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

The E2F2 transcription factor sustains hepatic glycerophospholipid homeostasis in mice.

Increasing evidence links metabolic signals to cell proliferation, but the molecular wiring that connects the two core machineries remains largely unknown. E2Fs are master regulators of cellular proliferation. We have recently shown that E2F2 activity facilitates the completion of liver regeneration after partial hepatectomy (PH) by regulating the expression of genes required for S-phase entry. Our study also revealed that E2F2 determines the duration of hepatectomy-induced hepatic steatosis. A transcriptomic analysis of normal adult liver identified "lipid metabolism regulation" as a major E2F2 functional target, suggesting that E2F2 has a role in lipid homeostasis. Here we use wild-type (E2F2+/+) and E2F2 deficient (E2F2-/-) mice to investigate the in vivo role of E2F2 in the composition of liver lipids and fatty acids in two metabolically different contexts: quiescence and 48-h post-PH, when cellular proliferation and anabolic demands are maximal. We show that liver regeneration is accompanied by large triglyceride and protein increases without changes in total phospholipids both in E2F2+/+ and E2F2-/- mice. Remarkably, we found that the phenotype of quiescent liver tissue from E2F2-/- mice resembles the phenotype of proliferating E2F2+/+ liver tissue, characterized by a decreased phosphatidylcholine to phosphatidylethanolamine ratio and a reprogramming of genes involved in generation of choline and ethanolamine derivatives. The diversity of fatty acids in total lipid, triglycerides and phospholipids was essentially preserved on E2F2 loss both in proliferating and non-proliferating liver tissue, although notable exceptions in inflammation-related fatty acids of defined phospholipid classes were detected. Overall, our results indicate that E2F2 activity sustains the hepatic homeostasis of major membrane glycerolipid components while it is dispensable for storage glycerolipid balance

The E2F2 Transcription Factor Sustains Hepatic Glycerophospholipid Homeostasis in Mice

Increasing evidence links metabolic signals to cell proliferation, but the molecular wiring that connects the two core machineries remains largely unknown. E2Fs are master regulators of cellular proliferation. We have recently shown that E2F2 activity facilitates the completion of liver regeneration after partial hepatectomy (PH) by regulating the expression of genes required for S-phase entry. Our study also revealed that E2F2 determines the duration of hepatectomy-induced hepatic steatosis. A transcriptomic analysis of normal adult liver identified "lipid metabolism regulation" as a major E2F2 functional target, suggesting that E2F2 has a role in lipid homeostasis. Here we use wild-type (E2F2+/+) and E2F2 deficient (E2F2-/-) mice to investigate the in vivo role of E2F2 in the composition of liver lipids and fatty acids in two metabolically different contexts: quiescence and 48-h post-PH, when cellular proliferation and anabolic demands are maximal. We show that liver regeneration is accompanied by large triglyceride and protein increases without changes in total phospholipids both in E2F2+/+ and E2F2-/- mice. Remarkably, we found that the phenotype of quiescent liver tissue from E2F2-/- mice resembles the phenotype of proliferating E2F2+/+ liver tissue, characterized by a decreased phosphatidylcholine to phosphatidylethanolamine ratio and a reprogramming of genes involved in generation of choline and ethanolamine derivatives. The diversity of fatty acids in total lipid, triglycerides and phospholipids was essentially preserved on E2F2 loss both in proliferating and non-proliferating liver tissue, although notable exceptions in inflammation-related fatty acids of defined phospholipid classes were detected. Overall, our results indicate that E2F2 activity sustains the hepatic homeostasis of major membrane glycerolipid components while it is dispensable for storage glycerolipid balance.Fil: Maldonado, Eduardo N.. Universidad del Pais Vasco; EspañaFil: Delgado, Igotz. University Of The Basque Country (upv/ehu). Department Of Chemical And Environmental Engineering, Polytechnic School; EspañaFil: Furland, Natalia Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET Bahía Blanca. Instituto de Investigaciones Bioquímicas Bahía Blanca (i); ArgentinaFil: Buqué, Xabier. Universidad del Pais Vasco; EspañaFil: Iglesias, Ainhoa. Universidad del Pais Vasco; EspañaFil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET Bahía Blanca. Instituto de Investigaciones Bioquímicas Bahía Blanca (i); ArgentinaFil: Zubiaga, Ana. Universidad del Pais Vasco; EspañaFil: Fresnedo, Olatz. Universidad del Pais Vasco; EspañaFil: Ocho, Begoña. Universidad del Pais Vasco; Españ

The E2F2 Transcription Factor Sustains Hepatic Glycerophospholipid Homeostasis in Mice

