Establishment Of Clinical Prognostic Model Of Ewing's Sarcoma And Mechanism Research Of Tumor Progression

BackgroundEwing's Sarcoma(ES)is the second most common malignant bone and soft tissue tumor during childhood and adolescence.ES has a predilection to occur among 10-20 years old with high malignancy and invasiveness,and the clinical prognosis for ES patients is considerably dismal.Statistically,the 5-year overall survival(OS)rate for localized ESs is approximately 65%,while the 5-year OS rate for spinal ESs descends to 42%.Up to 80%ES patients are detected with distant metastatic lesions at initial diagnosis,and the 5-year OS rate for such patients is below 30%.Osseous ESs have a predominance of metaphysis from upper and lower limbs,while the incidence of ESs originating from spine and pelvis is relatively low.Indeed,for the spine-pelvis ES patients,due to the limited cases,few clinical studies have reported the metastasis-related risk factors,as well as the establishment and validation of the prognostic model.Mechanistically,in order to screen out the potential critical proteins which were associated with ES tumorigenesis and progression,we performed systematic bioinformatic analysis and found extracellular matrix protein Tenascin-C(TNC)and deubiquitinases ZRANB1 were upregulated and downregulated in ES tumor tissues,respectively.However,it remains largely unclear regarding the function and related mechanism of TNC and ZRANB1 in regulating ES malignant biological behaviors.From both clinical and basic research aspects,this thesis was aimed at studying the prognostic model for spine-pelvis ESs,as well as the functions and related mechanisms of TNC and ZRANB1 in regulating ES tumor progression,respectively.Objectives1.To establish the clinical prognostic model for spine-pelvis ESs,and test the stability of the model via external cohort,as well as analyze the metastasis-related risk factors for the spine-pelvis ES patients.2.To study the function and related mechanism of TNC in regulating ESs:(1)To identify the expression and prognostic role of TNC in ESs.(2)To identify the functional role of TNC in regulating the proliferation,migration,and metastasis capability of ES cells,as well as angiogenesis in ES tumor microenvironment(TME).(3)To identify the relationship between TNC and EWS-FLI1 fusion protein.(4)To explore the potential mechanism of TNC in regulating ES tumor progression.3.To study the functional role and related mechanism of ZRANB1 in regulating ESs:(1)To detect ZRANB1 expression and its prognostic value in ESs.(2)To identify the functional role of ZRANB1 in regulating ES proliferation.(3)To explore the potential mechanism of ZRANB1 in regulating ES tumor progression.Material and methods1.The clinical information of 371 eligible patients with spine-pelvis ESs were collected from the “Surveillance,Epidemiology,and End Results”(SEER)database.The prognostic factors were analyzed by using Log-rank test and Cox proportional hazard modeling,and the nomogram was established based on the identified determinants.The spine-pelvis ES patients from our institution were enrolled as external cohort to validate the stability of the prognostic model.In addition,the risk factors affecting spine-pelvis ES metastasis were analyzed by logistic regression analysis.2.(1)TNC expression was analyzed by generating the data from the public databases,and detected in clinical ES specimens by immunohistochemical(IHC)staining and Western Blotting(WB).The prognostic role of TNC in ESs was also studied by incorporating the follow-up data.(2)The TNC-depleted monoclonal ES cell lines were constructed by CRISPR/Cas9 methods.The functional role of TNC in ES cell proliferation,migration,and angiogenesis was detected by CCK8 proliferation kit,Transwell migration and tube formation experiments,respectively.The pulmonary metastasis models of nude mice were also established to identify the role of TNC in ES metastasis.(3)By using WB,quantitative real-time PCR(q RT-PCR),Dual Luciferase Reporter assay and Chromatin immunoprecipitation(Ch IP)assay,we explored whether TNC expression was transcriptionally regulated by the EWS-FLI1 fusion protein.(4)The potential TNC-regulated signaling pathway in ES tumor progression was explored by RNA sequencing(RNA-seq),and the possible mechanism was further explored by WB,q RT-PCR,and immunocytochemistry(ICC).The single-cell transcriptome sequencing(Sc RNA-seq)was also performed to identify the cell subpopulations and detect TNC and its regulated signaling downstream genes expression by comparing with the normal cell cluster.3.(1)The potentially-significant gene in ES was screened out by weighted gene coexpression network analysis(WGCNA),and its expression and prognostic role were further confirmed by analyzing the tissue microarray from our institution.(2)The ZRANB1-knockdown and overexpression cell lines were constructed via lentivirus infection,then the functional role of ZRANB1 in ES proliferation was studied by CCK8 proliferation test,colony formation,and tumor-bearing nude mice experiments.