Based On Proteomics Studies On Signaling Pathway And Protein Of Radiosensitivity In NSCLC

Objective: Locally advanced non-small cell lung cancer(LA-NSCLC)is a highly heterogeneous disease.Radiotherapy is the main local treatment method for this group of diseases,but the overall effect is poor,the heterogeneous of radiotherapy response is obvious.Now,there is a lack of biological information to guide radiotherapy strategies,individualized treatment is more difficult.The purpose of this study was to explore the signal pathways and proteins related to radiosensitivity of NSCLC by combining the predictive analysis of relapse patterns in patients with LA-NSCLC after radical radiotherapy and chemotherapy and the proteomics information of cell lines and human samples,in order to lay a biological foundation for LA-NSCLC precise treatment.Methods:(1)Retrospectively collected data on patients with LA-NSCLC who underwent radical chemoradiotherapy in our hospital from 2013 to 2016.The study analysis endpoint was the first failure phenotype after radical chemoradiotherapy.Applying elastic-anet-cox regularization regression analysis,three-fold nested cross-validation,selecting the optimal model with better generalization ability,based on this,a nomogram prediction model of the first failure with local recurrence is established.ROC curve,calibration curve and K-M curve were used to verify the model(2)29 human NSCLC cell lines were subjected to protein quantitative detection by DIA mass spectrometry,biochemical analysis was used to screen proteomics information and related signal pathways related to radiotherapy sensitivity.At the same time,surgical paraffin sections of NSCLC patients with local recurrence after locally radical radiotherapy were collected for protein quantitative detection by DIA mass spectrometry.They were divided into two groups according to the differences in local control after radiotherapy,and the differences in proteomic expression between the two groups were analyzed to obtain proteomic information and signal pathways related to radiotherapy.The results of the NSCLC cells and patients' paraffin samples were further verified.On the one hand,we used cell lines and paraffin tissue samples to verify the expression and function of related proteins.On the other hand,according to the keywords(("radiation tolerance" [Mesh])OR "radiosensitivity")and("neoplasms" [Mesh]),we reviewed studies on radiosensitivity proteins in the past 10 years in the Pubmed database.The protein information in previous studies was extracted and called as "proteomics of previous studies",GO and KEGG functional annotation analysis was performed to obtain radiotherapy sensitivity-related signal pathways in previous studies to verify the results of this study.Finally,combining the cell line,paraffin pathway information in this study with previous studies,NSCLC radiosensitivity-related signaling pathways and proteins were obtained.Results:(1)With a median follow-up of 12 months,337 patients were included in the analysis.Among them,49% of patients occurred first failure with local recurrence,35%of patients occurred first failure with distant metastasis,11% patients had no recurrence until the last follow-up and 5% patients was lost to follow-up after 3 monthes after finishing radiotherapy.Predictive model including 5 factors(sex,weight loss,location,treatment mode and chemotherapy cycles)was finally built with the best performance.The first-year local recurrence predicting of the model performance was the best,the AUC of 3-fold nested cross-validation in test set was 0.691,which was better than any factors alone.The calibration curve revealed a satisfactory consistency between the predicted failure pattern and the actual observations.Based on these five characteristics of the prediction model,the test set data can be effectively divided into high-risk,medium-risk and low-risk populations with local recurrence(p = 0.016).(2)Analysis of the correlation between the quantitative expression of the proteome of the cell line and radiosensitivity factor-SF2,we obtained 27 proteins that were significantly correlated with SF2.Analysis of the correlation between paraffin proteome and local control showed that compared with the LPFS <1.5year group,16 proteins were up-regulated and 28 proteins were down-regulated in the LPFS ? 1.5year group.Signal pathway enrichment results suggest that there are 38 overlapping signal pathways between the targetted protein enrichment signal pathway of the cell line and the paraffin sample.Among the overlapping pathways,the top three signaling pathways including proteoglycans in cancer,focal adhesion and regulation of actin cytoskeleton,were verified in cell lines and paraffin samples.In addition,among the818 radiosensitivity-related protein enrichment signaling pathways reported in the past 10 years,there are 35 overlap with the radiosensitivity-related signaling pathways of the NSCLC cell line in this study,15 overlapped signaling pathways between previous studies and this stdudy.Finally,we obtained 22 NSCLC radiosensitivity-related proteins: IQGAP1,IQGAP2,ILK,HSPA2,BAG2,PAK1,MMP9,STAT1,STAT5 B,YWHAZ,GABARAPL2,NCBP1,SEC61 G,ISY1,HIF-1?,m TOR,EGFR,Akt1,ATM,Notch1,GLUT1,TGF?.Conclusions:(1)Clinicopathological parameters can predict the risk of local recurrence for the first failure of LA-NSCLC radical radiotherapy and chemotherapy patients.The prediction model based on these clinicopathological parameters has a certain predictive value in clinical practice.However,the prediction performance of the prediction model is slightly worse,and more information,such as biology,may be needed to improve the prediction efficiency of the model.(2)After 29 NSCLC cell lines and 13 NSCLC patients' paraffin samples were analyzed,we obtained a network of signal pathways related to radiosensitivity of NSCLC.The top three signaling pathway are proteoglycans in cancer,bacterial invasion of epithelial cells,focal adhesion,regulation of actin cytoskeleton.Inaddition,we combined our study with previous studies on radiosensitivity related signaling pathways and proteins,selected 22 proteins for clinical detection of NSCLC radiosensitivity-related protein panels: IQGAP1,IQGAP2,ILK,HSPA2,BAG2,PAK1,MMP9,STAT1,STAT5 B,YWHAZ,GABARAPL2,NCBP1,SEC61 G,ISY1,HIF-1?,m TOR,EGFR,Akt1,ATM,Notch1,GLUT1,TGF?.