AURKA-CXCL5 Promotes Radiation Resistance In Non-small Cell Lung Cancer By Regulating Autophagy

Objective: Aurora kinase A(AURKA)regulates apoptosis and autophagy in various diseases and has shown promising clinical effects.Nevertheless,the complex regulatory mechanism of AURKA and autophagy in non-small-cell lung cancer(NSCLC)radiosensitivity remains to be elucidated.The aim of the study was to explore the role of AURKA in regulating autophagy of non-small-cell-lung cancer and to investigate the mechanism of AURKA in regulating radiosensitivity in vivo and in vitro.Methods: Human normal lung epithelial cell HBE and non-small cell lung cancer cell lines A549,H1299,PC9,HCC827,H292 and H460 were used as the research objects.Western blot was used to detect the expression of AURKA in the above cell lines;Immunohistochemical staining of lung cancer tissue microarray and survival analysis were performed to explore the relationship between AURKA expression and the prognosis of lung cancer;The expression of AURKA in two types of lung cancer samples(lung adenocarcinoma and lung squamous cell carcinoma)and the correlation between AURKA and clinical parameters were detected by TCGA database.Small interfering RNAs(si RNAs)targeting AURKA were constructed to explore the effects on the growth and proliferation of NSCLC cells.AURKA stable knockdown cell lines were constructed and injected into nude mice to explore the effect of AURKA on tumor growth in vivo.Functional studies were carried out to explore the effects on NSCLC cell cycle,apoptosis,migration and invasion ability by targeting AURKA.Western blot was used to detect the changes in the expression of AURKA with or without radiotherapy.Clonogenic survival assays,γ-H2 AX assays,cell cycle and apoptosis assays were performed to evaluate the effect of AURKA-depletion on the sensitivity of NSCLC cells after receiving radiation.Changes in autophagy and sensitivity of NSCLC cells to radiation caused by AURKA-depletion was explored.CXCL5,a downstream effector of AURKA,was screened via RNA transcriptomics sequencing.Rescue experiments were performed to verify whether AURKA’s biological effects depend on CXCL5.Dual luciferase assays and Ch IP assays were carried out to explore the transcriptional regulation of AURKA on CXCL5.Mice experiments were performed to investigate the effect of combination treatment on tumor growth,Immunohistochemical staining was performed to analyze the expression of AURKA,CXCL5 and key proteins of autophagy signal.Results:(1).Western blot showed that the expression of AURKA in NSCLC cell lines was significantly higher than that of normal lung epithelial cell;Immunohistochemistry and survival analysis of lung cancer tissue microarray revealed that AURKA was mainly expressed in the nucleus,and its overexpression was significantly related to a poor prognosis and to tumor stage and lymph node metastasis in NSCLC.(2)Silencing of AURKA effectively inhibited cell growth and proliferation of A549 and H460 cells as well as the tumorigenesis of mice in vivo.(3)After silencing AURKA,the proportion of S-phase cells was decreased significantly,the ratio of apoptotic cells was increased and cell migration and invasion capability were significantly weakened.(4)Western blot revealed that the AURKA expression level was increased after 8Gy-irradiation in NSCLC cells.Moreover,the increase in A549,H1299,HCC827 and H460 cells was statistically significant.Clonogenic survival assays showed that inhibition of AURKA obviously increased the radiosensitivity of A549 and H460 cells.G2/M phase was significantly blocked in the AURKA knockdown combined with radiotherapy group.The ratio of apoptotic cells in the combination treatment group was significant increased,in addition,γ-H2 AX focis were significantly increased in the combination of X-ray and AURKA inhibition groups compared with that in X-ray treatment alone groups.(5)Cytotoxic autophagy was enhanced in si-AURKA cells,and the increase of autophagy level in cells with combination treatment(si AURKA combined with 8Gy radiation)was more pronounced.It was manifested as an increase in autophagy flux,an increase in the level of LC3II、p-Beclin 1,and a decrease in the level of P62.Moreover,inhibition of autophagy by ATG5 knockdown could partially reverse the radiosensitization effect and cell proliferation limitation induced by AURKA silencing.(6)CXCL5 was confirmed as the downstream target of AURKA,and rescue experiments revealed that the biological effects of AURKA was depend on CXCL5.(7)Cytotoxic autophagy was enhanced in si-CXCL5 cells,and the increase of autophagy level in cells with combination treatment(si CXCL5 combined with 8Gy radiation)was more pronounced.It was manifested as an increase in autophagy flux,an increase in the level of LC3II、p-Beclin 1,and a decrease in the level of P62.Moreover,inhibition of autophagy by ATG5 knockdown could partially reverse the radiosensitization effect and cell proliferation limitation induced by CXCL5-depletion.(8)Dual luciferase assays and Ch IP assays showed that AURKA could mediate the up-regulation of CXCL5 by promoting the ability of NF-κB to transcriptionally regulate CXCL5.(9)Combination of AURKA depletion and radiotherapy has better tumor killing effects than radiotherapy alone in vivo.Conclusions: In general,we revealed the role of the AURKA-CXCL5 axis and autophagy in regulating the sensitivity of NSCLC cells to radiotherapy,which may provide potential therapeutic targets and new ideas for combatting NSCLC resistance to radiotherapy.