The Role And Regulatory Pathways Of Histone H3Phosphorylation At Serine10in Epstein-Barr Virus Latent Membrane Protein-1-Induced Nasopharyngeal Carcinogenesis

Epigenetics refers to the heritable changes in gene expression without any alteration in DNA sequence, which reflects the interplay between environment and genetics. Like many other diseases, epigenetic changes play an important role in the tumorigenesis. DNA methylation and histone modification are the most common epigenetic changes in human tumors. Posttranslation modification (e.g. acetylation, methyaltion, phosphorylation and ubiquitination, etc.) of the N-terminal tail of histones disrupts DNA-histone interactions, destabilizes chromatin struction, and regulates gene transcription. Mutation of chromatin-modifying enzymes and regulatory proteins occur in many types of cancers.Phosphorylation of histone H3at Ser10is correlated closely with chromosome condensation, mitosis and gene expression. Phosphorylation of histone H3at Ser10induced by many tumor promotion agents, such as epidermal growth factor (EGF),12-O-tetradecanoyl phorbol-13-acetate (TPA), ultraviolet B (UVB) irradication or arsenite, and oncogene H-ras or v-Src, is associated with the transcriptional activation of immediate-early (IE) genes including c-fos、c-jun、c-myc and Cox-2gene. Increased phosphorylation of histone H3contributed to chromosome instability and was observed in many tumors, including colorectal, hepatocellular, cervical and gastric carcinomas, which is positively correlated with tumor malignancy. All described above imply that deregulation of histone H3phosphorylation may play an important role in neoplastic cell transformation.In recent years, many responsible H3kinases have been found. Accumulating evidences have demonstrated that phosphorylation of histone H3at Ser10is involved in different protein kinase signaling pathways depending on specific stimulation or stress, and cell type. The various protein kinases including Mitogen-and stress-activated kinase (MSK1), ribosomal subunit protein S6kinase2(RSK2), IkappaB kinase-alpha (IKK-a), cAMP dependent protein kinase C (PKC), Fyn and PIM1were found to mediate histone H3phosphorylation at Ser10under diverse stimuli, which is associated with transcription activation of responsive genes. It has been shown that MSK1is essential to mediate phosphorylation of histone H3at Ser10and activating protein-1(AP-1) activation induced by oncogene H-ras or v-Src, and EGF or TPA. Thus, it is very important to identify the responsible kinases and the circumstances under which histone H3becomes phosphorylated.Nasopharyngeal carcinoma (NPC) is a most common malignant tumor in southern China and some regions in Southeast Asia. Its occurrence involves the transaction between Epstein-Barr virus (EBV), environmental factors and genetic susceptibility, and is a multistep carcinogenic process. This specific etiological system of NPC refers that it is one of the best model for studying cancer epigenetics. EBV-encode latent membrane protein1(LMP1) is the only latent gene product with transformation properties, and is closely related with the development of several EBV-associated malignancies. Many of the oncogenic effects of LMP1are attributed to constitutively triggering a plethora of signaling pathways including NF-κB, AP-1and STAT pathways, which regulates the expression of downstream target genes and promotes cell proliferation, transformation invasion and metastasis. It has been shown that increased phosphorylation of histone H3at SerlO may contribute to the aberrant gene expressions and promote cell transformation. However, there is no evidence whether phosphorylation of histone H3at SerlO is involved in activation of transcription factors and cell malignant transformation induced by LMP1in NPC.In this study, the role of histone H3phosphorylation at ser10in nasopharyngeal carcinogenesis, especially in LMPl-induced CNE1cell transformation, is investigated. Then we further explore the protein kinases and signaling pathways responsible for phosphorylation