Wenya Huang

Hepatitis B virus pre-S mutants, endoplasmic reticulum stress and hepatocarcinogenesis

Although hepatitis B virus (HBV) has been documented to cause hepatocellular carcinoma (HCC), the exact role of HBV in the development of HCC remains enigmatic. Several hypotheses have been proposed to explain the potential mechanism, including insertional mutagenesis of HBV genomes and transcriptional activators of HBV gene products such as hepatitis B x protein (HBx) and truncated middle S mutants. In the past few years, we have identified two types of large HBV surface antigens (LHBs) with deletions at the pre‐S1 (ΔS1‐LHBs) and pre‐S2 (ΔS2‐LHBs) regions in ground glass hepatocytes. The pre‐S mutant LHBs are retained in the endoplasmic reticulum (ER) and escape from immune attack. The pre‐S mutants, particularly ΔS2‐LHBs, are increasingly prevalent in patients with hepatitis B e antigen (HBeAg)‐positive chronic HBV infection, ranging from 6% before the 3rd decade to 35% in the 6th decade. In HCC patients, the two pre‐S mutants were detected in 60% of HCC patients, in the serum and in HCC tissues. Pre‐S mutant LHBs can initiate ER stress to induce oxidative DNA damage and genomic instability. Furthermore, pre‐S mutant LHBs can upregulate cyclooxygenase‐2 and cyclin A to induce cell cycle progression and proliferation of hepatocytes. In transgenic mice, the pre‐S mutants can induce dysplasia of hepatocytes and development of HCC. In a nested control study, the presence of pre‐S mutants carried a high risk of developing HCC in HBV carriers. In summary, the findings we describe in this review suggest a potential role for HBV pre‐S mutants in HBV‐related hepatocarcinogenesis, providing a model of viral carcinogenesis associated with ER stress. (Cancer Sci 2006; 97: 683–688)

Hepatitis B virus pre‐S2 mutant upregulates cyclin A expression and induces nodular proliferation of hepatocytes

Naturally occurring mutants with a deletion in the pre‐S2 region of the large surface protein (ΔS2‐LHBs) are prevalent in serum and livers of patients with chronic hepatitis B virus (HBV) infection associated with cirrhosis. The ΔS2‐LHBs protein is retained in the endoplasmic reticulum (ER) and may induce ER stress. One interesting observation is the consistently clustered distribution of hepatocytes expressing ΔS2‐LHBs. In this study, complementary DNA microarray analysis identified cyclin A and several groups of genes as being significantly upregulated by ΔS2‐LHBs in the HuH‐7 cell line. This observation was confirmed in liver tissues. The induction of cyclin A expression may occur via the specific transactivator function of ΔS2‐LHBs independent of ER stress. In the presence of ΔS2‐LHBs, hepatocytes sustained cyclin A expression and cell cycle progression under ER stress and displayed increased BrdU incorporation with multinuclear formation. Furthermore, ΔS2‐LHBs could enhance anchorage‐independent cell growth in a nontransformed human hepatocyte line and induced nodular proliferation of hepatocytes in transgenic mice. In conclusion, these in vitro and in vivo data support a role for ΔS2‐LHBs in the hepatocyte hyperplasia and a likely role in the process of HBV‐related tumorigenesis. (HEPATOLOGY 2005.)

Enhanced expression of vascular endothelial growth factor‐A in ground glass hepatocytes and its implication in hepatitis B virus hepatocarcinogenesis

Ground glass hepatocytes (GGH) in chronic hepatitis B virus (HBV) infection harbor HBV pre‐S deletion mutants in endoplasmic reticulum (ER) and exhibit complex biologic features such as ER stress, DNA damage, and growth advantage. The presence of pre‐S mutants in serum has been shown to predict the development of hepatocellular carcinoma (HCC) in HBV carriers. GGHs hence represent a potentially preneoplastic lesion. Whether a specific growth factor is overexpressed and activated in GGHs remains to be clarified. In this study, growth factor(s) up‐regulated by pre‐S mutants was identified using a growth factor array in HuH‐7 cells. Immunohistochemistry, reverse‐transcriptase polymerase chain reaction, and Western blot analysis were performed to study the participation of these genes and their signal pathways in HuH‐7 cells and liver tissues. We demonstrate that vascular endothelial growth factor‐A (VEGF‐A) was up‐regulated by pre‐S mutants in HuH‐7 cells and further confirmed in GGHs by immunostaining. The VEGF‐A up‐regulation by pre‐S mutants could be suppressed by vomitoxin, an ER stress inhibitor. Furthermore, pre‐S mutants‐expressed HuH‐7 cells exhibited activation of Akt/mTOR (mammalian target of rapamycin) signaling and increased growth advantage, which could be inhibited by VEGF‐A neutralization. Consistent with this notion, enhanced expression of VEGF‐A and activation of Akt/mTOR signaling, comparable to the levels of paired HCC tissues, were also detected in HBV‐related nontumorous livers. Conclusion: The enhanced expression of VEGF‐A in GGHs provides potential mechanism to explain the progression from preneoplastic GGHs to HCC in chronic HBV infection. (HEPATOLOGY 2009;49:1962–1971.)

