Mechanistic Insight Into The SMAD9 To MYCN Pathway In Neuroblastoma

AimNeuroblastoma(NB),the most common extracranial childhood solid tumor,is characterized explicitly by aberrant neural crest development,posing a significant challenge in the high-risk NB patients with an approximate overall survival rate between 50 to 65%.MYCN is a critical oncogene in NB,and its DNA amplification serves as an absolute determinant of high risk.With the development of tumor omics and the NB public database,oncologists aimed to reveal biological and clinical characteristics of NB via previous and newly constructed datasets,and core regulatory circuit(CRC)is one of critical hypotheses in the analyses of NB omics.It indicates that a set of transcriptional factors(TFs)driven by MYCN are capable of binding to the enhancers,thus forming several oncogenic positive feedback loops.Of note,these TFs are characterized by specific high expression.Based on the characteristics of specific high expression in the pivotal NB genes,we combined previous public datasets and our self-constructed CRISPR-Cas9 screening and found that SMAD9 expression indicated specificity in NB.TGF-β/SMAD signaling is involved in the biological processes of tumor growth,drug resistance and metastasis,etc.Nevertheless,it remains controversial in the role of SMAD family in NB and to be elucidated how SMAD9 plays a role in NB.Therefore,we integrated bioinformatic and biological experiments to investigate SMAD9 concerning its clinical significance,biological role,functional mechanism and application potential.Methods(1)An integrative analysis of the datasets was performed,including functional genomics,transcriptomics of NB cells,and transcriptomics of NB tissues with their corresponding survival information.(2)The landscape of SMAD9 in many datasets was visualized,including profiles of expression,function,MYCN correlation and gene set enrichment analysis(GSEA).(3)Genetic sh RNA knockdown of SMAD9 was performed to observe the proliferative status of NB cells both in vitro and in vivo.(4)A combined analysis of RNA-sequencing after SMAD9 knockdown and chromatin immunoprecipitation sequencing(Ch IP-seq)after SMAD9 overexpression was employed.Downstream changes were validated.(5)Knockdown of transcriptional factors in CRC and upstream validation were performed.(6)Immune cell infiltration and drug target prediction were analyzed based on SMAD9 expression in the NB high-risk patients.Results(1)SMAD9 expression was highly specific in NB.(2)Highly specific SMAD9 was positively correlated with MYCN expression and poor prognosis.(3)SMAD9 induced MYCN-amplified NB cell proliferation and tumorgenicity both in vitro and in vivo.(4)Downstream analyses indicated that SMAD9 could bind to the MYCN promoter and transcriptionally regulate MYCN expression,thus sustaining proliferative signal of the cell cycle.(5)Upstream analyses suggested that MYCN,PHOX2 B,GATA3 and HAND2(TFs in CRC)could reciprocally bind to the SMAD9 enhancer and transactivate SMAD9,thus forming a positive feedback loop.(6)T-cell immunosuppression was exhibited in the high-risk NB patients with high expression of SMAD9.MEK inhibitors were predicted to possibly suppress the pathway.ConclusionIn summary,our data delineate that the CRC genes regulate the SMAD9 to MYCN pathway to drive the formation of a subset of high-risk NB.