Suberoylanilide hydroxamic acid induces viral lytic cycle in Epstein‐Barr virus‐positive epithelial malignancies and mediates enhanced cell death

In Epstein‐Barr virus (EBV)‐associated malignancies, the virus is harbored in every tumor cell and persists in tightly latent forms expressing a very limited number of viral latent proteins. Induction of EBV lytic cycle leads to expression of a much larger number of viral proteins, which may serve as potential therapeutic targets. We found that 4 histone deacetylase inhibitors, trichostatin A (TSA), sodium butyrate (SB), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), all significantly induced EBV lytic cycle in EBV‐positive gastric carcinoma cells (AGS/BX1, latency II) but only weakly induced in Burkitt lymphoma cells (AK2003, latency I) and did not induce in lymphoblastoid cells (LCLs, latency III). Interestingly, SAHA potently induced viral lytic cycle in AGS/BX1 cells at micromolar concentrations (evidenced by 8‐fold increase in viral DNA replication, strong expression of viral lytic proteins and production of infectious virus particles) and mediated enhanced cell death of EBV‐positive AGS/BX1 cells when compared with that of EBV‐negative AGS cells, possibly related to cell cycle arrest at G2/M phase. Furthermore, SAHA effected strong induction of EBV lytic cycle in nasopharyngeal carcinoma but not in NK lymphoma cells (both expressing EBV latency II pattern), indicating preferential viral lytic induction in epithelial rather than lymphoid malignancies. In conclusion, SAHA is found to be a potent EBV lytic cycle inducing agent, which warrants further investigation into its potential application as a novel virus‐targeted drug for treatment of EBV‐associated epithelial malignancies.     

Inhibition of class I histone deacetylases by romidepsin potently induces Epstein‐Barr virus lytic cycle and mediates enhanced cell death with ganciclovir

Pan‐histone deacetylase (HDAC) inhibitors, which inhibit 11 HDAC isoforms, are widely used to induce Epstein‐Barr virus (EBV) lytic cycle in EBV‐associated cancers in vitro and in clinical trials. Here, we hypothesized that inhibition of one or several specific HDAC isoforms by selective HDAC inhibitors could potently induce EBV lytic cycle in EBV‐associated malignancies such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). We found that inhibition of class I HDACs, particularly HDAC‐1, ‐2 and ‐3, was sufficient to induce EBV lytic cycle in NPC and GC cells in vitro and in vivo. Among a panel of selective HDAC inhibitors, the FDA‐approved HDAC inhibitor romidepsin was found to be the most potent lytic inducer, which could activate EBV lytic cycle at ～0.5 to 5 nM (versus ～800 nM achievable concentration in patients' plasma) in more than 75% of cells. Upregulation of p21^WAF1, which is negatively regulated by class I HDACs, was observed before the induction of EBV lytic cycle. The upregulation of p21^WAF1 and induction of lytic cycle were abrogated by a specific inhibitor of PKC‐δ but not the inhibitors of PI3K, MEK, p38 MAPK, JNK or ATM pathways. Interestingly, inhibition of HDAC‐1, ?2 and ?3 by romidepsin or shRNA knockdown could confer susceptibility of EBV‐positive epithelial cells to the treatment with ganciclovir (GCV). In conclusion, we demonstrated that inhibition of class I HDACs by romidepsin could potently induce EBV lytic cycle and mediate enhanced cell death with GCV, suggesting potential application of romidepsin for the treatment of EBV‐associated cancers.     

Anti‐tumor effects of suberoylanilide hydroxamic acid on Epstein–Barr virus‐associated T cell and natural killer cell lymphoma

The ubiquitous Epstein–Barr virus (EBV) infects not only B cells but also T cells and natural killer (NK) cells and is associated with various lymphoid malignancies. Recent studies have reported that histone deacetylase (HDAC) inhibitors exert anticancer effects against various tumor cells. In the present study, we have evaluated both the in vitro and in vivo effects of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on EBV‐positive and EBV‐negative T and NK lymphoma cells. Several EBV‐positive and EBV‐negative T and NK cell lines were treated with various concentrations of SAHA. SAHA suppressed the proliferation of T and NK cell lines, although no significant difference was observed between EBV‐positive and EBV‐negative cell lines. SAHA induced apoptosis and/or cell cycle arrest in several T and NK cell lines. In addition, SAHA increased the expression of EBV‐lytic genes and decreased the expression of EBV‐latent genes. Next, EBV‐positive NK cell lymphoma cells were subcutaneously inoculated into severely immunodeficient NOD/Shi‐scid/IL‐2Rγnull mice, and then SAHA was administered intraperitoneally. SAHA inhibited tumor progression and metastasis in the murine xenograft model. SAHA displayed a marked suppressive effect against EBV‐associated T and NK cell lymphomas through either induction of apoptosis or cell cycle arrest, and may represent an alternative treatment option.     

