically significant hyperphosphatemia or diarrhea.

TGFß-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma.

Cholangiocarcinoma is a devastating liver cancer characterized by high aggressiveness and therapy resistance, resulting in poor prognosis. Long non-coding RNAs and signals imposed by oncogenic pathways, such as transforming growth factor ß (TGFß), frequently contribute to cholangiocarcinogenesis. Here, we explore novel effectors of TGFß signalling in cholangiocarcinoma. LINC00313 is identified as a novel TGFß target gene. Gene expression and genome-wide chromatin accessibility profiling reveal that nuclear LINC00313 transcriptionally regulates genes involved in Wnt signalling, such as the transcriptional activator TCF7. LINC00313 gain-of-function enhances TCF/LEF-dependent transcription, promotes colony formation in vitro and accelerates tumour growth in vivo. Genes affected by LINC00313 over-expression in CCA tumours are associated with KRAS and TP53 mutations and reduce overall patient survival. Mechanistically, ACTL6A and BRG1, subunits of the SWI/SNF chromatin remodelling complex, interact with LINC00313 and affect TCF7 and SULF2 transcription. We propose a model whereby TGFß induces LINC00313 in order to regulate the expression of hallmark Wnt pathway genes, in co-operation with SWI/SNF. By modulating key genes of the Wnt pathway, LINC00313 fine-tunes Wnt/TCF/LEF-dependent transcriptional responses and promotes cholangiocarcinogenesis.

Fibroblast-Derived Lysyl Oxidase Increases Oxidative Phosphorylation and Stemness in Cholangiocarcinoma.

BACKGROUND & AIMS: Metabolic and transcriptional programs respond to extracellular matrix-derived cues in complex environments, such as the tumor microenvironment. Here, we demonstrate how lysyl oxidase (LOX), a known factor in collagen crosslinking, contributes to the development and progression of cholangiocarcinoma (CCA). METHODS: Transcriptomes of 209 human CCA tumors, 143 surrounding tissues, and single-cell data from 30 patients were analyzed. The recombinant protein and a small molecule inhibitor of the LOX activity were used on primary patient-derived CCA cultures to establish the role of LOX in migration, proliferation, colony formation, metabolic fitness, and the LOX interactome. The oncogenic role of LOX was further investigated by RNAscope and in vivo using the AKT/NICD genetically engineered murine CCA model. RESULTS: We traced LOX expression to hepatic stellate cells and specifically hepatic stellate cell-derived inflammatory cancer-associated fibroblasts and found that cancer-associated fibroblast-driven LOX increases oxidative phosphorylation and metabolic fitness of CCA, and regulates mitochondrial function through transcription factor A, mitochondrial. Inhibiting LOX activity in vivo impedes CCA development and progression. Our work highlights that LOX alters tumor microenvironment-directed transcriptional reprogramming of CCA cells by facilitating the expression of the oxidative phosphorylation pathway and by increasing stemness and mobility. CONCLUSIONS: Increased LOX is driven by stromal inflammatory cancer-associated fibroblasts and correlates with diminished survival of patients with CCA. Modulating the LOX activity can serve as a novel tumor microenvironment-directed therapeutic strategy in bile duct pathologies.

Liver-specific DICER1 syndrome model mice develop cystic liver tumors with defective primary cilia.

DICER1 syndrome is a tumor predisposition syndrome caused by familial genetic mutations in DICER1. Pathogenic variants of DICER1 have been discovered in many rare cancers, including cystic liver tumors. However, the molecular mechanisms underlying liver lesions induced by these variants remain unclear. In the present study, we sought to gain a better understanding of the pathogenesis of these variants by generating a mouse model of liver-specific DICER1 syndrome. The mouse model developed bile duct hyperplasia with fibrosis, similar to congenital hepatic fibrosis, as well as cystic liver tumors resembling those in Caroli's syndrome, intrahepatic cholangiocarcinoma, and hepatocellular carcinoma. Interestingly, the mouse model of DICER1 syndrome showed abnormal formation of primary cilia in the bile duct epithelium, which is a known cause of bile duct hyperplasia and cyst formation. These results indicated that DICER1 mutations contribute to cystic liver tumors by inducing defective primary cilia. The mouse model generated in this study will be useful for elucidating the potential mechanisms of tumorigenesis induced by DICER1 variants and for obtaining a comprehensive understanding of DICER1 syndrome. © 2024 The Pathological Society of Great Britain and Ireland.

Integrated analyses of the genetic and clinicopathological features of cholangiolocarcinoma: cholangiolocarcinoma may be characterized by mismatch-repair deficiency.

