Inhibition Of Nuclear Factor-kB Improves Sensitivity To Irradiation And EGFR-TKIs,and Decreases Irradiation-induced Lung Toxicity

BACKGROUND AND OBJECTIVEResistance to radiotherapy and to EGFR tyrosine kinase inhibitors(EGFR-TKIs),as well as therapy-related lung toxicity,are serious problems in the treatment of lung cancer.NF-?B has been reported to be associated with radio-resistance.Therefore,we evaluated its effects on sensitivity to irradiation and to EGFR-TKIs;irradiation induced lung toxicity;and the effects of irradiation on sensitivity to EGFR-TKIs.We used IKKP inhibitor IMD 0354 or p65 depletion to explore their effects on sensitivity to irradiation and to EGFR-TKIs in vitro and in vivo.We evaluated the efficacy of IMD 0354 in a radiation-induced pulmonary-fibrosis mouse model.Irradiation enhanced activation and expression of MET and therefore suppressed the sensitivity of lung cancer cells to irradiation or EGFR-TKIs.Inhibition of NF-?B by IMD 0354 or by p65 depletion reversed irradiation-induced MET activation and increased the sensitivity of lung cancer cells to irradiation,to EGFR-TKIs and to the combination thereof in vitro and in vivo.In addition,IMD 0354 significantly reduced lung toxicity in a murine model of irradiation-induced pneumonia and lung fibrosis.These findings indicated that NF-?B inhibition can improve sensitivity to irradiation and to EGFR-TKIs and can decrease irradiation-induced lung toxicity in lung cancer.METHODS1.IrradiationWe exposed cells(A549 and PC9)or mice to conventional X-rays(1-12 Gy,3 Gy/minute)emitted by a linear particle accelerator used for human radiotherapy operated at 6 MV and at room temperature.2.Cell growth assayWe measured cell growth by the(MTT)dye reduction method.We plated tumor cells into 96-well plates.After incubation for 24 hours,we added various reagents to each well and continued incubation for another 72 hours.3.Western blottingWe separated protein aliquots via(PVDF)membranes.We washed the membranes 3 times;incubated them with blocking solution for 1 hour at RT;and then incubated them overnight at 4?C with the primary antibodies.We then incubated the membranes with species-specific(HRP)-conjugated secondary antibodies for 1 hour at RT.We visualized immunoreactive bands.4.Ribonucleic acid(RNA)interference assayWe used Duplexed Stealth RNAi against p65 and MET,and Stealth RNAi Negative Control Low GC Duplex for RNA interference(RNAi)assays.We seeded cells into 6-well plates;24 hours later,we transfected them with short interfering RNA(siRNA)using Lipofectamine 3000.We confirmed knockdown of p65 and MET by Western blotting.5.Apoptosis analysisWe seeded cells in 6-well plates.After overnight incubation,we changed the media and then added DMSO or the indicated drugs 1 hour before irradiation.We harvested both cells floating in the media and adherent trypsinized cells in a single tube 48 hours after radiotherapy,pelleted the cells and washed them once with PBS.We measured apoptotic cells with a fluorescein isothiocyanate(FITC)Annexin V apoptosis detection kit and assayed them on a BD FACS Calibur flow cytometry using CellQuest Pro software to analyze(BD Biosciences).6.Cell immunofluorescenceWe plated PC9 and A549 cells that had been depleted of p65 in chamber slides,incubated them for 24 hours and then pretreated them with or without HGF(20 ng/ml)1 hour before irradiation respectively(2 or 10 Gy).We determined the number of residual foci 24 hours after irradiation.Then,we fixed cells and incubated them with a primary anti-y-H2AX antibody.Next,we washed off the primary antibody and applied a secondary,FITC-conjugated antibody to the slides.We visualized deoxyribonucleic acid(DNA)damage with a BX51 fluorescence microscope.7.Xenograft studies in nude miceWe injected suspensions of PC9 and A549 cells into the backs of 5-week-old male BALB/c-nu/nu nude mice.Ten days after cell inoculation(tumor diameter>5 mm),we randomized mice(n = 4-5 per group)and administered drugs to them once daily by oral gavage(AZD9291,5 mg/kg/d or 2 mg/kg/d)and