Epstein‐Barr virus strain variation and cancer

Epstein‐Barr virus (EBV) is a human tumor virus and is etiologically linked to various malignancies. Certain EBV‐associated diseases, such as Burkitt lymphomas and nasopharyngeal carcinomas, are endemic and exhibit biased geographic distribution worldwide. Recent advances in deep sequencing technology enabled high‐throughput sequencing of the EBV genome from clinical samples. Rapid cloning and sequencing of cancer‐derived EBV genomes, followed by reconstitution of infectious virus, have also become possible. These developments have revealed that various EBV strains are differentially distributed throughout the world, and that the behavior of cancer‐derived EBV strains is different from that of the prototype EBV strain of non‐cancerous origin. In this review, we summarize recent progress and future perspectives regarding the association between EBV strain variation and cancer.     

The role of oxidative stress in EBV lytic reactivation,radioresistance and the potential preventive and therapeutic implications

Epstein‐Barr virus (EBV) is an important cancer causing virus. Cancer associated with EBV account for approximately 1.5% of all cancers, and represent 1.8% of all cancer deaths worldwide. EBV reactivation plays an important role in the development of EBV‐related diseases and is closely related with patients' survival and clinical stages of EBV‐related cancers. The therapy regarding to EBV‐related cancers is very urgent, especially in endemic areas. Generating oxidative stress is a critical mechanism by which host cells defend against infection by virus. In addition, ROS‐mediated oxidative stress plays a significant but paradoxical role acting as a “double‐edged sword” to regulate cellular response to radiation, which is the main therapy strategy for EBV‐related cancers, especially nasopharyngeal carcinoma. Therefore, in this review we primarily discuss the possible interplay among the oxidative stress, EBV lytic reactivation and radioresistance. Understanding the role of oxidative stress in EBV lytic reactivation and radioresistance will assist in the development of effective strategies for prevention and treatment of EBV‐related cancers.     

Activation of lytic cycle of Epstein-Barr virus by suberoylanilide hydroxamic acid leads to apoptosis and tumor growth suppression of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). We reported that suberoylanilide hydroxamic acid (SAHA) induced EBV lytic cycle in EBV-positive gastric carcinoma cells and mediated enhanced cell death. However, expression of EBV lytic proteins was thought to exert antiapoptotic effect in EBV-infected cells. Here, we examined the in vitro and in vivo effects of SAHA on EBV lytic cycle induction in NPC cells and investigated the cellular consequences. Micromolar concentrations of SAHA significantly induced EBV lytic cycle in EBV-positive NPC cells. Increased apoptosis and proteolytic cleavage of poly(ADP-ribose) polymerase and caspase-3, -7 and -9 in EBV-positive versus EBV-negative NPC cells were observed. More than 85% of NPC cells expressing immediate-early (Zta), early (BMRF1) or late (gp350/220) lytic proteins coexpressed cleaved caspase-3. Tracking of expression of EBV lytic proteins and cleaved caspase-3 over time demonstrated that NPC cells proceeded to apoptosis following EBV lytic cycle induction. Inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA's induced cell death in NPC, indicating that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were also observed in NPC xenografts in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA leads to apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus-targeted therapy against EBV-positive cancer.     

Zinc finger E-box binding factor 1 plays a central role in regulating Epstein-Barr virus (EBV) latent-lytic switch and acts as a therapeutic target in EBV-associated gastric cancer

BACKGROUND:

The role of Epstein‐Barr virus (EBV) infection in gastric carcinogenesis remains largely unknown. The authors studied the effects of zinc finger E‐box binding factor 1 (ZEB1) on latent‐lytic switch of EBV infection in gastric cancer and explored the importance of EBV in gastric carcinogenesis.

METHODS:

Loss or gain of ZEB1 function was obtained by ZEB1 small‐interfering RNA (siRNA) knock‐down or forced ZEB1 re‐expression. Cell growth was evaluated by cell viability and colony formation assays, and the cell cycle was assessed by flow cytometry. EBV was detected using quantitative polymerase chain reaction (PCR) and in situ hybridization analyses.

