靶向mTOR促进CyclinD1经自噬-溶酶体途径降解及干预早期乳腺癌生长、侵袭的机制研究

The mammalian target of rapamycin (mTOR) is known to be involved in regulation of autophagy and G1 cell cycle process. However, it remains unknown whether rapalog-induced autophagy plays a critical role in its regulation of cell cycle. We, for the first time, report that rapalog everolimus could stimulate autophagy-mediated Cyclin D1 degradation in breast cancer cells. Inhibiting mTOR with everolimus rapidly increased the degradation of Cyclin D1 in transformed human mammary epithelial cells, while 3-Methyladenine, a classic autophagy inhibitor, could attenuate everolimus-induced Cyclin D1 degradation. Moreover, other classic autophagy inhibitors including chloroquine and bafilomycin A1 could also attenuate everolimus-induced down-regulation of Cyclin D1 protein. Similarly, knockdown of autophagy related gene 7 (Atg-7) also repressed everolimus-triggered degradation of Cyclin D1. In addition, everolimus-induced autophagy occurred earlier than its induction of G1 arrest and blockade of autophagy attenuated everolimus-induced G1 cell cycle arrest. We also found that everolimus stimulated autophagy and decreased Cyclin D1 protein levels in explant human breast tissue. These data support the conclusion that the autophagy induced by rapalogs in human mammary epithelial cells appears to cause Cyclin D1 protein degradation resulting in G1 cell cycle arrest. ..We also found the increased levels and accumulation of Cyclin D1 in the nucleus in human breast cancer tissues compared to adjacent non-tumor tissues, while rapamycin induced nuclear export of Cyclin D1 and decreased Cyclin D1 levels in human breast tissues ex vivo, which is consistent with previous data that mTOR inhibitors triggered Cyclin D1 nuclear export, ubiquitination and degradation by the 26S proteasome. Cyclin D1 expression level also is also a candidate biological markers predictive of invasive recurrence in ductal carcinoma in situ (DCIS). Next, we investigate the role of Cyclin D1 in rapalogs-induced inhibition of DCIS progress to invasive breast cancer...Our findings contribute to our knowledge of the interplay between autophagy and cell cycle regulation mediated by mTOR signaling, Cyclin D1 regulation and DCIS therapy.

雷帕霉素靶蛋白（mTOR）是乳腺癌细胞内最重要的自噬和周期调控通路之一，但其介导细胞自噬和周期进程之间的关系未见报道。本项目首次揭示mTOR抑制剂可通过自噬－溶酶体途径促进乳腺癌细胞Cyclin D1降解进而诱导G1期阻滞，后续拟在人源肿瘤异种移植瘤模型（PDTX）和类器官三维培养（3D-Organoid）的临床原代乳腺癌细胞中加以确证。Cyclin D1高表达为乳腺导管原位癌（DCIS）易发展为侵袭性乳腺癌的肿瘤标记。课题前期结果显示mTOR抑制剂可促进Cyclin D1蛋白由细胞核向细胞浆再分布，进而促进其在胞浆中降解。下一步拟探讨靶向mTOR信号通路干预Cyclin D1介导的乳房“癌前病变”的可能性。课题成果有助于揭示Cyclin D1调控新机制和研发mTOR抑制剂用于乳腺癌的早期治疗。

雷帕霉素靶蛋白（mTOR）是乳腺癌细胞内最重要的自噬和周期调控通路之一，但其介导细胞自噬和周期进程之间的关系未见报道。本研究首先验证了mTOR抑制剂依维莫司可诱导MCF-10DCIS.COM细胞自噬和细胞周期G1期阻滞，然而自噬抑制剂3-MA预处理显著拮抗依维莫司的周期阻滞作用。进一步研究发现，依维莫司显著降低周期素Cyclin D1蛋白水平，而对其他细胞周期相关蛋白Cyclin E、CDK4、CDK6、P21和P27没有影响，siRNA介导Cyclin D1蛋白水平下调拮抗依维莫司诱导细胞周期G1期阻滞作用。本研究首次揭示自噬抑制剂3-MA、Baf A1和CQ预处理拮抗依维莫司诱导的Cyclin D1降解，下调ATG7蛋白水平同样拮抗依维莫司诱导的Cyclin D1降解，初步证实依维莫司通过自噬-溶酶体途径诱导Cyclin D1降解。依维莫司作用于乳腺组织块培养，诱导自噬和促进Cyclin D1蛋白降低。临床乳腺导管原位癌组织切培养模型中，依维莫司处理24小时后，免疫组织化学实验检测显示依维莫司可显著降低Cyclin D1表达，同时上调LC3蛋白表达。乳腺导管原位移植瘤（Mouse Introductal Xenografts Model，MIND）中，依维莫司低剂量短时间（2 mg/kg X 7 day）作用可显著抑制DCIS生长和侵袭。综上所述，本研究揭示mTOR抑制剂通过自噬-溶酶体途径加速Cyclin D1降解的新机制，为依维莫司用于乳腺癌早期治疗提供了参考。

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