Cholangiolocarcinoma (CLC) is a primary liver carcinoma that resembles the canals of Hering and that has been reported to be associated with stem cell features. Due to its rarity, the nature of CLC remains unclear, and its pathological classification remains controversial. To clarify the positioning of CLC in primary liver cancers and identify characteristics that could distinguish CLC from other liver cancers, we performed integrated analyses using whole-exome sequencing (WES), immunohistochemistry, and a retrospective review of clinical information on eight CLC cases and two cases of recurrent CLC. WES demonstrated that CLC includes IDH1 and BAP1 mutations, which are characteristic of intrahepatic cholangiocarcinoma (iCCA). A mutational signature analysis showed a pattern similar to that of iCCA, which was different from that of hepatocellular carcinoma (HCC). CLC cells, including CK7, CK19, and EpCAM, were positive for cholangiocytic differentiation markers. However, the hepatocytic differentiation marker AFP and stem cell marker SALL4 were completely negative. The immunostaining patterns of CLC with CD56 and epithelial membrane antigen were similar to those of the noncancerous bile ductules. In contrast, mutational signature cluster analyses revealed that CLC formed a cluster associated with mismatch-repair deficiency (dMMR), which was separate from iCCA. Therefore, to evaluate MMR status, we performed immunostaining of four MMR proteins (PMS2, MSH6, MLH1, and MSH2) and detected dMMR in almost all CLCs. In conclusion, CLC had highly similar characteristics to iCCA but not to HCC. CLC can be categorized as a subtype of iCCA. In contrast, CLC has characteristics of dMMR tumors that are not found in iCCA, suggesting that it should be treated distinctly from iCCA. © 2024 The Pathological Society of Great Britain and Ireland.

Proteome Landscapes of Human Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma.

Liver cancer is among the top leading causes of cancer mortality worldwide. Particularly, hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA) have been extensively investigated from the aspect of tumor biology. However, a comprehensive and systematic understanding of the molecular characteristics of HCC and CCA remains absent. Here, we characterized the proteome landscapes of HCC and CCA using the data-independent acquisition (DIA) mass spectrometry (MS) method. By comparing the quantitative proteomes of HCC and CCA, we found several differences between the two cancer types. In particular, we found an abnormal lipid metabolism in HCC and activated extracellular matrix-related pathways in CCA. We next developed a three-protein classifier to distinguish CCA from HCC, achieving an area under the curve (AUC) of 0.92, and an accuracy of 90% in an independent validation cohort of 51 patients. The distinct molecular characteristics of HCC and CCA presented in this study provide new insights into the tumor biology of these two major important primary liver cancers. Our findings may help develop more efficient diagnostic approaches and new targeted drug treatments.

Identification of prognostic biomarkers for cholangiocarcinoma by combined analysis of molecular characteristics of clinical MVI subtypes and molecular subtypes.

Cholangiocarcinoma (CCA) is widely noted for its high degree of malignancy, rapid progression, and limited therapeutic options. This study was carried out on transcriptome data of 417 CCA samples from different anatomical locations. The effects of lipid metabolism related genes and immune related genes as CCA classifiers were compared. Key genes were derived from MVI subtypes and better molecular subtypes. Pathways such as epithelial mesenchymal transition (EMT) and cell cycle were significantly activated in MVI-positive group. CCA patients were classified into three (four) subtypes based on lipid metabolism (immune) related genes, with better prognosis observed in lipid metabolism-C1, immune-C2, and immune-C4. IPTW analysis found that the prognosis of lipid metabolism-C1 was significantly better than that of lipid metabolism-C2 + C3 before and after correction. KRT16 was finally selected as the key gene. And knockdown of KRT16 inhibited proliferation, migration and invasion of CCA cells.

Genomic and transcriptomic characteristics of 12 novel primary cell lines derived from three patients with cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive bile duct malignancy with poor prognosis. To improve our understanding of the biological characteristics of CCA and develop effective therapies, appropriate preclinical models are required. Here, we established and characterized 12 novel patient-derived primary cancer cell (PDPC) models using multi-region sampling. At the genomic level of PDPCs, we observed not only commonly mutated genes, such as TP53, JAK3, and KMT2C, consistent with the reports in CCA, but also specific mutation patterns in each cell line. In addition, specific expression patterns with distinct biological functions and pathways involved were also observed in the PDPCs at the transcriptomic level. Furthermore, the drug-sensitivity results revealed that the PDPCs exhibited different responses to the six commonly used compounds. Our findings indicate that the established PDPCs can serve as novel in vitro reliable models to provide a crucial molecular basis for improving the understanding of tumorigenesis and its treatment.

