The liver plays a central role in cholesterol homeostasis. It exclusively receives and metabolizes oxysterols, which are important metabolites of cholesterol and are more cytotoxic than free cholesterol, from all extrahepatic tissues. Hepatocellular carcinomas (HCCs) impair certain liver functions and cause pathological alterations in many processes including cholesterol metabolism. However, the link between an altered cholesterol metabolism and HCC development is unclear. Human ACAT2 is abundantly expressed in intestine and fetal liver. Our previous studies have shown that ACAT2 is induced in certain HCC tissues. Here, by investigating tissue samples from HCC patients and HCC cell lines, we report that a specific cholesterol metabolic pathway, involving induction of ACAT2 and esterification of excess oxysterols for secretion to avoid cytotoxicity, is established in a subset of HCCs for tumor growth. Inhibiting ACAT2 leads to the intracellular accumulation of unesterified oxysterols and suppresses the growth of both HCC cell lines and their xenograft tumors. Further mechanistic studies reveal that HCC-linked promoter hypomethylation is essential for the induction of ACAT2 gene expression. We postulate that specifically blocking this HCC-established cholesterol metabolic pathway may have potential therapeutic applications for HCC patients.

We have previously reported that human ACAT1 gene produces a chimeric mRNA through an interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG1397–1399 and GGC1274–1276 respectively as translation initiation codons to produce the normal 50-kD ACAT1 and a novel enzymatically active 56-kD isoform, which is authentically present in human cells including human monocyte-derived macrophages. In this work, we report that RNA secondary structures located in the vicinity of the GGC1274–1276 codon are required for producing the 56-kD isoform. The effects of the three predicted stem-loops (nt 1255–1268, 1286–1342 and 1355–1384) were tested individually by transfecting expression plasmids, which contain the wild-type, deleted or mutant stem-loop sequences linked with the partial ACAT1 AUG-open reading frame (ORF) or with the ORFs of other genes, into cells. The expression patterns were monitored by Western blot analyses. We found that the upstream stem-loop1255–1268 from chromosome 7 and downstream stem-loop1286–1342 from chromosome 1 were needed for production of the 56-kD isoform, whereas the last stem-loop1355–1384 from chromosome 1 was dispensable. The results of experiments using both the monocistronic and bicistronic vectors with a stable hairpin showed that the translation initiation from the GGC1274–1276 codon was mediated by internal ribosome entry site (IRES). Further experiments revealed that the translation initiation from the GGC1274–1276 codon required the upstream RNA secondary structure with AU-constitution and the downstream one with GC-richness. This mechanistic work further supports the biological significance that the chimeric human ACAT1 mRNA is expressed from two different chromosomes.