Hepatitis B virus X protein overcomes the growth-inhibitory potential of retinoic acid by downregulating retinoic acid receptor- 2 expression via DNA methylation

Abstract

Aberrant promoter methylation of retinoic acid receptor-b 2 (RAR-b 2 ) is frequently detected in hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC); however, the mechanism of methylation and its biological significance are unknown. This study showed that HBx, the principal oncogene product of HBV, induced promoter hypermethylation of RAR-b 2 via upregulation of DNA methyltransferases 1 and 3a, resulting in downregulation of its expression in human HCC cells. In addition, HBx abolished the potential of retinoic acid (RA) to downregulate levels of G 1 -checkpoint regulators including p16, p21 and p27, resulting in activation of E2F1 in the presence of RA. As a consequence, HBx-expressing cells were less susceptible to RA-induced cell growth inhibition compared with control cells. These effects almost completely disappeared when levels of RAR-b 2 in HBx-expressing cells were restored by treatment with a universal DNA methylation inhibitor, 5-aza-29-deoxycytidine. As RAR-b 2 is a major executor of the anti-tumour potential of RA, its epigenetic downregulation by HBx is likely to be an important step during HBV-mediated tumorigenesis. INTRODUCTIONRetinoids are vitamin A derivatives and analogues that are involved in many important biological processes, including vision, morphogenesis, differentiation, growth, metabolism and cellular homeostasis (De Luca, 1991). In addition, they act as inhibitors of carcinogenesis by blocking the promotion of initiated or transformed cells by three mechanisms: induction of apoptosis, arrest of further growth of abnormal cells and induction of abnormal cells to differentiate back to normal (Hansen et al., 2000). These effects are executed mainly by regulating gene expression primarily through two classes of nuclear receptors, including retinoic acid (RA) receptors (RARs) and retinoid X receptors (RXRs) (Chambon, 1996). As either homodimers or heterodimers, RARs and RXRs modulate the expression of target genes by acting on the RA response element (RARE) located on the promoter regions of target genes.RAR-b contains four isoforms with different affinities to retinoids and different biological functions (Chambon, 1996). In particular, RAR-b 2 has attracted attention as a major executor of the anti-tumour potential of retinoids in a wide variety of cancers (Houle et al., 1993;Xu, 2007). Loss of RAR-b 2 is closely associated with retinoid resistance during tumorigenesis (Gebert et al., 1991;Hoffman et al., 1996;Hu et al., 1991;Xu et al., 1994). In addition, exogenous introduction of RAR-b 2 into cervical, breast and lung cancer cells enhances cell responsiveness to growth inhibition and induction of apoptosis by retinoids (Seewaldt et al., 1995; Si et al., 1996). Therefore, its loss of function is now considered to play a critical role in tumorigenesis. Aberrant promoter methylation of RAR-b 2 , which can lead to transcriptional downregulation of its expression, is frequently detected in human malignant tumours including hepatocellular carcinoma (HCC) (Lee et al., 2003;Yang et...

Hepatitis B virus X protein overcomes all-trans retinoic acid-induced cellular senescence by downregulating levels of p16 and p21 via DNA methylation

“…In addition, levels of DNMT1 and DNMT3a were upregulated by HBx in the presence and absence of ATRA (Fig. 4c, lanes 2 and 4), which is consistent with previous reports (Jung et al, 2010; Park et al, 2007). HBx also suppressed the potential of ATRA to inhibit DNA methylation of p21, although it was unable to induce its DNA methylation (Fig.…”

“…To investigate whether HBx suppressed the antigrowth potential of RA in human hepatoma cells, we first compared the growth rate of two HepG2 stable cell lines with or without HBx expression (Kwun & Jang, 2004) in the presence of ATRA. Consistent with our recent report (Jung et al, 2010), HBx-expressing cells were less susceptible to ATRA-induced cell growth inhibition compared with control cells (Fig. 1a).…”

Hepatitis B virus X gene and hepatocarcinogenesis

“…Jung et al [120] also demonstrated that HBx induced promoter hypermethylation of retinoic acid receptor-beta (2) (RAR-beta (2)) via the upregulation of DNA methyltransferases 1 and 3a, thereby downregulating the expression of RAR-beta(2) in human HCC cells. HBx also abolished the potential of retinoic acid (RA) to downregulate levels of G1-checkpoint regulators including p16, p21, and p27, resulting in the activation of E2F1 in the presence of RA [120].…”

“…HBx also abolished the potential of retinoic acid (RA) to downregulate levels of G1-checkpoint regulators including p16, p21, and p27, resulting in the activation of E2F1 in the presence of RA [120]. Therefore, HBx-expressing cells were less susceptible to RA-induced cell growth inhibition, and it has been suggested that the epigenetic downregulation of RAR-beta2 by HBx can be an important step during hepatocarcinogenesis [120].…”

Epigenetic Dysregulation in Virus-Associated Neoplasms

“…In HBV-associated HCC and HBx expressing HepG2 cells promoter methylation mediated silencing of the retinoic acid receptor β 2 (RARβ2) tumor suppressor gene was observed (Jung et al 2010 ), whereas in HBV-positive HCC ASPP1, ASPP2 , the genes encoding the apoptosis regulator ankyrin-repeat-containing, SH3-domain-containing, proline-rich-regioncontaining proteins 1 and 2, were downregulated by promoter methylation (Zhao et al 2010 ). Zhao et al suggested that ASPP methylation was dependent on HBx expression and appeared to be an early event in tumor progression (Zhao et al 2010 ).…”