The Roles And Mechanisms Of Hes1 In Nasopharyngeal Carcinoma

Background and objectiveNasopharyngeal carcinoma（NPC）is a type of head and neck cancer,which is common in Southern China.The etiology study of NPC showed that EB virus infection and environmental factors（tumor promoters and/or carcinogens,such as TPA,butyric acid,nitrosamines,etc.）are likely to play an important role in the carcinogenic process.But so far,the etiology and pathogenesis of NPC is not yet clear,which brings great difficulties on the NPC primary prevention（prevention of etiology）.Therefore,it is still significant and urgent to continue to explore the etiology and pathogenesis of NPC.Currently,radiotherapy is the main treatment for NPC.With the development of IMRT technology,the local control rate of NPC patients has been significantly improved.But there are still 20-30%of patients with advanced NPC who suffer recurrence and distant metastases within five years.Recurrence and distant metastasis is the main cause for treatment failure in NPC.Therefore,it is very important to find new therapeutic target for NPC,to reduce recurrence and distant metastasis and to improve the therapeutic effect on NPC.Cancer stem cells（CSCs）are a small group of subpopulations cells existing in tumor tissues with stem cell properties.CSCs have important biological characteristics as follows:（1）self-renewal;（2）differentiation potential;（3）survival in a particular microenvironment;（4）highly tumorigenic;（5）resistance to chemotherapy and so on.CSCs are the root cause of tumorigenesis occurrence,tumor growth,recurrence,metastasis and treatment resistance.Tumor recurrence and metastasis is the main reason of death in cancer patients.Therefore,it is crucial to effectively remove CSCs in order to obtain the desired anti-cancer effect.The new anti-cancer therapeutic strategies for targeting CSCs bring new hope to cure cancer.It still has a long way for the treatment of targeting CSCs to get into clinical stage from search stage.Metastasis is the leading cause of death in cancer patients.However,the molecular mechanisms of metastasis are currently still poorly understood.It is particularly important to clarify the key mechanisms of tumor invasion and metastasis and to establish the appropriate way to block tumor metastasis.It may be a hope for cure cancer that to establish an efective molecules intervention in clinical practice before relapse and metastasis of the primary tumor.Epithelial-mesenchymal transition or transformation（EMT）is a complex developing process characterized by loss of cell adhesion,repression of E-cadherin expression,and increased cell motility.EMT is an important contributor to the invasion and metastasis of epithelial-derived cancers.It is very important to clarify the mechanisms of EMT for tumor at the early stage of diagnosis and treatment.miRNAs are small non-coding RNA molecules,which can participate in lots of physilollgical and pathological processes of organisms through a certain way.Our previous studies found that miR-9 targets CCNG1 and inhibits proliferation of NPC cells.miR-9 involved in inhibiting NPC CSCs self-renewal.Our studies confirmed that Hesl is a target gene of miR-9.Hesl may mediate miR-9 involved in inhibition NPC CSCs self-renewal function.Transcription factor Hesl（hairy and enhancer of split-1）is a protein that is encoded by the Hesl gene,and is the mammalian homolog of the hairy gene in Drosophila.Hesl is one of the seven members of the Hes gene family（Hesl-7）.Hes genes code nuclear proteins that suppress transcription.This protein belongs to the basic helix-loop-helix（bHLH）family of transcription factors.It is a transcriptional repressor of genes that require a bHLH protein for their transcription.There are three conserved domains in Hes genes that impart transcriptional functions:the bHLH domain,the Orange domain,and the WRPW motif.Hes genes differ from other bHLH factors in that they have a proline reside in the middle of the basic DNA binding