靶向糖原合酶激酶GSK-3β治疗MYCN扩增的神经母细胞瘤及其机制研究

Neuroblastoma (NB) is the most common extracranial solid tumor arising from the neural crest in childhood, accounting for ~7% of all pediatric malignancies and ~13% of all pediatric oncology deaths. MYCN (encodes N-Myc) is a driver gene of neuroblastoma and confers a particularly poor prognosis. Amplification of MYCN is found in ~22% of cases overall (up to ~50% of high-risk tumors). Glycogen synthase kinase-3 beta (GSK-3β) is recognized as a tumor suppressor because it promotes phosphorylation of many important oncoproteins including N-Myc, Cyclin E1 and Mcl1, leading to their ubiquitination and degradation. Recent studies show that inhibition of GSK-3β leads to cell death in melanoma and prostate cancer cells, indicating oncogenic role of GSK-3β. We found that pharmacological- or shRNA-mediated inhibition of GSK-3β decreased expression of N-Myc and Cyclin E1, but increased anti-apoptotic protein Mcl1 in neuroblastoma cells, leading to G0/G1 phase arrest and proliferation inhibition without significant cell apoptosis. Further research revealed that GSK-3β inhibition downregulated mRNA levels of MYCN and CCNE1 (encodes Cyclin E1). However, the molecular mechanism(s) by which GSK-3β activates MYCN and CCNE1 transcription needs to be identified. These data suggest that GSK-3β promotes neuroblastoma tumorigenesis by upregulating N-Myc and Cyclin E1 levels and mediating Mcl1 degradation. On the basis of above results, we hypothesize that combination of GSK-3β inhibitor and Mcl1 inhibitor may benefit MYCN-amplified neuroblastoma patients and we will investigate this in vitro and in vivo.

神经母细胞瘤是儿童最常见的颅外实体肿瘤，具有发病隐匿、进展快、恶性度高等特点。癌基因MYCN（编码N-Myc）是神经母细胞瘤的驱动基因，约22%的神经母细胞瘤患者伴有MYCN扩增。糖原合酶激酶GSK-3β能够促进N-Myc、Cyclin E1、Mcl1等癌蛋白降解，因而被认为是肿瘤抑制因子，但近期研究表明GSK-3β在黑色素瘤等肿瘤中具有促癌作用。申请者前期发现，在神经母细胞瘤细胞中抑制GSK-3β显著减慢细胞增殖却不引起凋亡，进一步研究发现N-Myc和Cyclin E1表达显著下降，Mcl1表达却显著升高。本项目旨在更加深入探究GSK-3β促进N-Myc和Cyclin E1表达的分子机制，阐明GSK-3β在神经母细胞瘤发生发展过程中的作用，并且在分子、细胞和动物水平探讨GSK-3β抑制剂和Mcl1抑制剂联用治疗MYCN扩增的神经母细胞瘤的可能性，从而为神经母细胞瘤的临床治疗提供新的思路。

MYC家族原癌基因在多种肿瘤中异常表达，MYCN扩增是神经母细胞瘤的典型特征，c-MYC高表达在急性T淋巴细胞白血病中较为常见，这类肿瘤恶性程度高、预后较差。深入探究MYC上下游调控网络对治疗MYC高表达的肿瘤具有重要意义。本项目旨在探讨Myc/GSK-3β调控网络的分子机制及潜在治疗策略，重点研究以下内容：（1）在神经母细胞瘤中，GSK-3β如何促进MYCN转录，进而发挥促癌作用；（2）在急性T淋巴细胞白血病中，AURKB如何抑制c-Myc/GSK-3β相互作用，促进c-Myc表达。通过系统和深入的研究，申请人证实了GSK-3β在神经母细胞瘤和急性T淋巴白血病中对Myc存在多重调节作用。在神经母细胞瘤中，虽然GSK-3β可介导N-Myc的磷酸化/泛素化降解，也能通过稳定E2F1间接促进MYCN转录，整体表现为促癌作用；将MCL-1抑制剂与GSK-3β抑制剂联用，既可抑制N-Myc表达，又可消除抑制GSK-3β引起的MCL-1上调，起到协同治疗作用。在急性T淋巴细胞白血病中，GSK-3β则主要介导c-Myc降解，而AURKB可直接磷酸化c-Myc，阻碍c-Myc/GSK-3β结合，抑制FBXW7介导的泛素化降解，促进c-Myc蛋白稳定性。申请人如期完成研究目标，已发表研究论文2篇，阐明了Myc/GSK-3β调控网络的分子机制，并提出靶向GSK-3β治疗神经母细胞瘤、靶向AURKB治疗急性T淋巴细胞白血病的新策略。