PO.TB07.01 · 肿瘤生物学
肌动蛋白细胞骨架调控可重编程耐药性GBM球体和GSC，在体外和体内增强对IR和temozolomide的应答
Actin cytoskeleton modulation reprograms resistant GBM spheroids and GSCs, enhancing response to IR and temozolomide in vitro and in vivo
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摘要 Abstract
中文摘要
肌动蛋白细胞骨架调控癌症进展中的关键过程，包括细胞分裂、迁移、侵袭和基因组稳定性。核内F-actin已成为DNA修复、染色质组织和转录的关键调控因子，代表一个潜在的治疗靶点。胶质母细胞瘤（GBM）是一种高度侵袭性肿瘤，对电离辐射（IR）和temozolomide（TMZ）具有强烈耐药性，常依赖GBM干样细胞（GSC）来维持复发。GSC表现出增强的DNA修复能力，其干性受与肌动蛋白动态相关的通路影响。我们研究了靶向肌动蛋白聚合是否可通过调节DNA损伤应答（DDR）和GSC表型来使GBM对治疗敏感。通过循环处理产生的IR耐药和TMZ耐药U87-MG细胞被培养为三维球体。耐药球体表现出增殖增加、细胞周期进程加快、核心内细胞死亡升高以及显著增强的自我更新，支持一种干样致瘤特征。有趣的是，耐药细胞表现出不同的侵袭模式：IR耐药细胞侵袭性更强，而TMZ耐药细胞迁移性更强，这与pFAK和vimentin免疫印迹结果一致，表明每个耐药群体内存在不同的GSC样亚型。值得注意的是，在F-actin破坏药物（cytochalasin D/B或latrunculin B）存在下组装球体，可降低治疗耐药性并减弱致瘤性。斑马鱼异种移植证实了耐药细胞的侵袭性增殖、死亡率和转移性播散，而所有这些在肌动蛋白靶向处理后均显著降低，从而提高了动物存活率。RNA-seq、qPCR、免疫印迹和免疫荧光揭示耐药球体中GSC标志物上调，而肌动蛋白破坏显著降低了它们的表达。我们提出核内F-actin调控维持干性和治疗耐药性的转录环路。与此一致，耐药细胞表现出核内肌动蛋白升高，其被基因毒性应激或药理学肌动蛋白破坏动态调节。正在进行的染色质组分蛋白质组学旨在鉴定损伤条件下与核内F-actin相关的蛋白质。最后，在PDX GSC23细胞中，抑制F-actin聚合可在自我更新条件下降低干性标志物，并阻止血清诱导的分化。Cytochalasin D增强了GSC23对IR和TMZ的敏感性，降低了增殖、活力和致瘤性。总之，这些发现揭示肌动蛋白细胞骨架调控，尤其是核内F-actin，是一个可被治疗性利用以克服GBM耐药性的脆弱点，支持将肌动蛋白调节化合物作为GBM治疗的有前景的辅助药物。
查看英文原文 English abstract
Actin cytoskeleton regulate essential processes in cancer progression, including cell division, migration, invasion, and genomic stability. Nuclear F-actin has emerged as a critical regulator of DNA repair, chromatin organization, and transcription, representing a potential therapeutic target. Glioblastoma (GBM), a highly aggressive tumor with strong resistance to ionizing radiation (IR) and temozolomide (TMZ), frequently relies on GBM stem-like cells (GSCs) to sustain recurrence. GSCs exhibit enhanced DNA repair, and their stemness is influenced by pathways linked to actin dynamics. We investigated whether targeting actin polymerization sensitizes GBM to therapy by modulating DNA damage response (DDR) and GSC phenotypes. IR- and TMZ-resistant U87-MG cells generated through cyclic treatments were grown as 3D spheroids. Resistant spheroids showed increased proliferation, faster cell-cycle progression, elevated cell death within the core, and markedly enhanced self-renewal, supporting a stem-like tumorigenic profile. Interestingly, resistant cells exhibited distinct invasion patterns: IR-resistant were more invasive, whereas TMZ-resistant were more migratory, consistent with pFAK and vimentin immunoblotting, indicating distinct GSC-like subtypes within each resistant population. Strikingly, spheroid assembly in the presence of F-actin-disrupting drugs (cytochalasin D/B or latrunculin B) reduced therapy resistance and diminished tumorigenicity. Zebrafish xenotransplantation confirmed the aggressive proliferation, mortality rates, and metastatic dissemination of resistant cells, all of which were significantly reduced upon actin-targeting treatments, resulting in increased animal survival. RNA-seq, qPCR, immunoblotting, and immunofluorescence revealed upregulation of GSC markers in resistant spheroids, while actin disruption markedly reduced their expression. We propose that nuclear F-actin regulates transcriptional circuits that maintain stemness and therapy resistance. Consistently, resistant cells exhibited elevated nuclear actin which was dynamically modulated by genotoxic stress or pharmacological actin disruption. Ongoing chromatin-fraction proteomics aim to identify nuclear F-actin-associated proteins under damage conditions. Finally, in PDX GSC23 cells, inhibition of F-actin polymerization reduced stemness markers under self-renewal conditions and prevented serum-induced differentiation. Cytochalasin D enhanced sensitivity of GSC23 to IR and TMZ, reducing proliferation, viability, and tumorigenicity. Collectively, these findings reveal actin cytoskeleton regulation, particularly nuclear F-actin, as a vulnerability that can be therapeutically exploited to overcome GBM resistance, supporting actin-modulating compounds as promising adjuvants for GBM therapy.
利益披露 Disclosure
Y. T. Magalhães, None..
L. Ramos Molica, None..
D. Joe, None.