Department of Anesthesiology, Jiangsu Provincial Key Laboratory of Geriatrics, the First Affiliated Hospital with Nanjing Medical University, Nanjing 210029, China.
Department of Geriatrics, First Affiliated Hospital with Nanjing Medical University, Nanjing 210029, China.
Theranostics. 2020 Jul 9;10(19):8573-8590. doi: 10.7150/thno.44321. eCollection 2020.
Metastasis accounts for 90% of cancer-associated mortality in patients with renal cell carcinoma (RCC). However, the clinical management of RCC metastasis is challenging. Lactate export is known to play an important role in cancer cell migration. This study investigated the role of heat shock protein A12A (HSPA12A) in RCC migration. HSPA12A expression was examined in 82 pairs of matched RCC tumors and corresponding normal kidney tissues from patients by immunoblotting and immunofluorescence analyses. The proliferation of RCC cells was analyzed using MTT and EdU incorporation assays. The migration of RCC cells was evaluated by wound healing and Transwell migration assays. Extracellular acidification was examined using Seahorse technology. Protein stability was determined following treatment with protein synthesis inhibitor cycloheximide and proteasome inhibitor MG132. Mass spectrometry, immunoprecipitation, and immunoblotting were employed to examine protein-protein interactions. RCC tumors from patients showed downregulation of HSPA12A, which was associated with advanced tumor node metastasis stage. Intriguingly, overexpression of HSPA12A in RCC cells inhibited migration, whereas HSPA12A knockdown had the opposite effect. Lactate export, glycolysis rate, and CD147 protein abundance were also inhibited by HSPA12A overexpression but promoted by HSPA12A knockdown. An interaction of HSPA12A with HRD1 ubiquitin E3 ligase was detected in RCC cells. Further studies demonstrated that CD147 ubiquitination and proteasomal degradation were promoted by HSPA12A overexpression whereas inhibited by HSPA12A knockdown. Notably, the HSPA12A overexpression-induced inhibition of lactate export and migration were abolished by CD147 overexpression. Human RCC shows downregulation of HSPA12A. Overexpression of HSPA12A in RCC cells unstabilizes CD147 through increasing its ubiquitin-proteasome degradation, thereby inhibits lactate export and glycolysis, and ultimately suppresses RCC cell migration. Our results demonstrate that overexpression of HSPA12A might represent a viable strategy for managing RCC metastasis.
转移是导致肾细胞癌（RCC）患者 90%癌症相关死亡的原因。然而，RCC 转移的临床管理极具挑战性。众所周知，乳酸外排在癌细胞迁移中发挥重要作用。本研究旨在探讨热休克蛋白 A12A（HSPA12A）在 RCC 迁移中的作用。通过免疫印迹和免疫荧光分析，检测了 82 对配对的 RCC 肿瘤和相应患者正常肾组织中的 HSPA12A 表达。通过 MTT 和 EdU 掺入测定分析 RCC 细胞的增殖。通过划痕愈合和 Transwell 迁移测定评估 RCC 细胞的迁移。通过 Seahorse 技术检测细胞外酸化。用蛋白质合成抑制剂环己酰亚胺和蛋白酶体抑制剂 MG132 处理后，测定蛋白质稳定性。通过质谱分析、免疫沉淀和免疫印迹检测蛋白质-蛋白质相互作用。患者的 RCC 肿瘤显示 HSPA12A 下调，与肿瘤进展分期有关。有趣的是，在 RCC 细胞中过表达 HSPA12A 抑制迁移，而 HSPA12A 敲低则产生相反的效果。HSPA12A 过表达还抑制乳酸外排、糖酵解率和 CD147 蛋白丰度，但促进 HSPA12A 敲低的上述过程。在 RCC 细胞中检测到 HSPA12A 与 HRD1 泛素 E3 连接酶的相互作用。进一步的研究表明，HSPA12A 过表达促进 CD147 的泛素化和蛋白酶体降解，而 HSPA12A 敲低则抑制上述过程。值得注意的是，CD147 过表达消除了 HSPA12A 过表达诱导的乳酸外排和迁移抑制。人 RCC 显示 HSPA12A 下调。HSPA12A 在 RCC 细胞中的过表达通过增加其泛素-蛋白酶体降解来稳定 CD147，从而抑制乳酸外排和糖酵解，最终抑制 RCC 细胞迁移。我们的研究结果表明，HSPA12A 的过表达可能是管理 RCC 转移的可行策略。
