Cholangiocarcinoma progression depends on the uptake and metabolization of extracellular lipids

[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.This work was supported by “Ayudas para apoyar grupos de investigación del sistema Universitario Vasco” (IT971‐16 to PA), MCIU/AEI/FEDER, UE (2018‐095134‐B‐100 to PA and by the University of Basque Country COLAB20/01 to PA; Spanish Carlos III Health Institute (ISCIII) (FIS PI15/01132, PI18/01075, PI21/00922, and Miguel Servet Program CON14/00129 and CPII19/00008 to JMB; FIS PI14/00399, PI17/00022 and PI20/00186 to MJP; Sara Borrell [CD19/00254 to PMR]) cofinanced by “Fondo Europeo de Desarrollo Regional” (FEDER); CIBERehd (ISCIII) to JMB, MJP, PMR, PA and LB); “Diputación Foral Gipuzkoa” (DFG15/010, DFG16/004 to JMB and 2020‐CIEN‐000067‐01 to PMR), Department of Health of the Basque Country (2019111024 to MJP, 2017111010 to JMB, and 2020111077 to JMB and PA), “Euskadi RIS3” (2016222001, 2017222014, 2018222029, 2019222054, 2020333010 to JMB), BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD to JMB) and Department of Industry of the Basque Country (Elkartek: KK‐2020/00008 to JMB); La Caixa Scientific Foundation (HR17‐00601 to JMB). “Fundación Científica de la Asociación Española Contra el Cáncer” (AECC Scientific Foundation, to JMB). AMMF‐The Cholangiocarcinoma Charity (EU/2019/AMMFt/001, to JMB and PMR). MRDG was funded by “Fundación Científica de la Asociación Española Contra el Cáncer” (AECC de Bizkaia), MJP was funded by the Spanish Ministry of Economy and Competitiveness (MINECO: “Ramón y Cajal” Program RYC‐2015‐17755), IL, AL and FG‐R by the Basque Government (PRE_2016_1_0152, PRE_2018_2_0195 and PRE 2020 2 02500, respectively), AN‐Z and BG‐S by the UPV/EHU, AB‐V by “Programa de especialización de Personal Investigador Doctor” at the UPV/EHU (2019‐2020) and MA by the MCIU/AEI/FEDER

Proteostasis disturbances and endoplasmic reticulum stress contribute to polycystic liver disease: New therapeutic targets

BACKGROUND & AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple biliary cysts. Recently, novel PLD-causative genes, encoding for endoplasmic reticulum (ER)-resident proteins involved in protein biogenesis and transport, were identified. We hypothesized that aberrant proteostasis contributes to PLD pathogenesis, representing a potential therapeutic target. METHODS: ER stress was analysed at transcriptional (qPCR), proteomic (mass spectrometry), morphological (transmission electron microscopy, TEM) and functional (proteasome activity) levels in different PLD models. The effect of ER stress inhibitors [4-phenylbutyric acid (4-PBA)] and/or activators [tunicamycin (TM)] was tested in polycystic (PCK) rats and cystic cholangiocytes in vitro. RESULTS: The expression levels of unfolded protein response (UPR) components were upregulated in liver tissue from PLD patients and PCK rats, as well as in primary cultures of human and rat cystic cholangiocytes, compared to normal controls. Cystic cholangiocytes showed altered proteomic profiles, mainly related to proteostasis (ie synthesis, folding, trafficking and degradation of proteins), marked enlargement of the ER lumen (by TEM) and hyperactivation of the proteasome. Notably, chronic treatment of PCK rats with 4-PBA decreased liver weight, as well as both liver and cystic volumes, of animals under baseline conditions or after TM administration compared to controls. In vitro, 4-PBA downregulated the expression (mRNA) of UPR effectors, normalized proteomic profiles related to protein synthesis, folding, trafficking and degradation and reduced the proteasome hyperactivity in cystic cholangiocytes, reducing their hyperproliferation and apoptosis. CONCLUSIONS: Restoration of proteostasis in cystic cholangiocytes with 4-PBA halts hepatic cystogenesis, emerging as a novel therapeutic strategy

A novel serum metabolomic profile for the differential diagnosis of distal cholangiocarcinoma and pancreatic ductal adenocarcinoma