of histone H3at Ser10iuduced by LMP1. These findings will contribute to elucidate the epigenetic mechanisms involved in LMP1-induced carcinogenesis of NPC and provide evidences for histone H3phosphorylation as a novel target for cancer diagnosis and therapy.Part I The role of histone H3phosphorylation at Ser10in LMP1-induced nasopharyngeal carcinogenesisChapter1Expression of phosphorylated histone H3at Ser10and its correlation with LMP1in NPC tissuesMethods1. The level of histone H3phosphorylation at Ser10was detected in NPC specimens, chronic nasopharyngitis specimens and adjacent/normal nasopharynx specimens using immunohistochemical staining.2. The expression of EBV-LMP1was detected in NPC specimens, and its correlation with phosphorylated histone H3at Ser10was analyzed.3. The level of histone H3phosphorylation at Ser10was detected in CNE2(poor-differentiated NPC cell line), CNE1(well-differentiated NPC cell line) and NP69(immortalized nasopharyngeal epithelial cell line) cells using immunocytochemical staining and Western blotting analysis.Results1. The level of histone H3phosphorylation at Ser10was significantly high in NPC tissuesThe positive labeling index (PLI) for histone H3phosphorylation at Ser10in48NPC specimens,15chronic nasopharyngitis specimens and36adjacent/normal nasopharynx specimens was22.43±10.57,14.87±6.04and8.10±4.78respectively, which indicated that the levels of histone H3phosphorylation at Ser10were significantly higher in NPC tissues than that in chronic nasopharyngitis tissues (P<0.05) and normal nasopharynx tissues (P<0.001). Moreover, the levels of histone H3phosphorylation were higher in chronic nasopharyngitis compared with normal nasopharynx tissues (P<0.005).2. The elevated level of histone H3phosphorylation in NPC tissues was positively related to LMP1expressionIn NPC tissues,28out of48(58.3%) cases showed LMP1expression. The correlation analysis revealed that there was a positive correlation between the level of histone H3phosphorylation at Ser10and LMP1expression in NPC tissues (Χ2=6.700, P=0.01;C=0.350).3. The level of histone H3phosphorylation at Ser10was significantly high in NPC cell linesImmunocytochemical staining and Western blotting analysis showed that the level of histone H3phosphorylation at Ser10was significantly higher in CNE2and CNE1cells than that in NP69cells. Moreover, the level of histone H3phosphorylation was higher in CNE2cells compared with CNE1cells.Chapter2EBV-LMP1induced phosphorylation of histone H3at Ser10in CNE1cellsMethods1. The level of histone H3phosphorylation at Ser10was detected in CNE1G and CNE1GL cells using immunocytochemical staining and Western blotting analysis.2. CNE1cells were transiently transfected with different amount of pcDNA3.0-LMP1, then the level of histone H3phosphorylation at Ser10was detected by Western blotting analysis.3. CNE1cells were transiently transfected with different amount of pcDNA3.0-LMP1, then AP-1transactivation activity was determined by luciferase reporter gene assay.Results1. LMP1induced phosphorylation of histone H3at Ser10in CNE1cells Immunocytochemical staining showed that phosphorylated histone H3at Ser10was more frequently observed in CNE1GL cells stably expressing LMP1compared with control CNE1G cells in the serum-starved condition. It was found that the most CNEIGL cells with p-H3Ser10expression did not belong to the G2/M phase of cell cycle. Similar results were also obtained by Western blotting analysis. In addition, CNE1cells were transiently transfected with increasing amounts of pcDNA3.0-LMP1, and Western blotting analysis showed that the level of histone H3phosphorylation at Ser10was increased in a dose-dependent manner with the expression of LMP1.2. LMP1induced AP-1transcriptional activation in CNE1cellsCNE1cells were transiently transfected with increasing amounts of pcDNA3.0-LMP1, and luciferase reporter gene assay indicated that AP-1transactivation was increased in a dose-dependent manner with the expression of LMP1.Chapter3The effect of histone H3knockdown on LMP1-promoted proliferation