Osteopontin Upregulates HIF-1? Expression Through The PI3K/AKT Pathway To Promote Radiotherapy Resistance In Esophageal Cancer

The new data of esophageal cancer evaluated in China was nearly half of the world,with an estimated 307,000 new cases and 283,000 deaths in 2018,while the number is estimated 572,000 and 508,000 all over the world.Neoadjuvant chemoradiotherapy followed by surgery as a preferred treatment has improved the survival of patients suitable for operation.For localized advanced esophageal cancer patients not suitable for operation,the standard therapy is radiation therapy plus concurrent chemotherapy.However,5-year survival rates are relatively low.The clearest evidence for hypoxia-mediated resistance to radiation therapy has been observed in esophageal squamous carcinoma(ESCC).Oxygen-mimetic nitroimidazoles acted to "fix”radiation damage selectively kill hypoxic tumor cells.While many individual trials failed to meet statistical end points.Hypoxia inducible factor-1?(HIF-1?)is the main factor for radiation resistance induced by hypoxia.It has been reported that osteopontin(OPN)can induce HIF-1? and vascular endothelial growth factor(VEGF)expression in breast cancer,resulting in increased cell invasion and metastasis.Our research team registered a clinical trial of combined sodium glycididazole(CMN)and standard chemo-radiation therapy for non-operation ESCC patients to evaluate the radiosensitization and side effects.To detect ESCC patient's status of hypoxia and the correlation with the radiosensitizing effect of the esophageal cancer,we designed the protocol with pretreatment plasma OPN to evaluate the correlation of pretreatment hypoxia status with radiosensitization effects in the study.The influence and mechanism of OPN on radiotherapy sensitivity of esophageal cancer were also verified in esophageal cancer cells in order to provide theoretical basis for individualized radiotherapy sensitization of patients with locally advanced esophageal squamous cell carcinoma.ObjectiveThis study analyzed the correlation between OPN expression and hypoxia microenvironment(HIF-1,VEGF)in patients with locally advanced esophageal squamous cell carcinoma,compared objective response rate(ORR)and survival data of radiotherapy sensitization in patients with different OPN expression,and evaluated the predictive value of OPN.The effect of OPN-induced HIF-1? expression on radiosensitivity and specific signaling pathways were elucidated in vitro in esophageal carcinoma cells.In vivo experiments,tumor-bearing mice were conducted to compare the differences in the growth curves of tumors with different OPN expressions after radiotherapy.Methods1.Patients with locally advanced esophageal cancer were prospectively enrolled in this study.Before treatment,serum OPN was detected,and immunohistochemistry was used to detect the expression of OPN,HIF-1? and VEGF.PET-CT examination was performed.The patients were randomly divided into two groups:glycidazole sodium combined with radical concurrent chemoradiotherapy(sensitization group)and radical concurrent chemoradiotherapy group(control group).The primary end point was ORR,and the secondary end point was survival.2.The data related to esophageal squamous cell carcinoma were extracted through The Cancer Genome Atlas(TCGA)database,and the differences in OPN expression between esophageal cancer and normal tissues,as well as esophageal cancer of different stages were compared.Gene Set Variation Analysis(GSVA)was used to analyze the signal pathways significantly related to OPN high expression.3.TE-1 cells with low OPN expression and KYSE150 cells with high OPN expression were screened.The apoptosis ability and the clonal formation ability of the two cells at baseline after radiotherapy were detected.The OPN transfected TE-1 oe-OPN cells,TE-1 NC cells,KYSE150 shOPN cells and KYSE150 NC cells were constructed respectively.The differences in the clonogenesis of esophageal carcinoma cells after OPN up-regulation and silencing were detected after radiotherapy.TUNEL assay was used to detect the difference of OPN up-regulated and silenced esophageal carcinoma cell apoptosis after radiotherapy.Protein western blot assay was used to detect the difference of apoptotic protein expression in esophageal cancer cells after OPN up-regulation and silencing.To determine the effect of OPN on HIF-la expression,different doses of recombinant human OPN were used under normal oxygen conditions,and the expression of HIF-1? and VEGF were detected by protein western blot assay in the two