RESULTS:

ZEB1 knock‐down in a latent EBV‐infected gastric cancer cell line (YCC10) increased lytic gene BamHI W leftward reading frame 1 (BZLF1) expression and decreased the expression of latent gene EB nuclear antigen 1 (EBNA1) concomitant with the inhibition of cell viability (P < .05) and S‐phase DNA synthesis (P < .01). ZEB1 depletion combined with ganciclovir revealed a further reduction in cell viability (P < .001). ZEB1 knock‐down induced cell apoptosis and the up‐regulation of caspase 3 and poly(adenosine diphosphate‐ribose) polymerase cleavage. Conversely, ectopic overexpression of ZEB1 in a lytic EBV‐infected gastric cancer cell line (AGS‐EBV) inhibited BZLF1 promoter (Zp) activity, BZLF1 expression, and apoptosis and promoted cell growth. EBV infection was detected in 11.3% (80 of 711) of gastric cancers. The presence of EBV was associated with age, men, and intestinal type cancer.

CONCLUSIONS:

ZEB1 was confirmed as a key mediator of the latent‐lytic switch of EBV‐associated gastric cancer, a distinct subtype with different clinicopathologic features. The current results indicated that inhibition of ZEB1 may be a potential target for EBV‐associated gastric cancer therapy. Cancer 2012;. © 2011 American Cancer Society.     

Zebularine reactivates silenced <Emphasis Type="Italic">E-cadherin</Emphasis> but unlike 5-Azacytidine does not induce switching from latent to lytic Epstein-Barr virus infection in Burkitt's lymphoma Akata cells

Epigenetic silencing of regulatory genes by aberrant methylation contributes to tumorigenesis. DNA methyltransferase inhibitors (DNMTI) represent promising new drugs for anti-cancer therapies. The DNMTI 5-Azacytidine is effective against myelodysplastic syndrome, but induces switching of latent to lytic Epstein-Barr virus (EBV) in vitro and results in EBV DNA demethylation with the potential of induction of lytic EBV in vivo. This is of considerable concern given that recurrent lytic EBV has been linked with an increased incidence of EBV-associated lymphomas. Based on the distinct properties of action we hypothesized that the newer DNMTI Zebularine might differ from 5-Azacytidine in its potential to induce switching from latent to lytic EBV. Here we show that both 5-Azacytidine and Zebularine are able to induce expression of E-cadherin, a cellular gene frequently silenced by hypermethylation in cancers, and thus demonstrate that both DNMTI are active in our experimental setting consisting of EBV-harboring Burkitt's lymphoma Akata cells. Quantification of mRNA expression of EBV genes revealed that 5-Azacytidine induces switching from latent to lytic EBV and, in addition, that the immediate-early lytic infection progresses to early and late lytic infection. Furthermore, 5-Azacytidine induced upregulation of the latent EBV genes LMP2A, LMP2B, and EBNA2 in a similar fashion as observed following switching of latent to lytic EBV upon cross-linking of the B-cell receptor. In striking contrast, Zebularine did not exhibit any effect neither on lytic nor on latent EBV gene expression. Thus, Zebularine might be safer than 5-Azacytidine for the treatment of cancers in EBV carriers and could also be applied against EBV-harboring tumors, since it does not induce switching from latent to lytic EBV which may result in secondary EBV-associated malignancies.     

Circulating Epstein‐Barr virus–encoded micro‐RNAs as potential biomarkers for nasal natural killer/T‐cell lymphoma

Nasal natural killer/T‐cell lymphoma (NNKTL) is an Epstein‐Barr virus (EBV)–associated malignancy and is characterized by local invasion and widespread dissemination, with a consequent poor prognosis. Micro‐RNAs (miRNAs) play roles in the pathogenesis of several malignancies by regulating gene expression and have been recently identified as stable entities in serum. Here, we investigated the value of circulating EBV‐miRNAs as biomarkers for NNKTL. Sera of patients with NNKTL were subjected to miRNA polymerase chain reaction (PCR)–array analysis, after which serum EBV‐miRNA levels were verified using quantitative PCR. The latter analysis revealed high miR‐BART2‐5p, miR‐BART7‐3p, miR‐BART13‐3p, and miR‐BART1‐5p expression levels in sera of patients with NNKTL and indicated accurate values for discriminating patients with NNKTL from healthy controls. Levels of these 4 EBV‐miRNAs, which were secreted from NNKTL cells, significantly decreased after treatment compared with those before treatment. Furthermore, a high circulating miR‐BART2‐5p level was associated with disease progression and poor prognosis in patients with NNKTL. Our findings demonstrate that circulating EBV‐miRNAs, particularly miR‐BART2‐5p, may serve as potential diagnostic and prognostic biomarkers in patients with NNKTL.     

