Development Of Multi-element Prognostic Prediction For GBM And Study Of Functional And Mechanistic Roles Of CDC45 In GBM

Objective:Glioblastoma Multiforme(GBM)is the most aggressive cancer in the central nervous system with invasive growth,high recurrence rate and short survival.Prognosis of patients can provide useful information for clinical decision and effective care on patients.However,traditional method of prognosis based on clinical experience often failed to accurately predict the prognosis of patients,and thus patients can not be treated effectively with immunotherapy.Overexpression of cell devision cycle 45(CDC45)that is as a biomarker for a variety of tumors has been found to be closely related to increased tumor size,advanced tumor grade and poor prognosis,but the role of CDC45 and mechanism by which CDC45 promotes tumor growth in GBM is still unclear.We analysized the expression of tumor-associated antigens(TAAs)and tumor microenviroment(TME)genes in clinical tumor tissue samples from 44 GBM patients,and developed a model to evaluate the prognosis for guiding clinical implementation of effective patient care.Furthermore,we investigated the roles of CDC45 in proliferation,migration and invasion of GBM.Finally,we identified a small molecule drug JQ-16 targeting CDC45 through virtual screening based on Molecular Dynamics(MD),and demonstrated that JQ-16 bound to CDC45 with high affinity,and inhibited tumor growth,inviasion and migration in vitro and in vivo with great potential for drug development for GBM therapy.Methods:1.A total of 54 clinical brain tissue samples including 44 brain tumor tissue samples from GBM patients and 10 normal brain tissues obtained as surgeric treatment for patients with non-malignant tumor or with trauma from the Guangdong 999 Brain Hospital and analyzed for the expression of a panel of 87 TAAs and 8 TME genes by qRT-PCR.Base on the analysis,we established a linear regression model based on the LASSO algorithm as executed in the R package,while applied the leave-one-out cross-validation to fit a COX regression model,and combined with the clinical characteristics one by one to improve the sensitivety,specificity and accuracy for prognosis.2.We next performed transcriptome sequencing to explore the role of CDC45 on cell proliferation,migration and invasion of tumor cell line by transwell culture and scratch experiments using the method of CCK8,and futher investgated the role of CDC45 in GBM xenograft in nude mice.Finally,we screened a pool of small molecule inhibitors(Chem Div,1.46million compounds)for bindimg to CDC45 by MD,Auto Dock,virtual screening to identify potential candiates for drug development for GBM therapy.Result:1.Development of of multi-element prognostic prediction for GBMWe investigated the m RNA expression levels of a panel of 87 TAAs and 8 immune TME-related genes in tumor tissue from 44 GBM patients.In spite of variations in given genes among different patients were found,we developed 5 formula(Y1-Y5)to accurately predict the prognosis based on the correlation analysis between gene signatures,clinical characteristics and the overall survival(OS).2.Functional and tumorgenestic role of CDC45 in GBMIncreased level of CDC45 expression was found in GBM and various maligant cancers and was associated with tumor size,tumor grade and poor prognosis.The ability of cell proliferation,migration and invasion of GBM cell lines(LN229 and U251)was positively correlated with the increased level of CDC45 gene expression.In contrast,when expression of CDC45 in these cell lines was knocked down by Si RNA and Sh RNA,we found that the ability of proliferation,migration and invasion of these cell lines were significantly reduced.Futhermore,similar effect of correlations of the level of CDC45 expression with tumor growth and migration in xenograft model in nude mice was also observed.Transcriptome sequencing analysis of LN229 and U251 cells with the overexpression of CDC45 revealed that expression of CDC45 in these cells was significantly correlated with the expression of genes mediated immune pathway.Further studies by GO(Gene Ontology Enrichment)and KEGG(Kyoto Encyclopedia of Genes and Genomes)showed that CDC45enhanced tumor growth and metastasis through NF-?B signaling pathway.Protein array analysis confirmed that IL-1?protein expression was significantly increased in GBM LN229 and U251cell lines,while GO and KEGG targeted the NF-?B signal pathway.These results suggests that CDC45 may regulate the NF-?B/IL-1?pathway and ultimately promoted tumor growth and metastasis as well as immune escape of tumor cells.JQ-16,a small molecule was identified by virtual screening based on Molecular Dynamics(MD)to bind CDC45 with highly affinity(KD=1.17×10^-11)was demonstrated to significantly inhibit the proliferation,migration and invasion of GBM tumor cells in vitro as well as in vivo xenograft animal models.Conclusion:We analyzed the expression of TAAs and TME genes in tumor tissues from GBM patients in comparison with the expression of these genes in normal brain tissue by qRT-PCR.We found that in spite of variations in given genes among different patients,some TAAs and TME genes were consistently found to be significantly increased in the level of expression in tumor tissue of GBM patients.We developed the mathematical model for evaluating the prognosis of GBM patients by linear regression based on LASSO,and verified the accuracy of the formula using clinical cases and TCGA database.CDC45 was found to have increased level of expression in GBM and various maligant cancers compared with normal tissues and is a biomarker of various malignant cancer.We found that CDC45 promoted the tumor proliferation,migration and invasion by activating NGk B/IL-1?pathway.It is well known that the NF-?B pathway and IL-1?play an important role in immune regulation.Results of transcriptome sequencing and protein array analysis suggest that CDC45may regulate the immune escape of tumor cells in the tumor microenvironment through NF-?B/IL-1?.By virtual screening a pool of small molecule inhibitors,we identified a total of 23 candidates that bound specifically to CDC45.Of these 23 inhibitors,JQ-16 was found to be most potent.It significantly inhibited the proliferation,invasion and migration of GBM tumor cells with high potency,and can be a valuable candidate for developing as chemic compound as targeted therapy for GBM.