Earwaker Philip, Anderson Caroline, Willenbrock Frances, Harris Adrian L, Protheroe Andrew S, Macaulay Valentine M
Department of Oncology, Oxford, United Kingdom.
Oxford Cancer and Haematology Centre, Oxford University Hospitals NHS Trust, Churchill Hospital, Oxford, United Kingdom.
PLoS One. 2018 Feb 1;13(2):e0191890. doi: 10.1371/journal.pone.0191890. eCollection 2018.
The outlook for patients with advanced renal cell cancer (RCC) has been improved by targeted agents including inhibitors of the PI3 kinase (PI3K)-AKT-mTOR axis, although treatment resistance is a major problem. Here, we aimed to understand how RCC cells acquire resistance to PI3K-mTOR inhibition. We used the RCC4 cell line to generate a model of in vitro resistance by continuous culture in PI3K-mTOR kinase inhibitor NVP-BEZ235 (BEZ235, Dactolisib). Resistant cells were cross-resistant to mTOR inhibitor AZD2014. Sensitivity was regained after 4 months drug withdrawal, and resistance was partially suppressed by HDAC inhibition, supporting an epigenetic mechanism. BEZ235-resistant cells up-regulated and/or activated numerous proteins including MET, ABL, Notch, IGF-1R, INSR and MEK/ERK. However, resistance was not reversed by inhibiting or depleting these pathways, suggesting that many induced changes were passengers not drivers of resistance. BEZ235 blocked phosphorylation of mTOR targets S6 and 4E-BP1 in parental cells, but 4E-BP1 remained phosphorylated in resistant cells, suggesting BEZ235-refractory mTORC1 activity. Consistent with this, resistant cells over-expressed mTORC1 component RAPTOR at the mRNA and protein level. Furthermore, BEZ235 resistance was suppressed by RAPTOR depletion, or allosteric mTORC1 inhibitor rapamycin. These data reveal that RAPTOR up-regulation contributes to PI3K-mTOR inhibitor resistance, and suggest that RAPTOR expression should be included in the pharmacodynamic assessment of mTOR kinase inhibitor trials.
包括PI3激酶（PI3K）-AKT-mTOR轴抑制剂在内的靶向药物改善了晚期肾细胞癌（RCC）患者的预后，尽管治疗耐药是一个主要问题。在此，我们旨在了解RCC细胞如何获得对PI3K-mTOR抑制的耐药性。我们使用RCC4细胞系，通过在PI3K-mTOR激酶抑制剂NVP-BEZ235（BEZ235，达可替尼）中连续培养来建立体外耐药模型。耐药细胞对mTOR抑制剂AZD2014具有交叉耐药性。停药4个月后敏感性恢复，HDAC抑制可部分抑制耐药性，支持一种表观遗传机制。BEZ235耐药细胞上调和/或激活了许多蛋白质，包括MET、ABL、Notch、IGF-1R、INSR和MEK/ERK。然而，抑制或消除这些途径并不能逆转耐药性，这表明许多诱导的变化是耐药的伴随现象而非驱动因素。BEZ235可阻断亲代细胞中mTOR靶点S6和4E-BP1的磷酸化，但4E-BP1在耐药细胞中仍保持磷酸化，提示存在对BEZ235难治的mTORC1活性。与此一致的是，耐药细胞在mRNA和蛋白质水平上过度表达mTORC1组分RAPTOR。此外，RAPTOR缺失或变构mTORC1抑制剂雷帕霉素可抑制BEZ235耐药性。这些数据表明RAPTOR上调促成了PI3K-mTOR抑制剂耐药性，并提示RAPTOR表达应纳入mTOR激酶抑制剂试验的药效学评估中。
