Metastatic castration-resistant (CR) prostate cancer (PCa) is a lethal disease for which no effective treatment is currently available. p66Shc is an oxidase previously shown to promote androgen-independent cell growth through generation of reactive oxygen species (ROS) and is elevated in clinical PCa and multiple CR PCa cell lines. We hypothesize p66Shc also increases the migratory activity of PCa cells through ROS and investigate the associated mechanism. Using the transwell assay, our study reveals that the level of p66Shc protein correlates with cell migratory ability across several PCa cell lines. Furthermore, we show hydrogen peroxide treatment induces migration of PCa cells that express low levels of p66Shc in a dose-dependent manner, while antioxidants inhibit migration. Conversely, PCa cells that express high levels of endogenous p66Shc or by cDNA transfection possess increased cell migration which is mitigated upon p66Shc shRNA transfection or expression of oxidase-deficient dominant-negative p66Shc W134F mutant. Protein microarray and immunoblot analyses reveal multiple proteins, including ErbB-2, AKT, mTOR, ERK, FOXM1, PYK2 and Rac1, are activated in p66Shc-elevated cells. Their involvement in PCa migration was examined using respective small-molecule inhibitors. The role of Rac1 was further validated using cDNA transfection and, significantly, p66Shc is found to promote lamellipodia formation through Rac1 activation. In summary, the results of our current studies clearly indicate p66Shc also regulates PCa cell migration through ROS-mediated activation of migration-associated proteins, notably Rac1.

转移性去势抵抗性（CR）前列腺癌（PCa）是一种致命疾病，目前尚无有效的治疗方法。p66Shc是一种氧化酶，以前被证明可以通过产生活性氧（ROS）来促进不依赖雄激素的细胞生长，并且在临床PCa和多种CR PCa细胞系中升高。我们假设p66Shc还可以通过ROS增强PCa细胞的迁移活性，并研究其相关机制。使用transwell分析，我们的研究表明p66Shc蛋白的水平与跨多个PCa细胞系的细胞迁移能力相关。此外，我们显示过氧化氢处理可诱导PCa细胞迁移，该PCa细胞以剂量依赖的方式表达低水平的p66Shc，而抗氧化剂可抑制迁移。反过来，表达高水平内源性p66Shc或通过cDNA转染的PCa细胞具有增加的细胞迁移，这种迁移在p66Shc shRNA转染或氧化酶缺陷型显性负性p66Shc W134F突变体表达后得到缓解。蛋白质微阵列和免疫印迹分析显示，在p66Shc升高的细胞中，多种蛋白质被激活，包括ErbB-2，AKT，mTOR，ERK，FOXM1，PYK2和Rac1。使用各自的小分子抑制剂检查了它们在PCa迁移中的参与。Rac1的作用已使用cDNA转染进一步验证，并且显着地，发现p66Shc通过Rac1激活促进了片状脂蛋白的形成。总而言之，我们当前研究的结果清楚地表明，p66Shc还通过ROS介导的与迁移相关的蛋白质（尤其是Rac1）的激活来调节PCa细胞的迁移。