The Effects And Mechanism Of HBV Infection On Diffuse Large B-cell Lymphoma

?Background?Diffuse large B-cell lymphoma(DLBCL)is the most common lymphoma in China with tumor heterogeneity.Combination rituximab with CHOP(cyclophosphamide,doxorubicin,vincristine,and prednisone),has dramatically improved the outcome in DLBCL,while approximately 30-40% of patients will develop relapsed or refractory.Increased age,advanced stages,elevated serum lactate dehydrogenase level(LDH),giant mass and poor molecular markers are generally acknowledged as the reasons of refractory and relapse of DLBCLs.Numbers of epidemiologic investigations about molecular markers that predict prognosis and guide treatment of DLBCLs have demonstrated that HBV infection is closely with the onset,progression,chemosensitivity and prognosis of DLBCL.However,the relationship and mechanisms of HBV infection and poor outcome remain to be further elucidated.?Objective?The purposes of this study are to explore the effects and mechanisms of HBV infection in DLBCL,to clarify the exact roles of HBX(a functional segment of HBV)in chemosensitivity of DLBCL cells,and to find potential molecular targets that benefit the DLBCL patients with HBV infection.?Methods?(1)Clinical data analysis: we collected clinical data of 428 DLBCL patients with or without HBV infection from January 2004 to July 2014 in Changhai Hospital(Shanghai,China).We used Chi-square test and non-parametric test to compare categorical variables.The Kaplan-Meier method and Log-rank test were used to generate and compare the OS and PFS between different groups.(2)Experiment methods: SUDHL-4 and DB cells were infected with lentivirus(HBX overexpression,CHK2 shRNAs,CHK2(WT)overexpression,and CHK2(T68A)overexpression)to overexpress or down-regulate the corresponding genes.Cell proliferation was detected by cell counting assay,and the effects on cell cycle and apoptosis were evaluated by PI staining and Annexin-V/PI double staining using flow cytometry respectively.RT-qPCR and Western blotting were used to detect the expression of mRNA and protein respectively.In order to observe the in vivo effect of HBX on chemosensitivity of DLBCL,transplanted tumor models were constructed by injecting tumor cells into subaxillary of NOD-SCID mice.Immunohistochemical staining was used to analyze the protein expression in the tumor specimens.?Results?(1)HBV infection was closely associated with poor outcome and chemoresistance of DLBCL patients,especially in germinal center B-cell-like(GCB)type,which was independent of HBV infection liver damage as an important prognostic factor.(2)HBX(an important functional segment of HBV)was ectopically overexpressed in DLBCL cell lines(SUDHL-4 and DB).Our results suggested that HBX had no significant effect on the proliferation,cell cycle distributions and apoptosis of DLBCL cells.It is further demonstrated that compared to Ctrl cells,SUDHL-4 and DB cells overexpressing HBX showed much less sensitive to methotrexate(MTX)and cytarabine(Ara-C),characterized by inducing S-phase arrest.Surprisingly,the enhancement of resistance was not observed in the other drugs such as epirubicin(EPI)and vindesine(VDS),which did not result cells in S-phase arrest.In addition,the HBX-expressed DLBCL cells(both SUDHL-4 and DB)presented a statistically significant less proportion of S-phase arrest cells compared to Ctrl cells.Together,these results suggested that HBX attenuated the proliferation inhibition of chemotherapeutics inducing S-phase arrest.(3)DNA damage response signals were reported to be closely related to chemoresistance.In order to gain mechanistic insight into possible molecules involved in the decreased chemosensitivity resulting from HBV infection,the changes of two major DDR pathways(ATM/CHK2 and ATR/CHK1)were assessed.The expression of P-CHK2 in Ctrl cells markedly was increased to a greater extent than that in HBX-expressed cells after treated with MTX,while neither baseline nor post-treatment levels of P-CHK1 was affected by overexpressing HBX.The expression level of P21 and P53,downstream targets of P-CHK2,were in parallel with the expression of P-CHK2.These results indicated that HBX overexpression appeared to specially inhibit CHK2 phosphorylation in DLBCL cells.(4)To further investigate the functional role of CHK2 in chemoresistance,we attempted to down-regulate CHK2 in SUDHL-4 cells using shRNA.CHK2 depletion obviously reduced the chemoresistance of DLBCL cells treated with agents inducing S-phase arrest,consistent with the effect of HBX overexpression.Furthermore,our data indicated that the chemosensitivity of HBX-expressed cells could be rescued by overexpressing wild type CHK2,but not the CHK2 unphosphorylated mutant(T68A),suggesting that HBX conferred resistance to chemotherapeutic inducing S-phase arrest via inhibiting CHK2 phosphorylation.(5)The in vivo findings indicated that HBX showed no effects on tumor volume and weight,but significantly reduced the response of DLBCL cells to MTX.And immunohistochemical staining showed that the expression level of P-CHK2 was mildly increased in HBX-expressed cells compared to Ctrl cells,while the expression of P-CHK1 was not affected by HBX overexpresssion.?Conclusion?HBV infection was closely associated with chemoresistance and poor OS and PFS as an independent prognostic factor.Our study uncovered the relevance between HBV infection and the resistance to MTX or Ara-C based chemotherapy mediated by the inhibition of CHK2 response signaling in DLBCL,which was the probable reason that HBV infection resulted in poor prognosis of DLBCL.Therefore,HBV infected DLBCL patients would not benefit from utilizing CHK2 inhibitors.