PTPRD Promotes Autophagy And Radiosensitivity Via Targeting The Jak/STAT3 Pathway In Nasopharyngeal Carcinoma

BackgroundNasopharyngeal carcinoma(NPC),a malignancy derived from nasopharyngeal epithelium,has a particularly high prevalence in Southern China and Southeast Asia.The etiologic factors for NPC include Epstein-Barr virus(EBV)infection,genetic susceptibility,and environmental factors,such as consumption of food with volatile nitrosamines.According to the NCCN guidelines,NPC patients with early stage should receive radiotherapy(RT),while both concurrent chemoradiotherapy with adjuvant chemotherapy or induction chemotherapy followed by concurrent chemoradiation is a crucial development for treating locoregionally advanced disease.However,despite the development of high-precision radiotherapy techniques and new chemotherapeutic drugs,a significant percentage of patients develop resistance to RT,and finally causing to treatment failure.The identification and therapy of radioresistant NPC remains an knotty clinical problem.Therefore,the mechanism under radiation resistance still need to be identified to develop novel strategies for enhancing the efficacy of RT in NPC patients.Protein tyrosine phosphatases(PTPs)play a vital role in regulating cancer cellular functions,such as cell proliferation,adhesion and apoptosis.PTP Receptor-type D(PTPRD)belongs to PTPs family and have been reported to be a tumor suppressor.Previous studies have demonstrated that PTPRD is downregulated by genetic(homozygous deletion,loss-of-function mutation,or copy number loss)or epigenetic(miRNA or methylation)modifications in different types of human cancers,including lung cancer,glioblastoma,breast cancer,melanoma,hepatocellular carcinoma and also head and neck squamous cell carcinoma(HNSCC).It has been reported that PTPRD cooperates with CD44 and β-catenin/TCF signaling to regulate cell migration in colon cancer.Additionally,another study demonstrated that phosphorylated STAT3(pSTAT3)is a substrate of PTPRD and PTPRD downregulation enhances stemness and promotes migration,invasion,and epithelial-mesenchymal transition(EMT)via Jak/STAT3 pathway in breast cancer.However,the molecular function and detailed mechanisms of PTPRD in NPC are still elusive.Research Objectives1.To clarify the expression levels and the underlying regulation mechanism of PTPRD in NPC.2.To explore the biological functions of PTPRD in NPC.3.To identify the interacting proteins and important downstream signaling pathways of P PTRD.4.To explore the molecular mechanism of PTPRD exerting its biological role through interacting proteins and downstream signaling pathways.5.To analyze the correlation between PTPRD and pathological features and survival prognosis of patients with NPC.Methods1.Based on the miRNA microarray、the methylation microarray and mRNA microarray data sets,candidate genes that was down-regulated by miRNA,hypermethylated in its promotor as well as down-expression were screened out,the expression levels of these genes were verified by qPCR and IHC in tissue samples,and the hypermethylated status of these genes were verified by bisulfite pyrosequencing analysis.2.Establishing NPC cell lines stably overexpressing PTPRD(HONE1-PTPRD and HK1-EBV-PTPRD)by the construction and infection of lentiviral vector,establishing NPC cell lines knockdown expressing PTPRD(CNE2-siPTPRD and 58F-siPTPRD)by the construction and transfection of siRNA.CCK8 and clone formation experiments were used to detect the effects of PTPRD on the radiotherapy sensitivity of NPC cells.3.Predicting the miRNA that directly targets PTPRD by using miRNA microarray and bioinformatics,further confirming whether miRNA directly targets PTPRD and regulates its expression by performing luciferase reporter assay,Western blot and qPCR assay.CCK8 and clone formation experiments were used to verify that PTPRD is a functional target of this miRNA.4.Predicting the downstream transcription factor(TF)as substrate for dephosphorylation of PTPRD by using literature retrieval and bioinformatics,the interaction of PTPRD with this downstream TF was detect by Coimmunoprecipitation and immunofluorescence.Predicting the downstream target genes of this TF by bioinformatics analysis,further confirming target genes of this TF by performing luciferase reporter assay and Western blot.And then detecting the alterations in phenotype,function and underlying mechanism after restored this TF expression in NPC cells with stable PTPRD overexpression.5.In vivo experiments(subcutaneous tumor formation in athymic nude mice)were used to verify the relationship between PTPRD and radiotherapy sensitivity;IHC experiments were used to detect the correlation between the expression of PTPRD and the expression of its downstream TF in subcutaneous tumor tissues and clinical patient