region.This proline has been proposed to give Hes proteins unique DNA binding capacity.While most bHLH factors bind to the E-box consensus sequence（CANNTG）that is present in the promoter region of target genes,Hes factors bind more preferentially to the Class C site or N box（CACNAG）.The Orange domain serves to regulate the choice of bHLH heterodimer partners.The C-terminal WRPW domain inhibits transcription.Recently,it has been reported that Hesl/Hes5 genes inhibits differentiation via down-regulating Hashl and promotes proliferation in cervical carcinoma cells.The Hairy and Enhancer of Split homologue-1（HES-1）mediates the proliferative effect of 17beta-estradiol on breast cancer cell lines.HES-1 inhibits 17beta-estradiol and heregulin-betal-mediated up-regulation of E2F-1.Hes1 is closely related with adult stem cells and CSCs.Studies have shown that,Hesl is often used as a stem cell marker,which is essential in the adult stem cells（e.g.,hematopoietic stem cells,intestinal stem cells,melanoma stem cells,pancreatic stem cells,etc.）of self-renewal and proliferation,maintaining sternness,survival and/or inhibition of stem cell differentiation.miR-199b cleares medulloblastoma CSCs by targeting Hesl.TNF increases oral squamous cell carcinoma CSCs by activating Notch-Hesl.Hesl maintains colon CSCs sternness and plays an important role in the development and evolution of colon cancer.Study on the relationship between Hes land NPC CSCs has reported that Hesl co-expressed with Oct4 and Sox2 in NPC cells.Our previous study found that Sternness-related genes expression were down-regulated by RNA interference Hesl expression in NPC cells.According to the above information and our previous studies,we hypothesized that,Hesl may also involve in regulating NPC CSCs biological characteristics（such as self-renewal,etc.）.But further experiments are still needed to confirm it.Based on the above studies,we identified characteristics of Hesl expression in NPC cells and tissues.We observed its effects on the biological characteristics of NPC cells by altering the expression levels of Hesl and exploring the possible mechanisms and regulatory networks.And this study may lay the foundation for in-depth understanding the molecular mechanisms of pathogenesis of NPC and identification for new molecular therapeutic targets.MethodsPart I Identification of expression characteristics of Hesl in NPC tissues and cell lines.1.Quantitative real-time PCR and Western bolt were used to identify the expression of Hesl in NPC cell lines（CNE1,CNE2,HNE1,SUNE1,HONE1,5-8F,6-10B and C666-1）and immortalized nasopharyngeal epithelial cells NP69,respectively.2.Identification of Hes1 expression in 29 noncancerous nasopharyngeal tissues and 103 NPC tissues by immunohistochemistry and its significance.Part II Roles and mechanisms of Hesl in NPC cell proliferation and NPC CSCs1.Establishment of Hes1 stably over-expression or knockdown NPC cells.Particles expressing vector along with packaging plasmids（psPAX2 and pMD2.G）were transfected into HEK293T cells（maintained in 10%FBS）using Lipofectamine 2000 reagent（Invitrogen）according to the manufacturers’ instruction.72h after transfection,virus supernatant was harvested from these cells,and subsequently used to infect NPC CNE2 and SUNE1 cells.Quantitative real-time PCR and Western bolt were used to identify the expression of Hesl in these cell lines,respectively.2.RNA isolation and quantitative real-time PCRFor mRNA analyses,total RNA from HCC cells was extracted using Trizol Reagent according to the protocol provided by the manufacturer.Total RNA was reversely transcribed with the PrimeScript RT reagent Kit（TaKaRa）,and quantitative real-time PCR（qRT-PCR）for the expression of mRNA analysis was performed using SYBR Green qRT-PCR master mix（TaKaRa）as described using GAPDH for normalization on a Stratagene Mx3005P qRT-PCR System.All of the samples were normalized to internal controls and fold changes were calculated through relative quantification（2-△△Ct）.3.CCK-8 