Abstract
In one-third of hepatocellular carcinomas (HCCs), cancer cells have mutations that activate β-catenin pathway. These cells have alterations in glutamine, bile, and lipid metabolism. We investigated whether positron emission tomography (PET) imaging allows identification of altered metabolic pathways that might be targeted therapeutically. We studied mice with activation of β-catenin in liver (Apcko-liv mice) and male C57Bl/6 mice given injections of diethylnitrosamine, which each develop HCCs. Mice were fed a conventional or a methionine- and choline-deficient diet or a choline-deficient (CD) diet. Choline uptake and metabolism in HCCs were analyzed by micro-PET imaging of mice; livers were collected and analyzed by histologic, metabolomic, messenger RNA quantification, and RNA-sequencing analyses. Fifty-two patients with HCC underwent PET imaging with 18F-fluorodeoxyglucose, followed by 18F-fluorocholine tracer metabolites. Human HCC specimens were analyzed by immunohistochemistry, quantitative polymerase chain reaction, and DNA sequencing. We used hepatocytes and mouse tumor explants for studies of incorporation of radiolabeled choline into phospholipids and its contribution to DNA methylation. We analyzed HCC progression in mice fed a CD diet. Livers and tumors from Apcko-liv mice had increased uptake of dietary choline, which contributes to phospholipid formation and DNA methylation in hepatocytes. In patients and in mice, HCCs with activated β-catenin were positive in 18F-fluorocholine PET, but not 18F-fluorodeoxyglucose PET, and they overexpressed the choline transporter organic cation transporter 3. The HCC cells from Apcko-liv mice incorporated radiolabeled methyl groups of choline into phospholipids and DNA. In Apcko-livmice, the methionine- and choline-deficient diet reduced proliferation and DNA hypermethylation of hepatocytes and HCC cells, and the CD diet reduced long-term progression of tumors. In mice and humans, HCCs with mutations that activate β-catenin are characterized by increased uptake of a fluorocholine tracer, but not 18F-fluorodeoxyglucose, revealed by PET. The increased uptake of choline by HCCs promotes phospholipid formation, DNA hypermethylation, andhepatocyte proliferation. In mice, the CD diet reverses theseeffects and promotes regression of HCCs that overexpress β-catenin.
Highlights
BACKGROUND & AIMSIn one-third of hepatocellular carcinomas (HCCs), cancer cells have mutations that activate bcatenin pathway
Using messenger RNAsequencing data for the same samples as for the analysis of metabolites, we showed that these deregulations were associated with higher mRNA levels for Oct[1], Oct[2], Oct[3], and Ctl[2] choline transporters, the Cept[1] enzyme of the Kennedy pathway, the Sgms enzymes involved in sphingolipid synthesis, and the Mat[2], Dnmt[1], and Dnmt3a enzymes (Figure 1B, Supplementary Table 4 for a full overview)
A similar result was obtained in vitro for primary cultures of hepatocytes: Apcko hepatocytes took up more methyl-14C-choline than control hepatocytes (Figure 2C). To assess whether this increased choline uptake was found in mouse liver tumors, we developed 2 mouse models of HCC: one is linked to adenomatous polyposis coli (Apc) inactivation and b-catenin gain-of-function[12]; the other is independent of b-catenin status, and HCC is induced by DEN treatment.[26]
Summary
The effects of the choline-deficient diet were tested only in mice. In humans, reduced dietary intake of choline can cause liver injuries such as steatosis. PET imaging with FCH might be used to identify HCCs with activated beta catenin These tumors might be susceptible to agents that block this signaling pathway, or to limitations on dietary choline. The 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) and its hybrid version associating computed tomography (PET/CT) have the clinical utility to detect many cancers, but are limited in HCC by low sensitivity (60% overall).[3] Positive results on FDG-PET indicate a dependence of the tumor cells on glucose due to the well-documented Warburg effect, in which high rates of glycolysis drive lactate production, conferring metabolic and growth advantages on the tumor.[4] The inability of FDG-PET to detect all HCCs may reflect heterogeneity in their metabolic and/or genetic traits, and intratumoral metabolism has been found to be driven by the oncogenic alteration involved. We demonstrated the importance of this increased choline uptake for hepatocarcinogenesis
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