BRUNOL5 as a Novel Oncogene Is Epigenetically Regulated by Stilbenoids in Primary Liver Cancer Cells

Abstract

Abstract Objectives We previously discovered that a novel gene, BRUNOL5, is hypomethylated at the promoter region and upregulated in patients with primary liver cancer. Since DNA hypomethylation was shown to underlie up-regulation of genes with oncogenic functions, BRUNOL5 could potentially act as an oncogene. Interestingly, certain dietary compounds such as polyphenols with a stilbenoid ring have been demonstrated by us and others to suppress hypomethylated cancer-driving genes. In the present study, we investigate BRUNOL5 oncogenic functions and the role of two stilbenoids, resveratrol (RSV) and pterostilbene (PTS), in BRUNOL5 transcriptional regulation. Methods Liver cancer cells, HepG2 and SkHep1, were treated with 15µM RSV or 10µM PTS for 9 days followed by the analysis of cell growth (trypan blue exclusion test), anchorage-independent growth (soft-agar assay), and invasiveness (Boyden chamber assay). The effect on BRUNOL5 promoter methylation and gene expression was assessed by pyrosequencing and QPCR, respectively. BRUNOL5 was then depleted in HepG2 cells using siRNA followed by RNA sequencing to establish gene expression profiles. Results BRUNOL5 was down-regulated by 95% in response to RSV and by 25–50% in response to PTS. This coincided with 10–15% hypermethylation of BRUNOL5 promoter. This effect on BRUNOL5 transcriptional regulation was associated with robust decrease in cell growth, anchorage independent growth and invasiveness. Interestingly, depletion of BRUNOL5 in HepG2 cells mimicked polyphenols’ anti-cancer effects. Further investigation by RNA sequencing in BRUNOL5-depleted cells established gene targets potentially regulated by BRUNOL5. We found 4,406 genes significantly differentially expressed in response to BRUNOL5 knockdown. The top downregulated genes included cancer-promoting genes such as FAIM2, AMOTL1, and MMP2; whereas tumor suppressor genes such as MT1G, CADH1, and ALDH1L1 were among genes with the highest upregulation. Conclusions Our findings demonstrate that BRUNOL5 is a novel target of stilbenoids and acts as an oncogene in liver cancer. RSV and PTS may exert their anti-cancer effects, at least partially, through BRUNOL5 downregulation. Funding Sources UBC VP Academic Award, CFI John. R. Evans Leaders Fund, and BC Knowledge Development Fund granted to BS