Ueno Hideaki, Tomiyama Arata, Yamaguchi Hideki, Uekita Takamasa, Shirakihara Takuya, Nakashima Katsuhiko, Otani Naoki, Wada Kojiro, Sakai Ryuichi, Arai Hajime, Mori Kentaro
Department of Neurosurgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan; Division of Metastasis and Invasion Signaling, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan; Department of Neurosurgery, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
Department of Neurosurgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan; Division of Metastasis and Invasion Signaling, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
Biochem Biophys Res Commun. 2015;468(1-2):240-7. doi: 10.1016/j.bbrc.2015.10.122. Epub 2015 Oct 27.
Temozolomide (TMZ) is one of the few effective anticancer agents against gliomas. However, acquisition of TMZ resistance or adaptation by gliomas is currently a crucial problem, especially increased invasiveness which is critical for the determination of clinical prognosis. This study investigated the molecular regulatory mechanisms of TMZ resistance in gliomas involved in invasiveness, particularly invadopodia formation, a molecular complex formed at the invasive front to cause extracellular matrix degradation during cellular local invasion. The TMZ-resistant clone of the U343 MG human glioma cell line (U343-R cells) was established. U343-R cells demonstrated higher invadopodia formation compared with U343 cells without TMZ resistance (U343-Con cells). Immunoblot analysis of DNA damage-related mitogen-activated protein kinase signals found increased phosphorylation of c-Jun terminal kinase (JNK) and higher activation of its downstream signaling in U343-R cells compared with U343-Con cells. Treatment of U343-R cells with specific inhibitors of JNK or siRNA targeting JNK suppressed up-regulation of invadopodia formation. In addition, paxillin, one of the known JNK effectors which is phosphorylated and affects cell migration, was phosphorylated at serine 178 in JNK activity-dependent manner. Expression of paxillin with mutation of the serine 178 phosphorylation site in U343-R cells blocked invadopodia formation. The present findings suggest that increased formation of invadopodia in U343-R cells is mediated by hyperactivation of JNK-paxillin signaling, and both JNK and paxillin might become targets of novel therapies against TMZ-resistant gliomas.
替莫唑胺（TMZ）是少数对胶质瘤有效的抗癌药物之一。然而，胶质瘤对TMZ产生耐药性或适应性目前是一个关键问题，尤其是侵袭性增加，这对临床预后的判定至关重要。本研究调查了胶质瘤中与侵袭性相关的TMZ耐药的分子调控机制，特别是侵袭伪足的形成，侵袭伪足是在细胞局部侵袭过程中于侵袭前沿形成的一种分子复合物，可导致细胞外基质降解。建立了人U343 MG胶质瘤细胞系的TMZ耐药克隆（U343-R细胞）。与无TMZ耐药性的U343细胞（U343-Con细胞）相比，U343-R细胞表现出更高的侵袭伪足形成。对DNA损伤相关的丝裂原活化蛋白激酶信号进行免疫印迹分析发现，与U343-Con细胞相比，U343-R细胞中c-Jun末端激酶（JNK）的磷酸化增加，其下游信号的激活程度更高。用JNK特异性抑制剂或靶向JNK的小干扰RNA处理U343-R细胞可抑制侵袭伪足形成的上调。此外，桩蛋白是已知的JNK效应器之一，可被磷酸化并影响细胞迁移，它以JNK活性依赖的方式在丝氨酸178位点被磷酸化。在U343-R细胞中表达丝氨酸178磷酸化位点发生突变的桩蛋白可阻断侵袭伪足的形成。目前的研究结果表明，U343-R细胞中侵袭伪足形成的增加是由JNK-桩蛋白信号过度激活介导 的，JNK和桩蛋白都可能成为抗TMZ耐药胶质瘤新疗法的靶点。
