Department of biological sciences, Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, Georgia.
Prostate. 2020 Apr;80(5):412-423. doi: 10.1002/pros.23956. Epub 2020 Jan 29.
Mammalian target of rapamycin (mTOR) is a downstream substrate activated by PI3K/AKT pathway and it is essential for cell migration. It exists as two complexes: mTORC1 and mTORC2. mTORC1 is known to be regulated by active AKT, but the activation of mTORC2 is poorly understood. In this study, we investigated the roles and differential activation of the two mTOR complexes during cell migration in prostate cancer cells.
We used small interfering RNA to silence the expression of Rac1 and the main components of mTOR complexes (regulatory associated protein of mTOR [RAPTOR] and rapamycin-insensitive companion of mTOR [RICTOR]) in LNCaP, DU145, and PC3 prostate cancer cell lines. We performed transwell migration assay to evaluate the migratory capability of the cells, and Western blot analysis to study the activation levels of mTOR complexes.
Specific knockdown of RAPTOR and RICTOR caused a decrease of cell migration, suggesting their essential role in prostate cancer cell movement. Furthermore, epidermal growth factor (EGF) treatments induced the activation of both the mTOR complexes. Lack of Rac1 activity in prostate cancer cells blocked EGF-induced activation of mTORC2, but had no effect on mTORC1 activation. Furthermore, the overexpression of constitutively active Rac1 resulted in significant increase in cell migration and activation of mTORC2 in PC3 cells, but had no effect on mTORC1 activation. Active Rac1 was localized in the plasma membrane and was found to be in a protein complex, with RICTOR, but not RAPTOR.
We suggest that EGF-induced activation of Rac1 causes the activation of mTORC2 via RICTOR. This mechanism plays a critical role in prostate cancer cell migration.
哺乳动物雷帕霉素靶蛋白（mTOR）是 PI3K/AKT 途径激活的下游底物，对细胞迁移至关重要。它存在于两种复合物中：mTORC1 和 mTORC2。mTORC1 的活性受 AKT 调节，但 mTORC2 的激活机制尚不清楚。在这项研究中，我们研究了在前列腺癌细胞迁移过程中两种 mTOR 复合物的作用和差异激活。
我们使用小干扰 RNA 沉默 Rac1 和 mTOR 复合物主要成分（mTOR 相关蛋白 [RAPTOR] 和雷帕霉素不敏感伴侣 [RICTOR]）在 LNCaP、DU145 和 PC3 前列腺癌细胞系中的表达。我们进行了 Transwell 迁移实验来评估细胞的迁移能力，并用 Western blot 分析来研究 mTOR 复合物的激活水平。
特异性敲低 RAPTOR 和 RICTOR 导致细胞迁移减少，表明它们在前列腺癌细胞运动中具有重要作用。此外，表皮生长因子（EGF）处理诱导两种 mTOR 复合物的激活。前列腺癌细胞中 Rac1 活性的缺失阻断了 EGF 诱导的 mTORC2 激活，但对 mTORC1 激活没有影响。此外，组成型激活的 Rac1 在 PC3 细胞中导致细胞迁移和 mTORC2 显著增加，但对 mTORC1 激活没有影响。活性 Rac1 位于质膜上，并与 RICTOR 而不是 RAPTOR 形成蛋白复合物。
我们认为 EGF 诱导的 Rac1 激活通过 RICTOR 引起 mTORC2 的激活。这种机制在前列腺癌细胞迁移中起着关键作用。