Ground glass hepatocytes contain pre-S mutants and represent preneoplastic lesions in chronic hepatitis B virus infection

The discovery of “ground glass” hepatocytes (GGH) that contain hepatitis B virus (HBV) surface antigens by Hadziyannis and Popper in 1973 represents a historical landmark in the pathology of chronic HBV infection. Different types of GGH have been correlated to the expression patterns of surface/core antigens and the stages of virus replication. The original two types (designated types I & II) of GGH were found to contain specific pre‐S mutants with deletions over either pre‐S1 or pre‐S2 regions, respectively. Type II GGH consistently harbor pre‐S2 deletion mutants, which can escape from immune attack and grow preferentially to form clusters. Both types of pre‐S mutants can induce endoplasmic reticulum (ER) stress and oxidative DNA damage. The pre‐S2 mutants, albeit inducing a weaker level of ER stress signals, could additionally initiate ER stress‐independent retinoblastoma/adenovirus E2 promoter binding factor/cyclin A signaling through their interaction with c‐Jun activation domain binding protein 1 to degrade p27, illustrating the growth advantage of type II GGH. The combined effects of genomic instability and the proliferation of hepatocytes harboring pre‐S mutants could potentially lead to hepatocarcinogenesis over the decades of chronic HBV infection. The presence of pre‐S mutants in sera was reported to carry a high risk of developing hepatocellular carcinoma (HCC). Furthermore, transgenic mice harboring pre‐S2 mutant plasmids have been shown to develop a dysplastic change of hepatocytes and HCC. Therefore, in addition to being a histological marker of chronic HBV infection, GGH, particularly type II GGH, may represent the preneoplastic lesions of HBV‐related HCC.

Hepatitis B virus Pre-S2 mutant surface antigen induces degradation of cyclin-dependent kinase inhibitor p27Kip1 through c-jun activation domain-binding protein 1

The hepatitis B virus (HBV) large surface antigen (LHBS) mutant with deletion at the pre-S2 region accumulates in endoplasmic reticulum (ER) and is associated with HBV-induced hepatocellular carcinogenesis. In this study, we found that the pre-S2 LHBS mutant directly interacts with the Jun activation domain–binding protein 1 (JAB1). Association of pre-S2 LHBS with JAB1 dissociated JAB1 from the JAB1/IRE1 complex in ER. The free (active) JAB1 then translocated into cell nuclei and rendered the Cdk inhibitor p27Kip1 to cytosolic proteasome for degradation. The pre-S2 LHBS mutant induced hyperphosphorylation of tumor suppressor retinoblastoma (RB) via cyclin-dependent kinase 2 (Cdk2), a downstream molecule regulated by p27Kip1. This effect is independent of the ER stress signaling pathway. The transgenic mice carrying the pre-S2 mutant LHBS gene also exhibited Cdk2 activation, p27Kip1 degradation, as well as RB hyperphosphorylation. The mouse hepatocytes exhibited morphologic abnormalities such as chromatin condensation, multinucleation, and dysplasia of hepatocytes. In summary, the pre-S2 LHBS mutant causes p27Kip1 degradation through direct interaction with JAB1. The pre-S2 mutant LHBS is suggested to be a potential oncoprotein for HBV-related hepatocellular carcinoma. (Mol Cancer Res 2007;5(10):1063–72)

Microarray profiling of gene expression patterns in bladder tumor cells treated with genistein.

Microarray technology was used to gain an insight into the molecular events of tumor cell growth inhibition mediated by the soy isoflavone genistein. For this, a susceptible bladder tumor line TCCSUP was treated with the inhibitory dose (50 microM) of genistein for various periods of time, followed by mRNA isolations, cDNA probe preparations, and hybridization individually to cDNA chips containing 884 sequence-verified known human genes. After analyzing the hybridization signals with a simple quantitative method developed by this study, we detected that egr-1, whose expression has been associated with proliferation and differentiation, was transiently induced and this expression pattern was later confirmed by RT-PCR. Thus, microarray technology is a reliable and powerful tool for profiling gene expression patterns in many biological systems related to cancer. We further detected many groups of genes with distinct expression profiles and most of them encode for proteins that regulate the signal transduction or the cell cycle pathways. These genes warrant further investigation as regards their roles in the susceptibility of the tumor cell line to the antitumor drug.