groups of cells.In order to further evaluate the molecular mechanism,TE-1 cells and KYSE150 cells were cultured in recombinant human OPN,PI3K and MAPK pathway inhibitors were added,and HIF-la expression was detected by protein western blot assay to determine which pathway molecule mainly affects HIF-1? expression.The phosphorylation of the pathway and its downstream molecules were further detected by western blot assay.Since hypoxia is a major regulatory factor for HIF-la expression,HIF-1? expression after OPN up-regulation and silencing was detected by protein western blot assay under hypoxia,and phosphorylation of pathway molecules was detected by protein western blot assay to clarify the synergistic effect of OPN and hypoxia on the regulation of HIF-1?.Subsequently,the pathway molecular inhibitor and the recombinant human OPN were applied respectively to rescue the clone formation ability of cells after radiotherapy.TUNEL detected the recovery of apoptosis ability of each cell line after radiotherapy.4.In this study,tumor formation in nude mice was carried out using TE-1 OE-OPN cells,KYSE150 shOPN cells and blank control cells constructed in vitro.Tumor diameter was recorded and tumor volume was calculated after radiotherapy for transplanted tumor,and growth rate and volume of tumor with different OPN expressions were compared after radiotherapy to verify the effect of OPN on radiotherapy sensitivity of esophageal cancer.Immunohistochemistry was used to detect the expression of HIF-1?,P-Akt,and VEGF in tumor tissues with different OPN expressions,and to analyze the correlation between OPN and tumor hypoxic microenvironment.ResultsFrom 2016 to 2018,a total of 78 patients were enrolled,66 of whom were included in further statistical analysis.Patients were randomly divided into CMN combined with concurrent chemoradiotherapy(sensitization group)and concurrent chemoradiotherapy(control group),with no significant difference in baseline characteristics between the two groups.The ORR of the sensitization group was 90.32%,while that of the concurrent chemoradiotherapy group was only 68.57%(P=0.027).Untill the time of follow-up up to November 2019,there was no statistically significant difference in overall survival(P=0.234)or progression-free survival(P=0.4635)between the sensitized group and the control group.The results of OPN subgroup analysis showed that the median serum OPN value of the enrolled patients was 94.87ng/mL.According to Receiver Operating characteristic Curve(ROC),the serum OPN threshold value is 84.72ng/ml.The ORR rate of the low OPN expression group was 89%,while that of the high OPN group was 72%(P=0.095).After the application of CMN,ORR in patients with high serum OPN expression was significantly improved compared with the control group(P=0.033).However,there was no significant difference in ORR between the patients in the low OPN expression group and the control group after the application of CMN(P=0.529).Survival analysis showed that OS(P<0.001)and PFS(P<0.001)of patients with high serum OPN were significantly lower than those with low serum OPN.After the application of CMN,there was no significant difference in OS between the groups with high and low OPN expression(P>0.05),but:in the control group,there was still a significant difference in OS between the groups with high and low OPN expression(P<0.001).The expressions of OPN,HIF-1?,VEGF in the esophageal carcinoma tissues were detected.The results showed that HIF-1? expression range and intensity were also significantly increased in patients with esophageal squamous cell carcinoma with high OPN expression,and VEGF expression was slightly increased,showing a significant positive correlation.Further analysis of OPN expression in different tissues of patients with clinical objective remission rate differences.The ORR rate of the OPN high expression group was significantly lower than that of the OPN low expression group(P<0.001).After the application of CMN,ORR of patients in the OPN high expression group could be significantly improved(P=0.020).Survival analysis showed that the OS time of Patients with high tissue OPN expression was significantly lower than that of patients with low OPN expression(P<0.001).After the application of CMN,there was no significant difference in OS between different patient groups with OPN expression(P>0.05).However,in the concurrent chemoradiotherapy group,OS of patients with different OPN expression was still significantly different(P<0.001).Univariate and multivariate analyses showed that serum OPN,tissue