Clinical Proteomics Analysis And Pre-clinical Therapeutic Target Verification Investigation Of Small Cell Lung Cancer

PART ? Clinical proteomics analysis of small cell lung cancer Objective:Small cell lung cancer(SCLC)is a highly invasive lung neuroendocrine tumor.It has the characteristics of rapid proliferation,early metastasis and drug resistance.The classification of SCLC has experienced a long process from the classic/variant based on morphology to the neuroendocrine/non-neuroendocrine based on the expression of neuroendocrine indicators,and then to the definition of transcription factor subtypes based on transcriptomics.However,the current classification system can not explain all the phenomena in the process of occurrence and development of SCLC,and its role in predicting the prognosis of patients and guiding clinical treatment is very limited.The purpose of this study is to elucidate the proteomic characteristics of SCLC and to establish a proteomic stratification method for treatment guidance of SCLC.Methods:This study included 75 cases of formalin fixed paraffin embedded small cell lung cancer specimens from 2012 to 2018 in our hospital.Through protein extraction,mass spectrometry,protein identification and quantitative analysis,the expression profile of SCLC characteristic proteins was analyzed.Based on the protein expression profile of SCLC,non negative matrix factorization method was used to cluster analysis for molecular typing.Combined with clinical information,the chemotherapy response and prognosis of each subtype were analyzed,and the molecular characteristics of different subtypes were analyzed to mine therapeutic targets.Results: A total of 11158 proteins were identified.After quality control and data filtering,445 non negative matrix factorization(NMF)consistent clustering proteins were obtained.NMF clustering results showed that they were related to the prognosis of SCLC patients and could be divided into three subgroups: S-I(n = 28,37%),S-II(n = 20,27%)and s-iii(n= 27,36%).S-I,S-II and s-III subtypes had a stratified relationship with the gradual deterioration of overall survival rate,and the K-M survival curve showed significant difference.Proteomic typing of small cell lung cancer is independent of transcriptome typing.High proliferative activity is a common characteristic of the three SCLC types.S-I type SCLC has strong DNA repair ability,good prognosis and the best response to chemotherapy;S-II subtype is an immune subtype with rich extracellular matrix and significant immune response,and macrophage infiltration is the most significant in S-II type.S-II and S-I have similar natural course,but S-II is unfavorable after chemotherapy;S-III has aggressive biological behavior,the worst prognosis and insufficient response to chemotherapy.Based on the common signaling pathways and high expression proteins among the three subtypes,the possible therapeutic targets include CDK1,CDK2,E2F1 and Sp1,etc.;for S-I subtype,DHPs and UBA5 may be potential targets.For S-II subtype,we identified 11 candidate genes,including DPEP1,VWF,MAOB,CASP1,TBXAS1,CEACAM6,C1 R,ICAM1,DPYD,S100A10 and NNMT.For potential targets of S-III subtype,FDA approved drugs include PRKAG1 and BCL2.Other candidates include PI4 KB,TRAF2,ATOX1,ATAD2,AKAP8,GSK3 A,RAC1,STK38,and ERMP1.Conclusion:This study found three subtypes of SCLC proteomics,which have guiding significance for the treatment of small cell lung cancer: S-I subtype,with good prognosis and sensitivity to chemotherapy,is the most suitable for the current clinical treatment guidelines;S-II subtype,with immune subtype,may be a candidate for immunotherapy;S-III subtype,with poor prognosis and insensitive to chemotherapy and insignificant immune microenvironment,is in urgent need of finding new therapeutic targets.The hierarchical model based on proteomics can predict the chemosensitivity and clinical prognosis of small cell lung cancer.PART ?Fasudil promotes differentiation,maturation and apoptosis of small cell lung cancerObjective: Small cell lung cancer(SCLC)is characterized by rapid proliferation,early metastasis and acquired chemotherapy resistance.In the past 40 years,more than 40large-scale phase III clinical studies have failed,and more than 60 drug explorations have failed.Therefore,we still need to continue to explore new drugs to bring patients longer survival and more choices.The activation of ROCK1/2 signaling pathway can inhibit the formation of nerve fibers and the process of repair and regeneration after nerve injury.We speculate that fasudil may be a potential drug for differentiation induction therapy of small cell lung cancer.Methods : The expression of ROCK1/2 was detected by immunohistochemistry in 113 cases of small cell lung cancer,17 cases of typical carcinoid,23 cases of atypical carcinoma and 35 cases of large cell neuroendocrine tumor.At the cellular level,CCK8 experiment,cell cycle experiment and apoptosis experiment were used to explore the changes of proliferation activity,cell cycle and apoptosis level of small cell lung cancer cells stimulated by Fasudil,and the morphological changes of cells were observed.We further explored the effect of Fasudil on the transcriptional regulation of small cell lung cancer cells by transcriptome sequencing.We explored the expression changes of differentiation and development related genes of SCLC cells by bioinformatics analysis.And q RT-PCR and Western blot were used to verify the changes of cyclins and differentiation and development related genes at the gene and protein levels.Cell line-derived xenograft nude mice model was used to evaluate the efficacy of Fasudil.Results: ROCK1 was negative in all TC,AC and LCNEC samples,and the positive rate in SCLC was 77/113(68.1%).ROCK2 was negative in all AC and LCNEC samples,but positive in 14/17,82.4% TC and 94/113,83.2% SCLC samples.The positive rate of ROCK1/2 in SCLC was much higher than that in other three kinds of pulmonary neuroendocrine tumors.At the cellular level,Fasudil induced synaptic morphological changes and inhibited cell activity in small cell lung cancer,with IC50 of 76.04 ?g/ml.Cell cycle analysis showed that compared with the control group,the number of S-phase cells in Fasudil group was significantly reduced after stimulation with 100 ?g/ml Fasudil for 24 and48 hours.The number of cells in G0/G1 phase increased slightly,while that in G2/M phase increased significantly.After 72 hours of Fasudil concentration gradient stimulation,flow cytometry showed that the early apoptotic cells increased in a concentration dependent manner.In the transcriptome sequencing analysis,on the one hand,the down-regulation of tumor proliferation related biological processes,such as cell cycle regulation,DNA replication and chromosome segregation,was found through time sequence analysis;on the other hand,a large number of differentially expressed genes were found to be involved in the positive regulation of neuronal differentiation,stem cell differentiation,cell development and nervous system development.Next,neural related genes and cell cycle related genes were selected for q RT-PCR verification.At the same time,the expression levels of ASCL1,CDK4 and CDK6 decreased,while the expression levels of SOX11,PLK5 and SLIT2 increased after Fasudil stimulation.The expression patterns of CDK6,SOX11,PLK5 and SLIT2 were consistent with the trend of DEGs in RNA-seq and q RT-PCR.Besides,Fasudil can inhibit the growth of tumor in nude mice.Morphologically,the tumor tissue in Fasudil group was rich in stroma and loose in cell arrangement.Gordon sweet reticular fiber staining showed that Fasudil treatment group had more fibrous reticular septa,suggesting that Fasudil can promote the structural maturity.TUNEL assay further confirmed that Fasudil could induce apoptosis in vivo.Conclusion:This study indicates that ROCK1/2 is highly expressed and may be a potential therapeutic target for SCLC.Fasudil,a ROCK1/2 inhibitor commonly used in cardiovascular and cerebrovascular diseases,may have a new application in the treatment of SCLC induced differentiation.