and transformation of CNE1cellsMethods1. The small interfering RNA (siRNA) expression vectors targeting histone H3gene (si-H3) and an irrelevant sequence (si-mock) were constructed and co-transfected with pcDNA6-Myc/HisB plasmid into CNE1cells, then stably transfected CNE1cell line silencing histone H3gene and its negative control cell line were established via blasticidin screening. The knockdown efficiency was assessed by real-time quantitative RT-PCR and Western blotting analysis.2. The effect of histone H3knockdown on LMP1-promoted CNE1cell proliferation and transformation was evaluated by CCK-8assay, plate colony formation assay and soft agar assay respectively.3. The effect of histone H3knockdown on AP-1transactivation induced by LMP1was detected by luciferase reporter gene assay.Results1. Establishment and identification of siRNA-mediated histone H3knockdown CNE1cell lineThe stably transfected CNE1cell line silencing histone H3and its negative control cell line were obstained via blasticidin screening. The results from real-time quantitative RT-PCR and Western blotting analysis showed that both mRNA and protein levels of histone H3were suppressed by60%-70%in CNE1cells stably transfected with si-H3compared with negative control cells.2. The knockdown of histone H3suppressed LMP1-promoted cell proliferation and transformationAs indicated by previous studies, our results also showed that expression of LMP1in CNE1cells promoted cell proliferation and colony formation. However, CNE1cells stably expressing si-H3showed a marked decrease in the rate of proliferation after culture for48h compared with negative control cells. Both plate colony formation assay and soft agar assay revealed that knockdown of histone H3in CNE1cells significantly inhibited LMP1-promoted colony formation compared with negative control cells. The inhibition was evident not only in colony number (P<0.005) but also in colony size.3. The knockdown of histone H3suppressed AP-1transcriptional activation induced by LMP1The luciferase reporter gene assay indicated that CNE1cells stably transfected with si-H3showed a suppression of AP-1transactivation activity induced by LMP1up to62.49%compared with negative control cells.Chapter4The effect of overexpressing wild type and mutant histone H3on LMP1-promoted proliferation and transformation of CNE1cellsMethods1. A cDNA fragment encoding human histone H3was recombined into the pcDNA6-Myc/HisB vector to construct the wild-type histone H3expression vector (H3WT). Take H3WT as template, Ser10of histone H3was replaced with alanine by site-directed mutagenesis (31T�'G) to generate the mutant histone H3 expression vector (pcDNA6-H3S10A)2. The wild-type and mutant histone H3expression vectors were respectively transfected into CNE1cells, then stably transfected CNE1cell lines were established via blasticidin screening. The expression of transfected gene was confirmed by Western blotting analysis.3. The effect of overexpressing wild type and mutant histone H3on LMP1-promoted CNE1cell proliferation and transformation was evaluated by CCK-8assay, plate colony formation assay and soft agar assay.4. The effect of overexpressing wild type and mutant histone H3on c-Jun, c-Fos protein levels and AP-1transcriptional activation induced by LMP1were assessed by Western blotting analysis and luciferase reporter gene assay respectively.Results1. Establishment and identification of overexpressing wild type or mutant histone H3CNE1cell linesWestern blotting analysis showed that the expressions of His-H3fusion protein were detected in CNE1cells stably transfected with histone H3WT or H3S10A expression vectors, which indicated that CNE1cell lines overexpressing wild type or mutant histone H3were successfully established.2. The overexpression of histone H3WT enhanced LMP1-promoted cell proliferation and transformationThe CCK-8assay showed that histone H3WT-overexpressing CNE1cells proliferated at higher rate after culture for72h compared with mock control cells. Plate colony formation assay showed that overexpression of histone