The diagnosis of adenocarcinomas located in the pancreas head, i.e., distal cholangiocarcinoma (dCCA) and pancreatic ductal adenocarcinoma (PDAC), constitutes a clinical challenge because they share many symptoms, are not easily distinguishable using imaging techniques and accurate biomarkers are not available. Searching for biomarkers with potential usefulness in the differential diagnosis of these tumors, we have determined serum metabolomic profiles in healthy controls and patients with dCCA, PDAC or benign pancreatic diseases (BPD). Ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) analysis was performed in serum samples from dCCA (n = 34), PDAC (n = 38), BPD (n = 42) and control (n = 25) individuals, divided into discovery and validation cohorts. This approach permitted 484 metabolites to be determined, mainly lipids and amino acids. The analysis of the results led to the proposal of a logistic regression model able to discriminate patients with dCCA and PDAC (AUC value of 0.888) based on the combination of serum levels of nine metabolites (acylcarnitine AC(16:0), ceramide Cer(d18:1/24:0), phosphatidylcholines PC(20:0/0:0) and PC(O-16:0/20:3), lysophosphatidylcholines PC(20:0/0:0) and PC(0:0/20:0), lysophosphatidylethanolamine PE(P-18:2/0:0), and sphingomyelins SM(d18:2/22:0) and SM(d18:2/23:0)) and CA 19-9. In conclusion, we propose a novel specific panel of serum metabolites that can help in the differential diagnosis of dCCA and PDAC. Further validation of their clinical usefulness in prospective studies is required

A novel serum metabolomic profile for the differential diagnosis of distal cholangiocarcinoma and pancreatic ductal adenocarcinoma

The diagnosis of adenocarcinomas located in the pancreas head, i.e., distal cholangiocarcinoma (dCCA) and pancreatic ductal adenocarcinoma (PDAC), constitutes a clinical challenge because they share many symptoms, are not easily distinguishable using imaging techniques and accurate biomarkers are not available. Searching for biomarkers with potential usefulness in the differential diagnosis of these tumors, we have determined serum metabolomic profiles in healthy controls and patients with dCCA, PDAC or benign pancreatic diseases (BPD). Ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) analysis was performed in serum samples from dCCA (n = 34), PDAC (n = 38), BPD (n = 42) and control (n = 25) individuals, divided into discovery and validation cohorts. This approach permitted 484 metabolites to be determined, mainly lipids and amino acids. The analysis of the results led to the proposal of a logistic regression model able to discriminate patients with dCCA and PDAC (AUC value of 0.888) based on the combination of serum levels of nine metabolites (acylcarnitine AC(16:0), ceramide Cer(d18:1/24:0), phosphatidylcholines PC(20:0/0:0) and PC(O-16:0/20:3), lysophosphatidylcholines PC(20:0/0:0) and PC(0:0/20:0), lysophosphatidylethanolamine PE(P-18:2/0:0), and sphingomyelins SM(d18:2/22:0) and SM(d18:2/23:0)) and CA 19-9. In conclusion, we propose a novel specific panel of serum metabolites that can help in the differential diagnosis of dCCA and PDAC. Further validation of their clinical usefulness in prospective studies is required

A novel serum metabolomic profile for the differential diagnosis of distal cholangiocarcinoma and pancreatic ductal adenocarcinoma

The diagnosis of adenocarcinomas located in the pancreas head, i.e., distal cholangiocarcinoma (dCCA) and pancreatic ductal adenocarcinoma (PDAC), constitutes a clinical challenge because they share many symptoms, are not easily distinguishable using imaging techniques and accurate biomarkers are not available. Searching for biomarkers with potential usefulness in the differential diagnosis of these tumors, we have determined serum metabolomic profiles in healthy controls and patients with dCCA, PDAC or benign pancreatic diseases (BPD). Ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) analysis was performed in serum samples from dCCA (n = 34), PDAC (n = 38), BPD (n = 42) and control (n = 25) individuals, divided into discovery and validation cohorts. This approach permitted 484 metabolites to be determined, mainly lipids and amino acids. The analysis of the results led to the proposal of a logistic regression model able to discriminate patients with dCCA and PDAC (AUC value of 0.888) based on the combination of serum levels of nine metabolites (acylcarnitine AC(16:0), ceramide Cer(d18:1/24:0), phosphatidylcholines PC(20:0/0:0) and PC(O-16:0/20:3), lysophosphatidylcholines PC(20:0/0:0) and PC(0:0/20:0), lysophosphatidylethanolamine PE(P-18:2/0:0), and sphingomyelins SM(d18:2/22:0) and SM(d18:2/23:0)) and CA 19-9. In conclusion, we propose a novel specific panel of serum metabolites that can help in the differential diagnosis of dCCA and PDAC. Further validation of their clinical usefulness in prospective studies is required

Serum metabolites as diagnostic biomarkers for cholangiocarcinoma, hepatocellular carcinoma and primary sclerosing cholangitis

