The Mechanism Of NF-κB Enhancing The Malignant Progression In Glioma

Background:Glioma is the most common primary intracranial tumor,nearly half of which are highly invasive glioblastomas.It is the most aggressive tumor in the central nervous system.Even with aggressive combination therapy,it is still incurable,and the overall survival(OS)of patients with glioblastoma is only 4.9 months.With the widespread use of next-generation sequencing,the World Health Organization has changed the classification of glioma to molecular typing based on genetic and epigenetic features,and these abnormal molecules are also potential therapeutic targets for drug development.In the past decades,great efforts have been made to find suitable therapeutic targets.However,due to the high heterogeneity of glioma,these drugs have no long-term clinical effect on patients with glioma,and so far,no effective targeted therapeutic strategy has been developed in clinical practice.Therefore,we need to further study the mechanism of action between these molecules to find new therapeutic targets and develop more effective combined treatment strategies.Previous studies have shown that chronic inflammation is a major feature of glioblastoma biology.The blood-brain barrier is disrupted by glioblastoma,leading to chronic neuroinflammation.Inflammatory microenvironment is an important component of malignant progression in most tumors.Therefore,the internal mechanisms of inflammation and tumorigenesis have been the main focus of cancer research.NF-κB is an important link between chronic inflammation and cancer.Reliable and direct genetic evidence suggests that NF-κB signaling activation is an important factor in tumor promotion.The mammalian NF-κB family consists of five subunits,NFKB1(P105),RELA(p65),NFKB2(P100),RELB and c-rel,which form various homodimers or heterodimers.The most common active form is NFKB1/RELA,which regulates the canonical NFκB pathway.NF-κB dimers bind to the binding site(kappB sites)in regulatory regions of genes involved in a variety of cellular processes,including inflammation,cell proliferation,and apoptosis in some tumors,particularly in glioblastoma.Like other tumors,glioblastoma shows high NF-κB activity,and a variety of central carcinogenic pathways have certain correlation with NF-κB.Studies over the past few decades have shown that NF-κB inhibition alone does not significantly affect solid tumors in the majority of patients.However,the inhibition of apoptosis by NF-κB reminds us that it makes tumors resistant to other therapies.Therefore,inhibition of NF-κB may be a suitable option to improve the efficiency of conventional anticancer strategies.Epigenetic modifications are at the heart of many common diseases and are one of the central hallmarks of cancer.Histone methylation is a reversible post-translational modification that plays an important role in a variety of biological processes.Histone lysine methylation is one of the most characterized histone modifications,and its regulatory function depends on the location of its residues and the degree of methylation(monomethylation,dimethylation,or trimethylation)of its tail structure.Histone methylation sites that have been extensively studied include H3K4,H3K9,H3K27,and H3K36.H3K27me3 is considered to be an important epigenetic modification during differentiation and proliferation,and its aberrant regulation is common in many types of cancer,including glioma.The most important reason for the abnormal regulation of H3K27me3 is the abnormal expression of its methyltransferase EZH2.EZH2 is an important methyltransferase that can regulate the methylation of histones.EZH2 is overexpressed in a variety of cancers and promotes the development of these tumors,including glioblastoma.In a variety of cancers,EZH2 is often overactivated to promote tumorigenesis,making it a promising drug target for cancer therapy.However,the efficacy of EZH2 inhibitors as single agents has not met their early clinical high expectations.EZH2 inhibitors alone are ineffective or lead to multidrug resistance in some solid tumors with EZH2 overexpression.Nonetheless,further preclinical studies have also shown synergistic anticancer effects of combining EZH2 inhibitors with other therapies,revealing that cancer cells can become more sensitive to EZH2 inhibitors in the context of multifactorial interventions.Chronic inflammation and epigenetic reprogramming are both important markers of cancer.However,the mechanism of their interaction and their function have not been elucidated in glioblastoma.Therefore,we need to explore this and hope that our research can provide a reliable theoretical basis for the diagnosis and treatment of glioma.Methods:First of all,we explored the histone lysine methylation changes regulated by drugs and small interference RNA by inhibiting and activating NF-κB pathway.Then,we screened the NF-κB regulatory gene EZH2 by RNA-seq experiment,and verified its transcriptional regulation mechanism by double luciferase reporter gene experiment and ChIP experiment.Then we use CRISPR-Cas9 technology to knock out EZH2 gene,and through rescue experiments to verify whether the effect of NF-κB on the malignant progression of glioma depends on EZH2.Furthermore,through tissue microarray analysis,we confirmed that the co-expression of NF-κB and EZH2 can be used as a biomarker of glioblastoma molecular grade.Finally,we verified that NF-κB inhibitor and EZH2 inhibitor had synergistic inhibitory effect,respectively.Results:By exploring the correlation between chronic inflammation and epigenetic reprogramming,we found that H3K27me3 is specifically regulated by NF-κB.Next,in the process of screening histone methyltransferases and demethylases,we identified EZH2 as a key methyltransferase driving epigenetic reprogramming by inflammation.The ChIP and ChIP-seq results suggest that NF-κB regulates EZH2 expression by activating its transcription,thereby driving H3K27me3 epigenetic reprogramming.Meanwhile,our rescue experiments showed that the regulation of proliferation and migration by NF-κB was dependent on EZH2 to a certain extent,but the regulation of cycle and apoptosis was less dependent on EZH2.In addition,we found that coactivation of NF-κB and EZH2 resulted in the worst clinical outcomes and accurate molecular grading of glioblastoma by NF-κB and EZH2.Most importantly,we demonstrated a synergistic inhibitory effect on glioma cells by combined inhibition of NF-κB and EZH2,both of which are often overactivated in glioblastoma.Conclusion:We found that NF-κB classical pathway drives H3K27me3 reprogramming through EZH2.From the perspective of mechanism,NF-κB regulates the expression of EZH2 by activating the transcription of EZH2,thus affecting the expression and distribution of H3K27me3.In addition,NF-κB and EZH2 can accurately classify the risk grade of glioma.Most importantly,we demonstrated the synergistic effect of the combined inhibition of NF-κB and EZH2 on tumor cells.In summary,we have identified the mechanism between chronic inflammation and epigenetic reprogramming in malignant gliomas and provide a safe and effective potential strategy for the treatment of this fatal disease.