tissues;Kaplan-Meier survival analysis and Univariate/Multivariate Cox regression analysis were used to explore the relationship between PTPRD and mortality in patients with NPC.Results1.Based on the combined analysis of the miRNA microarray(GSE42945),the methylation microarray(GSE52068)and the mRNA microarray(GSE12452),six candidate genes that are hypermethylated in nasopharyngeal carcinoma and may be targeted by miRNAs are initially selected.PTPRD was identified as the most differentially candidate gene and further confirmed by real-time quantitative PCR analysis in 5 primary fresh NPC samples and 5 non-cancerous fresh nasopharyngeal samples.PTPRD protein level was significantly decreased in 117 NPC tissues when compared with 20 normal nasopharyngeal epithelium tissues by IHC,and the hypermethylated status of PTPRD was confirmed in 7 NPC tissues when compared with 7 normal nasopharyngeal epithelium tissues by bisulfite pyrosequencing analysis.2.In order to explore the role of PTPRD in NPC,we constructed HONE1 and HK1-EBV cells stably expressing PTPRD or control vector,whereas knocked down PTPRD gene expression in CNE2 and 5-8F cells with PTPRD-specific siRNA oligos.The infection and transfection efficiency were validated using real-time RT-PCR or Western blot assays.CCK8 assay and colony formation assays were performed in NPC cells exposed to radiation(0-10 Gy),and the results indicate that overexpression of PTPRD increases radiation sensitivity in NPC cells.3.The analysis of miRNA microarray study and bioinformatics prediction show that miR-454-3p interacts with the 3’UTR of the PTPRD mRNA well.The luciferase reporter assays experiment showed that miR-454-3p directly targeted and inhibited the transcriptional activity of PTPRD.Western blot and qPCR showed that the expression level of PTPRD was down-regulated in miR-454-3p overexpressing NPC cells.CCK8 and clone formation experiments show that miR-454-3p regulates the radiotherapy sensitivity of nasopharyngeal carcinoma cells by targeting PTPRD.4.GSEA enrichment analysis showed that the Jak/STAT3 signaling pathway was enriched in the PRPRD low expression group.Western blot experiments showed that the expression level of PTPRD was negatively correlated with STAT3 phosphorylation.Co-immunoprecipitation experiments showed that p-STAT3 antibody can precipitate Flag-PTPRD protein,while Flag antibody can precipitate p-STAT3 protein.Immunofluorescence experiments showed co-localization of PTPRD and STAT3 in nasopharyngeal carcinoma cells.This indicates that PTPRD directly targeted and dephosphorylated p-STAT3 in nasopharyngeal carcinoma cells.5.Bioinformatics predicts that the transcription factor STAT3 may target autophagy-related genes ATG5 and BECN1.Western blot experiments showed that the expression of ATG5 was negatively correlated with STAT3 phosphorylation,while the expression of BECN1 stayed not alter.The luciferase reporter assays showed that pSTAT3 directly binded to the A and B sites of the ATG5 promoter region and inhibited transcriptional activity of ATG5.6.Western blot assay and Immunofluorescence assay showed that overexpression of PTPRD can promote autophagy of NPC cells exposed to radiation(0-10 Gy).We restored p-STAT3 expression in NPC cells with stable PTPRD overexpression and subsequently exposed radiation,Western blot showed that ATG5 was down-regulated,LC3 Ⅱ/LC3 Ⅰ levels were down-regulated,P62 was up-regulated,BECN1 remained unchanged,and radiotherapy-induced autophagy phenotype was partially reversed.7.NPC cells were inoculated subcutaneously into athymic nude mice and exposed to radiotherapy,the results shown that PTPRD promotes radiotherapy sensitivity of NPC cells in vivo.IHC assays showed that PTPRD expression was negative correlation with p-STAT3 expression in subcutaneous tumor tissues as well as clinical patient tissues.Downregulation of PTPRD and hyperphosphorylation of STAT3 were associated with poor overall survival and progression-free survival in patients with NPC.Conclusions1.PTPRD is downregulated in clinical NPC tissues with promoter hypermethylated.2.Overexpression of PTPRD sensitizes NPC cells to radiation,miR-454-3p directly targets PTPRD and regulates radiation sensitivity in NPC cells.3.PTPRD directly targets and dephosphorylate p-STAT3,promotes p-STAT3dependent ATG5 transcription and radiation-induced autophagy in NPC cells,thereby promoting the radiosensitivity of NPC cells.4.PTPRD promotes radiosensitivity of NPC cells in vivo,and its expression is negative correlation with p-STAT3 expression.5.Patients with PTPRD low expression and STAT3 high phosphorylation exhibited poor OS and DFS.Elevated PTPRD expression in the primary nasopharyngeal carcinoma was an independent unfavorable prognostic factor for overall survival(OS),disease-free survival(DFS)in nasopharyngeal carcinoma patients.