The Study Of Inhibiting The Invasion-metastasis Cascade Of HNSCC By Targeting EZH2/STAT3/VEGFR2 Axis

Objective:About half a million head and neck carcinomas are diagnosed annually worldwide,and most of the cases are squamous cell carcinoma(HNSCC).In recent years,comprehensive treatment including surgery,radiation and chemotherapy has made some progress,but the prognosis of some HNSCC is still unsatisfactory.The poor prognosis is associated with lymph node metastasis and tumor recurrence.Epithelial mesenchymal transition(EMT)has been reported to be one of the most important mechanisms of tumor metastasis.Accumulating evidence shows that the enhancer of Zeste Homologue 2(EZH2)is overexpressed in several cancers and functions as an oncogene by activating tumor angiogenesis as well as promoting cell metastasis.EZH2 could enhance signal transducer and activator of transcription 3(STAT3)activity via methylating tyrosine phosphorylation of STAT3.And STAT3 level is positively correlated with EMT.STAT3 could also directly regulate vascular endothelial growth factor receptor 2(VEGFR2,also named as KDR)expression.VEGFR2 signaling activates several downstream signaling pathways including STAT3.However the mechanism of EZH2 regulating tumor cell metastasis in HNSCC is unclear,and there's no reports about EZH2/STAT3/VEGFR2 axis inducing EMT in HNSCC.Method:We chose two head and neck squamous cell lines,UM1 and CAL27.We used DZNEP to inhibit the expression of EZH2,EZH2 overexpression plasmid to elevate the expression of EZH2,meanwhile,siRNA which function as anti-STAT3 and anti-VEGFR2 were used to knockdown the expression of STAT3 and VEGFR2,respectively.There are two parts in the study:Part I: In UM1 and CAL27 cell lines,western blot assay was used to detect the expression of EZH2,STAT3,p-STAT3,VEGF,VEGFR2 and EMT related markers after the treatment of DZNEP or transfection of the plasmid of EZH2,siSTAT3 and siKDR;cell wound scratch assay was used to explore the migration ability;transwell assay was to detect the migration and invasion ability;immunofluorescence(IF)valued the expression of EMT related markers after the treatment of DZNEP or transfection of siSTAT3 and siKDR;furthermore,F-actin was used to value the change of cytoskeleton.Part II: CAL27 xenograft tumor models were established.Each group was treated once every three days for 21 days.Each time we measured the volume of the tumor.IHC was used to investigate the influence of inhibiting EZH2 on the expression of STAT3,p-STAT3,VEGF,VEGFR2 and EMT related markers in HNSCC.Result:EZH2 overexpression elevated the expression of p-STAT3,VEGF,VEGFR2.After using DZNEP,si STAT3 and si KDR to knockdown EZH2,STAT3,VEGFR2 respectively,the expression of EZH2,p-STAT3,VEGF,VEGFR2 were all inhibited.Meanwhile,EMT related markers N-cadherin,Vimentin were also attenuated,while the expression of E-cadherin was elevated.The inhibition of EZH2,STAT3,VEGFR2 had reduced the migration,invasion ability and cellular deformability of tumor cells.The CAL27 xenograft tumor model indicated that knockdown of EZH2 could decrease the expression of p-STAT3,VEGFR2,N-cadherin,Vimentin and increase the expression of E-cadherin.Conclusions:1.The EZH2/STAT3/VEGFR2 axis was involved in EMT in HNSCC cell lines.Inhibition of EZH2,STAT3,VEGFR2 respectively could decrease the expression of the other two proteins,inhibited EMT progression,restrain the migration and invasion ability of tumor cells.2.In xenograft tumor model,EZH2 inhibition decreased the expression of p-STAT3,VEGFR2,inhibited EMT progression.EZH2/STAT3/VEGFR2 axis might serve as a new target for the treatment of HNSCC.