Qing Deng

Exome sequencing of hepatitis B virus-associated hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and shows a propensity to metastasize and infiltrate adjacent and more distant tissues. HCC is associated with multiple risk factors, including hepatitis B virus (HBV) infection, which is especially prevalent in China. Here, we used exome sequencing to identify somatic mutations in ten HBV-positive individuals with HCC with portal vein tumor thromboses (PVTTs), intrahepatic metastases. Both C:G>A:T and T:A>A:T transversions were frequently found among the 331 non-silent mutations. Notably, ARID1A, which encodes a component of the SWI/SNF chromatin remodeling complex, was mutated in 14 of 110 (13%) HBV-associated HCC specimens. We used RNA interference to assess the roles of 91 of the confirmed mutated genes in cellular survival. The results suggest that seven of these genes, including VCAM1 and CDK14, may confer growth and infiltration capacity to HCC cells. This study provides a view of the landscape of somatic mutations that may be implicated in advanced HCC.

Epigenetic modification induced by hepatitis B virus X protein via interaction with de novo DNA methyltransferase DNMT3A

BACKGROUND/AIMS The hepatitis B virus X protein (HBx) has been implicated as a potential trigger of the epigenetic deregulation of some genes, but the underlying mechanisms remain unknown. The aim of this study was to identify underlying mechanisms involved in HBx-mediated epigenetic modification. METHODS Interactions between HBx and DNA methyltransferase (DNMT) or histone deacetylase-1 (HDAC1) were assessed by co-immunoprecipitation. DNA methylation of gene promoters was detected by bisulfite sequencing, and HBx-mediated protein binding to gene regulatory elements was evaluated by chromatin immunoprecipitation. Target gene transcriptional activity was measured by real-time polymerase chain reaction. RESULTS HBx can interact directly with DNMT3A and HDAC1. HBx recruited DNMT3A to the regulatory promoters of interleukin-4 receptor and metallothionein-1F and subsequently silenced their transcription via de novo DNA methylation. By contrast, the transcription of CDH6 and IGFBP3 was triggered by HBx through the deprivation of DNMT3A from their promoters. Transcriptional levels of target genes in hepatocellular carcinoma (HCC) specimens were strongly correlated with the occurrence of HBx. CONCLUSIONS The interaction of HBx and DNMT3A facilitates cellular epigenetic modification (via regional hypermethylation or hypomethylation) at distinct genomic loci, providing an alternative mechanism within HBx-mediated transcriptional regulation, and a profound understanding of hepatitis and HCC pathogenesis.

E2F8 Contributes to Human Hepatocellular Carcinoma via Regulating Cell Proliferation

The E2F family member of transcription factors includes the atypical member E2F8, which has been little studied in cancer. We report that E2F8 is strongly upregulated in human hepatocellular carcinoma (HCC), where it was evidenced to contribute to oncogenesis and progression. Ectopic overexpression of E2F8 promoted cell proliferation, colony formation, and tumorigenicity, whereas E2F8 knockdown inhibited these phenotypes, as documented in Huh-7, Focus, Hep3B, and YY-8103 HCC cell lines. Mechanistic analyses indicated that E2F8 could bind to regulatory elements of cyclin D1, regulating its transcription and promoting accumulation of S-phase cells. Together, our findings suggest that E2F8 contributes to the oncogenic potential of HCC and may constitute a potential therapeutic target in this disease.

SRPK1 contributes to malignancy of hepatocellular carcinoma through a possible mechanism involving PI3K/Akt

