Han Chieh Wu

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.

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.

The emerging role of hepatitis B virus Pre-S2 deletion mutant proteins in HBV tumorigenesis

Chronic hepatitis B virus (HBV) infection can cause hepatocellular carcinoma (HCC). Several hypotheses have been proposed to explain the mechanisms of HBV tumorigenesis, including inflammation and liver regeneration associated with cytotoxic immune injuries and transcriptional activators of mutant HBV gene products. The mutant viral oncoprotein-driven tumorigenesis is prevailed at the advanced stage or anti-HBe-positive phase of chronic HBV infection. Besides HBx, the pre-S2 (deletion) mutant protein represents a newly recognized oncoprotein that is accumulated in the endoplasmic reticulum (ER) and manifests as type II ground glass hepatocytes (GGH). The retention of pre-S2 mutant protein in ER can induce ER stress and initiate an ER stress-dependent VEGF/Akt/mTOR and NFκB/COX-2 signal pathway. Additionally, the pre-S2 mutant large surface protein can induce an ER stress-independent pathway to transactivate JAB-1/p27/RB/cyclin A,D pathway, leading to growth advantage of type II GGH. The pre-S2 mutant protein-induced ER stress can also cause DNA damage, centrosome overduplication, and genomic instability. In 5-10% of type II GGHs, there is co-expression of pre-S2 mutant protein and HBx antigen which exhibited enhanced oncogenic effects in transgenic mice. The mTOR signal cascade is consistently activated throughout the course of pre-S2 mutant transgenic livers and in human HCC tissues, leading to metabolic disorders and HCC tumorigenesis. Clinically, the presence of pre-S2 deletion mutants in sera frequently develop resistance to nucleoside analogues anti-virals and predict HCC development. The pre-S2 deletion mutants and type II GGHs therefore represent novel biomarkers of HBV-related HCCs. A versatile DNA array chip has been developed to detect pre-S2 mutants in serum. Overall, the presence of pre-S2 mutants in serum has implications for anti-viral treatment and can predict HCC development. Targeting at pre-S2 mutant protein-induced, ER stress-dependent, mTOR signal cascade and metabolic disorders may offer potential strategy for chemoprevention or therapy in high risk chronic HBV carriers.

Milrinone therapy for enterovirus 71-induced pulmonary edema and/or neurogenic shock in children: a randomized controlled trial.

Objective:Enterovirus 71-induced brainstem encephalitis with pulmonary edema and/or neurogenic shock (stage 3B) is associated with rapid mortality in children. In a small pilot study, we found that milrinone reduced early mortality compared with historical controls. This prospective, randomized control trial was designed to provide more definitive evidence of the ability of milrinone to reduce the 1-week mortality of stage 3B enterovirus 71 infections. Design:Prospective, unicenter, open-label, randomized, controlled study. Setting:Inpatient ward of a large tertiary teaching hospital in Ho Chi Minh City, Vietnam. Patients:Children (⩽18 yr old) admitted with proven enterovirus 71-induced pulmonary edema and/or neurogenic shock. Interventions:Patients were randomly assigned to receive intravenous milrinone (0.5 &mgr;g/kg/min) (n = 22) or conventional management (n = 19). Both groups received dopamine or dobutamine and intravenous immunoglobulin. Measurements and Main Results:The primary endpoint was 1-week mortality. The secondary endpoints included length of ventilator dependence and hospital stay and adverse events. The median age was 2 years with a predominance of boys in both groups. The 1-week mortality was significantly lower, 18.2% (4/22) in the milrinone compared with 57.9% (11/19) in the conventional management group (relative risk = 0.314 [95% CI, 0.12–0.83], p = 0.01). The median duration of ventilator-free days was longer in the milrinone treatment group (p = 0.01). There was no apparent neurologic sequela in the survivors in either group, and no drug-related adverse events were documented. Conclusions:Milrinone significantly reduced the 1-week mortality of enterovirus 71-induced pulmonary edema and/or neurogenic shock without adverse effects. Further studies are needed to determine whether milrinone might be useful to prevent progression of earlier stages of brainstem encephalitis.

