| Background and objective: Glioblastoma(GBM)is the most common primary malignant tumor of the central nervous system in adults with the worst prognosis.The recurrence rate is almost 100%.Currently,the average survival time of GBM patients after receiving standardized treatment regimens including surgery,radiotherapy and chemotherapy is only 15 months,so there is an urgent need to find effective treatment strategies.Recent studies in oncology have revealed extensive heterogeneity in cancer,which is reflected in the presence of a large number of tumor cells with different differentiation states within the tumor.Although more work is needed to differentiate tumor cell hierarchies,cancer stem cells appear to be key drivers of maintenance of cancer heterogeneity as well as malignant progression.Glioma stem cells(GSCs)have remarkable proliferative and self-renewal capabilities,which are located at the top of the GBM tumor cell differentiation hierarchy and are closely related to GBM onset and progression.They are thought to be a key factor in tumor recurrence and treatment resistance.Under the intervention of therapeutic means,the cellular state of GSCs is remodeled and promotes the development of treatment resistance.At the same time,GSCs interact with other cells in the tumor microenvironment to promote the malignant progression of GBM.As one of the most important targets for drug development in the21 st century,protein kinases are intimately involved in the development and progression of tumors as well as other types of diseases.Of particular importance,dysfunction of kinases has also been shown to be involved in the functional maintenance of cancer stem cells.Therefore,exploring the key kinase molecules and related mechanisms that regulate stemness and cell state changes in GSCs is conducive to the research and development of GBM precise treatment scheme,the development of new targeted therapeutic drugs,the promotion of the synchronization of glioma cell state,and the improvement of the sensitivity of GBM patients to targeted treatment.Methods:(1)Clinical tissue specimen collection and database data information: The tissue samples used in this study were obtained from the First Affiliated Hospital of China Medical University.The expression of STK32 B was clarified by immunohistochemical(IHC)staining of the tumor tissue samples.The datasets involved in this study included in-house datasets as well as public databases.The in-house dataset included transcriptome sequencing data from GBM tumor tissue specimens,non-tumor tissue specimens,and patient-derived paired primary GSCs and differentiated glioma cells(DGCs).Public database data in this study included The Cancer Genome Atlas(TCGA)data,Genotype-Tissue Expression Project(GTEx)data,Chinese Glioma Genome Atlas(CGGA)data,and GSCs-related GEO database datasets.We performed a comprehensive analysis of in-house datasets as well as the transcriptome sequencing of the public database to clarify key kinase genes in GSCs.(2)Bioinformatics analysis methods: DESeq2 package as well as limma package were used to analyze differential genes between different groups of samples;the expression of STK32 B in different kinds of cells in GBM was determined by single cell sequencing analysis;Gene Set Enrichment Analysis(GSEA)was applied to probe the differentially activated pathways;single sample gene set enrichment analysis(ss GSEA)was used to evaluate the biological process activity scores for individual samples.(3)Cell lines as well as glioma stem cell models: normal human astrocyte(NHA)were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.Patient-derived primary GSCs(GSC0117,GSC4,GSC21,GSC62)were extracted from GBM tumor tissues.The murine glioma stem cells(m GSCs)were extracted from a mouse GBM tumor model established by Sleeping Beauty(SB)vector.(4)Molecular biology experiments: The expression of STK32 B and other stemnessrelated markers in primary GSCs of patients and tumor tissue specimens were examined using Real Time PCR,Western Blot,immunohistochemical staining and other assays.Lentiviral transfection was used to construct GSCs with stable knockdown or overexpression of STK32 B for further functional assays and mechanistic investigations.The knockdown and overexpression efficiency of lentiviral transfection was verified by RT PCR and Western Blot.The proliferation,sphere-forming and self-renewal abilities of GSCs under different treatment conditions were clarified by MTS assay,in vitro neurosphere sphere-forming assay,and in vitro ELDA assay.In vivo limited dilution assay was used to assess the effect of STK32 B on the tumorigenic ability of GSCs.Cellular immunofluorescence assays were used to analyze the intracellular expression as well as the localization of different indicators.Western Blot were used to elucidate the expression of downstream related molecules affected by STK32 B.Co-IP were used to clarify the interaction between STK32 B and SCD1.In vitro kinase activity assays were used to clarify the phosphorylation of SCD1 by STK32 B.The occurrence of ferroptosis in GSCs was clarified by the detection of reactive oxygen species(ROS)levels as well as electron microscopic analysis.The effect of STK32 B on the tumorigenic ability of GSCs in vivo was investigated by constructing a human-derived GSCs-associated immunodeficient mice in situ tumorigenic model and m GSCs-associated in situ tumorigenic model.