assay and colony formation assay were used to detect cell growth and proliferation.4.Cell-cycle analysis detected by flow cytometry.5.Stemness-related genes expression in NPC cells with Hesl over-expression or knockdown detected by Western blot.6.Tumor sphere formation and ratio of side populations（SP）cells detected by flow cytometry to evaluate the "stemness" of NPC cells.7.Subcutaneous tumor formation in nude mice:Cells were injected subcutaneously and tumors formed in subcutaneous were measured every 3 days,tumor volumes were calculated as follows:Dxdxd/2（D meant the longest diameter and d meant the shortest diameter）.8.Histopathology of xenograft tumors:The tumor sections were under H&E staining and IHC staining using antibodies against Hesl,BrdU,Ki-67 and P21.9.Immunohistochemical staining of Hes1,Nanog and Oct4 in the same NPC samples.10.ChIP was performed by using anti-Hes1 antibody or IgG antibody to identify Hes1 binding sites on the PTEN promoter in CNE2 cells.11.Establishment of PTEN and Hes1/PTEN stably over-expression or knockdown CNE2 cells.Western blot were used to identify protein expression of genes.Flow cytometry was used to analyze the ratio of SP cells in these cell lines.Part III Roles and mechanisms of Hesl involved in EMT,invasion and metastasis in NPC1.EMT-related genes expression affected by Hesl in NPC cells detected by qRT-PCR and Westen blot,respectively.2.Effects of Hes1 over-expression or knockdown on cell migration and invasion were evaluated using a transwell chamber and a Matrigel-coated Boyden chamber,respectively.3.Subcapsular liver transplantation in nude mice:Cells were injected into hepatic subcapsular of nude mice.Tumor formation of subcapsular liver transplantation and lung sections were under H&E staining.The number of spontaneous lung metastatic lesions in mice was analyzed by counting ten serial sections from each sample.4.Clinical associations of Hesl with EMT marker expression by immunohistochemistry.ResultsPart I Identification of expression characteristics of Hes1 in NPC tissues and cell lines.1.Identification the expression of Hes1 in NPC cell linesQuantitative real-time PCR were used to identify the expression of Hesl in NPC cell lines and immortalized nasopharyngeal epithelial cells NP69,respectively.The results of Welch analysis showed that the expression of Hesl in 8 cell lines was significantly different from each other（F=48.630,P=0.000）.Dunnett T3 analysis showed that the expression of Hes1 in SUNE1 and 5-8F was higher than that in NP69（P<0.05）,and Hesl expression in 6-10B with low metastasis ability were lower than that in NP69（P<0.05）.Western blot showed that,compared with NP69 cells,the expression of Hesl were up-regulated in the CNE1,CNE2,HNE1 and SUNE1.Hes1 expression in 6-1 OB was lower than that in NP69,and there were no significantly difference between C666-1 and NP69.2.Expression of Hes1 in 29 non-cancerous epithelial tissues and 103 NPC tissuesImmunohistochemistry was used to identify the expression of Hesl in 29 noncancerous nasopharyngeal tissues and 103 NPC tissues.High Hesl expression was found in 57.3%（59/103）cases of NPC,which is higher then 34.5%（10/29）cases of non-cancerous nasopharyngeal epithelial tissue samples（χ2=4.715,P=0.030）.High cytoplasmic Hesl staining in tumor-adjacent epithelium and strong nuclei Hesl in tumor cells were mostly observed.Strong nuclei and cytoplasmic Hes1 expression were both observed in some NPC tissues.3.Correlation between the clinicopathological features and expression of HeslThe relationship between the clinicopathological features and Hesl expression in NPC was analyzed with Pearson χ2 test.No significant associations were found between Hesl expression and age（P=0.882）,gender（P=0.162）,histological type（P--0.079）and tumor recurrence（P--0.718）.Interestingly,we observed that Hesl expression was positively correlated with T classification（P=0.018）,N classification（P=0.006）,distant metastasis（M classification,P=0.021）and clinical