Chronic hepatitis B virus (HBV) infection is a major factor in hepatocellular carcinoma (HCC) pathogenesis by a mechanism not yet understood. Elucidating mechanisms of HBV‐mediated hepatocarcinogenesis is needed to gain insights into classification and treatment of HCC. In HBV replicating cells, including virus‐associated HCCs, suppressor of zeste 12 homolog (SUZ12), a core subunit of Polycomb repressive complex2 (PRC2), undergoes proteasomal degradation. This process requires the long noncoding RNA, Hox transcript antisense intergenic RNA (HOTAIR). Intriguingly, HOTAIR interacts with PRC2 and also binds RNA‐binding E3 ligases, serving as a ubiquitination scaffold. Herein, we identified the RNA helicase, DEAD box protein 5 (DDX5), as a regulator of SUZ12 stability and PRC2‐mediated gene repression, acting by regulating RNA‐protein complexes formed with HOTAIR. Specifically, knockdown of DDX5 and/or HOTAIR enabled reexpression of PRC2‐repressed genes epithelial cell adhesion molecule (EpCAM) and pluripotency genes. Also, knockdown of DDX5 enhanced transcription from the HBV minichromosome. The helicase activity of DDX5 stabilized SUZ12‐ and PRC2‐mediated gene silencing, by displacing the RNA‐binding E3 ligase, Mex‐3 RNA‐binding family member B (Mex3b), from HOTAIR. Conversely, ectopic expression of Mex3b ubiquitinated SUZ12, displaced DDX5 from HOTAIR, and induced SUZ12 down‐regulation. In G2 phase of cells expressing the HBV X protein (HBx), SUZ12 preferentially associated with Mex3b, but not DDX5, resulting in de‐repression of PRC2 targets, including EpCAM and pluripotency genes. Significantly, liver tumors from HBx/c‐myc bitransgenic mice and chronically HBV‐infected patients exhibited a strong negative correlation between DDX5 messenger RNA levels, pluripotency gene expression, and liver tumor differentiation. Notably, chronically infected HBV patients with HCC expressing reduced DDX5 exhibited poor prognosis after tumor resection, identifying DDX5 as an important player in poor prognosis HCC. Conclusion: The RNA helicase DDX5, and E3 ligase Mex3b, are important cellular targets for the design of novel, epigenetic therapies to combat HBV infection and poor prognosis HBV‐associated liver cancer. (Hepatology 2016;64:1033‐1048)

G-quadruplexes (G4) are noncanonical secondary structures formed in guanine-rich DNA and RNA sequences. MYC, one of the most critical oncogenes, forms a DNA G4 in its proximal promoter region (MycG4) that functions as a transcriptional silencer. However, MycG4 is highly stable in vitro and its regulatory role would require active unfolding. Here we report that DDX5, one of the founding members of the DEAD-box RNA helicase family, is extremely proficient at unfolding MycG4-DNA. Our results show that DDX5 is a highly active G4-resolvase that does not require a single-stranded overhang and that ATP hydrolysis is not directly coupled to G4-unfolding of DDX5. The chromatin binding sites of DDX5 are G-rich sequences. In cancer cells, DDX5 is enriched at the MYC promoter and activates MYC transcription. The DDX5 interaction with the MYC promoter and DDX5-mediated MYC activation is inhibited by G4-interactive small molecules. Our results uncover a function of DDX5 in resolving DNA and RNA G4s and suggest a molecular target to suppress MYC for cancer intervention.

The mammalian DEAD-box RNA helicase DDX5, its paralog DDX17, and their orthologs in Saccharomyces cerevisiae and Drosophila melanogaster, namely Dbp2 and Rm62, define a subfamily of DEAD-box proteins. Members from this subfamily share highly conserved protein sequences and cellular functions. They are involved in multiple steps of RNA metabolism including mRNA processing, microRNA processing, ribosome biogenesis, RNA decay, and regulation of long non-coding RNA activities. The DDX5/Dbp2 subfamily is also implicated in transcription regulation, cellular signalling pathways, and energy metabolism. One emerging theme underlying the diverse cellular functions is that the DDX5/Dbp2 subfamily of DEAD-box helicases act as chaperones for complexes formed by RNA molecules and proteins (RNP) in vivo. This RNP chaperone activity governs the functions of various RNA species through their life time. Importantly, mammalian DDX5 and DDX17 are involved in cancer progression when overexpressed through alteration of transcription and signalling pathways, meaning that they are possible targets for cancer therapy.