Molecular portraits of patients with intrahepatic cholangiocarcinoma who diverge as rapid progressors or long survivors on chemotherapy.

OBJECTIVE: Cytotoxic agents are the cornerstone of treatment for patients with advanced intrahepatic cholangiocarcinoma (iCCA), despite heterogeneous benefit. We hypothesised that the pretreatment molecular profiles of diagnostic biopsies can predict patient benefit from chemotherapy and define molecular bases of innate chemoresistance. DESIGN: We identified a cohort of advanced iCCA patients with comparable baseline characteristics who diverged as extreme outliers on chemotherapy (survival <6 m in rapid progressors, RP; survival >23 m in long survivors, LS). Diagnostic biopsies were characterised by digital pathology, then subjected to whole-transcriptome profiling of bulk and geospatially macrodissected tissue regions. Spatial transcriptomics of tumour-infiltrating myeloid cells was performed using targeted digital spatial profiling (GeoMx). Transcriptome signatures were evaluated in multiple cohorts of resected cancers. Signatures were also characterised using in vitro cell lines, in vivo mouse models and single cell RNA-sequencing data. RESULTS: Pretreatment transcriptome profiles differentiated patients who would become RPs or LSs on chemotherapy. Biologically, this signature originated from altered tumour-myeloid dynamics, implicating tumour-induced immune tolerogenicity with poor response to chemotherapy. The central role of the liver microenviroment was confrmed by the association of the RPLS transcriptome signature with clinical outcome in iCCA but not extrahepatic CCA, and in liver metastasis from colorectal cancer, but not in the matched primary bowel tumours. CONCLUSIONS: The RPLS signature could be a novel metric of chemotherapy outcome in iCCA. Further development and validation of this transcriptomic signature is warranted to develop precision chemotherapy strategies in these settings.

PARP-1 selectively impairs KRAS-driven phenotypic and molecular features in intrahepatic cholangiocarcinoma.

OBJECTIVE: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer with limited therapeutic options. KRAS mutations are among the most abundant genetic alterations in iCCA associated with poor clinical outcome and treatment response. Recent findings indicate that Poly(ADP-ribose)polymerase1 (PARP-1) is implicated in KRAS-driven cancers, but its exact role in cholangiocarcinogenesis remains undefined. DESIGN: PARP-1 inhibition was performed in patient-derived and established iCCA cells using RNAi, CRISPR/Cas9 and pharmacological inhibition in KRAS-mutant, non-mutant cells. In addition, Parp-1 knockout mice were combined with iCCA induction by hydrodynamic tail vein injection to evaluate an impact on phenotypic and molecular features of Kras-driven and Kras-wildtype iCCA. Clinical implications were confirmed in authentic human iCCA. RESULTS: PARP-1 was significantly enhanced in KRAS-mutant human iCCA. PARP-1-based interventions preferentially impaired cell viability and tumourigenicity in human KRAS-mutant cell lines. Consistently, loss of Parp-1 provoked distinct phenotype in Kras/Tp53-induced versus Akt/Nicd-induced iCCA and abolished Kras-dependent cholangiocarcinogenesis. Transcriptome analyses confirmed preferential impairment of DNA damage response pathways and replicative stress response mediated by CHK1. Consistently, inhibition of CHK1 effectively reversed PARP-1 mediated effects. Finally, Parp-1 depletion induced molecular switch of KRAS-mutant iCCA recapitulating good prognostic human iCCA patients. CONCLUSION: Our findings identify the novel prognostic and therapeutic role of PARP-1 in iCCA patients with activation of oncogenic KRAS signalling.

Function of mast cell and bile-cholangiocarcinoma interplay in cholangiocarcinoma microenvironment.

