Study On The Mechanism Of RKIP Regulating The Radiosensitivity Of Non-Small Cell Lung Cancer

Objective:According to WHO's International Agency for Research on Cancer criteria,lung cancer is the leading cause of cancer-related morbidity,and non-small-cell lung cancer(NSCLC)accounts for 80%–85% of all lung cancer cases.Approximately 2/3 of the patients were diagnosed with an advanced stage of cancer and the main therapy was radiation treatment combined with chemotherapy.Among these patients,however,only10% achieved a complete response,and the total 5-year survival rate remained a dismal15%.Radiation resistance is the major biological factor that affects the efficacy of radiotherapy.Raf kinase inhibitory protein(RKIP),also known as PEBP-1,was first found in the bovine brain in 1984 and is a member of the phosphatidylethanolamine-binding protein(PEBP)family.RKIP is an inhibitory protein in the Raf/mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase(ERK)pathway and acts via direct interaction with Raf-1 kinase.Therefore,RKIP is a crucial modulator in the MAPK signaling pathway that controls many cellular functions such as cellular proliferation,cell differentiation,cell survival and angiogenesis.RKIP also plays an important role in suppressing tumor progression and metastasis through the coordination of intracellular signals such as the phosphoinositide3-kinase-AKT pathway or the nuclear factor nuclear factor ?B pathway.In addition to modulating various cellular functions,RKIP also proves to be a good prognostic marker for disease-free survival in cancer patients.Survival curves suggest that low RKIP expression is associated with worse prognosis and easier relapse of the tumor focus.In addition to enhancing metastasis and affecting prognosis,RKIP depletion is also closely related to tumor chemotherapy sensitivity and radiosensitivity.However,the mechanism of how RKIP affects the radiosensitivity of non-small cell lung cancer by regulating various signal transduction pathways is not yet clear.The sonic hedgehog(Shh)signaling pathway is highly conserved in the human genome and plays an important role in tissue patterning,cell proliferation and differentiation.It also contributes to tumorigenesis when it is mutated or misregulated.Increasing research evidence has demonstrated that the Shh signaling pathway plays a key role in multiple types of cancers including basal cell carcinoma,pancreatic cancer,colon carcinoma,gastric cancer,breast cancer and non-small cell lung cancer.The hedgehog pathway has been implicated in the regulation of CSCs in various cancers CSCs are highly undiffer-entiated cells that are resistant to both drugs and irradiation.Our hypothesis,which is consistent with the RKIP-regulated changes in radiosensitivity,is that RKIP silencing inappropriately activates the Shh–Gli1 pathway in the stem cells of NSCLC and that such cells,with almost unlimited replicative potential,would significantly improve tumor resistance to irradiation.In order to explore the effect and mechanism of RKIP on radiosensitivity of non-small cell lung cancer,we have conducted the following studies.Methods:1.Specimens were obtained from 93 NSCLC patients of the radiation oncology department of the First Affiliated Hospital,China Medical University,between 2011 and2016(under fibrous bronchoscopy or interventional guided puncture before the treatment).Each of the patients underwent a spiral contrast-enhanced computed tomography(CT)simulation before the treatment began,and radical radiotherapy was administered at 2 Gy per fraction per day,5 days per week,for a total dose of 60 Gy.When the therapeutic dose reached 40 Gy(20 times),the patient underwent another CT scan,which was combined with the initial one on the treatment planning system to compare the tumor volume and evaluate the radiation response.The expression of RKIP in non-small cell lung cancer tissues was detected by immunohistochemistry.The correlation between RKIP expression and clinical pathological factors and prognosis was analyzed.2.Human lung adenocarcinoma cell line A549 and human lung squamous cell carcinoma cell line SK-MES-1 were cultured in vitro and transfected into shRNA by lentivirus.Cells stably transfected with silenced RKIP were obtained by puromycin selection.Western blot was used to detect the expression of RKIP protein in two kinds of cells and their transfected cells.The clonogenic assay was used to evaluate the effect of RKIP on radiosensitivity under different irradiation doses.The survival curve was fitted by the multi-target single-hit model.D0,Dq,SF2,k,N and other biomarkers were calculated to evaluate the effect of silencing RKIP on radiosensitivity.MTT assay wasused to evaluate the effect of RKIP on cell viability under different irradiation doses.The wound-healing assay,Transwell migration and invasion assay were used to evaluate the effect of RKIP expression on cell migration and invasion ability;The effect of RKIP expression on apoptosis was analyzed by TUNEL staining;The effect of RKIP expression on apoptosis and cell cycle distribution was analyzed by flow cytometry;Western blot were used to analyze the effect of RKIP expression on Shh-Gli1 signaling pathway and cancer stem cell marker CD133.3.Statistical analysis was performed using GraphPad Prism and the SPSS 20.0software,and the data were represented as the mean ± SD from at least three independent experiments.The data were tested for normal distribution.Student's t-test was used for comparisons between groups and one-way analysis of variance(ANOVA)used for comparisons in the same group at different doses or time points.The Spearman's