AMPK-dependent epigenetic regulation of metabolism mediates the anti-cancer action of pterostilbene in hepatocellular carcinoma.

Scope: Disrupted metabolism, often implicated in hepatocellular carcinoma (HCC), is linked to aberrant epigenetic patterns. Dietary polyphenols, including pterostilbene (PTS), have been demonstrated to remodel epigenetic landscapes and restore metabolic homeostasis by regulating the activity of AMP-activated protein kinase (AMPK), a protein recently shown to orchestrate a diverse set of networks to epigenetically mediate transcription. We therefore explored the mechanistic involvement of AMPK in the epigenetic effects of PTS in HCC. Methods and Results: We incorporated PTS into a choline-deficient amino acid defined HCC-inducing diet (CDAA) in male Fisher-344 rats and found significant attenuation in HCC development compared to CDAA alone. Transcriptomics by RNA-sequencing revealed PTS-upregulated targets, that were enriched in key metabolic processes, including the folate (Aldh1l1), methionine (Bhmt) and sarcosine (Dmdgh) cycles. PTS-mediated gene upregulation was linked to lower levels of histone H3-methylation at lysine 27 (H3K27me3) at gene promoters. Mechanistic studies in HCC HepG2 cells revealed that AMPK inhibition abolished epigenetic gene activation in response to PTS, which was accompanied by diminished binding of H3K27me3-demethylase KDM6A at promoters of PTS-target genes. Conclusion: Our findings provide evidence for new disease vulnerabilities that arise from epigenetic/metabolic changes and constitute novel opportunities for preventative and therapeutic success in HCC. Overall design: Male F344 rats were housed two per cage in a temperature-controlled environment (24°C) with a 12-hour light/dark cycle and were provided ad libitum access to water and food. Upon arrival at the animal facility at 4 weeks of age, the rats underwent one week acclimation period during which they were maintained on a choline-sufficient L-amino acid-defined (CSAA) diet. Following acclimation, the rats were randomly assigned to one of four experimental groups: Group 1 (CSAA), Group 2 (choline-deficient L-amino acid-defined, CDAA), Group 3 (CDAA supplemented with RSV, CDAA+PTS), and Group 4 (CDAA supplemented with PTS, CDAA+PTS), with n=6 rats per group. As shown in the scheme in Figure 1A, Group 1 and 2 were on CSAA diet for first 3 weeks, whereas Group 3 and 4 were on CSAA diet supplemented with RSV or PTS, respectively, for first 3 weeks of the experiment. The CSAA and CDAA diets were obtained from Dyets Inc. (Bethlehem, PA, USA) and prepared in pellet form. RSV and PTS were obtained from Biotang Inc. (Albuquerque, NM, USA) and incorporated into the respective diets by Dyets Inc. at a dose of 1.2 and 1.34 g/kg of diet, respectively. The concentration of RSV and PTS in diets was measured using liquid chromatography-mass spectrometry (LC-MS) as described by Moser et al. [29], with modification of calibration with authentic standards for RSV and PTS. Body weight and food intake were recorded weekly throughout the 60-week experimental period. At the conclusion of the study, the rats were killed by carbon dioxide asphyxiation followed by cervical dislocation. Liver tissues, including tumors, nodules, or normal tissue in the absence of tumors, were collected for subsequent analyses. Tumor and nodule volumes were calculated using the formula: volume = (length × width^2)/2. The study design and all procedures followed strictly the animal use protocol #1112000342 approved by the Institutional Animal Care and Use Committee at the involved institutions. RNA-seq data on CDAA and CSAA is deposited under GSE199443.