H3WT in CNE1cells promoted colony formation in a reduced serum condition compared with mock control cells (P<0.01). In addition, the results from soft agar assay indicated that increasing colony numbers were observed in CNE1cells overexpressing H3WT promoted by LMP1compared with mock control cells (P<0.05). However, overexpression of histone H3S10A did not cause an obvious increase in colony formation. These results revealed that phosphorylation of histone H3at Ser10was most likely a critical site for regulating LMP1-induced cell proliferation and transformation.3. The overexpression of histone H3WT increased c-Jun or c-Fos protein levels and AP-1activation induced by LMP1Western blotting analysis showed that the endogenous c-Jun or c-Fos protein levels were dramatically increased in histone H3WT-overexpressing CNE1cells promoted by LMP1compared with mock control cells. However, no significant ganis of c-Jun or c-Fos protein levels were observed in histone H3S10A-overexpressing cells. In additional, the luciferase reporter gene assay indicated that only histone H3WT, but not H3S10A, enhanced AP-1transcriptional activation induced by LMP1compared with mock control cells (P<0.05).Part Ⅱ The protein kinase signaling pathways regulating LMP1-induced histone H3phosphorylation at Ser10Chapter1MSK1mediated LMPl-induced phosphorylation of histone H3at Ser10in CNE1cellsMethods1. The level of MSK1phosphorylation at Thr581was detected in NPC specimens and adjacent/normal nasopharynx specimens using immunohistochemical staining, and its correlations with LMP1expression and histone H3phosphorylation at Ser10were analyzed.2. The levels of ERK1/2and MSK1phosphorylation were detected in CNE1G and CNE1GL cells by Western blotting analysis. CNE1cells were transiently transfected with different amount of pcDNA3.0-LMP1, then the levels of ERK1/2and MSK1phosphorylation were detected by Western blotting analysis.3. In vitro kinase assay with histone H3protein as a substrate was used to examine the histone H3kinase activities of CNE1G and CNE1GL cell extracts in the presence or absence of H89.4. MSK1was immunoprecipitated from the cell extracts isolated from CNE1G and CNE1GL cells with anti-phospho-MSK1antibody, and then MSK1kinase activity was determined by immunecomplex kinase assay using histone H3protein as a substrate.5. ERK1/2inhibitor PD98059and MSK1inhibitor H89were used to treat LMP1-transfected CNE1cells, then the level of histone H3phosphorylation at Ser10was detected by Western blotting analysis.6. The siRNA expression vector targeting MSK1gene was constructed (si-MSK1) and co-transfected with pcDNA6-Myc/HisB plasmid into CNE1cells, then stably transfected CNE1cell line silencing MSK1was established via blasticidin screening. The knockdown efficiency was assessed by real-time quantitative RT-PCR and Western blotting analysis.7. The effect of MSK1knockdown on LMP1-induced phosphorylation of histone H3at Ser10was detected by Western blotting analysis.Results1. The level of MSK1phosphorylation at Thr581was significantly high in NPC tissuesThe immunohistochemical staining from48NPC specimens and36adjacent/normal nasopharynx specimens revealed that the level of MSK1phosphorylation at Thr581was significantly higher in NPC tissues than that in adjacent/normal nasopharynx tissues (P<0.001).2. The elevated level of MSK1phosphorylation in NPC tissues was positively related to LMP1expressionIn48NPC tissues, the correlation analysis revealed that there was a significantly positive correlation between the level of MSK1phosphorylation and LMP1expression (Χ2=9.600, P=0.002; C=0.408). In addition, the level of MSK1phosphorylation in NPC tissues was positively correlated with the level of histone H3phosphorylation at Ser10(Χ2=11.959, P=0.001; C=0.447)3. LMP1induced phosphorylation of ERK1/2and MSK1in CNE1cellsWestern blotting analysis showed that levels of ERK1/2and MSK1phosphorylation in CNE1GL cells stably expressing LMP1were higher than that in control CNE1G cells. In addition, CNE1cells were transiently transfected with increasing amounts