rank correlation coefficient was used to determine the correlation between two parameters.P-values <0.05 were considered to be statistically significant.Results:1.RKIP reduction is associated with NSCLC response to irradiation and poor prognosisImmunohistochemistry was performed in 44 radiosensitive NSCLC tissues and 49 radioresistant ones.We observed that RKIP expression was significantly reduced in the radioresistant NSCLC tissues compared with that in the radiosensitive ones.As shown in the table,the expression levels of RKIP were positively correlated with NSCLC radiosensitivity(r=0.378,P<0.001),and tumors with high RKIP expression were frequently radiosensitive.In addition,the expression level of RKIP was related to the T stage of the tumor.Kaplan–Meier analysis revealed that lower RKIP expression in NSCLC tissues is significantly correlated with clearly decreased overall survival.A univariate Cox proportional hazards regression analysis indicated that both clinical T stage and RKIP expression level significantly affected the overall survival of NSCLC patients.A multivariate Cox proportional hazards regression analysis has confirmed that a low expression level of RKIP is an independent predictor for decreased patient overall survival.The relative risk of RKIP expression to prognosis was 1.830.2.RKIP reduction increases the radioresistance of NSCLCcell lines in vitroA methylthiazol tetrazolium(MTT)assay was performed to determine the impact of RKIP depletion on the viability of NSCLC cells under irradiation.The cell viability rate was clearly higher in cell lines with low RKIP expression subjected to varying degrees of radiotherapy(P<0.05);The radiosensitivity of the cell lines was also detected using the colony formation assays.The results showed that NSCLC cell lines with downregulated RKIP expression hada higher survival fraction at 2 Gy irradiation and an obvious increase in quasi-threshold dose compared to RKIP-positive NSCLC cell lines(P<0.05).Taken together,the results revealed that RKIP reduction enhances NSCLC radioresistance in vitro.3.RKIP reduction enhances the migration and invasion of NSCLC cellsTo explore how RKIP reduction could affect the metastatic behaviors of NSCLC cell lines,Transwell chambers were applied to test the migration and invasion abilities of the cancer cells.As shown in the data,cell lines with low RKIP expression have stronger migration and invasion abilities(P<0.05).Furthermore,a wound-healing assay was conducted to determine the influence of RKIP reduction on the metastatic ability of cancer cells.The results revealed that reduced RKIP expression clearly increased the metastatic abilities of NSCLC cells.Overall,RKIP reduction enhanced the migration and invasion of NSCLC cell lines,which means that NSCLC cells with low RKIP expression will exhibit a less satisfactory response to radiotherapy.4.RKIP reduction decreases radiotherapy-mediated apoptosisThe flow cytometry data showed significantly lower radiation-induced apoptosis in the cell lines with lower RKIP expression than in the corresponding control cell lines under different doses of irradiation.The TUNEL assay results were consistent with the flow cytometry data.Fewer apoptotic cells were observed in RKIP low expression cell lines under 4 Gy irradiation.5.RKIP reduction enhances radioresistance by affecting cell cycle progressionCompared with cell lines with high RKIP expression,distribution trends such as the cell cycle arrest of the G2-M phase and the G1 phase depletion caused by irradiation were reversed in the cell lines with reduced RKIP expression.In conclusion,RKIP reduction could promote the radioresistance of NSCLC by preventing cancer cells from entering the G2-M phase,which is relatively sensitive to irradiation.6.RKIP reduction enhances the radioresistance of NSCLCcell lines through activation of the Shh–Gli1–CD133 signaling pathway The Western blot results showed that the expression levels of Smo and Gli1 in the RKIP knock-down cell lines were clearly higher than in their corresponding control cell lines as well as that of CD133.When the Shh signaling pathway status was altered by N-shh and cyclopamine,the RKIP expression level of the cell lines did not change correspondingly,which indicated that RKIP occupied an upstream position in the pathway.Furthermore,when N-shh was added to RKIP knock-down cell lines,SMO,Gli1 and CD133 all increased.This result shows that RKIP reduction could activate the Shh–Gli1–CD133signaling pathway just as N-shh did.Similarly,when cyclopamine was added to the RKIP-KD cell lines,the activated Shh–Gli1–CD133 pathway went silent again,as in the RKIP-positive cell lines.Taken together,these data suggested that RKIP regulates the radiation sensitivity by activating the Shh–Gli1 signaling pathway and increasing the number of CSCs among NSCLC cells.Conclusion: 1.The expression of RKIP in patients with non-small cell lung cancer was associated with the prognosis.The patients with low expression of RKIP have lower sensitivity to radiotherapy.The expression of RKIP is an independent prognostic factor in patients with non-small cell lung cancer.2.The decrease of RKIP expression can reduce the radiosensitivity of non small cell lung cancer cell lines and enhance their migration and invasion ability.The decrease of RKIP can reduce the apoptosis of non-small cell lung cancer cell lines and regulate their radiosensitivity by changing the distribution of cell cycle.3.RKIP may change the radiosensitivity of tumor cells by regulating the Shh-Gli1 signal transduction pathway and the characteristics of cancer stem cells in non-small cell lung cancer.