The Combination of Sorafenib and Everolimus Abrogates mTORC1 and mTORC2 Upregulation in Osteosarcoma Preclinical Models
The Combination of Sorafenib and Everolimus Abrogates mTORC1 and mTORC2 Upregulation in Osteosarcoma Preclinical Models
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DOI：
10.1158/1078-0432.ccr-12-2293
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发表时间：
2013-04-15
影响因子：
11.5
通讯作者：
Grignani, Giovanni
中科院分区：
文献类型：
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作者：
Pignochino, Ymera;Dell'Aglio, Carmine;Grignani, Giovanni
目的：多激酶抑制剂索拉非尼在骨肉瘤临床前模型中显示出抗肿瘤活性。然而，在索拉非尼治疗的转移复发性骨肉瘤患者中，疾病稳定和肿瘤缩小是短暂的，并且发生耐药性。实验设计：采用免疫沉淀、蛋白质印迹和免疫组织化学方法分析mTOR通路[mTOR复合物1（mTORC 1）和mTOR复合物2（mTORC 2）]。在不同的骨肉瘤细胞系（MNNG-HOS、HOS、KHOS/NP、MG 63、U-2 OS、SJSA-1和SAOS-2）中，用索拉非尼（10-0.625 μ mol/L）、雷帕霉素类似物依维莫司（100-6.25 nmol/L）和两者的组合进行标量剂量处理后，评价细胞活力、集落生长和细胞迁移。流式细胞仪检测细胞周期、活性氧（ROS）产生和凋亡。非肥胖糖尿病/严重联合免疫缺陷（NOD/SCID）小鼠注射MNNG-HOS细胞，以确定抗肿瘤和抗转移作用。血管生成和血管化进行了评估，在体外利用内皮分支形态发生测定和在体内异种移植小鼠和chorioallantoic membranes.Results：索拉非尼治疗后，mTORC 1信号减少（下游目标P-S6），而mTORC 2增加（磷酸mTOR Ser 2481）在MNNG-HOS异种移植物与车辆治疗的小鼠相比。索拉非尼与依维莫司联合使用导致mTORC 1 [通过ROS介导的AMP激活激酶（AMPK）激活]和mTORC 2（通过复合物分解）完全消除。索拉非尼/依维莫司组合产生：（i）增强抗增殖和促凋亡作用，（ii）受损的肿瘤生长，（iii）增强抗血管生成，和（iv）降低迁移和转移potential.Conclusion：mTORC 2激活是索拉非尼治疗的逃逸机制。当索拉非尼与依维莫司组合时，其抗肿瘤活性通过在临床前环境中完全抑制mTOR通路而增加。临床癌症研究; 19（8）; 2117-31。(C)2013年AACR。
Purpose: The multikinase inhibitor sorafenib displays antitumor activity in preclinical models of osteosarcoma. However, in sorafenib-treated patients with metastatic-relapsed osteosarcoma, disease stabilization and tumor shrinkage were short-lived and drug resistance occurred. We explored the sorafenib treatment escape mechanisms to overcome their drawbacks.Experimental Design: Immunoprecipitation, Western blotting, and immunohistochemistry were used to analyze the mTOR pathway [mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2)]. Cell viability, colony growth, and cell migration were evaluated in different osteosarcoma cell lines (MNNG-HOS, HOS, KHOS/NP, MG63, U-2OS, SJSA-1, and SAOS-2) after scalar dose treatment with sorafenib (10-0.625 mu mol/L), rapamycin-analog everolimus (100-6.25 nmol/L), and combinations of the two. Cell cycle, reactive oxygen species (ROS) production, and apoptosis were assessed by flow cytometry. Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice injected with MNNG-HOS cells were used to determine antitumor and antimetastatic effects. Angiogenesis and vascularization were evaluated in vitro by exploiting endothelial branching morphogenesis assays and in vivo in xenografted mice and chorioallantoic membranes.Results: After sorafenib treatment, mTORC1 signaling was reduced (downstream target P-S6), whereas mTORC2 was increased (phospho-mTOR Ser2481) in MNNG-HOS xenografts compared with vehicle-treated mice. Combining sorafenib with everolimus resulted in complete abrogation of both mTORC1 [through ROS-mediated AMP-activated kinase (AMPK) activation] and mTORC2 (through complex disassembly). The sorafenib/everolimus combination yielded: (i) enhanced antiproliferative and proapoptotic effects, (ii) impaired tumor growth, (iii) potentiated antiangiogenesis, and (iv) reduced migratory and metastatic potential.Conclusion: mTORC2 activation is an escape mechanism from sorafenib treatment. When sorafenib is combined with everolimus, its antitumor activity is increased by complete inhibition of the mTOR pathway in the preclinical setting. Clin Cancer Res; 19(8); 2117-31. (C) 2013 AACR.