Increasing evidence links metabolic signals to cell proliferation, but the molecular wiring that connects the two core machineries remains largely unknown. E2Fs are master regulators of cellular proliferation. We have recently shown that E2F2 activity facilitates the completion of liver regeneration after partial hepatectomy (PH) by regulating the expression of genes required for S-phase entry. Our study also revealed that E2F2 determines the duration of hepatectomy-induced hepatic steatosis. A transcriptomic analysis of normal adult liver identified "lipid metabolism regulation" as a major E2F2 functional target, suggesting that E2F2 has a role in lipid homeostasis. Here we use wild-type (E2F2(+/+)) and E2F2 deficient (E2F2(-/-)) mice to investigate the in vivo role of E2F2 in the composition of liver lipids and fatty acids in two metabolically different contexts: quiescence and 48-h post-PH, when cellular proliferation and anabolic demands are maximal. We show that liver regeneration is accompanied by large triglyceride and protein increases without changes in total phospholipids both in E2F2(+/+) and E2F2(-/-) mice. Remarkably, we found that the phenotype of quiescent liver tissue from E2F2(-/-) mice resembles the phenotype of proliferating E2F2(+/+) liver tissue, characterized by a decreased phosphatidylcholine to phosphatidylethanolamine ratio and a reprogramming of genes involved in generation of choline and ethanolamine derivatives. The diversity of fatty acids in total lipid, triglycerides and phospholipids was essentially preserved on E2F2 loss both in proliferating and non-proliferating liver tissue, although notable exceptions in inflammation-related fatty acids of defined phospholipid classes were detected. Overall, our results indicate that E2F2 activity sustains the hepatic homeostasis of major membrane glycerolipid components while it is dispensable for storage glycerolipid balance.The study design, data collection and analysis and decision to publish were funded by the Department of Education, Universities and Research of the Basque Goverment (grants IT336/10 to BO and OF and IT634/2013 to AZ) and the Department of Industry of the Basque Government(grants PE13UN139 to BO and IE12-331 to AZ). The preparation of the manuscript and the publication costs were funded by the University of the Basque Country (grant UFI11/20 to AZ, AI, BO, OF and XB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Scheme representing E2F2 as a regulator of the major metabolic pathways that generate phosphatidylcholine and phosphatidylethanolamine in the mammalian liver.

<p>The name in bold between parentheses corresponds to the gene coding for the dominant isoform in adult liver. Constitutive deletion of E2F2 gene promotes increased phosphatidylethanolamine (PE) relative content, a low phosphatidylcholine (PC):PE ratio, increased expression of genes required for PE formation and deregulated expression of some genes involved both in the Kennedy pathway for PC synthesis and the deacylation:reacylation arm of PC turnover. Such permanent decrease in the PC:PE ratio may impact phospholipid-related biological responses. CTP, cytidinetriphosphate; CDP, cytidinediphosphate; LPC, lysophosphatidylcholine, PA, phosphatidic acid; TAG, triacylglycerol. The Lands (deacylation-reacylation) cycle consists of phospholipases A₂ (or A₁) and acyl:CoA lysophospholipid acyltransferases. We only show here the deacylation/reacylation of phosphatidylcholine. Transcript upregulations are shown in red and downregulations in green.</p

E2F2 gene deletion does not promote changes in total triacylglycerol, phospholipid and protein content in quiescent liver or at 48-h after 70% partial hepatectomy.

<p>Partial hepatectomy was performed on E2F2^+/+ (wild-type, WT) and E2F2^-/- mice, and were sacrificed 48 hours later. Quiescent (0-h, solid bars) and regenerating (48-h, open bars) livers were harvested and homogenized and the triacylglycerol (TAG), phospholipid (PL) and protein content of homogenates were quantified as described in Materials and Methods. Data are shown as means ± SD and are representative of three independent experiments using cohorts of 6 WT and 6 E2F2^-/- mice each. Statistical differences between the regenerating and the quiescent tissue of a genotype are denoted by * <i>P</i>≤0.05, *** <i>P</i>≤0.001 (unpaired, 2-tailed Student's <i>t</i>-test).</p

Fatty acid composition of total lipids in wild-type (WT) and E2F2^-/- mice liver before (0-h) and 48 hours (48-h) post-partial hepatectomy.

<p>The results are expressed as a percentage of the fatty acids measured in the corresponding total lipid sample and are the average ± SD of two independent experiments using cohorts of 6 E2F2^+/+ (wild-type, WT) and 6 E2F2^-/- mice each. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Student's <i>t</i>-test for paired data: * <i>P</i>≤0.05 for comparisons of 48-h <i>vs</i> 0-h within the same genotype; # <i>P</i>≤.05 for comparisons of the same condition between E2F2^-/- and WT groups.</p><p>Fatty acid composition of total lipids in wild-type (WT) and E2F2^-/- mice liver before (0-h) and 48 hours (48-h) post-partial hepatectomy.</p

E2F2 gene deletion promotes changes in transcript expression of selected genes involved in phospholipid and fatty acid metabolism.

<p>Partial hepatectomy was performed on E2F2^+/+ (wild-type, WT) and E2F2^-/- mice, and were sacrificed 48 hours later. Quiescent (0-h, solid bars) and regenerating (48-h, open bars) livers were harvested and total RNA from each mouse was examined in triplicate by quantitative real-time PCR for the following genes: Pemt, phosphatidylethanolamine N-methyltransferase; Chpt1, choline phosphotransferase 1; Cept1, choline/ethanolamine phosphotransferase 1; Pebp1, phosphatidylethanolamine binding protein 1; Sptlc2, serine palmitoyltransferase, long chain base subunit 2; Fads2, fatty acid desaturase 2; Scd1, stearoyl-CoA desaturase 1. Relative transcript abundance was calculated for each mouse using a normalization factor computed by Genorm software for pyruvate carboxylase, transferrin receptor and vascular endothelial zinc finger containing factor 1 mRNAs. Values are referred to 0-h WT mice samples (100%). Results are presented as means ± SD (n = 8 mice per group), except for Scd1 transcript levels, which were estimated in 3 pools of eight mice each from each genotype (n = 3). Statistical differences between regenerating and quiescent liver of a genotype are denoted by ** <i>P</i>≤0.01, *** <i>P</i>≤0.001, and between the same tissue condition of different genotypes are denoted by # <i>P</i>≤0.05, ## <i>P</i>≤0.01, ### <i>P</i>≤0.001 (unpaired, 2-tailed Student's <i>t</i>-test).</p