PO.TB05.01 · 肿瘤生物学
PP2A再激活上调神经母细胞瘤中的FGA基因
PP2A reactivation upregulates FGA gene in neuroblastoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：由MYCN扩增驱动的高危神经母细胞瘤（NBL）对许多儿童仍然是致命的。我们此前的工作证明，药理学PP2A再激活通过减少MYCN磷酸化和启动子乙酰化来抑制MYCN的表达和稳定性，同时伴随BRD4和RNA Pol II去磷酸化，从而降低NBL体内肿瘤生长。当前研究是此前工作的延伸，我们进行了功能缺失实验以验证先前的发现，并在这一PP2A调控的网络中鉴定出一个由MYCN调控的下游靶点。
方法：用PP2A激活剂ATUX-1215或PP2A抑制剂冈田酸（OA）处理MYCN扩增的人NBL细胞系SK-N-BE(2)。免疫印迹定量磷酸化-BRD4、BRD4、MYCN、PP2Aalphabeta-C亚基、乙酰化组蛋白（H3K27ac、H3ac）、总H3和GAPDH。以酶学方法测量PP2A活性。批量RNA测序鉴定转录改变，并用qRT-PCR确认结果。分析了公共NBL数据集（TARGET和Capasso）。
结果：OA处理增加了磷酸化-BRD4、MYCN、H3K27ac和H3ac，同时降低了PP2A的表达和激活。免疫印迹的密度定量分析验证了这些转录和表观遗传标志物的变化。批量RNA-seq揭示纤维蛋白原alpha链（FGA）为用ATUX-1215激活PP2A后显著上调的基因。基因集富集分析（GSEA）鉴定出MYCN相关通路的富集，特别是WEI_MYCN_TARGETS_WITH_E_BOX基因集，FGA位列上调靶点之中。在ATUX-1215处理的NBL细胞中（24小时至72小时），qRT-PCR确认了FGA mRNA丰度的增加。公共数据集分析表明，FGA表达升高与MYCN扩增及4期NBL患者总生存期的改善相关。
结论：我们发现PP2A再激活涉及BRD4介导的表观遗传调控和组蛋白乙酰化，上调FGA基因，且FGA增加与NBL生存改善相关。这些发现提示PP2A再激活可能恢复有益的MYCN相关转录程序，为高危NBL提供潜在的治疗策略。
查看英文原文 English abstract
Background: High-risk neuroblastoma (NBL), driven by MYCN amplification, remains fatal for many children. Our previous work demonstrated that pharmacologic PP2A reactivation decreases NBL in vivo tumor growth through suppression of MYCN expression and stability by reducing MYCN phosphorylation and promoter acetylation, concurrent with BRD4 and RNA Pol II dephosphorylation. The current study is an extension of this previous work, in that we performed loss-of function tests validating the previous findings, and identified a downstream target regulated by MYCN within this PP2A-controlled network.
Methods: The MYCN amplified human NBL cell line SK-N-BE(2) was treated with the PP2A activator, ATUX-1215, or the PP2A inhibitor, okadaic acid (OA). Immunoblotting quantified phospho-BRD4, BRD4, MYCN, PP2Aalphabeta-C subunit, acetylated histones (H3K27ac, H3ac), total H3, and GAPDH. PP2A activity was measured enzymatically. Bulk RNA sequencing identified transcriptional alterations, and qRT-PCR confirmed the findings. Public NBL datasets (TARGET and Capasso) were analyzed.
Results: OA treatment increased phospho-BRD4, MYCN, H3K27ac, and H3ac while decreasing PP2A expression and activation. Densitometric analysis of immunoblots validated these transcriptional and epigenetic marker changes. Bulk RNA-seq revealed fibrinogen alpha chain ( FGA ) as a significantly upregulated gene following PP2A activation with ATUX-1215. Gene Set Enrichment Analysis (GSEA) identified enrichment of MYCN associated pathways, specifically the WEI_MYCN_TARGETS_WITH_E_BOX gene set, with FGA among upregulated targets. Increased FGA mRNA abundance was confirmed with qRT-PCR in ATUX-1215 treated NBL cells (24 h - 72 h). Public dataset analysis demonstrated that elevated FGA expression correlated with improved overall survival in MYCN amplified and stage 4 NBL patients.
Conclusion: We have found that PP2A reactivation involves BRD4-mediated epigenetic regulation and histone acetylation, upregulates FGA gene, and that increased FGA is associated with improved NBL survival. These findings suggest that PP2A reactivation may restore beneficial MYCN-linked transcriptional programs, offering potential therapeutic strategy for high-risk NBL.
利益披露 Disclosure
N. Nazam, None..
S. Manzoor, None..
M. Ghufran, None..
M. L. Brown, None..
A. M. Eakes, None..
P. Nande, None..
J. C. Opara, None..
A. Kaimari, None..
M. Ohlmeyer, None..
E. A. Beierle, None.