Epstein‐Barr virus infection induces miR‐21 in terminally differentiated malignant B cells

The association of Epstein‐Barr virus (EBV) with plasmacytoid malignancies is now well established but how the virus influences microRNA expression in such cells is not known. We have used multiple myeloma (MM) cell lines to address this issue and find that an oncomiR, miR‐21 is induced after in vitro EBV infection. The PU.1 binding site in miR‐21 promoter was essential for its activation by the virus. In accordance with its noted oncogenic functions, miR‐21 induction in EBV infected MM cells caused downregulation of p21 and an increase in cyclin D3 expression. EBV infected MM cells were highly tumorigenic in SCID mice. Given the importance of miR‐21 in plasmacytoid malignancies, our findings that EBV could further exacerbate the disease by inducing miR‐21 has interesting implications both in terms of diagnosis and future miR based therapeutical approaches for the virus associated plasmacytoid tumors.     

Antitumor activity of cyclin‐dependent kinase inhibitor alsterpaullone in Epstein‐Barr virus‐associated lymphoproliferative disorders

Epstein‐Barr virus (EBV) is a well‐established tumor virus that has been implicated in a wide range of immunodeficiency‐associated lymphoproliferative disorders (LPDs). Although rituximab, a CD20 mAb, has proven effective against EBV‐associated LPDs, prolonged use of this drug could lead to resistance due to the selective expansion of CD20^? cells. We have previously shown that cyclin‐dependent kinase (CDK) inhibitors are able to specifically suppress the expression of viral late genes, particularly those encoding structural proteins; however, the therapeutic effect of CDK inhibitors against EBV‐associated LPDs is not clear. In this study, we examined whether CDK inhibitors confer a therapeutic effect against LPDs in vivo. Treatment with alsterpaullone, an inhibitor of the CDK2 complex, resulted in a survival benefit and suppressed tumor invasion in a mouse model of LPDs. Inhibition of CDK efficiently induced G₁ cell cycle arrest and apoptosis in EBV‐positive B cells. These results suggest that alsterpaullone suppresses cell cycle progression, resulting in the antitumor effect observed in vivo.     

Re‐expression of microRNA‐150 induces EBV‐positive Burkitt lymphoma differentiation by modulating c‐Myb in vitro

Burkitt lymphoma (BL) is a highly aggressive B‐cell lymphoma that includes two forms of BL differing in Epstein–Barr virus (EBV) infection status, EBV‐positive and EBV‐negative. Although many efforts, such as high‐intensity, short‐duration combination chemotherapy, have been devoted to improving therapy for this rapidly proliferating neoplasm, there are still significant treatment‐associated toxicities. Therefore, there remains a need for novel effective therapeutic strategies. MicroRNAs play a role in “fine tuning” the physiological and pathological differentiation process, by which cells can rapidly regulate dynamic events such as cell‐lineage decisions and morphogenesis. This unique miRNA feature shifts the traditional one drug target paradigm to a novel one drug multiple targets paradigm. Here, we found that BL cell lines showed an extremely low expression of microRNA‐150 (miR‐150), and then restored miR‐150 expression at physiologic levels in BL cell lines Daudi, Raji, BJAB, and Ramos. The results showed that re‐expression of miR‐150 reduced proliferation of Daudi and Raji cells. Furthermore, Daudi and Raji, both of which are of EBV‐positive germinal center B‐cell origin, transduced with miR‐150 can be rescued to differentiate toward B‐cell terminal stage. However, no significant changes were observed in BJAB or Ramos cells, which are of EBV‐negative germinal center B‐cell origin. Of note, re‐expression of miR‐150 also resulted in decreasing c‐Myb protein levels. Additionally, c‐Myb knockdown in Daudi and Raji cell lines recapitulated the partial characteristics similar to that caused by re‐expression of miR‐150. Taken together, our findings show that miR‐150 can induce EBV‐positive BL differentiation by targeting c‐Myb.     