stage（P=0.010）of NPC patients.Wiih the evolvement of TNM stage and clinical stage,the positive rate of Hesl expression in NPC tissues increased.4.Survival analysis of 103 NPC patients with different Hesl expressionUnivariate analysis showed that the overall survival rate of NPC patients with Hesl high expression was significantly lower than the low Hesl expression group（P=0.003）.In addition,N classification（P=0.019）,distant metastasis（M classification,P=0.000）,clinical stage（P=0.010）and tumor recurrence（P=0.000）can also have an effect on the overall survival rate of NPC patients.No significant associations were found between the overall survival rate and age,gender,histological type and T classification（P>0.05）.Cox multivariate regression analysis showed that distant metastasis（P=0.01 1）and tumor recurrence（P=0.000）can be used as an important independent factor to affect survival and prognostic of NPC patients.But there are not statistically significant for Hesl expression as an independent factor（p=0.053）.Part II Roles and mechanisms of Hesl in NPC cell proliferation and NPC CSCs1.Establishment of Hes1 stably over-expression or knockdown NPC cells.We established Hesl stably over-expression or knockdown NPC（CNE2 and SUNE1）cell lines.Expression of Hesl in these cells was detected by qRT-PCR.The results showed that the levels of Hesl in Hesl over-expression CNE2 and SUNE1 cells were higher than that in the control vector cells（t=8.965,21.017,P=0.001,0.000）.While the levers of Hesl in the Hesl knockdown cells were lower than that in the vector cells（t=-10.756,-13.287,P=0.000,0.000）.Western blot showed the expression levels of Hesl protein were consistent with the expression levels of mRNA.2.Hesl promotes cell growth in NPC cells.CCK-8 assay showed that Hesl-expressing CNE2 and SUNE1 cells grew faster than the control cells（F=97.234,29.590,P=0.000,0.000）,whereas the proliferation of NPC cells with Hesl knockdown was inhibited（F=48.112,27.193,P=0.000,0.000）.Flat cloning formation experiments also indicated that the ability of proliferation was significantly increased in Hesl-expressing groups（t=11.533,5.859,P=0.000,0.004）.While the number of colony of NPC cells with Hesl knockdown was decreased（t=-9.717,-4.990,P=0.001,0.008）.In sum,the above two experiments illustrates that Hes1 promotes the proliferation of NPC cells in vitro.3.Hesl affects cell-cycle distribution.We studied the effects of Hesl on cell cycle using FACS.The results showed that compare to the control group,the ratio of S phase in Hesl-expressing cells was significantly increased（t=6.713,5.889,P=0.003,0.004）,while the ratio of S phase in NPC cells with Hesl knockdown was significantly decreased（t=-6.042,-2.821,P=0.004,0.048）,indicating that Hesl may promoted cell proliferation by increasing the number of S phase cell.4.Effects of Hesl on NPC CSCsHesl up-regulates the expression of stemness-related genes in NPC cells.Western blot showed that Nanog and ABCG2 were up-expressed in Hesl-expressing CNE2 and SUNE1 cells,and down-expressed in Hesl knockdown cells.The ability of tumor sphere formation was enhanced in Hesl-expressing CNE2 and SUNE1 cells（r=12.559,15.336,P=0.000,0.000）,while the number of spheres was decreased in Hesl knockdown CNE2 and SUNE1 cells（t=-18.601,-21.870,P=0.000,0.000）.The ratio of SP cells in Hesl-expressing CNE2 and SUNE1 cells was increased（t=30.559,16.286,P=0.000,0.000）,while the ratio was decreased in Hesl-silenced cells compared to the control（t=-6.030,-32.199,P=0.024,0.000）.5.Hes1 promotes tumorigenesis of CNE2 cells in vivo.Hesl-expressing CNE2 cells（LV-Hesl）and the control cells（LV-con）were subcutaneously injected into the dorsal flank of nude mice at a dose of 1.0×106（n==6）,1.0×105（n=8）,1.0×104（n=10）and 1.0×103（n=10）,respectively.The tumors of the group with Hesl-expressing were larger than the controls.All the mice injected with 1.0×106 cells developed tumors.When injected with 1×105,1×104 and 1×103 Hesl-expressing NPC cells,76.5%of the nude