ETV4/IGFBP2/PKM2 Mediate Aerobic Glycolysis To Promote Vasculogenic Mimicry In Head And Neck Rhabdomyosarcoma

Background:Rhabdomyosarcoma（RMS）is the most common soft tissue tumor in children,with the head and neck being its most common site,accounting for 40%of RMS cases.Due to the insidious onset of head and neck RMS,nearly 50%of patients have already experienced local progression,and even extensive infiltration or metastasis by the time of initial diagnosis.Once metastasis occurs,the 5-year survival rate is only 20-30%,which is a major cause of death in head and neck RMS patients.Hematogenous spread is one of the main pathways for tumor dissemination,and tumor angiogenesis is a crucial step necessary for tumor growth and metastasis.However,long-term clinical studies have shown that anti-angiogenic therapy does not achieve the expected clinical efficacy in head and neck tumors.Some scholars believe that this may be due to the existence of other blood supply pathways in tumors,namely vasculogenic mimicry（VM）.Studies have shown that VM as a new blood supply mechanism can provide nutrients for tumors.Unlike traditional vessels composed of endothelial cells,VM is a vascular-like structure formed based on changes in tumor cells themselves and the reconstruction of extracellular matrix,providing a new pathway for tumor growth and metastasis.Therefore,VM has become a promising potential target in cancer treatment.However,the specific mechanisms underlying VM formation in tumors remain unclear.Aerobic glycolysis,as a typical metabolic reprogramming phenomenon,is the primary way for tumors to acquire energy and plays an important role in tumor development.During the rapid growth phase of tumors,reliance on glycolysis for energy supply increases due to the shortage of vascular systems.Studies have reported that glycolysis can promote angiogenesis by meeting the glycolytic needs of endothelial cells and creating an acidic environment.Currently,research on the relationship between glycolysis and VM formation is scarce.ETV4,as a member of the ETS transcription factor family,is aberrantly expressed in various types of tumors and is associated with poor prognosis in patients.It participates in the regulation of various biological processes in tumors,including angiogenesis,metabolic reprogramming,and epithelial-mesenchymal transition（EMT）.However,the function and mechanism of ETV4 in RMS have not been elucidated.Objectives:1.Utilize single-cell transcriptome sequencing（ScRNA-seq）and spatial transcriptome sequencing（ST RNA-seq）to screen for key genes regulating VM formation in head and neck RMS.2.Determine the expression of ETV4 in head and neck RMS and its correlation with adverse clinical features.3.Elucidate the role of ETV4 in VM formation and metastasis of RMS.4.Determine whether ETV4 promotes VM formation in RMS by transcriptionally regulating the expression of IGFBP2.5.Determine whether ETV4/IGFBP2 promotes VM formation in RMS by mediating aerobic glycolysis.6.Elucidate the detailed molecular mechanism by which ETV4/IGFBP2/PKM2 promotes VM formation in RMS.Methods:1.Construct nude mouse xenograft models（subcutaneous tumor experiments）and perform single-cell transcriptome sequencing and spatial transcriptome sequencing on RMS tumors.Perform dimensionality reduction and clustering analysis on the ScRNA-seq sequencing data to obtain tumor cell subgroups with different characteristics.Subsequently,select the tumor cell subgroups with the highest match with VM formation cells through combined ST RNA-seq,and then analyze the differentially expressed genes in the selected subgroups.2.Analyze the expression of ETV4 in normal head and neck tissues and head and neck RMS patient tissues using Western blot and immunohistochemical staining.Additionally,analyze the expression of ETV4 in human skeletal muscle cells（HSkMC）and RMS cell lines（RD,A204,A673）using qRT-PCR and Western blot.3.Construct stable knockdown and overexpression RMS cell lines of ETV4 and analyze the effect of ETV4 on RMS VM formation and metastasis through in vivo experiments（transwell,scratch assay,tube formation,3D spheroid assay,spheroid invasion assay）and in vitro experiments（nude mouse subcutaneous tumor,nude mouse lung vein metastasis model,zebrafish xenograft model）.4.Conduct combined analysis using ChIP-seq,RNA-seq,and GEO data to screen for downstream target genes regulated by ETV4 transcription.Predict potential binding sites of ETV4 in the IGFBP2 promoter region through bioinformatics analysis.Analyze the active binding sites of ETV4 in the IGFBP2 promoter region using dual luciferase reporter gene,ChIP-PCR,and agarose gel electrophoresis experiments.5.Validate the regulatory effect of ETV4 on IGFBP2 expression using qRT-PCR and Western blot,and verify