Virally targeted therapies for EBV-associated malignancies

In Epstein-Barr virus (EBV)-positive lymphomas, the presence of the EBV genome in virtually all tumor cells, but very few normal cells, suggests that novel, EBV-targeted therapies could be used to treat these malignancies. In this paper, we review a variety of different approaches currently under development that specifically target EBV-infected cells for destruction. EBV-based strategies for treating cancer include prevention of viral oncogene expression, inducing loss of the EBV episome, the purposeful induction of the lytic form of EBV infection, and enhancing the host immune response to virally encoded antigens.     

The biology of EBV infection in human epithelial cells

EBV-associated human malignancies may originate from B cells and epithelial cells. EBV readily infects B cells in vitro and transforms them into proliferative lymphoblastoid cell lines. In contrast, infection of human epithelial cells in vitro with EBV has been difficult to achieve. The lack of experimental human epithelial cell systems for EBV infection has hampered the understanding of biology of EBV infection in epithelial cells. The recent success to infect human epithelial cells with EBV in vitro has allowed systematic investigations into routes of EBV entry, regulation of latent and lytic EBV infection, and persistence of EBV infection in infected epithelial cells. Understanding the biology of EBV infection in human epithelial cells will provide important insights to the role of EBV infection in the pathogenesis of EBV-associated epithelial malignancies including nasopharyngeal carcinoma and gastric carcinoma.     

Infectious agents in human cancers: lessons in immunity and immunomodulation from gammaherpesviruses EBV and KSHV

Members of the herpesvirus family have evolved the ability to persist in their hosts by establishing a reservoir of latently infected cells each carrying the viral genome with reduced levels of viral protein synthesis. In order to spread within and between hosts, in some cells, the quiescent virus will reactivate and enter lytic cycle replication to generate and release new infectious virus particles. To allow the efficient generation of progeny viruses, all herpesviruses have evolved a wide variety of immunomodulatory mechanisms to limit the exposure of cells undergoing lytic cycle replication to the immune system. Here we have focused on the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) that, uniquely among the eight human herpesviruses identified to date, have growth transforming potential. Most people infected with these viruses will not develop cancer, viral growth-transforming activity being kept under control by the host's antigen-specific immune responses. Nonetheless, EBV and KSHV are associated with several malignancies in which various viral proteins, either predominantly or exclusively latency-associated, are expressed; at least some of these proteins also have immunomodulatory activities. Of these malignancies, some are the result of a disrupted virus/immune balance through genetic, infectious or iatrogenic immune suppression. Others develop in people that are not overtly immune suppressed and likely modulate the immunological response. This latter aspect of immune modulation by EBV and KSHV forms the basis of this review.     

Recurrent chemical reactivations of EBV promotes genome instability and enhances tumor progression of nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) is an endemic malignancy prevalent in South East Asia. Epidemiological studies have associated this disease closely with Epstein‐Barr virus (EBV) infection. Previous studies also showed that EBV reactivation is implicated in the progression of NPC. Thus, we proposed that recurrent reactivations of EBV may be important for its pathogenic role. In this study, NPC cell lines latently infected with EBV, NA and HA, and the corresponding EBV‐negative NPC cell lines, NPC‐TW01 (TW01) and HONE‐1, were treated with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and sodium n‐butyrate (SB) for lytic cycle induction. A single treatment with TPA/SB revealed that DNA double‐strand breaks and formation of micronuclei (a marker for genome instability) were associated with EBV reactivation in NA and HA cells. Examination of EBV early genes had identified several lytic proteins, particularly EBV DNase, as potent activators that induced DNA double‐strand breaks and contribute to genome instability. Recurrent reactivations of EBV in NA and HA cells resulted in a marked increase of genome instability. In addition, the degree of chromosomal aberrations, as shown by chromosome structural variants and DNA copy‐number alterations, is proportional to the frequency of TPA/SB‐induced EBV reactivation. Whereas these DNA abnormalities were limited in EBV‐negative TW01 cells with mock or TPA/SB treatment, and were few in mock‐treated NA cells. The invasiveness and tumorigenesis assays also revealed a profound increase in both characteristics of the repeatedly reactivated NA cells. These results suggest that recurrent EBV reactivations may result in accumulation of genome instability and promote the tumor progression of NPC. © 2008 Wiley‐Liss, Inc.