Sirt3 inhibits hepatocellular carcinoma cell growth through reducing Mdm2-mediated p53 degradation

Sirt3 is a member of the mammalian sirtuin family that is localized to mitochondria and plays a role in the control of the metabolic activity. Recently, Sirt3 has been reported to be associated with the deregulating metabolism of cancer cells. However, the role of Sirt3 in hepatocellular carcinoma (HCC) has never been studied. In this study, we found that Sirt3 protein expression was downregulated in human HCC tissue. We also showed that overexpression of Sirt3 using adenovirus inhibited HCC cell growth (two cell lines: HepG2 and HuH-7 cells) and induced apoptosis, which was evidenced by the increase of LDH leakage, enhancement of TUNEL-positive cells number and promotion of AIF translocation to nuclei. Sirt3 overexpression reduced the intracellular NAD(+) level, repressed the ERK1/2 signaling pathway, and activated the Akt and JNK signaling pathways. Furthermore, Sirt3 overexpression upregulated p53 protein level through downregulating Mdm2 and thereby slowing p53 degradation. Collectively, our data suggests that Sirt3 may play an important role in HCC development and progression and may be a promising therapeutic target for HCC.     

COX-2 mediates hepatitis B virus X protein abrogation of p53-induced apoptosis

The oncogenic hepatitis B virus X protein (HBx) and cyclooxygenase (COX)-2 are highly co-expressed in chronic hepatitis, cirrhosis and well-differentiated hepatocellular carcinoma (HCC). Although HBx is shown to activate COX-2, the functional consequences of this interaction in hepatocarcinogenesis remain unknown. Using an engineered hepatoma cell system in which the expression of wild-type p53 can be chemically modulated, we show here that COX-2 mediates HBx actions in opposing p53. Enforced expression of HBx sequestrates p53 in the cytoplasm and significantly abolishes p53-induced apoptosis. The anti-apoptotic Mcl-1 protein is suppressed by p53 but reactivated by HBx. The abrogation of apoptosis is completely reversed by specific COX-2 inhibition, suggesting that HBx blocks p53-induced apoptosis via activation of COX-2/PGE₂ pathway. We further show that COX-2 inhibition blocks HBx reactivation of Mcl-1, linking this protein to the anti-apoptotic function of COX-2. These results demonstrate that COX-2 is an important survival factor mediating the oncogenic actions of HBx. Over-expression of HBx and COX-2 may provide a selective clonal advantage for preneoplastic or neoplastic hepatocytes and contribute to the initiation and progression of HCC.     

Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation

Deficiency in autophagy, a lysosome-dependent cell degradation pathway, has been associated with a variety of diseases especially cancer. Recently, the activation of autophagy by hepatitis B virus X (HBx) protein, which is implicated in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), has been identified in hepatic cells. However, the underlying mechanism and the relevance of HBx-activated autophagy to the carcinogenesis caused by HBV remain elusive. Here, by transfection of HBV genomic DNA and HBx in hepatic and hepatoma cells, we showed that HBV- or HBx-induced autophagosome formation was accompanied by unchanged MTOR (mechanistic target of rapamycin) activity and decreased degradation of LC3 and SQSTM1/p62, the typical autophagic cargo proteins. Further functional and morphological analysis indicated that HBx dramatically impaired lysosomal acidification leading to a drop in lysosomal degradative capacity and the accumulation of immature lysosomes possibly through interaction with V-ATPase affecting its lysosome targeting. Moreover, clinical specimen test showed increased SQSTM1 and immature lysosomal hydrolase CTSD (cathepsin D) in human liver tissues with chronic HBV infection and HBV-associated liver cancer. These data suggest that a repressive effect of HBx on lysosomal function is responsible for the inhibition of autophagic degradation, and this may be critical to the development of HBV-associated HCC.     