(3)The potential mechanism of ZRANB1 in regulating ES growth was analyzed by RNA-seq,and further explored by IHC staining,WB,cycloheximide(CHX)test,coimmunoprecipitation(Co-IP),in vivo ubiquitination test,and flow cytometry analysis.Results1.The univariate and multivariate survival analysis revealed that the independent prognostic factors for spine-pelvis ES patients were age,tumor extension,tumor size,and primary tumor surgery.The nomogram was further built based on the identified factors,and the internal and external validations indicated favorable predictive value and stability of the model.In addition,the logistic regression analysis revealed tumor size>59mm and lymph node involvement were the risk factors of metastasis for spine-pelvis ES patients.2.TNC promoted ES progression:(1)High expression level of TNC was detected in ES specimens and cell lines,and the TNC expression was correlated with the metastasis-free survival and overall survival for patients with ESs.(2)TNC promoted the proliferation,migration,and metastasis capability of ES cells,and also enhanced angiogenesis in TME.(3)TNC expression was associated with EWS-FLI1 expression in ES cells,and EWSFLI1 transcriptionally activated TNC by directly binding to the promoter region(nt-360?368)of TNC.(4)The bulk RNA-seq data indicated that the differentiated genes were partly enriched in Hippo-YAP signaling pathway after TNC depletion.The Sc RNA-seq analysis revealed ES tumor was composed of massive tumorous cells and fibroblasts,a small number of endothelial cells and vascular smooth muscle cells.Meanwhile,by comparing with the normal bone marrow-derived mesenchymal stem cell cluster,TNC expression was upregulated remarkably in the identified tumorous cell cluster.Moreover,the expression of YAP positive downstream genes(CYR61 and CTGF)was upregulated,while YAP negative downstream genes(DDIT4 and TNFSF10)was downregulated in the tumorous cell cluster.Collectively,the sequencing data demonstrated TNC may promote ES tumor progression by regulating Hippo-YAP signaling pathway.Further differentiated genes analysis revealed MALAT1 was prominently downregulated when TNC was depleted.By the bioinformatic analysis of public databases and RNA Fluorescence immune-in-situ hybridization,MALAT1 upregulation was found in ES tissues compared with normal ones.Positive correlation was found between MALAT1 and TNC expression.When TNC was depleted,the phosphorylation expression of YAP at Ser127 was decreased,and the nuclear localization of YAP was remarkably elevated,together with the increased expression of the downstream genes of Hippo-YAP(CYR61&CTGF).Administration of Verteporfin inhibited MALAT1 expression by blocking the YAP-TEAD complex.The expression of Integrin ?5,?V and ?1,as TNC-associated receptors,was upregulated in ES specimens compared to the normal tissue.Blockade of Integrin ?5,but not Integrin?V,promoted YAP phosphorylation(Ser127)and cytoplasmic retention,together with the decreased MALAT1 expression.The expression of Integrin downstream kinases revealed the phosphorylation expression of Src(Tyr 416)was downregulated upon TNC depletion.Inhibition of Src activity by Saracatinib blocked the nuclear translocation of YAP and decreased MYC expression.3.ZRANB1 inhibited ES cell growth:(1)ZRANB1 expression was decreased in ESs by WGCNA and IHC staining of tissue microarray,and low ZRANB1 expression was correlated with poor prognosis for ES patients.(2)The ES cell viability was increased after silencing ZRANB1,while reintroduction of ZRANB1 inhibited ES proliferation.(3)The RNA-seq analysis indicated the lysosome-related signaling pathways might be involved in regulation of ZRANB1 in ES proliferation,and WB results showed ZRANB1 was capable of inhibiting the amino acid-dependent m TORC1 activity.Meanwhile,the IHC staining revealed p-S6(Ser235/236)was elevated when ZRANB1 expression decreased.The flow cytometry results also demonstrated increased cell size in ZRANB1-knockdown cell lines,but decreased cell size in ZRANB1-overexpressed cell lines.ZRANB1 promoted NPRL2 expression by enhancing its stability,which indicated by CHX test.The further Co-IP results showed interaction of ZRANB1 with NPRL2,which might be associated with the OUT domain of ZRANB1.Finally,the in vivo ubiquitination test revealed ZRANB1 might inhibit m TORC1 activity by cleaving the K48-linked polyubiquitinated chains of NPRL2.Conclusions1.The clinical predicting model for spine-pelvis ES patients has favorable stability and feasibility,which can be introduced to evaluate the prognosis for spine-pelvis ES patients at initial diagnosis.In the clinical practice,distant metastasis may develop for spine-pelvis ES patients when tumor size>59mm and/or lymph node is infiltrated.2.TNC is prominently upregulated in ES tissue and cell lines,and high TNC expression is associated with poor prognosis for ES patients.Functionally,TNC promotes ES cells proliferation,migration,and distant metastasis,and enhances angiogenesis in TME as well.Mechanistically,the EWS-FLI1 fusion protein transcriptionally promotes TNC expression by directly binding to its promoter region(nt-360?368).Meanwhile,TNC promotes ES tumor progression by inducing YAP dephosphorylation and nuclear translocation through Integrin ?5/?1-meadiated Src activation,and activated YAP increased the expression of MALAT1 and other Hippo-YAP downstream genes.3.ZRANB1 expression was decreased in ES,and ES patients with low ZRANB1 expression might have worse prognosis.ZRANB1 inhibited ES cell growth by blocking the m TORC1 activity through cleaving the K48-linked poly-ubiquitinated chains of NPRL2.