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.

Dual Targeting of G9a and DNA Methyltransferase-1 for the Treatment of Experimental Cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. APPROACH AND RESULTS: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. CONCLUSIONS: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.

In Vivo Tissue Lipid Uptake in Antisense Oligonucleotide (ASO)-Treated Mice.

The prevalence of obesity has increased to pandemic levels over the past years. Associated comorbidities linked with the accumulation of lipids in different tissues and blood are responsible for the high mortality in these patients. The increased dietary lipid uptake contributes to these metabolic diseases. Identifying which pathways might be dysregulated in these patients will contribute to find new therapeutic targets. Thus, here, a protocol to follow up the distribution of dietary lipids in blood and tissues is provided. For this, radiolabeled triglyceride in olive oil is administered by oral gavage. To ascertain more precisely the capacity of each tissue for fatty acid uptake, not considering the intestinal barrier, the intravenous (IV) administration of radiolabeled lipids is also described.

Methionine adenosyltransferase 1a antisense oligonucleotides activate the liver-brown adipose tissue axis preventing obesity and associated hepatosteatosis.

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.