intraperitoneal injection(IMD 0354,20 mg/kg/day).The therapeutic irradiation dose was 3 Gy/day ×3 days and 2 Gy/day × 3 days in the A549 and PC9 xenograft models,respectively.We measured tumor volume and mouse body weight twice per week.8.Irradiation-induced lung injury modelWe divided 80 adult male C57BL6 mice into 3 groups:control(n = 20),and irradiation with or without IMD 0354 therapy(n = 30 per group).After anesthetizing mice with chloral hydrate,we locally irradiated the whole thorax at 12 Gy at a dose rate of approximately 95 cGy/minute.Mice were exposed to irradiation once,after which we treated them with or without IMD 0354.They were then killed at several time points for further experiments.9.Histological analyses of tumorsWe deparaffinized formalin-fixed,paraffin-embedded tissue sections(4?m thick).We detected proliferating cells by incubating tissue sections with Ki-67 antibody,and we determined apoptotic cells using TUNEL assay.For MET detection,the sections were reacted with anti-total-MET antibody(Abcam).All sections were stained with hematoxylin and eosin(H&E)for routine histological examinations.10.Histological analyses of lung tissueWe stained paraffin-embedded sections of pulmonary tissues with H&E and Masson's trichrome and fibronectin and Collagen I for F4/80 morphological studies per the manufacturer's instructions.For the quantitative histological analysis,we used a numeric fibrotic scale(Ashcroft score).11.Statistical analysisData were presented as the mean ± standard deviation(SD)of 3 independent experiments.We performed all statistical analyses using GraphPad Prism software version 4.01(GraphPad Software,La Jolla,California,US).We analyzed between-group differences by 2-tailed Student's t tests or 1-way analysis of'variance(ANOVA).P<0.05 was considered statistically significant.Results:1.Irradiation induces activation of NF-?B,MET and EGFR and upregulates MET expression,while NF-?B inhibition can reverse activation of NF-?B and MET but not of EGFRFirst,we examined changes to several cell signaling pathways within 24 hours after irradiation.Irradiation enhanced the activation of MET,EGFR and I?B?,as well as MET expression.We then evaluated the effects of NF-?B inhibition on the aforementioned irradiation-activated signaling pathways.Stimulation with 1 or 3 ?M IMD 0354 suppressed the activated MET and IKBa but not the activated EGFR or its downstream signaling molecules.Irradiation induced the translocation of p65 from the cytoplasm to the nucleus,while IMD 0354 successfully diminished this phenomenon in PC9 and A549 cells2.Inhibition of NF-?B signaling increased radiosensitivity and irradiation-induced apoptosisWe determined the effect of depleting NF-?B by pharmacological inhibition or siRNA knockdown on radiosensitivity of NSCLC cells.Although IMD 0354 did not at first enhance irradiation-induced ?-H2AX,a second round of irradiation increased?-H2AX levels.This was further confirmed by our cell apoptosis assay.Interestingly,NF-?B depletion by p65 siRNA increased the levels of y-H2AX and cell apoptosis in response to the first round of irradiation.On the other hand,p65 knockdown directly downregulated the expression of MET and phosphorylated MET,not affect EGFR expression or activation.These results suggested that inhibiting NF-?B signaling could increase the radiosensitivity of NSCLC cells by inhibiting MET but not EGFR activation.3.HGF induced MET activation independently and partially reversed the effects of irradiation-induced apoptosis and DNA damage after p65 knockdownWe evaluated whether HGF could influence the effect of p65 depletion on MET activation and radiosensitivity.As expected,the depletion of p65 after irradiation suppressed MET expression.In spite of this,however,HGF activated MET,as well as reduced irradiation-induced cell apoptosis and DNA fracture damage after p65 depletion.These results strongly indicated that HGF-stimulated MET activation decreased cell radiosensitivity that had been augmented by NF-?B depletion,further verifying that NF-?B depletion