PO.TB05.03 · 肿瘤生物学
靶向ALKBH5介导的ChREBP信号通路损害髓母细胞瘤中癌症干细胞代谢和肿瘤生长
Targeting ALKBH5 mediated ChREBP signaling impairs cancer stem cell metabolism and tumor growth in medulloblastoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：髓母细胞瘤（MB）是最常见的恶性儿童脑肿瘤，也是儿童癌症死亡的主要原因。它包含四个分子亚组：WNT、SHH、Group 3和Group 4。Group 3 MB通常以c-MYC扩增为特征，预后最差。尽管接受手术、放疗和化疗，高危患者仍面临较低的5年生存率和频繁的复发。目前的治疗方法会造成严重副作用。癌症干细胞（CSC）驱动MB的侵袭性、治疗耐药和复发。本项目旨在揭示维持CSC和MB进展的机制。
方法：对具有ALKBH5过表达、敲低（siRNA）或敲除（CRISPR）的MB细胞系（HD-MB03、D556、D425、DAOY）及对照进行分析，采用活力、迁移、侵袭、集落形成、细胞周期和凋亡检测。RNA测序鉴定ALKBH5调控的基因，并通过RT-qPCR、蛋白质印迹和RNA免疫沉淀验证。原位颅内异种移植模型评估了ALKBH5在体内的促肿瘤作用。
结果：为探索m6A RNA甲基化在髓母细胞瘤（MB）中的作用，我们在MB细胞系中沉默了关键调节因子（写入者：METTL3、METTL14；擦除者：ALKBH5、FTO）。ALKBH5缺失导致增殖降低最为显著，提示其治疗潜力。对儿童癌症数据集和组织微阵列的分析证实了ALKBH5在MB中的过表达和扩增。ALKBH5敲低增加了整体m6A水平，并降低了MB细胞的活力、迁移、侵袭和干性（髓球形成以及NANOG、OCT4、SOX9表达）。凋亡检测显示Annexin V阳性细胞升高，体内研究证明原位异种移植中肿瘤生长受到抑制。ALKBH5缺失还增加了DNA损伤标志物（gammaH2AX、53BP1）。RNA-seq和IPA揭示了涉及糖酵解、脂质生成和c-MYC靶标（包括ChREBP）的基因下调。蛋白质印迹证实了这些通路中蛋白水平的降低。
结论：ALKBH5通过m6A RNA去甲基化成为MB肿瘤发生和CSC维持的关键调节因子。靶向ALKBH5可能提供一种有前景的治疗策略，以抑制MB进展、克服治疗耐药并改善高危MB患者的结局。
查看英文原文 English abstract
Background: Medulloblastoma (MB) is the most common malignant pediatric brain tumor and a major cause of childhood cancer mortality. It comprises four molecular subgroups: WNT, SHH, Group 3, and Group 4. Group 3 MB, often marked by c-MYC amplification, has the worst prognosis. Despite surgery, radiation, and chemotherapy, high-risk patients face low 5-year survival and frequent relapse. Current treatments cause severe side effects. Cancer stem cells (CSCs) drive MB aggressiveness, therapy resistance, and recurrence. This project aims to uncover mechanisms sustaining CSCs and MB progression.
Methods: MB cell lines (HD-MB03, D556, D425, DAOY) with ALKBH5 overexpression, knockdown (siRNA), or knockout (CRISPR), along with controls, were analyzed using viability, migration, invasion, colony formation, cell cycle, and apoptosis assays. RNA sequencing identified ALKBH5-regulated genes, validated by RT-qPCR, western blot, and RNA immunoprecipitation. An orthotopic intracranial xenograft model assessed ALKBH5's tumor-promoting role in vivo.
Results: To explore the role of m6A RNA methylation in medulloblastoma (MB), we silenced key regulators (writers: METTL3, METTL14; erasers: ALKBH5, FTO) in MB cell lines. ALKBH5 depletion caused the greatest reduction in proliferation, suggesting its therapeutic potential. Analysis of pediatric cancer datasets and tissue microarrays confirmed ALKBH5 overexpression and amplification in MB. Knockdown of ALKBH5 increased global m6A levels and reduced MB cell viability, migration, invasion, and stemness (medullosphere formation and NANOG, OCT4, SOX9 expression). Apoptosis assays showed elevated Annexin V-positive cells, and in vivo studies demonstrated suppressed tumor growth in orthotopic xenografts. ALKBH5 loss also increased DNA damage markers (gammaH2AX, 53BP1). RNA-seq and IPA revealed downregulation of genes involved in glycolysis, lipogenesis, and c-MYC targets, including ChREBP. Western blot confirmed decreased protein levels in these pathways.
Conclusion: ALKBH5 is a critical regulator of MB tumorigenesis and CSC maintenance via m6A RNA demethylation. Targeting ALKBH5 may offer a promising therapeutic strategy to inhibit MB progression, overcome therapy resistance, and improve outcomes for high-risk MB patients.
利益披露 Disclosure
P. Subbarayalu, None..
D. Medina, None..
P. Pitta Venkata, None..
S. Abdulsahib, None..
S. Nirzhor, None..
S. Timilsina, None..
D. Singh, None..
P. Do, None..
D. Denman, None..
K. P. Evani, None..
D. Billa, None..
M. Nair, None..
E. Reddy, None..
Y. Gupta, None..
P. Houghton, None..
Y. Chen, None..
S. Viswanadhapalli, None..
G. R. Sareddy, None..
A. Brenner, None..
R. K. Vadlamudi, None..
M. Rao, None.