Cholangiocarcinoma (CCA) is characterized by resistance to chemotherapy and a poor prognosis. Therefore, treatments that can effectively suppress tumor growth are urgently needed. Aber-rant activation of hedgehog (HH) signaling has been implicated in several cancers, including those of the hepatobiliary tract. However, the role of HH signaling in intrahepatic CCA (iCCA) has not been completely elucidated. In this study, we addressed the function of the main transducer Smoothened (SMO) and the transcription factors (TFs) GLI1 and GLI2 in iCCA. In addition, we evaluated the potential benefits of the combined inhibition of SMO and the DNA damage kinase WEE1. Transcriptomic analysis of 152 human iCCA samples showed increased expression of GLI1, GLI2, and Patched 1 (PTCH1) in tumor tissues compared with nontumor tissues. Genetic silencing of SMO, GLI1, and GLI2 inhibited the growth, survival, invasiveness, and self-renewal of iCCA cells. Pharmacologic inhibition of SMO reduced iCCA growth and viability in vitro, by inducing double-strand break DNA damage, leading to mitotic arrest and apoptotic cell death. Importantly, SMO inhibition resulted in the activation of the G2-M checkpoint and DNA damage kinase WEE1, increasing the vulnerability to WEE1 inhibition. Hence, the combination of MRT-92 with the WEE1 inhibitor AZD-1775 showed increased antitumor activity in vitro and in iCCA xenografts compared with single treatments. These data indicate that combined inhibition of SMO and WEE1 reduces tumor burden and may represent a strategy for the clinical devel-opment of novel therapeutic approaches in iCCA.

Background & Aims: Cholangiocarcinoma (CCA) is a heterogeneous and lethal malignancy, the molecular origins of which remain poorly understood. MicroRNAs (miRs) target diverse signalling pathways, functioning as potent epigenetic regulators of tran-scriptional output. We aimed to characterise miRNome dysregulation in CCA, including its impact on transcriptome homeostasis and cell behaviour.Methods: Small RNA sequencing was performed on 119 resected CCAs, 63 surrounding liver tissues, and 22 normal livers. High -throughput miR mimic screens were performed in three primary human cholangiocyte cultures. Integration of patient tran-scriptomes and miRseq together with miR screening data identified an oncogenic miR for characterization. MiR-mRNA in-teractions were investigated by a luciferase assay. MiR-CRISPR knockout cells were generated and phenotypically characterized in vitro (proliferation, migration, colony, mitochondrial function, glycolysis) and in vivo using subcutaneous xenografts.Results: In total, 13% (140/1,049) of detected miRs were differentially expressed between CCA and surrounding liver tissues, including 135 that were upregulated in tumours. CCA tissues were characterised by higher miRNome heterogeneity and miR biogenesis pathway expression. Unsupervised hierarchical clustering of tumour miRNomes identified three subgroups, including distal CCA-enriched and IDH1 mutant-enriched subgroups. High-throughput screening of miR mimics uncovered 71 miRs that consistently increased proliferation of three primary cholangiocyte models and were upregulated in CCA tissues regardless of anatomical location, among which only miR-27a-3p had consistently increased expression and activity in several cohorts. FoxO signalling was predominantly downregulated by miR-27a-3p in CCA, partially through targeting of FOXO1. MiR-27a knockout increased FOXO1 levels in vitro and in vivo, impeding tumour behaviour and growth.Conclusions: The miRNomes of CCA tissues are highly remodelled, impacting transcriptome homeostasis in part through regulation of transcription factors like FOXO1. MiR-27a-3p arises as an oncogenic vulnerability in CCA.