enhanced radiosensitivity via MET signaling.4.IMD 0354 increased radiosensitivity in lung cancer in vivoWe next examined the effect of combined irradiation and IMD 0354 in an A549 subcutaneous-tumor model.Although IMD 0354 or irradiation monotherapy had a minimal or moderate effect on hindering tumor growth,the combination thereof caused tumors to regress dramatically.Through Ki-67 and TUNEL assays,we found that the combination therapy dramatically inhibited cell proliferation and increased cell apoptosis compared with irradiation alone.These results demonstrated that the combination of IMD 0354 and irradiation may be a promising strategy for NSCLC treatment.5.Irradiation decreased susceptibility to EGFR-TKIs,which could be overcome by inhibition of NF-?B or METWe used EGFR-mutant PC9 cells pretreated with irradiation to examine susceptibility to AZD9291,a third-generation EGFR-TKI.PC9 cells exhibited less sensitivity to AZD9291 after irradiation compared with non-treated cells,whereas downregulation of p65 reversed this hyposensitivity.We detected a similar effect with MET siRNA.These data revealed that irradiation-induced activation of the NF-?B/MET pathway resulted in lung cancer cells being less sensitive to an EGFR-TKI.To further verify these results,we explored the effect of irradiation,AZD9291 and the combination thereof in a PC9 subcutaneous-tumor model.Consistent with the in vitro findings,although a low single dose of AZD9291 prevented tumor enlargement,combined irradiation with AZD9291,it did not result in further tumor regression.In contrast,the combination of irradiation,AZD9291 and IMD 0354 resulted in marked tumor regression.More importantly,MET activation was significantly increased in both the irradiation group and the irradiation +AZD9291 group.Interestingly,triple therapy successfully inhibited MET activation,even though it included irradiation and AZD9291(Fig.5E).These in vivo data strongly suggested that NF-?B/MET signaling plays an important role in radioresistance and irradiation-induced EGFR-TKI resistance,and that irradiation +AZD9291 + IMD 0354 triple therapy may be a promising approach to the treatment of NSCLC with EGFR mutation.6.The effect of IMD 0354 on irradiation-induced lung injury in a murine modelWe next explored its effect on irradiation-induced lung injury in a murine model.We observed skin injury in irradiated mice;IMD 0354 remarkably delayed time to occurrence and severity of skin injury.On the other hand,it diminished irradiation-induced translocation of p65 from the cytoplasm to the nucleus in the lung tissue of mice treated by irradiation with or without IMD 0354.In the irradiation-induced lung injury model,we observed bleeding in lung tissue and recruitment of inflammatory cells at 2 and 6 weeks after irradiation,respectively.At 24 weeks after irradiation,we detected severe pulmonary fibrosis by H&E,Masson,fibronectin and Collagen I staining.Surprisingly,IMD 0354 dramatically prevented bleeding,inflammatory-cell accumulation and pulmonary fibrosis after irradiation.Macrophages have been reported to play a key role in pulmonary fibrosis;we evaluated the effect of IMD 0354 on macrophages after irradiation.In the murine model,IMD 0354 markedly inhibited monocyte and macrophage migration out of the alveolar septum at 12 weeks and accumulation in areas of lung consolidation at 24 weeks after irradiation.Conclusion:1.Irradiation induces activation of NF-?B,MET and EGFR and upregulates MET expression,while NF-?B inhibition can reverse activation of NF-?B and MET but not of EGFR.2.Inhibition of NF-?B signaling increased radiosensitivity and irradiation-induced apoptosis.3.HGF induced MET activation independently and partially reversed the effects of irradiation-induced apoptosis and DNA damage after p65 knockdown.4.IMD 0354 increased radiosensitivity in lung cancer in vivo.5.Irradiation decreased susceptibility to EGFR-TKIs,which could be overcome by inhibition of NF-?B or MET.6.The effect of IMD 0354 on irradiation-induced lung injury in a murine model.