mice（26/34）developed tumors,whereas only 38.2%of these mice（13/34）did so when injected with the control cells.In addition,the first palpable tumor in the Hesl groups injected with 1×103 cells appeared within 16 days,while it happened at 34 days after injected the control cells.The tumor sections were under H&E staining and IHC staining using antibodies against Hesl,BrdU,Ki-67 and P21.We found that Hesl-expressing tumors cells showed higher positive Hesl,BrdU and Ki67 staining and lower p21 positive staining than the controls.Hence,Hesl promotes tumorigenesis of CNE2 cells in vivo.6.The relationship of Hesl expression with stem cells-like markers in 103 NPCsImmunohistochemistry showed the expression of Hesl was highly correlated with the expression of ALDH1,Oct4 and Nanog in NPC tissues（P<0.05）.7.Hesl represses PTEN by binding to the PTEN promoter locus.To explore the mechanism of Hesl in NPC,we want to identify the target gene of Hesl.Firstly,we found that Hes1 over-expression down-regulated PTEN expression in CNE2 and SUNE1 cells（t=-17.195,-26.532,P=0.000,0.000）,while Hesl interference up-regulated PTEN expression（t=3.529,5.174,P=0.024,0.007）.Then we performed ChIP assay to determine whether Hesl could bind to the PTEN promoter locus in CNE2 cells.The full lenth of PTEN promoter sequences is（-2000bp₊1032bp）.Precipitated DNA was amplified by PCR using primers specific for regions A-D（A:₁492bp_-1343bp,B:-1186bp_-1085bp,C:-385bp_-286bp,D:+119bp₊278bp）.Quantitative ChIP analysis showed Hesl could bind to the PTEN promoter locus（A,p=0.000,B,p=0.025,C,p=0.002,D,p=0.021）in CNE2 cells.Taken together,all these results strongly suggest that Hesl represses PTEN by binding to the PTEN promoter locus.8.Hesl increases NPC CSCs by repressing PTEN and activating PTEN/AKT pathway.To investigate whether PTEN mediated the promoting effect of Hes1 on NPC CSCs,first we established PTEN and Hesl/PTEN stably over-expression or knockdown in CNE2 cells named LV-PTEN,LV-shPTEN,LV-Hesl+LV-PTEN and LV-shHes1+LV-shPTEN,respectively.Then we identified the expression of related genes by Western blot and analyzed the ratio of SP cells by Flow cytometry,respectively.The results showed that Hesl over-expression up-regulated the expression of p-AKT and sternness-related genes and increased the number of tumor spheres and SP cells,similar to these effects induced by the PTEN knockdown in CNE2 cells.As expected,PTEN over-expression in Hesl-expressing CNE2 cells or cells treated with LY294002（used as an AKT inhibitor）could reverse these alters induced by Hesl in CNE2 cells.While Hesl knockdown down-regulated the expression of p-AKT and sternness-related genes and decreased the ratio of SP cells,similar to the suppressive effect induced by the PTEN over-expression.PTEN silenced in Hes1 knockdown CNE2 cells could rescues the suppressive effect induced by Hesl knockdown in CNE2 cells.Take together,Hesl increases NPC CSCs by repressing PTEN and activating PTEN/AKT pathway.Part III Roles and mechanisms of Hesl involved in EMT,invasion and metastasis in NPC1.Hes1 regulated EMT-related genes expression in NPC cellsqRT-PCR analysis showed the significantly reduced expression of epithelial markers（E-cadherin and β-catenin）,and the significantly increased expression of mesenchymal markers（N-cadherin and vimentin）in Hesl-expressing CNE2 and SUNE1 cells（p<0.05）.While when Hesl was silenced in CNE2 and SUNE1 cells,E-cadherin and β-catenin were up-regulated and N-cadherin and vimentin were down-regulated.Western blot analysis validated the decrease in E-cadherin,β-catenin and a-catenin,and the increase in N-cadherin and vimentin in Hes1-expressing CNE2 and SUNE1 cells.When Hesl was silenced in CNE2 and SUNE1 cells,the expression of epithelial markers（E-cadherin,β-catenin and a-catenin）were increased and mesenchymal markers（N-cadherin,vimentin and Fibronectin）were down-regulated.Therefore,our results illustrate that Hes1 induces EMT-like cellular marker alterations in NPC cells.2.Hesl promotes NPC