Abstract
BackgroundThe 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.MethodsStably 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.ResultsCHIP 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.ConclusionsOur 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.
Highlights
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
CHIP silencing inhibits cell growth of DLD‐1 cells To evaluate the expression of CHIP in colorectal cancer (CRC) cell lines, quantitative real-time RT-PCR (qRT-PCR) and western blot analysis were carried out
In conclusion, we considered that CHIP is a novel potential unfavorable independent predictive biomarker for CRC
Summary
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. The exact function and underlying mechanism of CHIP in colorectal cancer (CRC) is not yet clear. More efforts are needed to elucidate of the underlying molecular mechanism of the CRC progression and metastasis, and to find the new effective therapeutic target for the metastatic CRC. E-cadherin, encoded by the CDH1 gene, is the hallmark of EMT. It is a transmembrane glycoprotein, which is localized to adjacent cell membranes, and responsible for cell–cell interactions. Downregulation or loss of the E-cadherin, is reported to be involved in the invasion and metastatic progression of several malignancies, including CRC [9,10,11,12]. Glycogen synthase kinase 3β (GSK-3β) could promote the phosphorylation and degradation of Slug, and subsequently trigger EMT and tumor metastasis [21]
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