Ground-glass hepatocytes co-expressing hepatitis B virus X protein and surface antigens exhibit enhanced oncogenic effects and tumorigenesis

Hepatitis B virus (HBV) X protein (HBx) and pre-S2 deletion mutant large surface antigens are oncoproteins that induce hepatocellular carcinoma (HCC). The interaction of these two oncoproteins in hepatocytes and its significance in tumorigenesis remain to be elucidated. In this study, we observed the co-expression of HBx with surface antigens in ground-glass hepatocytes in 5 of 20 hepatitis B surface antigen-positive livers. In vitro, hepatocytes co-expressing HBx and a pre-S2 mutant showed enhanced expression of vascular endothelial growth factor-A, phosphorylated Akt 1/2/3, phosphorylated extracellular signal-regulated kinase 1/2, and phosphorylated mammalian target of rapamycin signals. Transgenic mice harboring both HBx and pre-S2 mutant construct plasmids developed HCCs at an average of 15.1 months, earlier than animals carrying either HBx (16.9 months) or pre-S2 mutant (24.5 months) alone. The oncogenic signals of vascular endothelial growth factor-A, phosphorylated Akt 1/2/3, phosphorylated extracellular signal-regulated kinase 1/2, and phosphorylated mammalian target of rapamycin were sequentially and differentially activated at different stages in tumorigenesis. Phosphorylated mTOR was consistently activated in transgenic and human HCCs. We conclude that ground-glass hepatocytes co-expressing HBx and surface antigens exhibit enhanced oncogenic effects and tumorigenesis in chronic HBV infections. The mTOR signal cascade may be the key regulator in HBV tumorigenesis and may be useful targets in the design of HCC therapy.

Histone deacetylase inhibitor suberoylanilide hydroxamic acid suppresses the pro-oncogenic effects induced by hepatitis b virus pre-S2 mutant oncoprotein and represents a potential chemopreventive agent in high-risk chronic HBV patients

Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S(2) mutant large HBV surface antigen (LHBS) in type II ground glass hepatocytes (GGHs) has been recognized as an emerging viral oncoprotein; it directly interacts with the c-Jun activation domain-binding protein 1 (JAB1) and subsequently causes hyperphosphorylation of the tumor-suppressor retinoblastoma and, consequently, leads to disturbed cell cycle progression. The interaction of the pre-S(2) mutant LHBS with JAB1 could provide a potential target for chemoprevention. In this study, we found that the preneoplastic type II GGHs showed a significant decrease of the cyclin-dependent kinase inhibitor p27(Kip1), which serves as a marker for pre-S(2) mutant-JAB1 complex formation. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) elevated expression of the tumor-suppressor thioredoxin-binding protein 2 (TBP2), which subsequently enhanced the JAB1-TBP2 interaction and abolished the pre-S(2) mutant LHBS-induced degradation of p27(Kip1), which, in turn, recovered the normal cell cycle checkpoint. The pre-S(2) mutant LHBS-induced pro-oncogenic effects: increased cell proliferation, nuclear/cytoplasmic ratio and proliferating cell nuclear antigen expression, were all greatly ameliorated after SAHA treatments, which suggested SAHA as a promising chemopreventive agent for the pre-S(2) mutant oncoprotein-induced HCC. In conclusion, this study provides the mechanism of histone deacetylase (HDAC) inhibitor in preventing the pre-S(2) mutant-induced oncogenic phenotype. The HDAC inhibitor SAHA is therefore a potential chemopreventive agent for high-risk chronic HBV patients who may develop HCC.

A pre-S gene chip to detect pre-S deletions in hepatitis B virus large surface antigen as a predictive marker for hepatoma risk in chronic hepatitis B virus carriers

BackgroundChronic hepatitis B virus (HBV) infection is an important cause of hepatocellular carcinoma (HCC) worldwide. The pre-S1 and -S2 mutant large HBV surface antigen (LHBS), in which the pre-S1 and -S2 regions of the LHBS gene are partially deleted, are highly associated with HBV-related HCC.MethodsThe pre-S region of the LHBS gene in two hundred and one HBV-positive serum samples was PCR-amplified and sequenced. A pre-S oligonucleotide gene chip was developed to efficiently detect pre-S deletions in chronic HBV carriers. Twenty serum samples from chronic HBV carriers were analyzed using the chip.ResultsThe pre-S deletion rates were relatively low (7%) in the sera of patients with acute HBV infection. They gradually increased in periods of persistent HBV infection: pre-S mutation rates were 37% in chronic HBV carriers, and as high as 60% in HCC patients. The Pre-S Gene Chip offers a highly sensitive and specific method for pre-S deletion detection and is less expensive and more efficient (turnaround time 3 days) than DNA sequencing analysis.ConclusionThe pre-S1/2 mutants may emerge during the long-term persistence of the HBV genome in carriers and facilitate HCC development. Combined detection of pre-S mutations, other markers of HBV replication, and viral titers, offers a reliable predictive method for HCC risks in chronic HBV carriers.