Protein kinases are important regulators in biologic processes. Aberrant expression of protein kinases often causes diseases including cancer. In the present study, we found that the serine-arginine protein kinase 1 (SRPK1) might be involved in hepatocellular carcinoma (HCC) proliferation from a kinome screen using a loss-of-function approach. In clinical samples, SRPK1 was frequently up-regulated in HCCs as compared with adjacent non-tumor tissues at both mRNA and protein levels. Functional studies indicated that overexpression of wild-type SRPK1 promoted HCC cell proliferation, while forced expression of the kinase-dead mutant of SRPK1 or RNA interference against SRPK1 suppressed cell growth and malignancy as measured in soft agar assay. The kinase-dead mutant of SRPK1 also inhibited subcutaneous xenografts’ growth of HCC cells in nude mice. Furthermore, western bolt analysis showed overexpression of wild-type SRPK1 enhanced Akt phosphorylation and knockdown of SRPK1 by RNA interference attenuated Akt phosphorylation induced by epidermal growth factor. Meanwhile, overexpression of wild-type SRPK1 also induced a concurrent increase in the total tyrosine phosphorylation of phosphotidylinositol-3 kinase p110α subunit, indicating a functional link between SRPK1 and PI3K/Akt signaling. Our findings suggest that SRPK1 plays an oncogenic role and could be a potential therapeutic target in HCC.

RNA interference against cancer/testis genes identifies dual specificity phosphatase 21 as a potential therapeutic target in human hepatocellular carcinoma

Cancer/testis (CT) antigens have been considered therapeutic targets for treating cancers. However, a central question is whether their expression contributes to tumorigenesis or if they are functionally irrelevant by‐products derived from the process of cellular transformation. In any case, these CT antigens are essential for cancer cell survival and may serve as potential therapeutic targets. Recently, the cell‐based RNA interference (RNAi) screen has proven to be a powerful approach for identifying potential therapeutic targets. In this study we sought to identify new CT antigens as potential therapeutic targets for human hepatocellular carcinoma (HCC), and 179 potential CT genes on the X chromosome were screened through a bioinformatics analysis of gene expression profiles. Then an RNAi screen against these potential CT genes identified nine that were required for sustaining the survival of Focus and PLC/PRF/5 cells. Among the nine genes, the physiologically testis‐restricted dual specificity phosphatase 21 (DUSP21) encoding a dual specificity phosphatase was up‐regulated in 39 (33%) of 118 human HCC specimens. Ectopic DUSP21 had no obvious impact on proliferation and colony formation in HCC cells. However, DUSP21 silencing significantly suppressed cell proliferation, colony formation, and in vivo tumorigenicity in HCC cells. The administration of adenovirus‐mediated RNAi and an atelocollagen/siRNA mixture against endogenous DUSP21 significantly suppressed xenograft HCC tumors in mice. Further investigations showed that DUSP21 knockdown led to arrest of the cell cycle in G1 phase, cell senescence, and expression changes of some factors with functions in the cell cycle and/or senescence. Furthermore, the antiproliferative role of DUSP21 knockdown is through activation of p38 mitogen‐activated protein kinase in HCC. Conclusion: DUSP21 plays an important role in sustaining HCC cell proliferation and may thus act as a potential therapeutic target in HCC treatment. (Hepatology 2014;59:518–530)

SAMD9L Inactivation Promotes Cell Proliferation via Facilitating G1-S Transition in Hepatitis B Virus-associated Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a highly malignant cancer with poor prognosis, and driver genes harboring genetic lesions and/or expression dysregulation contribute to hepatocarcinogenesis. Sterile Alpha Motif Domain-containing 9-like (SAMD9L) was a novel identified mutated gene in our previous study on exome sequencing of hepatitis B virus (HBV)-associated HCC, but its expression and role in HCC remain unknown. Here, we demonstrated that SAMD9L was frequently inactivated by somatic mutations, and that its expression was deregulated in HCC patients with hepatitis B virus (HBV) infection. SAMD9L knockdown significantly promoted cell proliferation, colony formation of SK-hep-1, QGY-7701, BEL-7721 and MHCC-97H HCC cells. Furthermore, SK-hep-1 and MHCC-97H cells with stable SAMD9L knockdown exhibited enhanced tumorigenicity in athymic mice. Interestingly, SAMD9L silence facilitated G1-S transition of cell cycle progression and led to the elevated activity of Wnt/β-catenin pathway. Collectively, these findings highlight a novel tumor-suppressive role of SAMD9L inactivation by somatic mutation and decreased expression in human HBV-related HCC.