Simple Summary A potential relationship between cholangiocarcinoma and metabolic disorders has been suggested, but there is a lack of published data. This study aimed to describe the prevalence of metabolic disorders in a cohort of 122 patients with cholangiocarcinoma and report clinical outcomes. We found a prevalence of 42.6% of metabolic disorders. There was no significant difference in overall survival between patients with or without metabolic disorders, although there was a better survival in the subgroup of patients undergoing surgical resection. This indicates a need to better explore the association between cholangiocarcinoma in a metabolic background. Introduction and objectives: The incidence of cholangiocarcinoma (CCA) has been increasing globally. Although a concomitant increase in the incidence of metabolic disorders might suggest a causal relationship, the data are scarce. We aimed to describe the prevalence of metabolic disorders in patients with CCA and report the clinical features and outcomes. Patients and Methods: Retrospective study including patients with CCA. Patients were divided into: (1) past history of diabetes or/and overweight/obesity (metabolic disorder group) and (2) without any of these features (non-metabolic-disorder group). A Cox regression model was used to determine the prognostic factors. Results: 122 patients were included. In total, 36 (29.5%) had overweight/obesity, 24 (19.7%) had diabetes, and 8 (6.6%) had both. A total of 29 (23.8%) patients had resectable disease and received upfront surgery. A total of 104 (85.2%) received chemotherapy for advanced/recurrent disease. The overall survival of the cohort was 14.3 months (95% CI: 10.1-17.3). ECOG-PS 0 (p < 0.0001), resectable disease (p = 0.018) and absence of vascular invasion (p = 0.048) were independently associated with better prognosis. The metabolic disorder group (n = 52) had a median survival of 15.5 months (95% CI 10.9-33.9) vs. 11.5 months (95% CI 8.4-16.5) in the non-metabolic-disorder group (n = 70) (HR: 1.10; 95% CI 0.62-1.94). Patients with resectable disease in the metabolic group had longer survival than patients in the non-metabolic group (43.4 months (95% CI 33.9-NR) vs. 21.8 months (95% CI 8.6-26.9); HR = 0.12, 95% CI 0.03-0.59). Conclusion: Metabolic disorders are frequent among CCA patients. Underlying metabolic comorbidities may be associated with prognosis in resectable CCA. There is a need to explore the mechanism that drives CCA carcinogenesis in a metabolic background.

Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-x(L), Mcl-1) is particularly important. In this study, we investigated whether antiapoptotic protein patterns are prognostically relevant and potential therapeutic targets in CCA. Bcl-2 proteins were analysed in a pan-cancer cohort from the NCT/DKFZ/DKTK MASTER registry trial (n = 1140, CCA n = 72) via RNA-sequencing and transcriptome-based protein activity interference revealing high ranks of CCA for Bcl-x(L) and Mcl-1. Expression of Bcl-x(L), Mcl-1, and Bcl-2 was assessed in human CCA tissue and cell lines compared with cholangiocytes by immunohistochemistry, immunoblotting, and quantitative-RT-PCR. Immunohistochemistry confirmed the upregulation of Bcl-x(L) and Mcl-1 in iCCA tissues. Cell death of CCA cell lines upon treatemnt with specific small molecule inhibitors of Bcl-x(L) (Wehi-539), of Mcl-1 (S63845), and Bcl-2 (ABT-199), either alone, in combination with each other or together with chemotherapeutics was assessed by flow cytometry. Targeting Bcl-x(L) induced cell death and augmented the effect of chemotherapy in CCA cells. Combined inhibition of Bcl-x(L) and Mcl-1 led to a synergistic increase in cell death in CCA cell lines. Correlation between Bcl-2 protein expression and survival was analysed within three independent patient cohorts from cancer centers in Germany comprising 656 CCA cases indicating a prognostic value of Bcl-x(L) in CCA depending on the CCA subtype. Collectively, these observations identify Bcl-x(L) as a key protein in cell death resistance of CCA and may pave the way for clinical application.

Background and Aims Cholangiocarcinoma (CCA) includes a heterogeneous group of biliary cancers with a dismal prognosis. We investigated if lipid metabolism is disrupted in CCA and its role in tumor proliferation. Approach and Results The in vitro and in vivo tumorigenic capacity of five human CCA cell lines was analyzed. Proteome, lipid content, and metabolic fluxes were evaluated in CCA cells and compared with normal human cholangiocytes (NHC). The Akt1/NOTCH1 intracellular cytoplasmic domain (Nicd1)-driven CCA mouse model was also evaluated. The proteome of CCA cells was enriched in pathways involved in lipid and lipoprotein metabolism. The EGI1 CCA cell line presented the highest tumorigenic capacity. Metabolic studies in high (EGI1) versus low (HUCCT1) proliferative CCA cells in vitro showed that both EGI1 and HUCCT1 incorporated more fatty acids (FA) than NHC, leading to increased triglyceride storage, also observed in Akt1/Nicd1-driven CCA mouse model. The highly proliferative EGI1 CCA cells showed greater uptake of very-low-density and HDLs than NHC and HUCCT1 CCA cells and increased cholesteryl ester content. The FA oxidation (FAO) and related proteome enrichment were specifically up-regulated in EGI1, and consequently, pharmacological blockade of FAO induced more pronounced inhibition of their tumorigenic capacity compared with HUCCT1. The expression of acyl-CoA dehydrogenase ACADM, the first enzyme involved in FAO, was increased in human CCA tissues and correlated with the proliferation marker PCNA. Conclusions Highly proliferative human CCA cells rely on lipid and lipoprotein uptake to fuel FA catabolism, suggesting that inhibition of FAO and/or lipid uptake could represent a therapeutic strategy for this CCA subclass.