the role of ETV4/IGFBP2 in RMS VM formation and metastasis through a series of in vivo and in vitro experiments.6.Perform KEGG and GSEA bioinformatics analysis based on the results of high-throughput sequencing to explore the biological functions and signaling pathways potentially regulated by ETV4/IGFBP2.Investigate the role of ETV4/IGFBP2 in aerobic glycolysis in RMS using transmission electron microscopy,lactate content measurement,and extracellular acidification rate（ECAR）detection.7.Further clarify whether ETV4/IGFBP2 promotes RMS VM formation and metastasis by mediating aerobic glycolysis through a series of in vitro experiments and the use of glycolysis inhibitor（2-DG）.8.Identify proteins that may bind to IGFBP2 through immunoprecipitation（Co-IP）and tandem mass spectrometry.Validate the binding relationship between IGBP2 and PKM2 through GST-pulldown experiments.Investigate whether ETV4/IGFBP2 regulates PKM2 ubiquitination using Co-IP experiments with MG132 and CHX-treated RMS cell lines.9.Confirm whether the ETV4/PKM2/IGFBP2 axis affects RMS VM formation,metastasis,and growth by accelerating aerobic glycolysis through in vivo experiments（transwell,scratch assay,ECAR,tube formation,3D spheroid assay,spheroid invasion assay）and in vitro experiments（nude mouse subcutaneous tumor,nude mouse lung vein metastasis model）.Results:1.Based on single-cell transcriptome sequencing and spatial transcriptome sequencing,tumor heterogeneity in RMS tissue was analyzed,and different tumor cell subgroups were identified.ClusterCancerCells₅ was identified as the subgroup of cells with the highest correlation to VM formation.The transcription factor ETV4,highly expressed in ClusterCancerCells₅,was selected based on differential gene expression analysis.2.ETV4 was found to be highly expressed in head and neck RMS tissues compared to normal tissues and in RMS cells,correlating with adverse clinical features.In vivo experiments and in vitro experiments confirmed that ETV4 promotes VM formation,growth,and metastasis of RMS.3.Through ChIP-seq,RNA-seq,and joint analysis of GEO databases,potential downstream target genes of the transcription factor ETV4,including IGFBP2,were screened.qRT-PCR,Western Blot,and gel electrophoresis experiments confirmed that ETV4 upregulates the expression of IGFBP2 by transcriptional activation.The active binding site of ETV4 in the IGFBP2 promoter region was confirmed through ChIP-PCR and dual luciferase reporter assays.4.In vivo experiments and in vitro experiments demonstrated that ETV4 promotes VM formation,growth,and metastasis of RMS by upregulating IGFBP2 expression.High-throughput sequencing bioinformatics analysis suggested that aerobic glycolysis is a potential downstream biological function regulated by ETV4/IGFBP2.Electron microscopy,lactate content measurement,and extracellular acidification rate（ECAR）experiments confirmed that ETV4/IGFBP2 promotes aerobic glycolysis in RMS.In vitro experiments further confirmed that ETV4/IGFBP2 promotes VM formation,growth,and metastasis of RMS by enhancing aerobic glycolysis.6.IHC,qRT-PCR,and Western Blot experiments confirmed the high expression of IGFBP2 in RMS,closely associated with adverse clinical features.GST-pulldown and co-immunoprecipitation（Co-IP）experiments confirmed the direct interaction between IGFBP2 and PKM2 in RMS.Subsequent Western Blot and Co-IP experiments demonstrated that ETV4/IGFBP2 regulates the ubiquitination level to inhibit PKM2 protein degradation.7.Through in vivo and in vitro experiments,it was verified that the ETV4/IGFBP2/PKM2 axis promotes VM formation,growth,and metastasis of RMS by mediating aerobic glycolysis.Conclusion:In summary,this study elucidates the mechanism by which ETV4/IGFBP2/PKM2 mediates glycolysis to promote the formation of vasculogenic mimicry（VM）in head and neck rhabdomyosarcoma（RMS）.Due to glycolysis and VM formation play an important role in the malignant progression of tumors,interventions targeting the ETV4/IGFBP2/PKM2 signaling axis hold promise for the treatment of head and neck RMS patients.The data indicate that ETV4 is highly expressed in head and neck RMS and promotes VM formation,growth,and metastasis.IGFBP2,as a downstream target gene activated by ETV4 transcription,effectively enhances glycolysis and promotes RMS VM formation in a glycolysis-dependent manner.ETV4/IGFBP2,through regulating ubiquitination to inhibit PKM2 degradation,accelerates aerobic glycolysis,thereby promoting RMS VM formation,growth,and metastasis.This study reveals a novel mechanism of VM formation in head and neck RMS,facilitating the identification of new biological markers and potential therapeutic targets reflecting RMS metastasis and providing a theoretical basis for guiding precise clinical treatment of RMS.