Role of Hydroxy acid oxidase 2 (Hao2) in hepatocellular carcinoma

Background: Hydroxy acid oxidases are flavin mononucleotide (FMN)-dependent peroxisomal enzymes. Although it is unclear whether 2-hydroxy acid oxidases contribute to a general mechanism of fatty acid α-oxidation, these enzymes are capable of oxidizing a broad range of 2-hydroxy acids, ranging from glycolate to long chain 2-hydroxy fatty acids, such as 2-hydroxypalmitate, to 2-keto acids; this reaction results in hydrogen peroxide (H2O2) formation at the expense of molecular oxygen. Aim: Since no data concerning Hao2, a member of this family, and cancer are available in the literature, we analyzed the expression of this enzyme in mouse, rat and human hepatocellular carcinoma (HCC). Results: Our microarray analysis, performed in the liver of rats subjected to the Resistant Hepatocyte (R-H) model, revealed that Hao2 was among the most down-regulated genes in HCCs developed, 14 months after initiation with a single dose of the hepatocarcinogen Diethylnitrosamine (DENA). Next, we investigated whether Hao2 down-regulation is an early event during liver carcinogenesis; to this aim, we analyzed the expression of Hao2 by qRT-PCR in preneoplastic lesions and HCCs generated 10 weeks, and 14 months after initiation, respectively. Interestingly, qRT-PCR showed down-regulation of Hao2 already in rat early preneoplastic lesions, especially in those positive for the putative progenitor cell marker KRT-19, considered to be the precursor of HCC in this model of hepatocarcinogenesis. Western blot analysis showed Hao2 down-regulation also at protein level. To determine whether this down-regulation is a general phenomenon in liver tumorigenesis or is specific only for rat liver, we analyze the expression of Hao2 in a chemically-induced mouse model of hepatocarcinogenesis, consisting of a single injection of DENA followed by treatment with TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)] benzene), a ligand of the nuclear receptor CAR (Constitutive androstane receptor), that causes massive hepatomegaly and then HCC. Notably, similar to what found in rat HCC, Hao2 was strongly down-regulated also in TCP-induced mouse HCC. Finally, we investigated the expression levels of Hao2 in two distinct series of human HCCs. Interestingly, we found a strong down-regulation of Hao2 gene in human HCCs when compared to both normal and cirrhotic peri-tumoral liver. Furthermore, the levels of Hao2 were inversely correlated with time of recurrence, overall survival and occurrence of metastases. Conclusions: These results describe, for the first time, that Hao2 deregulation is severely impaired in HCCs generated in three different species and by different etiological agents. They also demonstrate that down-regulation of Hao2 is a very early event in the development of HCC, and may represent a useful diagnostic tool and a marker of poor prognosis

High frequency of β-catenin mutations in mouse hepatocellular carcinomas induced by a nongenotoxic constitutive androstane receptor agonist

Activation of Wnt/β-catenin signaling is frequent in human and rodent hepatocarcinogenesis. Although in mice the tumor-promoting activity of agonists of constitutive androstane receptor (CAR) occurs by selection of carcinogen-initiated cells harboring β-catenin mutations, the molecular alterations leading to hepatocellular carcinoma (HCC) development by the CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCP) in the absence of genotoxic injury are unknown. Here, we show that CAR activation per se induced HCC in mice and that 91% of them carried β-catenin point mutations or large in-frame deletions/exon skipping targeting Ctnnb1 exon 3. Point mutations in HCCs induced by TCP alone displayed different nucleotide substitutions compared with those found in HCCs from mice pretreated with diethylnitrosamine. Moreover, unlike those occurring in HCCs from diethylnitrosamine + TCP mice, they did not result in increased expression of β-catenin target genes, such as Glul, Lgr5, Rgn, Lect2, Tbx3, Axin2, and Ccnd1, or nuclear translocation of β-catenin compared with the control liver. Remarkably, in the nontumoral liver tissue, chronic CAR activation led to down-regulation of these genes and to a partial loss of glutamine synthetase–positive hepatocytes. These results show that, although chronic CAR activation per se induces HCCs carrying β-catenin mutations, it concurrently down-regulates the Wnt/β-catenin pathway in nontumoral liver. They also indicate that the relationship between CAR and β-catenin may be profoundly different between normal and neoplastic hepatocytes

The thyromimetic KB2115 (Eprotirome) induces rat hepatocyte proliferation

Although the hepatomitogenic activity of T3 is well established, the wide range of harmful effects exerted by this hormone precludes its use in regenerative therapy. The aim of this study was to investigate whether an agonist of TR, KB2115 (Eprotirome) could exert a mitogenic effect in the liver, without most of the adverse T3/TR-dependent side effects. F-344 rats treated with KB2115 for one week displayed a massive increase in bromodeoxyuridine incorporation (from 20 to 40% vs. 5% of controls), which was associated with increased mitotic activity in the absence of significant signs of liver toxicity. Noteworthy, while cardiac hypertrophy typical of T3 was not observed, beneficial effects, such as lowering blood cholesterol levels, were associated to KB2115 administration. Following a single dose of KB2115, hepatocyte proliferation was evident as early as 18 hours demonstrating its direct mitogenic effect. No increase of serum transaminase levels or apoptosis was observed prior to- or concomitantly with S phase. While KB2115-induced mitogenesis was not associated to enhanced expression of c-fos, c-jun and c-myc, cyclin D1 levels rapidly increased. In conclusion, KB2115 induces hepatocyte proliferation without overt toxicity. Hence this agent may be useful for regenerative therapies in liver transplantation or other surgical settings

Nrf2 Mutation/Activation Is Dispensable for the Development of Chemically Induced Mouse HCC

Background & Aims: Activation of the kelch-like ECH-associated protein 1 (Keap1)–nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway has been associated with metabolic reprogramming in many tumors, including hepatocellular carcinoma (HCC). However, the contribution of Nrf2 mutations in this process remains elusive. Here, we investigated the occurrence of Nrf2 mutations in distinct models of mouse hepatocarcinogenesis. Methods: HCCs were generated by experimental protocols consisting of the following: (1) a single dose of diethylnitrosamine (DEN), followed by repeated treatments with the nuclear-receptor agonist 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene; (2) repeated treatments with 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene alone; (3) a single dose of DEN followed by exposure to a choline-deficient L-amino acid–defined diet; and (4) a single dose of DEN with no further treatment. All of these protocols led to HCC development within 28–42 weeks. Activation of the Keap1-Nrf2 pathway was investigated by analyzing the presence of Nrf2 gene mutations, and the expression of Nrf2 target genes. Metabolic reprogramming was assessed by evaluating the expression of genes involved in glycolysis, the pentose phosphate pathway, and glutaminolysis. Results: No Nrf2 mutations were found in any of the models of hepatocarcinogenesis analyzed. Intriguingly, despite the described cooperation between β-catenin and the Nrf2 pathway, we found no evidence of Nrf2 activation in both early dysplastic nodules and HCCs, characterized by the presence of up to 80%–90% β-catenin mutations. No HCC metabolic reprogramming was observed either. Conclusions: These results show that, unlike rat hepatocarcinogenesis, Nrf2 mutations do not occur in 4 distinct models of chemically induced mouse HCC. Interestingly, in the same models, metabolic reprogramming also was minimal or absent, supporting the concept that Nrf2 activation is critical for the switch from oxidative to glycolytic metabolism

Increased Lipogenesis, Induced by AKT-mTORC1-RPS6 Signaling, Promotes Development of Human Hepatocellular Carcinoma

BACKGROUND & AIMS: De novo lipogenesis is believed to be involved in oncogenesis. We investigated the role of aberrant lipid biosynthesis in the pathogenesis of human hepatocellular carcinoma (HCC). METHODS: We evaluated expression of enzymes that regulate lipogenesis in human normal liver tissues and HCC and surrounding, nontumor, liver tissues from patients using real-time reverse transcription polymerase chain reaction, immunoblotting, immunohistochemistry, and biochemical assays. Effects of lipogenic enzymes on human HCC cell lines were evaluated using inhibitors and overexpression experiments. The lipogenic role of the proto-oncogene AKT was assessed in vitro and in vivo. RESULTS: In human liver samples, de novo lipogenesis was progressively induced from nontumorous liver tissue toward the HCC. Extent of aberrant lipogenesis correlated with clinical aggressiveness, activation of the AKT-mammalian target of rapamycin signaling pathway, and suppression of adenosine monophosphate-activated protein kinases. In HCC cell lines, the AKT-mammalian target of rapamycin complex 1-ribosomal protein S6 pathway promoted lipogenesis via transcriptional and post-transcriptional mechanisms that included inhibition of fatty acid synthase ubiquitination by the USP2a de-ubiquitinase and disruption of the SREBP1 and SREBP2 degradation complexes. Suppression of the genes adenosine triphosphate citrate lyase, acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, or sterol regulatory element-binding protein 1, which are involved in lipogenesis, reduced proliferation, and survival of HCC cell lines and AKT-dependent cell proliferation. Overexpression of an activated form of AKT in livers of mice induced lipogenesis and tumor development. CONCLUSIONS: De novo lipogenesis has pathogenic and prognostic significance for HCC. Inhibitors of lipogenic signaling, including those that inhibit the AKT pathway, might be useful as therapeutics for patients with liver cancer