Hepatitis B virus X protein modulates upregulation of DHX9 to promote viral DNA replication

Hepatitis B virus (HBV) infection is a major cause of acute and chronic liver diseases. During the HBV life cycle, HBV hijacks various host factors to assist viral replication. In this research, we find that the HBV regulatory protein X (HBx) can induce the upregulation of DExH‐box RNA helicase 9 (DHX9) expression by repressing proteasome‐dependent degradation mediated by MDM2. Furthermore, we demonstrate that DHX9 contributes to viral DNA replication in dependence on its helicase activity and nuclear localization. In addition, the promotion of viral DNA replication by DHX9 is dependent on its interaction with Nup98. Our findings reveal that HBx‐mediated DHX9 upregulation is essential for HBV DNA replication.     

p53 inhibits tumor cell invasion via the degradation of snail protein in hepatocellular carcinoma

The tumor suppressor protein p53 is a key regulator of cell cycle arrest and apoptosis. Snail protein regulates cancer-associated malignancies. However, the relationship between p53 and Snail proteins in hepatocellular carcinoma (HCC) has not been completely understood. To determine whether Snail and p53 contribute to hepatocarcinogenesis, we analyzed the expression of Snail proteins in p53-overexpressing HCC cells. We found that p53 wild-type (WT) induced the degradation of Snail protein via murine double minute 2-mediated ubiquitination, whereas p53 mutant did not induce Snail degradation. As we expected, only p53WT induced endogenous Snail protein degradation and inhibited tumor cell invasion. These findings contribute to a better understanding of the role of p53 mutation and Snail overexpression as a late event in hepatocarcinogenesis.

Structured summary

MINT-7718917: p53 (uniprotkb:P04637) physically interacts (MI:0915) with Snai1 (uniprotkb:O95863) by anti bait coimmunoprecipitation (MI:0006)MINT-7719877: Snai1 (uniprotkb:O95863) physically interacts (MI:0915) with ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7718928: Snai1 (uniprotkb:O95863) physically interacts (MI:0915) with p53 (uniprotkb:P04637) by anti tag coimmunoprecipitation (MI:0007)MINT-7718939: Snai1 (uniprotkb:O95863) physically interacts (MI:0915) with MDM2 (uniprotkb:Q00987) by anti tag coimmunoprecipitation (MI:0007)     

Hepatitis B virus X protein upregulates alpha-fetoprotein to promote hepatocellular carcinoma by targeting miR-1236 and miR-329

Hepatitis B virus (HBV) infection is the most common cause of hepatocellular carcinoma (HCC) worldwide, wherein the expression of alpha-fetoprotein (AFP) is reactivated to promote tumorgenesis. Hepatitis B virus X protein (HBx) protein encoded by the HBV virus X gene has been considered to be oncogenic and implicated in hepatocarcinogenesis. However, the relationship between HBx and abnormal AFP expression in HCC is yet to be fully understood. To explore the potential regulation of HBx on AFP re-expression in HCC, 97 HCC samples of different etiologies were analyzed, and extremely higher serum AFP levels were found in patients with HBsAg⁺. Analyses of HBV-related HCC specimens showed that the expression of AFP was negatively correlated with the levels of miR-1236 and miR-329. Further analyses indicated that HBx promotes the expression of AFP by orchestrating the levels of miR-1236 and miR-329 both in vitro and in vivo. Specifically, miR-1236 and miR-329 bind to the potential target sequences in AFP mRNA 3′-untranslated region to suppress its expression. HBx transfection resulted in the significant decrement of these microRNAs and increment of AFP expression. Moreover, AFP promotes the proliferation of hepatoma cells and attenuates the proapoptotic effect of chemotherapy agents. These findings revealed a novel regulatory mechanism of HBx on the abnormal AFP expression in HCC, which may provide a therapeutic approach for combating HBV-related HCC by targeting the regulation of AFP expression.     

Hepatitis B virus X protein activates the p38 mitogen-activated protein kinase pathway in dedifferentiated hepatocytes

Hepatitis B virus X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. Employing a cellular model linked to pX-mediated transformation, we investigated the role of the previously reported Stat3 activation by pX in hepatocyte transformation. Our model is composed of a differentiated hepatocyte (AML12) 3pX-1 cell line that undergoes pX-dependent transformation and a dedifferentiated hepatocyte (AML12) 4pX-1 cell line that does not exhibit transformation by pX. We report that pX-dependent Stat3 activation occurs only in non-pX-transforming 4pX-1 cells and conclude that Stat3 activation is not linked to pX-mediated transformation. Maximum Stat3 transactivation requires Ser727 phosphorylation, mediated by mitogenic pathway activation. Employing dominant negative mutants and inhibitors of mitogenic pathways, we demonstrate that maximum, pX-dependent Stat3 transactivation is inhibited by the p38 mitogen-activated protein kinase (MAPK)-specific inhibitor SB 203580. Using transient-transreporter and in vitro kinase assays, we demonstrate for the first time that pX activates the p38 MAPK pathway only in 4pX-1 cells. pX-mediated Stat3 and p38 MAPK activation is Ca(2+) and c-Src dependent, in agreement with the established cellular action of pX. Importantly, pX-dependent activation of p38 MAPK inactivates Cdc25C by phosphorylation of Ser216, thus initiating activation of the G(2)/M checkpoint, resulting in 4pX-1 cell growth retardation. Interestingly, pX expression in the less differentiated hepatocyte 4pX-1 cells activates signaling pathways known to be active in regenerating hepatocytes. These results suggest that pX expression in the infected liver effects distinct mitogenic pathway activation in less differentiated versus differentiated hepatocytes.     