of pcDNA3.0-LMP1, and the results indicated that the levels of ERK1/2and MSK1phosphorylation were increased in a dose-dependent manner with the expression of LMP1.4. LMP1increased histone H3kinase activity in CNE1cellsAn in vitro H3kinase assay showed that H3kinase activity in CNE1GL cell extracts was greater than that from CNE1G cells. However, pretreatment of MSK1inhibitor H89significantly reduced H3kinase activity in both cell extracts.5. LMP1increased MSK1kinase activity for histone H3in CNE1cellsWestern blotting analysis showed that MSK1was isolated from cell extracts by anti-phospho-MSK1antibody using immunoprecipitation assay. An in vitro MSK1kinase assay revealed that MSK1kinase activity for histone H3was greater in the immunoprecipitated CNE1GL fraction than that from CNE1G cell extracts.6. PD98059or H89suppressed LMP1-induced phosphorylation of histone H3at Ser10Western blotting analysis showed that a relatively low concentration PD98059(10μmol/L) and H89(5μmol/L) significantly inhibited LMP1-induced phosphorylation of histone H3at Ser10, which was a dose-dependent manner.7. Establishment and identification of siRNA-mediated MSK1-knockdown CNE1cell lineThe stably transfected CNE1cell line silencing MSK1was obstained via blasticidin screening. The results from real-time quantitative RT-PCR and Western blotting analysis showed that both mRNA and protein levels of MSK1were suppressed by70%-80%in CNE1cells stably transfected with si-MSK1compared with negative control cells.8. The knockdown of MSK1inhibited LMP1-induced phosphorylation of histone H3at Ser10Consistent to the effect of treatment with H89, LMP1-induced phosphorylation of histone H3at Ser10was effectively inhibited in MSK1knockdown CNE1cells compared with negative control cells. Chapter2The role of MSK1in LMP1-promoted proliferation and transformation of CNE1cellsMethods1. The effect of H89or MSK1knockdown on LMP1-promoted CNE1cell proliferation and transformation was evaluated by CCK-8assay, flow cytometry assay, plate colony formation assay and soft agar assay.2. The effect of H89and MSK1knockdown on AP-1transcriptional activation induced by LMP1was assessed by luciferase reporter gene assay.Results1. H89or MSK1knockdown suppressed LMP1-promoted CNE1cell proliferation and transformationThe CCK-8assay showed that si-MSK1stably transfected cells promoted by LMP1showed a marked decrease in the rate of proliferation after culture for48h compared with negative control cells. Moreover, cell cycle distributions were analyzed by flow cytometry, our data revealed that knockdown of MSK1resulted in less cells occupying S phase and a greater accumulation of cells in G0/G1phase compared with negative control cells (P<0.001). These results indicated that knockdown of MSK1suppressed LMP1-induced cell proliferation because of an impaired Gl-S cell cycle transition. In addition, both plate colony formation assay and soft agar assay indicated that decreased colony numbers as well as colony sizes were observed in cells treated by H89or in MSK1knockdown cells compared with control cells.2. H89and MSK1knockdown inhibited AP-1activation induced by LMP1The results from luciferase reporter gene assay indicated that AP-1transactivation activity induced by LMP1was significantly decreased by treatment with H89in a dose-dependent manner. In addition, AP-1activation was also suppressed by57.21%in MSK1knockdown cells compared with negative control cells (P<0.005) Chapter3TAKl regulated LMP1-induced phosphorylation of histone H3at Ser10by activating p38/MSK1signaling pathwayMethod1. TAK1was immunoprecipitated from the cell extracts with anti-TAKl antibody, and then TAK1kinase activity was determined by immunecomplex kinase assay using MKK6recombination protein as a substrate.2.Active TAK1-TAB1was incubated with histone H3protein, and then tested whether TAK1directly phosphorylated histone H3in vitro.3. Immunofluorescence and immunoprecipitation assays were used to detect whether they have co-localization and interaction between TAK1and phosphorylated histone H3at Ser10.4. The