cells migration and invasion in vitro.Transwell assay and Boy den chamber assay displayed that Hesl-expressing CNE2 and SUNE1 cells exhibited significantly enhanced cell migration and invasion compared with vector control cells.While CNE2 and SUNE cells with Hesl knockdown demonstrated the significantly reduced cell migration and invasion ability compared with control cells.Collectively,Hes1 promotes NPC cells migration and invasion in vitro.3.Hesl increases lung metastasis in nude mice.To further evaluate the effects of Hes1 on metastasis in vivo,we injected 1×106 CNE2 cells infected with LV-Hes1 or the control plasmid（LV-con）subcapsularly into the livers of nude mice,8 mice for each group.Mice were sacrificed at 30 days after injection.All the mice developed tumors in liver at the end of the experiment.The tumors and lungs were then photographed and the sections were under H&E staining.We found 7/8 mice in LV-Hesl group developed lung metastases,while 3/8 mice in control group developed lung node metastases.The number of spontaneous lung metastatic lesions in mice was analyzed by counting ten serial sections from each sample.The results showed the number of spontaneous lung metastatic lesions in LV-Hesl group was larger than the control group（t=2.478,P=0.036）.It suggested that Hesl can promote tumor metastasis in vivo.4.Hesl triggers EMT and induced cell motility&invasion by down-regulation of PTEN and activating PTEN/AKT/GSK3β/Snail pathway.It has been illustrated that PTEN/AKT/GSK3β pathway participates in regulating EMT.And we has previous confirmed that PTEN is a target inhibition gene of Hes1 in NPC cells.This prompted us to detect the expression of PTEN/AKT/GSK3(3 pathway related genes.Western blot analysis validated p-AKT and p-GSK3β was up-regulated in Hesl-expressing CNE2 and SUNE1 cells,and down-regulated in Hesl knockdown CNE2 and SUNE1 cells.To elucidate whether the Hesl-induced EMT and inhibition of cell motility&invasion were mediated by repression of PTEN in NPC cells,we performed gain-of-function and loss-of-function studies.We examined the expression of EMT-related genes in NPC cells.When PTEN was over-expressed in CNE2 cells,the expression levels of epithelial markers（E-cadherin and a-catenin）increased,while the levels of mesenchymal markers（N-cadherin,vimentin,Fibronectin and Snail）decreased,compared with that in control cells,similar to the MET effect induced by the Hesl knockdown in CNE2 cells.PTEN silenced in Hesl knockdown CNE2 cells could reverse the MET effect induced by Hes1 knockdown in CNE2 cells.Subsequently,we evaluated motile and invasive capacity of HCC cells by transwell migration assay and Boy den invasion assay.The results showed that PTEN interference induced cell migration and invasion,compared with the control cells,similar to the effect induced by Hesl over-expression in CNE2 cells.And PTEN over-expression decreased cell migration and invasion,similar to the suppressive effect on the cells with Hesl knockdown.In addition,both PTEN over-expressed in Hesl-expressing CNE2 cells and PTEN silenced in Hesl knockdown CNE2 cells could rescues the effect induced by Hesl over-expression or knockdown in CNE2 cells.Taken together,these results provide evidence suggesting that Hesl induces EMT,invasion and metastasis by down-regulation of PTEN and activation of PTEN/AKT GSK3β/Snail pathway.5.The relationship of Hesl expression with EMT markers in 103 NPCsImmunohistochemistry showed the expression of Hesl was negatively correlated with the expression of E-cadherin（P=0.006）,and positively correlated with the expression of Fibronectin（P=0.000）and Snail（P=0.001）in NPC tissues.Conclusions1.The expression of Hesl is up-regulated in NPC and correlates statistically with the malignant status of NPC.2.Hesl promotes cell proliferation and increases NPC CSCs by repressing PTEN and activating PTEN/AKT pathway.3.Hesl induces EMT,invasion and metastasis by down-regulation of PTEN and activation of PTEN/AKT GSK3β/Snail pathway.