OBJECTIVE: The correlation between cholangiocarcinoma (CCA) progression and bile is rarely studied. Here, we aimed to identify differential metabolites in benign and malignant bile ducts and elucidate the generation, function and degradation of bile metabolites. DESIGN: Differential metabolites in the bile from CCA and benign biliary stenosis were identified by metabonomics. Biliary molecules able to induce mast cell (MC) degranulation were revealed by in vitro and in vivo experiments, including liquid chromatography-mass spectrometry (MS)/MS and bioluminescence resonance energy transfer assays. Histamine (HA) receptor expression in CCA was mapped using a single-cell mRNA sequence. HA receptor functions were elucidated by patient-derived xenografts (PDX) in humanised mice and orthotopic models in MC-deficient mice. Genes involved in HA-induced proliferation were screened by CRISPR/Cas9. RESULTS: Bile HA was elevated in CCA and indicated poorer prognoses. Cancer-associated fibroblasts (CAFs)-derived stem cell factor (SCF) recruited MCs, and bile N,N-dimethyl-1,4-phenylenediamine (DMPD) stimulated MCs to release HA through G protein-coupled receptor subtype 2 (MRGPRX2)-Gαq signalling. Bile-induced MCs released platelet-derived growth factor subunit B (PDGF-B) and angiopoietin 1/2 (ANGPT1/2), which enhanced CCA angiogenesis and lymphangiogenesis. Histamine receptor H1 (HRH1) and HRH2 were predominantly expressed in CCA cells and CAFs, respectively. HA promoted CCA cell proliferation by activating HRH1-Gαq signalling and hastened CAFs to secrete hepatocyte growth factor by stimulating HRH2-Gαs signalling. Solute carrier family 22 member 3 (SLC22A3) inhibited HA-induced CCA proliferation by importing bile HA into cells for degradation, and SLC22A3 deletion resulted in HA accumulation. CONCLUSION: Bile HA is released from MCs through DMPD stimulation and degraded via SLC22A3 import. Different HA receptors exhibit a distinct expression profile in CCA and produce different oncogenic effects. MCs promote CCA progression in a CCA-bile interplay pattern.

Strategies to enhance the response of liver cancer to pharmacological treatments.

In contrast to other types of cancers, there is no available efficient pharmacological treatment to improve the outcomes of patients suffering from major primary liver cancers, i.e., hepatocellular carcinoma and cholangiocarcinoma. This dismal situation is partly due to the existence in these tumors of many different and synergistic mechanisms of resistance, accounting for the lack of response of these patients, not only to classical chemotherapy but also to more modern pharmacological agents based on the inhibition of tyrosine kinase receptors (TKIs) and the stimulation of the immune response against the tumor using immune checkpoint inhibitors (ICIs). This review summarizes the efforts to develop strategies to overcome this severe limitation, including searching for novel drugs derived from synthetic, semisynthetic, or natural products with vectorial properties against therapeutic targets to increase drug uptake or reduce drug export from cancer cells. Besides, immunotherapy is a promising line of research that is already starting to be implemented in clinical practice. Although less successful than in other cancers, the foreseen future for this strategy in treating liver cancers is considerable. Similarly, the pharmacological inhibition of epigenetic targets is highly promising. Many novel "epidrugs," able to act on "writer," "reader," and "eraser" epigenetic players, are currently being evaluated in preclinical and clinical studies. Finally, gene therapy is a broad field of research in the fight against liver cancer chemoresistance, based on the impressive advances recently achieved in gene manipulation. In sum, although the present is still dismal, there is reason for hope in the non-too-distant future.

Degradation of AZGP1 suppresses apoptosis and facilitates cholangiocarcinoma tumorigenesis via TRIM25.

Alpha-2-Glycoprotein 1, Zinc-binding (AZGP1, ZAG) is a secreted protein that is synthesized by adipocytes and epithelial cells; it is downregulated in several malignancies such as breast, prostate, liver and lung cancers. However, its function remains unclear in cholangiocarcinoma (CCA). Here, we evaluated the impact AZGP1 in CCA using Gene Expression Omnibus (GEO) and GEPIA. In addition, we analysed AZGP1 expression using quantitative reverse transcription PCR and western blotting. Expression of AZGP1 was nearly deficient in CCA patients and cell lines and was associated with poor prognosis. AZGP1 overexpression upregulated apoptosis markers. Co-immunoprecipitation experiments showed that AZGP1 interacts with tripartite motif-containing protein 25 (TRIM25), and tissue microarray and bioinformatic analysis showed that AZGP1 is negatively correlated with TRIM25 expression in CCA. Thereafter, TRIM25 knockdown led to AZGP1 upregulation and induced cancer cell apoptosis. TRIM25 targets AZGP1 for degradation by catalysing its ubiquitination. AZGP1 overexpression significantly suppressed tumour growth in a xenograft mouse model. This study findings suggest that AZGP1 is a potential therapeutic target or a diagnostic biomarker for treating patients with CCA.

Downregulation of JAM3 occurs in cholangiocarcinoma by hypermethylation: A potential molecular marker for diagnosis and prognosis.

