PO.MCB03.01 · 分子与细胞生物学
双重靶向Pyk2/FAK和MEK/ERK通路增强对胶质母细胞瘤的生长抑制
Dual targeting of Pyk2/FAK and MEK/ERK pathways enhances growth suppression in glioblastoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
胶质母细胞瘤(GBM)是一种高度侵袭性的脑肿瘤，其特征是对化疗和靶向治疗干预均具有强烈耐药性。我们之前的研究表明，在小鼠胶质瘤模型中,药理学抑制黏着斑激酶(FAK)和富含脯氨酸的酪氨酸激酶2(Pyk2)信号传导可显著减少肿瘤生长，但仅使总生存期略有增加，提示代偿性耐药机制的产生。此外，抑制FAK/Pyk2后观察到MEK/ERK信号通路的代偿性激活。因此，我们假设同时靶向FAK/Pyk2和MEK/ERK信号通路可以克服MEK/ERK激活并增强FAK/Pyk2抑制在GBM中的治疗疗效。
对原代人GBM细胞系和C57BL/6小鼠GL261胶质瘤模型的Western blot分析显示，使用双重Pyk2/FAK抑制剂defactinib(体外5 µM；体内50 mg/kg)处理72小时后，MEK和ERK的磷酸化增加。与MEK/ERK抑制剂avutometinib(体外1 µM；体内0.3 mg/kg)联合处理消除了这种代偿性激活，导致体外ERK磷酸化减少10倍，体内减少3倍。细胞周期分析表明，用defactinib处理72小时后细胞在G2/M期积累并诱导凋亡，提示细胞周期停滞。相比之下，avutometinib处理72小时导致细胞在G0期积累，伴随cyclin D1表达降低(经Western blot分析证实)，与细胞抑制作用一致。联合处理在有丝分裂(G2/M)期和G0期均诱导细胞周期停滞，反映了两种抑制剂的相加效应。细胞活力检测支持这些发现，显示defactinib和avutometinib单药治疗分别诱导约50%和20%的细胞死亡，同时与未处理对照相比活细胞群分别减少60%和25%。
总之，联合抑制Pyk2/FAK和MEK/ERK信号传导在胶质瘤细胞中产生了相加的抗肿瘤效应，抑制了代偿性MEK/ERK激活并增强了生长抑制。
查看英文原文 English abstract
Glioblastoma (GBM) is a highly aggressive brain tumor characterized by strong resistance to both chemotherapeutic and targeted therapeutic interventions. Our previous studies demonstrated that pharmacological inhibition of Focal Adhesion Kinase (FAK) and Proline-rich Tyrosine Kinase 2 (Pyk2) signaling in mouse glioma models significantly reduces tumor growth but results in only a modest increase in overall survival, suggesting the development of compensatory resistance mechanisms. Additionally, compensatory activation of the MEK/ERK signaling pathway was observed following the inhibition of FAK/Pyk2. We therefore hypothesized that concurrent targeting of FAK/Pyk2 and MEK/ERK signaling pathways could overcome MEK/ERK activation and enhance the therapeutic efficacy of FAK/Pyk2 inhibition in GBM.
Western blot analysis of primary human GBM cell lines and the GL261 glioma model in C57BL/6 mice revealed increased phosphorylation of MEK and ERK following treatment with the dual Pyk2/FAK inhibitor defactinib (5 µM in vitro; 50 mg/kg in vivo) for 72 hours. Combinatorial treatment with the MEK/ERK inhibitor avutometinib (1 µM in vitro; 0.3 mg/kg in vivo) abolished this compensatory activation, resulting in a 10-fold reduction in ERK phosphorylation in vitro and a 3-fold reduction in vivo. Cell cycle analysis demonstrated an accumulation of cells in the G2/M phase and induction of apoptosis following 72-hour treatment with defactinib, indicating cell cycle arrest. In contrast, avutometinib treatment for 72 hours resulted in cell accumulation in the G0 phase, accompanied by a decrease in cyclin D1 expression, as confirmed by Western blot analysis, consistent with a cytostatic effect. Combined treatment induced cell cycle arrest at both the mitotic (G2/M) and G0 phases, reflecting the additive effects of both inhibitors. Cell viability assays supported these findings, showing that defactinib and avutometinib monotherapies induced approximately 50% and 20% cell death, respectively, together with 60% and 25% reductions in live cell populations compared to untreated controls.
In conclusion, combined inhibition of Pyk2/FAK and MEK/ERK signaling produced an additive anti-tumor effect in glioma cells, suppressing compensatory MEK/ERK activation and enhancing growth inhibition.
利益披露 Disclosure
E. Morales-Colon, None..
K. A. Rosa- Gonzalez, None.