OPN and tissue HIF-1? expression were all predictors of poor prognosis in patients with esophageal squamous cell carcinoma.OPN gene(SPP1)expression analysis in the TCGA database of esophageal cancer tissues and normal tissues showed that SPP1 gene expression was significantly higher in esophageal cancer tissues than in normal tissues(P<0.001).SPP1 gene expression in patients with locally advanced esophageal cancer was higher than that in patients with early stage and metastasis.The survival rate of patients with high SPP1 expression level was significantly lower than that of patients with low SPP1 expression level,P=0.004.When SPP1 expression was increased,it was significantly correlated with the activation of hypoxia and PI3K/AKT pathways.Four esophageal squamous cell lines with different OPN expression were screened,and TE-1 cells with low OPN expression and KYSE150 cell lines with high OPN expression were selected.After radiotherapy,the ability of cell clone formation was significantly reduced and apoptosis was significantly increased in both groups.TE-1 oe-OPN cells and TE-1 NC cells with high OPN expression were successfully constructed,and KYSE150-shOPN and KYSE150 NC cells were simultaneously constructed.The clone formation rate of TE-1 oe-OPN was significantly increased at the dose of 2Gy,4Gy,6Gy and 8Gy,and the apoptosis rate was significantly decreased after radiotherapy.The clonal forming ability of KYSE150 shOPN cells was significantly decreased,and the apoptosis rate was significantly increased after radiotherapy.Apoptosis promoting protein Bax was decreased and apoptosis inhibiting protein Bcl-2 was increased after radiotherapy in TE-1 oe-OPN cells.After radiotherapy,the expression of Bax protein and Bcl-2 protein in KYSE 150-shOPN cells were significantly increased and decreased.Under normal oxygen and hypoxic conditions,TE-1 cells and KYSE150 cells were treated with human recombinant OPN at different doses,respectively.HIF-1?and VEGF protein expression increased,and showed a positive correlation with OPN dose.HIF-1? expression in stable cells was consistent with the trend of OPN.PI3K inhibitor LY294002 can significantly reduce OPN-induced HIF-1 expression,and the PI3K/AKT pathway p-ILK,P-Akt,and P-P65 activities are enhanced followed with high expression of OPN.Rescue experiments showed that after the application of LY294002,the clone formation ability of TE-1 oe-OPN cells after radiotherapy was reduced,and the apoptosis level was increased,and the cell was restored to the baseline of TE-1 NC.The addition of recombinant human OPN in KYSE150 cells restored the clonal formation ability and apoptosis level to KYSE150 NC level after radiotherapy.In vivo experimental results showed that the growth rate and tumor volume of KYSE150 shOPN tumor after radiotherapy were significantly lower than those of the KYSE150 control group and the KYSE150 radiotherapy group.The growth rate and tumor volume of TE-1 oe-OPN tumor after radiotherapy were significantly greater than that of THE TE-1 tumor radiotherapy group.Immunohistochemical results showed that the expression intensity and range of HIF-1?,VEGF,P-Akt and CD34 in TE-1 oe-OPN tumor were significantly enhanced compared with those in TE-1 cell tumor.The expression of HIF-1?,VEGF,P-Akt and CD34 in KYSE150 tumor was significantly decreased.ConclusionOPN expression is associated with hypoxic microenvironment(HIF-1? and VEGF)in patients with locally advanced esophageal squamous cell carcinoma.Patients with high OPN expression had poor ORR and OS,and the application of CMN sensitization could significantly improve the objective remission rate and survival.OPN has predictive value for the efficacy and prognosis of concurrent chemoradiotherapy sensitization in patients with esophageal squamous cell carcinoma.In esophageal cancer TE-1 cells and KYSE150 cells,the high expression of OPN can lead to the enhanced phosphorylation of PI3K/AKT pathway in esophageal squamous cell carcinoma cells,and induce the significant increase of HIF-1? protein expression,leading to radiotherapy resistance.In hypoxic state,OPN induced HIF-1?hyperexpression was enhanced.PI3K inhibitor LY294002 can improve the radiotherapy resistance of esophageal squamous cell carcinoma cells caused by high OPN expression.OPN and hypoxia microenvironmental factors HIF-1?,VEGF,p-AKT and CD34 showed consistent expression trends in the nude mouse esophageal cancer model.The high expression of OPN can promote the radiotherapy resistance of esophageal squamous cell carcinoma tumor-bearing mice.