Junctional adhesion molecular 3 (JAM3) is downregulated by hypermethylation in cancers but is unclear in cholangiocarcinoma. The JAM3 expression level was checked in cholangiocarcinoma cell lines and tissues. Methylated JAM3 was detected in cell lines, tissues and plasma cell-free DNAs (cfDNA). The roles of JAM3 in cholangiocarcinoma were studied by transfection of siRNA and pCMV3-JAM3. The survival analysis was based on the Gene Set Cancer Analysis (GSCA) database. JAM3 was downregulated in HCCC-9810 and HuCCT1 cell lines and tissues by hypermethylation. Methylated JAM3 was detected in cfDNAs with 53.3% sensitivity and 96.6% specificity. Transfection of pCMV3-JAM3 into HCCC-9810 and HuCCT1 induced apoptosis and suppressed cell proliferation, migration and invasion. The depletion of JAM3 in RBE cells using siRNA decreased apoptosis and increased cell proliferation, migration and invasion. Hypermethylation of JAM3 was associated with tumour differentiation, metastasis and TNM stage. Downregulation and hypermethylation of JAM3 were related to poor progression-free survival. Junctional adhesion molecular 3 may function as a tumour suppressor in cholangiocarcinoma. Methylated JAM3 DNA may represent a non-invasive molecular marker for the early detection of cholangiocarcinoma and prognosis.

Tumour burden score combined with albumin-to-alkaline phosphatase ratio predicts prognosis in patients with intrahepatic cholangiocarcinoma.

Tumour morphology (tumour burden score (TBS)) and liver function (albumin-to-alkaline phosphatase ratio (AAPR)) have been shown to correlate with outcomes in intrahepatic cholangiocarcinoma (ICC). This study aimed to evaluate the combined predictive effect of TBS and AAPR on survival outcomes in ICC patients. We conducted a retrospective analysis using a multicentre database of ICC patients who underwent curative surgery from 2011 to 2018. The Kaplan-Meier method was employed to examine the relationship between a new index (combining TBS and AAPR) and long-term outcomes. The predictive efficacy of this index was compared to other conventional indicators. A total of 560 patients were included in the study. Based on TBS and AAPR stratification, patients were classified into three groups. Kaplan-Meier curves demonstrated that 124 patients with low TBS and high AAPR had the best overall survival (OS) and recurrence-free survival (RFS), while 170 patients with high TBS and low AAPR had the worst outcomes (log-rank p < 0.001). Multivariate analyses identified the combined index as an independent predictor of OS and RFS. Furthermore, the index showed superior accuracy in predicting OS and RFS compared to other conventional indicators. Collectively, this study demonstrated that the combination of liver function and tumour morphology provides a synergistic effect in evaluating the prognosis of ICC patients. The novel index combining TBS and AAPR effectively stratified postoperative survival outcomes in ICC patients undergoing curative resection.

Combined analysis of secreted proteins and glycosylation identifies prognostic features in cholangiocarcinoma.

Secreted proteins are overexpressed in cholangiocarcinoma (CCA) and actively involved in promoting metastatic spread. Many of these proteins possess one or more sites of glycosylation and their various glycoforms have potential utility as prognostic or diagnostic biomarkers. To evaluate the effects of secretome glycosylation on patient outcome, we elucidated the glycosylation patterns of proteins secreted by parental and metastatic CCA cells using liquid chromatography-mass spectrometry. Our analysis showed that the secretome of CCA cells was dominated by fucosylated and fucosialylated glycoforms. Based on the glycan and protein profiles, we evaluated the combined prognostic significance of glycosyltransferases and secretory proteins. Significantly, genes encoding fucosyltransferases and sialyltransferases showed favorable prognostic effects when combined with secretory protein-coding gene expression, particularly thrombospondin-1. Combining these measures may provide improved risk assessment for CCA and be used to indicate stages of disease progression.

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 locally invasive IPNB tissues (LI-IPNB) and IPNB tissues without local invasion from the same patients. We identified a protein-truncating variant in an E3 ubiquitin ligase, RNF213 (c.6967C>T; p.Gln2323X; chr17: 78,319,102 [hg19], exon 29), as the most common protein-truncating variant 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 vs control cells was then performed in the HuCCT1, YSCCC, and KKU-100 cells. Gene ontology enrichment analysis of the common differentially expressed genes revealed significantly altered cytokine and oxidoreductase-oxidizing metal ion activities, as confirmed by Western blotting. Gene Set Enrichment Analysis identified the most enriched pathways being oxidative phosphorylation, fatty acid metabolism, reactive oxygen species, adipogenesis, and angiogenesis. In sum, loss-of-function mutation 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 to inflammation and energy metabolisms.

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

BACKGROUND: circular 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. METHODS: circPCNXL2 (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. RESULTS: circPCNXL2 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. CONCLUSION: circPCNXL2 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.