siRNA expression vector targeting TAK1gene was constructed (si-TAK1) and co-transfected with pcDNA6-Myc/HisB plasmid into CNE1cells, then stably transfected CNE1cell line was established via blasticidin screening. The knockdown efficiency was assessed by real-time quantitative RT-PCR and Western blotting analysis.5. The effect of TAK1inhibitor5z-7-oxozeaenol or TAK1knockdown on LMP1-induced phosphorylation of histone H3at Ser10was detected by Western blotting analysis.6. The effect of5z-7-oxozeaenol and TAK1knockdown on AP-1transcriptional activation induced by LMP1were analyzed by luciferase reporter gene assay.7. The effect of5z-7-oxozeaenol and TAK1knockdown on LMP1-induced phosphorylation of ERK1/2, p38, and MSK1was detected by Western blotting analysis.Results1. LMP1increased TAKl kinase activity in CNE1cellsWestern blotting results showed that TAK1was isolated from cell extracts by anti-TAK1antibody using immunoprecipitation assay. An in vitro TAK1kinase assay revealed that elevated TAK1activity was observed in immunoprecipitated fraction from CNE1cells transfected with LMP1plasmid compared with that from mock control cells.2. Active TAK1-TAB1phosphorylated histone H3in vitroActive TAK1-TAB1was incubated with histone H3protein, and immunoblot results showed that TAK1-TAB1directly phosphorylated histone H3at Ser10in vitro in a dose-dependent manner.3. TAKl did not co-localize with phosphorylated histone H3in the nucleusImmunofluorescence results showed that LMP1did not induce nuclear translocation of TAK1, and TAKl did not co-localize with phosphorylated histone H3in the nucleus. The results from co-immunoprecipitation assay revealed that there was no significant interaction between TAK1and phosphorylated histone H3.4. Establishment and identification of siRNA-mediated TAK1-knockdown CNE1cell lineThe stably transfected CNE1cell line silencing TAK1was obstained via blasticidin screening. The results from real-time quantitative RT-PCR and Western blotting analysis showed that both mRNA and protein levels of TAKl were suppressed by70%-80%in CNE1cells stably transfected with si-TAK1compared with negative control cells.5.5z-7-oxozeaenol or TAKl knockdown suppressed LMP1-induced phosphorylation of histone H3at Ser10Western blotting analysis showed that LMP1-induced phosphorylation of histone H3at Ser10was suppressed by treatment with5z-7-oxozeaenol in a dose-dependent manner, whereas phosphorylation of histone H3at Ser28was not affected. Similar results were obtained with TAK1-specific siRNA.6.5z-7-oxozeaenol and TAK1knockdown inhibited AP-1activation induced by LMP1The results from luciferase reporter gene assay showed that AP-1transactivation activity induced by LMP1was decreased by treatment with5z-7-oxozeaenol in a dose-dependent manner. In addition, the knockdown of TAK1inhibited LMP-induced AP-1activation by59.38%compared with negative control cells (P<0.005). 7.5z-7-oxozeaenol and TAKl knockdown repressed LMP1-induced phosphorylation of p38and MSKlWestern blotting analysis showed that the phosphorylation of p38and MSK1promoted by LMP1was repressed by treatment with5z-7-oxozeaenol in a dose-dependent manner, whereas phosphorylation of ERK1/2was not affected. Similar results were obtained with TAK1-specific siRNA. These results indicated that TAK1regulated LMP1-induced MSK1activation via p38MAPK pathway, but not ERK1/2. Conclusions1. The elevated level of histone H3phosphorylation at Ser10was found in NPC tissues. LMP1induced phosphorylation of histone H3at Ser10in CNE1cells.2. Histone H3, especially the phosphorylation at Ser10, played an essential role in LMP1-induced CNE1cells proliferation and transformation through regulating AP-1transcriptional activation.3. As a histone H3kinase, MSK1directly mediated LMP1-induced phosphorylation of histone H3at Ser10in CNE1cells, and regulating LMP1-induced CNE1cells proliferation and transformation. It may consider as a crucial target of therapy in the future.4. TAK1regulated LMP1-induced phosphorylation of histone H3at Ser10by activating p38/MSK1signaling pathway...