Primary biliary cholangitis (PBC) is a rare chronic cholestatic and immune-mediated liver disease of unknown aetiology that targets intrahepatic bile duct cells (cholangiocytes) and primarily affects postmenopausal women, when their estrogen levels sharply decrease. An impaired cholangiocyte response to estrogen characterizes the terminal stage of the disease, as this is when an inefficiency of cholangiocyte proliferation, in balancing the loss of intrahepatic bile ducts, is observed. Here, we report that the estrogen precursor dehydroepiandrosterone (DHEA) and its sulfate metabolites, DHEA-S and 17 beta-estradiol, enhance the proliferation of cholangiocytes and hepatocytes in vitro. Flow cytometry analysis showed that DHEA and DHEA-S decreased glyco-chenodeoxycholic acid (GCDC)-driven apoptosis in cholangiocytes. Cell viability assay (MTT) indicated that ER-alpha, -beta, and the G-protein-coupled estrogen receptor, are involved in the protection of DHEA against oxidative stress in cholangiocytes. Finally, immunoblot analysis showed an elevated level of steroid sulfatase and a reduced level of sulfotransferase 1E1 enzymes, involved in the desulfation/sulfation process of estrogens in cirrhotic PBC, and primary sclerosis cholangitis (PSC) liver tissues, another type of chronic cholestatic and immune-mediated liver disease. Taken together, these results suggest that DHEA can prevent the deleterious effects of certain potentially toxic bile acids and reactive oxygen species, delaying the onset of liver disease.

Simple Summary Lymph node metastasis and positive resection margins have been reported to be major determinants of overall survival (OS) and poor recurrence-free survival (RFS) for patients after resection for perihilar cholangiocarcinoma (pCCA). The aim of current study was to assess the prognostic value of positive lymph nodes and resection margin status on OS. We found a major negative effect of positive lymph nodes on survival and recurrence, which, surprisingly, was independent of margin status. ECOG performance status, positive lymph nodes, and the grade of tumor differentiation were found as the main independent indicators of oncological outcome. This suggests that tumor biology and performance status have a higher impact on survival than extended or radical surgery. Background: Lymph node metastasis and positive resection margins have been reported to be major determinants of overall survival (OS) and poor recurrence-free survival (RFS) for patients who underwent resection for perihilar cholangiocarcinoma (pCCA). However, the prognostic value of positive lymph nodes independently from resection margin status on OS has not been evaluated. Methods: From the European Cholangiocarcinoma (ENSCCA) registry, patients who underwent resection for pCCA between 1994 and 2021 were included in this retrospective cohort study. The primary outcome was OS stratified for resection margin and lymph node status. The secondary outcome was recurrence-free survival. Results: A total of 325 patients from 11 different centers and six European countries were included. Of these, 194 (59.7%) patients had negative resection margins. In 113 (34.8%) patients, positive lymph nodes were found. Lymph node status, histological grade, and ECOG performance status were independent prognostic factors for survival. The median OS for N0R0, N0R1, N+R0, and N+R1 was 38, 30, 18, and 12 months, respectively (p < 0.001). Conclusion: These data indicate that in the presence of positive regional lymph nodes, resection margin status does not determine OS or RFS in patients with pCCA. Achieving negative margins in patients with positive nodes should not come at the expense of more extensive surgery and associated higher mortality.

Altered methionine metabolism is associated with weight gain in obesity. The methionine adenosyltransferase (MAT), catalyzing the first reaction of the methionine cycle, plays an important role regulating lipid metabolism. However, its role in obesity, when a plethora of metabolic diseases occurs, is still unknown. By using antisense oligonucleotides (ASO) and genetic depletion of Mat1a, here, we demonstrate that Mat1a deficiency in diet-induce obese or genetically obese mice prevented and reversed obesity and obesity-associated insulin resistance and hepatosteatosis by increasing energy expenditure in a hepatocyte FGF21 dependent fashion. The increased NRF2-mediated FGF21 secretion induced by targeting Mat1a, mobilized plasma lipids towards the BAT to be catabolized, induced thermogenesis and reduced body weight, inhibiting hepatic de novo lipogenesis. The beneficial effects of Mat1a ASO were abolished following FGF21 depletion in hepatocytes. Thus, targeting Mat1a activates the liver-BAT axis by increasing NRF2-mediated FGF21 secretion, which prevents obesity, insulin resistance and hepatosteatosis. High methionine and S-adenosylmethionine serum levels are related with obesity. Here the authors show that knockdown of methionine adenosyltransferase by using antisense oligonucleotides provides beneficial effects in obesity and comorbidities.

Background Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA. Methods Cholangiocarcinogenesis was induced in rats (TAA) and mice (Jnk(Delta hepa) + CCl4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRAS(G12D) cells. Cell signaling, growth, gene regulation and [U-C-13]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model. Results Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRAS(G12D) can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRAS(G12D) promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRAS(G12D) CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression. Conclusions In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA.