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.