Radiation-induced JAK2/STAT3 Activation Mediates Lipid Metabolism Reprogramming To Promote Radiation Resistance Of NSCLC

ObjectiveRadiotherapy is an effective local treatment,which plays a more and more important role in the treatment of non-small cell lung cancer(NSCLC).However,the local recurrence and progression of NSCLC caused by radiotherapy resistance are still one of the main reasons for failure of radiotherapy.Exploring metabolic reprogramming and its regulatory mechanism in radiotherapy of NSCLC is helpful to elucidate the metabolic mechanism of radiotherapy resistance,to find new effective targets to increase radiotherapy sensitivity,and to overcome NSCLC radiotherapy resistance finally.Methods1.With clinical conventional fractionated radiotherapy,we constructed human lung cancer cell lines(A549,H520,H460)irradiated with repeated equidifferent gradient dose(2Gy×20F,2Gy×30F,2Gy×40F).Three equidifferent gradient dose cell lines were obtained after stably passaging.The radiotherapy sensitivity detected by clone formation assay,EDU cell proliferation assay,neutral comet assay and y-H2AX immunofluorescence staining.2.We also establish NSCLC subcutaneous xenografts in nude mice.The mice were randomly divided into two groups:radiotherapy group(dose and fraction are:10Gy/5F)and control group.Then we recorded and summarized the changes of tumor volume of subcutaneously xenografts.3.Two parental/resistant cell lines H460,H460-40F?H520,H520-40F and subcutaneous xenograft(control group,the 0h of radiotherapy and 72h after radiotherapy)were performed transcriptome sequence.The differential gene expression in each group was detected and the GO function cluster analysis was carried out.The results of transcriptome sequence in vitro and in vivo were analyzed integratedly to search the metabolic pathway that most likely to cause radiotherapy resistance in metabolic reprogramming.4.The expression of key metabolic targets were further verified by western blotting,the relationship between the expression of fatty acid ?-oxidation rate-limiting enzyme CPT1A?glyceride metabolism rate-limiting enzyme ATGL and fatty acid synthesis rate-limiting enzyme FASN in fatty acid metabolism and the prognosis of NSCLC was analyzed with TCGA database.The ability of lipid synthesis and storage of lipid droplets was detected by oil red O staining.The cell oxygen consumption rate(OCR)was detected by Seahorse experiment.After intervention with fatty acid oxidation inhibitor Etomoxir and fat triglyceride lipase inhibitor Atglistatin,the radiosensitivity and proliferation efficiency of cells were further detected.5.The parental and radiation resistance cell line transcriptome sequencing result was further analysed by KEGG pathway enrichment.The expression of related signaling pathway molecule sequenced in vivo and in vitro verifed by Western blotting.After overexpression and knockdown JAK2 gene,the expression of lipid metabolism targets ATGL and CPT1A were detected by Western blotting.Luciferase reporter experiment and chromatin immunoprecipitation assay(CHIP)were used to verified whether JAK2/STAT3 signaling pathway transcriptional regulated ATGL and CPT1A.With JAK2 small molecule inhibitor AZD1480,the expression of lipid metabolism target enzymes were detected by Western blotting;oil red O staining and Seahorse test detected the effect of AZD1480 on lipid metabolism;colony formation and EDU cell proliferation experiment detected NSCLC cells radiosensitivity after AZD1480 intervention.Results1.We successfully established the lung cancer cell model of radiation survival/resistance by conventional fractionation of equidifferent gradient dose in A549,H520 and H460.In A549,H520,H460 and their derived cell lines,the radiation resistance and proliferation rate of each radiation survival/resistance cell line was dose-dependent.Compared with the parental cells,with the increase of the radiation dose received by these cells,DNA double-strand breakage(DSB)gradually decreased,suggesting that the cell lines radiation resistance to more and more tolerant.we also successfully constructed the subcutaneous xenograft tumor model of A549 and H520 in nude mice,and summed up the macroscopic rules between radiation therapy and the inherent tumor growth in nude mice:the tumor volume increased most significantly at the end of radiotherapy(2Gy×5F),and then began to shrink,and the tumor volume remained unchanged or slightly increased on 72h after radiotherapy.The results of RNA-seq in vivo and vitro showed that there are many significant differently expressed genes in lung cancer cells after different doses of irradiation.The functional clustering enrichment GO pathways is mainly manifested in various lipid metabolism,glucose metabolism,JAK/STAT,MAPK and TGF-? signal pathways.2.The expression of glucose and lipid metabolic proteins detected by Western blotting,the results were consistent with RNA-seq.TCGA survival curve indicates that the high expression of CPT1A,ATGL and FASN,which are the key genes of fatty acid metabolism,are related to poor prognosis of NSCLC.Oil red O staining showed that fat deposition was significantly increased in a dose-dependent manner in each dose of radiation survival/resistance cell lines compared with parental cell lines.With the increase of the radiation dose received by these cells,the OCR increased gradually.After intervention of Etomoxir and Atglistatin,the radio sensitivity and proliferation efficiency of resistant cells were reversed.3.The transcriptomic sequencing result of parental and radiation resistance cell line was further analyzed with KEGG pathway enrichment,and the expression of the signaling pathway in vitro and vivo was verified by Western blotting.After JAK2 was overexpressed and knocked down,the expression of lipid metabolism targets ATGL and CPT1A was evaluated by Western blotting.Luciferase report assay and CHIP demonstrated that lipid metabolism targets ATGL and CPT1A were transcriptional regulated by JAK2/STAT3 signaling pathway.Then Western blotting was used to verify the influence of JAK2 small molecule inhibitor AZD1480 on the expression of key enzymes in lipid metabolism.Oil red O staining and Seahorse test were used to detect the regulation of AZD1480 on lipid metabolism.After AZD1480 intervention,the radiosensitivity and proliferation efficiency of NSCLC cells were determined by clony formation and EDU cell proliferation assay.Conclusion1.The equidifferent gradient dose radiation resistant lung cancer cell lines established in this study provides a preclinical research model for exploring the mechanism of dose-dependent radiation resistance.2.Radiation dose-dependent fatty acid metabolism reprogramming enhances the radiation resistance of NSCLC cells.3.Radiotherapy activates the JAK2/STAT3 signaling pathway in NSCLC cells.ATGL and CPT1A were transcriptional upregulated through JAK2/STAT3 signaling pathway.4.Small molecule inhibitors targeting the JAK2/STAT3 pathway and key enzymes in lipid metabolism can effectively sensitize radiotherapy,which may be a potential treatment for reversing radiotherapy resistance of NSCLC.