(5)RNA sequencing: RNA-Seq of STK32B-silenced GSCs was performed to clarify the altered gene expression related to STK32 B maintaining the stemness of GSCs.(6)Liquid chromatography-mass spectrometry(LC-MS): Liquid chromatographymass spectrometry(LC-MS)experiments using Flag-tagged STK32 B overexpressing GSCs were performed to clarify the potential proteins in GSCs which could bind STK32 B,and to investigate the specific mechanism of STK32 B in maintaining the stemness of cancer stem cells.(7)Statistical analysis: All experimental results were performed at least three independent experiments.Student’s t-test,one-way ANOVA was used to assess the statistical significance of differences between groups.All statistical tests were two-tailed.Differences in survival between different subgroups were assessed by Log-rank test and Kaplan-Meier analysis.Graph Pad Prism 9 software was used for statistical analysis.Bioinformatics analysis was performed by R software.A two-tailed p-value less than0.05 was defined as a statistically significant difference.Results:Part I: First,through analyzing transcriptome sequencing data from in-house datasets as well as public databases,we confirmed that STK32 B is a key kinase gene highly expressed in GBM as well as GSCs.TCGA and CGGA databases analyses as well as clinical specimens also confirmed that high STK32 B expression is significantly and positively associated with malignant progression of GBM and the poor prognosis of patients.Most importantly,we confirmed by single-cell sequencing analysis that GBM tumor cells with high STK32 B expression were significantly enriched in oligodendrocyte progenitor-like cell states,elucidating its close relationship with GSCs.After using lentivirus to construct stably knockdown and overexpressed STK32 B GSCs,a series of in vivo and in vitro experiments confirmed that STK32 B can promote the proliferation,sphere formation and self-renewal ability of GSCs.The above experimental results confirmed that STK32 B is a key kinase molecule for maintaining the stemness of GSCs and is involved in the malignant progression of GBM.Part II: In order to explore the specific molecular mechanism by which STK32 B maintains the stemness of GSCs,we performed RNA-Seq of the constructed stable GSCs with knockdown STK32 B and analyzed them together with RNA-Seq data from GBM tissues and found that high expression of STK32 B could cause abnormal activation of Wnt/β-catenin signaling pathway in GSCs.In the screening of potential proteins that interact with STK32 B by mass spectrometry analysis,we found that SCD1 was able to interact directly with STK32 B.Further experiments showed that STK32 B was able to phosphorylate SCD1 and maintain its protein stability.Taken together,we confirm that STK32 B maintains the stemness of GSCs is regulated by phosphorylating SCD1 and promoting its protein stability to activate the Wnt/β-catenin signaling pathway.Part III: Using previously constructed ferroptosis scores we found that DGCs are more prone to ferroptosis compared to GSCs and that patients with high STK32 B expression are more likely to develop ferroptosis resistance.It was confirmed that targeting STK32 B significantly enhanced the sensitivity of GSCs to ferroptosis by means of electron microscope morphological detection and molecular biology detection.We hypothesized that STK32B-related regulatory mechanisms in GSCs could promote the survival of GSCs under therapeutic interventions and reduce their chances of ferroptosis,causing GBM malignant progression and treatment resistance.The clinical transformation potential of STK32B/SCD1 signaling axis blockade in GBM was clarified by the construction of a preclinical model in mice,and the combination of targeted inhibition of STK32 B with Erastin is a novel strategy for GBM treatment that warrants further clinical investigation.Conclusions:Through a series of in vitro and in vivo experiments,we have confirmed that STK32 B is a key kinase gene that maintains the stemness of GSCs.STK32 B promotes GBM malignant progression by activating Wnt/β-catenin signaling via phosphorylation of SCD1 in GSCs.Using the constructed mouse GBM model,we found that blocking the STK32B/SCD1 signaling axis can inhibit the malignant progression of GBM and enhance the sensitivity to ferroptosis. |
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