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Abstract：
Neuroblastoma (NB) is the most common extracranial malignancy in childhood; however, the mechanisms underlying its aggressive characteristics still remain elusive.
Integrative data analysis was performed to reveal tumour-driving transcriptional regulators. Co-immunoprecipitation and mass spectrometry assays were applied for protein interaction studies. Real-time reverse transcription-polymerase chain reaction, western blotting, sequential chromatin immunoprecipitation and dual-luciferase reporter assays were carried out to explore gene expression regulation. The biological characteristics of NB cell lines were examined via gain- and loss-of-function assays. For survival analysis, the Cox regression model and log-rank tests were used.
Cellular nucleic acid-binding protein (CNBP) was found to be an independent factor affecting NB outcome, which exerted oncogenic roles in ribosome biogenesis, tumourigenesis and aggressiveness. Mechanistically, karyopherin subunit beta 1 (KPNB1) was responsible for nuclear transport of CNBP, whereas liquid condensates of CNBP repressed the activity of switch/sucrose-nonfermentable (SWI/SNF) core subunits (SMARCC2/SMARCC1/SMARCA4) via interaction with SMARCC2, leading to alternatively increased activity of SMARCC1/SMARCA4 binary complex in facilitating gene expression essential for 18S ribosomal RNA (rRNA) processing in tumour cells, extracellular vesicle-mediated delivery of 18S rRNA and subsequent M2 macrophage polarisation. A cell-penetrating peptide blocking phase separation and interaction of CNBP with SMARCC2 inhibited ribosome biogenesis and NB progression. High KPNB1, CNBP, SMARCC1 or SMARCA4 expression or low SMARCC2 levels were associated with poor survival of NB patients.
These findings suggest that CNBP phase separation is a target for inhibiting ribosome biogenesis and tumour progression in NB via modulating SWI/SNF complex activity.
Integrative data analysis was performed to reveal tumour-driving transcriptional regulators. Co-immunoprecipitation and mass spectrometry assays were applied for protein interaction studies. Real-time reverse transcription-polymerase chain reaction, western blotting, sequential chromatin immunoprecipitation and dual-luciferase reporter assays were carried out to explore gene expression regulation. The biological characteristics of NB cell lines were examined via gain- and loss-of-function assays. For survival analysis, the Cox regression model and log-rank tests were used.
Cellular nucleic acid-binding protein (CNBP) was found to be an independent factor affecting NB outcome, which exerted oncogenic roles in ribosome biogenesis, tumourigenesis and aggressiveness. Mechanistically, karyopherin subunit beta 1 (KPNB1) was responsible for nuclear transport of CNBP, whereas liquid condensates of CNBP repressed the activity of switch/sucrose-nonfermentable (SWI/SNF) core subunits (SMARCC2/SMARCC1/SMARCA4) via interaction with SMARCC2, leading to alternatively increased activity of SMARCC1/SMARCA4 binary complex in facilitating gene expression essential for 18S ribosomal RNA (rRNA) processing in tumour cells, extracellular vesicle-mediated delivery of 18S rRNA and subsequent M2 macrophage polarisation. A cell-penetrating peptide blocking phase separation and interaction of CNBP with SMARCC2 inhibited ribosome biogenesis and NB progression. High KPNB1, CNBP, SMARCC1 or SMARCA4 expression or low SMARCC2 levels were associated with poor survival of NB patients.
These findings suggest that CNBP phase separation is a target for inhibiting ribosome biogenesis and tumour progression in NB via modulating SWI/SNF complex activity.
摘要：
背景：神经母细胞瘤（NB）是儿童时期最常见的颅外恶性肿瘤；然而，其侵略性特征背后的机制仍然难以捉摸。
方法：进行整合数据分析以揭示肿瘤驱动转录调节因子。免疫共沉淀和质谱测定用于蛋白质相互作用研究。实时逆转录-聚合酶链反应,西方印迹,连续进行染色质免疫沉淀和双荧光素酶报告基因测定以探索基因表达调控。通过功能增益和功能丧失测定法检查了NB细胞系的生物学特性。对于生存分析，采用Cox回归模型和对数秩检验.
结果：发现细胞核酸结合蛋白（CNBP）是影响NB结局的独立因素，在核糖体生物发生中发挥致癌作用，肿瘤发生和侵略性。机械上,核蛋白亚基β1（KPNB1）负责CNBP的核转运，而CNBP的液体冷凝物通过与SMARCC2的相互作用抑制了开关/蔗糖不可发酵（SWI/SNF）核心亚基（SMARCC2/SMARCC1/SMARCA4）的活性，导致SMARCC1/SMARCA4二元复合物在促进肿瘤细胞中18S核糖体（rRNA）加工所必需的基因表达方面的选择性活性增加，细胞外囊泡介导的18SrRNA递送和随后的M2巨噬细胞极化。细胞穿透肽阻断CNBP与SMARCC2的相分离和相互作用抑制核糖体生物发生和NB进展。高KPNB1、CNBP、SMARCC1或SMARCA4表达或低SMARCC2水平与NB患者的低生存率相关。
结论：这些发现表明，CNBP相分离是通过调节SWI/SNF复合物活性来抑制NB中核糖体生物发生和肿瘤进展的靶标。
方法：进行整合数据分析以揭示肿瘤驱动转录调节因子。免疫共沉淀和质谱测定用于蛋白质相互作用研究。实时逆转录-聚合酶链反应,西方印迹,连续进行染色质免疫沉淀和双荧光素酶报告基因测定以探索基因表达调控。通过功能增益和功能丧失测定法检查了NB细胞系的生物学特性。对于生存分析，采用Cox回归模型和对数秩检验.
结果：发现细胞核酸结合蛋白（CNBP）是影响NB结局的独立因素，在核糖体生物发生中发挥致癌作用，肿瘤发生和侵略性。机械上,核蛋白亚基β1（KPNB1）负责CNBP的核转运，而CNBP的液体冷凝物通过与SMARCC2的相互作用抑制了开关/蔗糖不可发酵（SWI/SNF）核心亚基（SMARCC2/SMARCC1/SMARCA4）的活性，导致SMARCC1/SMARCA4二元复合物在促进肿瘤细胞中18S核糖体（rRNA）加工所必需的基因表达方面的选择性活性增加，细胞外囊泡介导的18SrRNA递送和随后的M2巨噬细胞极化。细胞穿透肽阻断CNBP与SMARCC2的相分离和相互作用抑制核糖体生物发生和NB进展。高KPNB1、CNBP、SMARCC1或SMARCA4表达或低SMARCC2水平与NB患者的低生存率相关。
结论：这些发现表明，CNBP相分离是通过调节SWI/SNF复合物活性来抑制NB中核糖体生物发生和肿瘤进展的靶标。
