METHODS: We explored the effects of NC on mTORC1 signaling by immunoblotting and fluorescence microscopy in wild-type and gene knockout cell lines generated by the CRISPR/Cas9 gene editing technique. We identified IGF2R as a direct target of NC via the drug affinity-responsive target stability (DARTS) method. We investigated the antitumor effects of NC using a mouse melanoma B16 tumor xenograft model.
RESULTS: NC inhibits mTORC1 activity by targeting amino acid-sensing signaling through activating transcription factor 4 (ATF4)-mediated Sestrin2 induction. NC directly binds to IGF2R and promotes its lysosomal degradation. Moreover, NC displayed potent cytotoxicity against various cancer cells and inhibited B16 tumor xenografts.
CONCLUSIONS: NC inhibits mTORC1 signaling through nutrient sensing and directly targets IGF2R for lysosomal degradation, providing mechanistic insights into the MOA of NC.
方法:我们通过免疫印迹和荧光显微镜在CRISPR/Cas9基因编辑技术产生的野生型和基因敲除细胞系中探索了NC对mTORC1信号传导的影响。我们通过药物亲和响应靶标稳定性(DARTS)方法将IGF2R鉴定为NC的直接靶标。我们使用小鼠黑色素瘤B16肿瘤异种移植模型研究了NC的抗肿瘤作用。
结果:NC通过激活转录因子4(ATF4)介导的Sestrin2诱导靶向氨基酸感应信号来抑制mTORC1活性。NC直接结合IGF2R并促进其溶酶体降解。此外,NC对各种癌细胞表现出有效的细胞毒性并抑制B16肿瘤异种移植物。
结论:NC通过营养感知抑制mTORC1信号传导,并直接靶向IGF2R进行溶酶体降解,提供对NCMOA的机械学见解。