Early and differential diagnosis of intrahepatic cholangiocarcinoma (iCCA) and hepatocellular carcinoma (HCC) by noninvasive methods represents a current clinical challenge. The analysis of low-molecular-weight metabolites by new high-throughput techniques is a strategy for identifying biomarkers. Here, we have investigated whether serum metabolome can provide useful biomarkers in the diagnosis of iCCA and HCC and could discriminate iCCA from HCC. Because primary sclerosing cholangitis (PSC) is a risk factor for CCA, serum metabolic profiles of PSC and CCA have also been compared. The analysis of the levels of lipids and amino acids in the serum of patients with iCCA, HCC, and PSC and healthy individuals (n = 20/group) showed differential profiles. Several metabolites presented high diagnostic value for iCCA versus control, HCC versus control, and PSC versus control, with areas under the receiver operating characteristic curve (AUC) greater than those found in serum for the nonspecific tumor markers carbohydrate antigen 19-9 (CA 19-9) and alpha-fetoprotein (AFP), commonly used to help in the diagnosis of iCCA and HCC, respectively. The development of an algorithm combining glycine, aspartic acid, SM(42:3), and SM(43:2) permitted to accurately differentiate in the diagnosis of both types of tumors (biopsy-proven). The proposed model yielded 0.890 AUC, 75% sensitivity, and 90% specificity. Another algorithm by combination of PC(34:3) and histidine accurately permitted to differentiate PSC from iCCA, with an AUC of 0.990, 100% sensitivity, and 70% specificity. These results were validated in independent cohorts of 14-15 patients per group and compared with profiles found in patients with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Conclusion: Specific changes in serum concentrations of certain metabolites are useful to differentiate iCCA from HCC or PSC, and could help in the early diagnosis of these diseases

A novel serum metabolomic profile for the differential diagnosis of distal cholangiocarcinoma and pancreatic ductal adenocarcinoma

The diagnosis of adenocarcinomas located in the pancreas head, i.e., distal cholangiocarcinoma (dCCA) and pancreatic ductal adenocarcinoma (PDAC), constitutes a clinical challenge because they share many symptoms, are not easily distinguishable using imaging techniques and accurate biomarkers are not available. Searching for biomarkers with potential usefulness in the differential diagnosis of these tumors, we have determined serum metabolomic profiles in healthy controls and patients with dCCA, PDAC or benign pancreatic diseases (BPD). Ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) analysis was performed in serum samples from dCCA (n = 34), PDAC (n = 38), BPD (n = 42) and control (n = 25) individuals, divided into discovery and validation cohorts. This approach permitted 484 metabolites to be determined, mainly lipids and amino acids. The analysis of the results led to the proposal of a logistic regression model able to discriminate patients with dCCA and PDAC (AUC value of 0.888) based on the combination of serum levels of nine metabolites (acylcarnitine AC(16:0), ceramide Cer(d18:1/24:0), phosphatidylcholines PC(20:0/0:0) and PC(O-16:0/20:3), lysophosphatidylcholines PC(20:0/0:0) and PC(0:0/20:0), lysophosphatidylethanolamine PE(P-18:2/0:0), and sphingomyelins SM(d18:2/22:0) and SM(d18:2/23:0)) and CA 19-9. In conclusion, we propose a novel specific panel of serum metabolites that can help in the differential diagnosis of dCCA and PDAC. Further validation of their clinical usefulness in prospective studies is required

Serum metabolites as diagnostic biomarkers for cholangiocarcinoma, hepatocellular carcinoma and primary sclerosing cholangitis

Early and differential diagnosis of intrahepatic cholangiocarcinoma (iCCA) and hepatocellular carcinoma (HCC) by noninvasive methods represents a current clinical challenge. The analysis of low-molecular-weight metabolites by new high-throughput techniques is a strategy for identifying biomarkers. Here, we have investigated whether serum metabolome can provide useful biomarkers in the diagnosis of iCCA and HCC and could discriminate iCCA from HCC. Because primary sclerosing cholangitis (PSC) is a risk factor for CCA, serum metabolic profiles of PSC and CCA have also been compared. The analysis of the levels of lipids and amino acids in the serum of patients with iCCA, HCC, and PSC and healthy individuals (n = 20/group) showed differential profiles. Several metabolites presented high diagnostic value for iCCA versus control, HCC versus control, and PSC versus control, with areas under the receiver operating characteristic curve (AUC) greater than those found in serum for the nonspecific tumor markers carbohydrate antigen 19-9 (CA 19-9) and alpha-fetoprotein (AFP), commonly used to help in the diagnosis of iCCA and HCC, respectively. The development of an algorithm combining glycine, aspartic acid, SM(42:3), and SM(43:2) permitted to accurately differentiate in the diagnosis of both types of tumors (biopsy-proven). The proposed model yielded 0.890 AUC, 75% sensitivity, and 90% specificity. Another algorithm by combination of PC(34:3) and histidine accurately permitted to differentiate PSC from iCCA, with an AUC of 0.990, 100% sensitivity, and 70% specificity. These results were validated in independent cohorts of 14-15 patients per group and compared with profiles found in patients with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Conclusion: Specific changes in serum concentrations of certain metabolites are useful to differentiate iCCA from HCC or PSC, and could help in the early diagnosis of these diseases