ATF4 destabilizes RET through nonclassical GRP78 inhibition to enhance chemosensitivity to bortezomib in human osteosarcoma
ATF4 destabilizes RET through nonclassical GRP78 inhibition to enhance chemosensitivity to bortezomib in human osteosarcoma
复制标题
ATF4 通过非经典 GRP78 抑制来破坏 RET 的稳定性，从而增强人骨肉瘤对硼替佐米的化疗敏感性
DOI：
10.7150/thno.36818
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发表时间：
2019-01-01
期刊：
影响因子：
12.4
通讯作者：
Kong, Lingyi
中科院分区：
文献类型：
--
作者：
Luo, Jie;Xia, Yuanzheng;Kong, Lingyi
原理：激活转录因子4(ATF4)是细胞应激反应的中心调节因子，通过控制与诱导细胞凋亡有关的靶基因的表达来减轻肿瘤负担。有证据表明，蛋白酶体抑制剂Bortezomib(BTZ)诱导骨肉瘤细胞死亡与ATF4激活有关。然而，在长期的治疗干预过程中，这种抑制功能是如何受损的仍不清楚。方法：建立稳定的细胞和体内移植瘤模型，揭示ATF4在细胞凋亡和肿瘤生长中的重要作用。应用荧光原位杂交(FISH)和免疫组织化学方法检测ATF4在骨肉瘤标本中的表达及意义。通过增殖、凋亡、实时定量聚合酶链式反应、免疫印迹和免疫荧光检测耐药癌细胞和化疗敏感癌细胞的生化差异。用荧光素酶报告实验分析启动子活性。免疫沉淀用于研究蛋白质与其他蛋白质或DNA的相互作用。结果：ATF4可显著抑制OS的致瘤作用，而ATF4基因敲除可阻断BTZ的抗肿瘤作用。正常成骨细胞被认为优先表达ATF4，但在OS临床标本和BTZ耐药亚系(OS/BTZ)中都检测到ATF4沉默。我们发现ATF4的下调与RET的异常表达密切相关，这主要是由于RET在OS/BTZ细胞中的稳定。RET的缺失上调了ATF4的表达，并增强了对BTZ的细胞凋亡反应。ATF4通过招募其反式激活的E3连接酶Cbl-c来识别RET的TK结构域，以加速RET蛋白酶体的更新，从而防止对BTZ的耐药性。相反，伴侣蛋白GRP78与RET结合并干扰ATF4/RET相互作用，促进RET的稳定。有趣的是，ATF4通过第一次ERSE抑制了OS/BTZ细胞中GRP78的转录，而不是通过经典的Cre元件在野生型OS中反式激活GRP78，揭示了RET和GRP78的双重靶向以克服化疗耐药。结论：研究结果揭示了ATF4在阻断RET/GRP78阳性人骨肉瘤的进展和耐药反应中的关键作用。
Rationale: Activating transcription factor 4 (ATF4) is a central regulator of the cellular stress response and reduces tumor burden by controlling the expression of target genes implicated in the induction of apoptosis. Evidence shows ATF4 activation is responsible for proteasome inhibitor bortezomib (BTZ)-induced osteosarcoma (OS) cell death. However, it remains unclear how such suppressive function is impaired during prolonged therapeutic interventions. Methods: Stable cells and in vivo xenograft models were generated to reveal the essential role of ATF4 in cell apoptosis and tumor growth. Fluorescence in situ hybridization (FISH) and immunohistochemistry were employed to detect the expression and significance of ATF4 in the specimens from osteosarcoma patients. Biochemical differences between chemoresistant and chemosensitive cancer cells were determined by proliferation, apoptosis, real-time PCR, immunoblotting and immunofluorescence. Promoter activity was analysed using the luciferase reporter assay. Immunoprecipitation was used to explore the interaction of proteins with other proteins or DNAs. Results: ATF4 significantly inhibited OS tumorigenesis, whereas knockdown of ATF4 prevented the antitumor effects of BTZ. Normal osteoblasts are supposed to preferentially express ATF4, but ATF4 silencing was detected in both OS clinical samples and BTZ-resistant sublines (OS/BTZ). We found that ATF4 downregulation was tightly linked to the aberrant expression of RET, primarily due to RET stabilization in OS/BTZ cells. Loss of RET upregulated ATF4 and potentiated the apoptotic response to BTZ. ATF4 recognized the TK domain of RET by recruiting its transactivated E3 ligase Cbl-c to accelerate RET proteasomal turnover, which in turn prevented BTZ resistance. In contrast, the chaperone GRP78 bound to RET and interfered with ATF4/RET interactions, promoted RET stabilization. Intriguingly, ATF4 repressed GRP78 transcription in OS/BTZ cells via the first ERSE, instead of transactivating GRP78 in wild-type OS via classical CRE element, revealing a dual targeting of RET and GRP78 to overcome chemoresistance. Conclusion: The results uncover a crucial role for ATF4 in blocking the progression and resistance response in RET/GRP78-positive human osteosarcoma.