CHIP 通过激活 MAPK 和 AKT 信号通路和抑制 E-钙黏蛋白来促进结直肠癌转移，从而发挥癌基因的作用。
CHIP functions as an oncogene by promoting colorectal cancer metastasis via activation of MAPK and AKT signaling and suppression of E-cadherin.
机构信息
Center for Clinical Laboratory, The First Affiliated Hospital of Soochow University, Suzhou, 215006, China.
Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou, 215006, China.
出版信息
J Transl Med. 2018 Jun 19;16(1):169. doi: 10.1186/s12967-018-1540-5.
BACKGROUND
The carboxyl terminus of Hsc70-interacting protein (CHIP) is an E3 ubiquitin ligase that plays a controversial role in different cancers, either as a tumor suppressor or a tumor promoter. To date, the exact function and underlying mechanism of CHIP in colorectal cancer (CRC) is not yet clear. Here we aimed to determine whether CHIP could affect the biological behaviors of CRC cells and its underlying mechanisms.
METHODS
Stably transfected CHIP overexpression and depletion DLD-1 and HT-29 cells were established using Lipofectamine 2000. Cell growth was monitored by x-Celligence system. Cell proliferation was detected using CCK-8 and Brdu proliferation assay. Cell apoptosis and cell cycle were detected by flow cytometry analysis. Cell migration and invasion abilities were monitored by x-Celligence system, wound healing assay and transwell assay. In vivo intraperitoneal metastasis assay was performed to investigate the influence of CHIP on the tumor metastasis of CRC cells in nude mice. The expression of ERK, AKT, NF-кB signaling subunits and EMT related proteins were detected by Western blotting. The influence and function of CHIP on the protein expression of CRC cells were also elucidated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. CRC microarray tissue was analyzed to investigate the CHIP expression and its clinical significance.
RESULTS
CHIP depletion inhibited cell growth, migration and invasion potential of CRC cells, accompanied by downregulation of MAPK and AKT signaling activities and upregulation of E-cadherin. CHIP overexpression dramatically enhanced the migration and invasion abilities, due to the upregulation of MAPK and AKT signaling and downregulation of E-cadherin. The proteomic analysis confirmed that E-cadherin was decreased in CHIP-overexpressing CRC cells. Furthermore, clinical tissue data revealed that CHIP expression was upregulated in CRC samples and was significantly correlated with poor survival of CRC patients. Mechanically, CHIP probably activated the MAPK and AKT signaling, which inactivated GSK-3β. The GSK-3β inactivation, in turn, upregulated Slug and led to E-cadherin downregulation and EMT initiation.
CONCLUSIONS
Our finding suggested that CHIP functions as an oncogene in the migration and metastasis of CRC, and is a potential unfavorable independent predictive biomarker for CRC. CHIP activates the AKT pathway to promote EMT and metastasis in CRC through the CHIP-MAPK/AKT-GSK-3β-Slug-E-cadherin pathways.
背景
Hsc70 相互作用蛋白（CHIP）的羧基末端是一种 E3 泛素连接酶，在不同的癌症中发挥着有争议的作用，既是肿瘤抑制因子，又是肿瘤促进因子。迄今为止，CHIP 在结直肠癌（CRC）中的确切功能和潜在机制尚不清楚。在这里，我们旨在确定 CHIP 是否会影响 CRC 细胞的生物学行为及其潜在机制。
方法
使用 Lipofectamine 2000 稳定转染 CHIP 过表达和耗尽 DLD-1 和 HT-29 细胞。使用 x-Celligence 系统监测细胞生长。使用 CCK-8 和 Brdu 增殖测定法检测细胞增殖。通过流式细胞术分析检测细胞凋亡和细胞周期。通过 x-Celligence 系统、划痕愈合测定和 Transwell 测定监测细胞迁移和侵袭能力。进行体内腹腔转移实验以研究 CHIP 对裸鼠 CRC 细胞肿瘤转移的影响。通过 Western blot 检测 ERK、AKT、NF-кB 信号亚基和 EMT 相关蛋白的表达。通过液相色谱-串联质谱（LC-MS/MS）分析阐明 CHIP 对 CRC 细胞蛋白表达的影响和功能。分析 CRC 微阵列组织以研究 CHIP 的表达及其临床意义。
结果
CHIP 耗竭抑制 CRC 细胞的生长、迁移和侵袭能力，同时下调 MAPK 和 AKT 信号活性并上调 E-钙粘蛋白。CHIP 过表达显著增强了迁移和侵袭能力，这是由于 MAPK 和 AKT 信号的上调和 E-钙粘蛋白的下调。蛋白质组学分析证实 E-钙粘蛋白在 CHIP 过表达的 CRC 细胞中减少。此外，临床组织数据显示 CHIP 表达在 CRC 样本中上调，并与 CRC 患者的不良生存显著相关。在机制上，CHIP 可能激活 MAPK 和 AKT 信号，使 GSK-3β失活。GSK-3β 的失活反过来又上调 Slug，导致 E-钙粘蛋白下调和 EMT 起始。
结论
我们的发现表明 CHIP 在 CRC 的迁移和转移中起癌基因作用，是 CRC 潜在的不利独立预后生物标志物。CHIP 通过 CHIP-MAPK/AKT-GSK-3β-Slug-E-cadherin 途径激活 AKT 通路促进 EMT 和 CRC 转移。
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