Hepatitis B virus x protein induces epithelial-mesenchymal transition of hepatocellular carcinoma cells by regulating long non-coding RNA

Background

It has been widely accepted that hepatitis B virus X protein (HBx) plays an important role in hepatocellular carcinoma (HCC). This study aimed to explore the function of long non-coding RNAs (lncRNAs) in the epithelial-mesenchymal transition (EMT) induced by HBx.

Methods

The association between HBx and EMT markers was detected using immunohistochemistry in HCC tissues. The effect of HBx on HCC EMT was assessed through morphological analysis, transwell assay, metastatic in vivo study and detection of EMT markers. LncRNA microarray was used to screen the differently expressed lncRNAs. Small interfering RNA and Western blot were used to analyse the function and mechanism of the locked lncRNA.

Results

HBx was negatively correlated with the epithelial marker E-cadherin but positively correlated with the mesenchymal marker vimentin in HCC tissues. HBx induced the mesenchymal phenotype and improved the metastatic ability of HCC cells. Meanwhile, HBx down-regulated E-cadherin, whereas it up-regulated vimentin. In HCC cells, HBx altered the expression of 2002 lncRNAs by more than 2-fold. One of them was ZEB2-AS1. Inhibition of ZEB2-AS1 can compensate for the EMT phenotype and reverse the expression of EMT markers regulated by HBx. Additionally, HBx affected the Wnt signalling pathway.

Conclusions

HBx promotes HCC cell metastasis by inducing EMT, which is at least partly mediated by lncRNAs.

     

TRIAD1 inhibits MDM2-mediated p53 ubiquitination and degradation

Murine double minute (MDM2) is an E3 ligase that promotes ubiquitination and degradation of tumor suppressor protein 53 (p53). MDM2-mediated regulation of p53 has been investigated as a classical tumorigenesis pathway. Here, we describe TRIAD1 as a novel modulator of the p53-MDM2 axis that induces p53 activation by inhibiting its regulation by MDM2. Ablation of TRIAD1 attenuates p53 levels activity upon DNA damage, whereas ectopic expression of TRIAD1 promotes p53 stability by inhibiting MDM2-mediated ubiquitination/degradation. Moreover, TRIAD1 binds to the C-terminus of p53 to promote its dissociation from MDM2. These results implicate TRIAD1 as a novel regulatory factor of p53-MDM2.Structured summary of protein interactions:p53 physically interacts with Mdm2 and Triad1 by anti tag coimmunoprecipitation (View Interaction: 1, 2, 3)Mdm2physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Mdm2 by anti tag coimmunoprecipitation (View interaction)Triad1binds to p53 by pull down (View interaction)Mdm2physically interacts with p53 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)     

Expression of Jagged1 and its association with hepatitis B virus X protein in hepatocellular carcinoma

Jagged1 is one of the ligands of Notch signaling pathway, which controls cellular proliferation and differentiation, and also plays important roles in various malignant tumors. However, the expression of Jagged1 in hepatocellular carcinoma (HCC) has not been elucidated, nor whether it is associated with hepatitis B virus X protein (HBx). In this study, we found that Jagged1 was highly expressed in 79.2% (42/53) of HCC tissues compared with adjacent nontumor liver (P <0.05), and its expression was found to be closely related with HBx (rs=0.522, P <0.001) in HCC tissues. Our in vitro study also showed that alteration of HBx expression in HCC cell lines led to a consistent change of Jagged1. Moreover, Jagged1 was found to co-localize and directly interact with HBx in HCC tissues and HBx expressed HCC cell lines. Our results reveal that Jagged1, which is regulated by HBx, may contribute to the development of HCC.