Aberrant cyclin A expression and centrosome overduplication induced by hepatitis B virus Pre-S2 mutants and its implication in hepatocarcinogenesis

Ground glass hepatocytes harboring hepatitis B virus (HBV) pre-S2 mutants have been recognized as pre-neoplastic lesions of hepatocellular carcinoma (HCC). The pre-S2 mutants accumulated in endoplasmic reticulum (ER) can induce ER stress, upregulate cyclin A and promote hepatocyte proliferation. Notably, cyclin A was aberrantly detected in the cytoplasm, instead of nucleus, of pre-S2 mutant-transgenic mice livers, thereby raising the potential role of cytoplasmic cyclin A in HBV hepatocarcinogenesis. In this study, we confirmed that cyclin A was detected in the cytoplasm in the majority of HBV-related HCC tissues. In vitro, the pre-S2 mutant-initiated ER stress could induce cytoplasmic cyclin A mediated via cleavage by the calcium-dependent protease μ-calpain, resulting in an N-terminal truncated product which was preferentially located in the cytoplasm. The aberrant cyclin A expression subsequently induced centrosome overduplication, and this effect was abolished by calpain-specific inhibitors or RNA interference targeting to cyclin A. Overall, our data indicate that HBV pre-S2 mutant may elicit aberrant cyclin A expression and centrosome overduplication through ER stress induction and thereby represent a potential mechanism for the chromosome instability in HBV hepatocarcinogenesis.

Novel feedback inhibition of surface antigen synthesis by mammalian target of rapamycin (mTOR) signal and its implication for hepatitis B virus tumorigenesis and therapy

Ground glass hepatocytes (GGHs) harboring hepatitis B virus (HBV) pre‐S mutants have been recognized as precursor lesions of hepatocellular carcinoma (HCC). Previously, we observed the activation of mammalian target of rapamycin (mTOR) in GGHs and HCCs, together with a decreased expression of HBV surface antigen (HBsAg) in HCC tissues. It is, therefore, hypothesized that the activation of mTOR during HBV tumorigenesis may potentially down‐regulate HBsAg expression. In this study, we verified an inverse relationship between the expression of HBsAg and phosphorylated mTOR (p‐mTOR) in 13 of 20 paired nontumorous liver and HCC tissues. In vitro, wild‐type or mutant pre‐S proteins could activate mTOR in the HuH‐7 cell line. Interestingly, the up‐regulated mTOR, in turn, suppressed HBsAg synthesis at the transcriptional level via the transcription factor, Yin Yang 1 (YY1), which bound to nucleotide 2812‐2816 of the pre‐S1 promoter. This inhibitory effect by the mTOR signal could be abolished by the knockdown of histone deacetylase 1 (HDAC1). Furthermore, YY1 was physically associated with HDAC1 in a manner dependent on mTOR activation. Collectively, pre‐S protein‐induced mTOR activation may recruit the YY1‐HDAC1 complex to feedback suppress transcription from the pre‐S1 promoter. Conclusion: The activation of mTOR signal in GGHs may feedback suppress HBsAg synthesis during HBV tumorigenesis and explain the observed decrease or absence of HBsAg in HCC tissues. Therapy using mTOR inhibitors for HCCs may potentially activate HBV replication in patients with chronic HBV infection. (HEPATOLOGY 2011 )

A clustered ground‐glass hepatocyte pattern represents a new prognostic marker for the recurrence of hepatocellular carcinoma after surgery

The recurrence of hepatocellular carcinoma (HCC) after hepatectomy is a serious event. It has been demonstrated that different ground‐glass hepatocyte (GGH) patterns harbor specific hepatitis B virus (HBV) pre‐S deletion mutants and represent preneoplastic lesions in chronic HBV infection. In the current study, the authors investigated whether a specific GGH pattern in nontumorous liver tissues was associated with the recurrence of HBV‐related HCC after surgery.

Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris.

We report the culture conditions for successful amino‐acid‐type selective (AATS) isotope labeling of protein expressed in Pichia pastoris (P. pastoris). Rhodostomin (Rho), a six disulfide‐bonded protein expressed in P. pastoris with the correct fold, was used to optimize the culture conditions. The concentrations of [α‐15N] selective amino acid, nonlabeled amino acids, and ammonium chloride, as well as induction time, were optimized to avoid scrambling and to increase the incorporation rate and protein yield. The optimized protocol was successfully applied to produce AATS isotope‐labeled Rho. The labeling of [α‐15N]Cys has a 50% incorporation rate, and all 12 cysteine resonances were observed in HSQC spectrum. The labeling of [α‐15N]Leu, ‐Lys, and ‐Met amino acids has an incorporation rate greater than 65%, and the expected number of resonances in the HSQC spectra were observed. In contrast, the labeling of [α‐15N]Asp and ‐Gly amino acids has a low incorporation rate and the scrambling problem. In addition, the culture condition was successfully applied to label dendroaspin (Den), a four disulfide‐bonded protein expressed in P. pastoris. Therefore, the described condition should be generally applicable to other proteins produced in the P. pastoris expression system. This is the first report to present a protocol for AATS isotope labeling of protein expressed in P. pastoris for NMR study. Proteins 2006.