CHIP Regulates The Cancer Stem-like Cell Behaviors Of Oral Squamous Cancer By Ubiquitin Degradation Of CD166

Objectives:Several independent studies have reported the roles of the E3 ubiquitin ligase,carboxy-terminus HSC70 interacting protein(CHIP)in some cancers.But the biological effects of CHIP vary on different cancers and the roles of CHIP in oral squamous cancer remain unknown.Material and methods:In this study,CHIP overexpression plasmids and CHIP knockdown lentivirus were constructed to affect the expression levels of CHIP and biological behaviors in oral squamous cancer cell lines bilaterally.The biological behaviors of CHIP in oral squamous cancer were investigated both in vivo and in vitro with a series of assays and analysis.The biological roles and the underlying mechanisms of E3 ubiquitin ligase CHIP in the regulation of cancer stem-like cell(CSC)behaviors were investigated by microsphere culture,immunoblotting,co-immunoprecipitation,and confocal observation.A tissue microarray was stained and analyzed for the clinical significance of CHIP expression in oral squamous cancer.The expression levels of CHIP,CD166,and other involved protein were detected with immunohistochemistry for clinical samples,and xenograft tumors.A systematical review of CD166 related expression and cancers was aimed to clarify its clinical significance for potential translation as a cancer biomarker.Results:We identified that CHIP could suppress the malignant behaviors of oral squamous cancer in a series of in vitro and in vivo experiments.Besides,we observed a changing expression pattern of CHIP from well-differentiation,moderate-differentiation,to poor-differentiation pathological status in oral squamous cancer specimens.In a retrospective cohort of oral squamous cancer,lower expression of CHIP indicates poor differentiation status and lower overall survival rate.Besides,we found that CHIP represses CSC characteristics of oral squamous cancer.The above date indicated that CHIP functions as a candidate of tumor suppressor and shows great potential to regulate the CSC properties of oral squamous cancer.We found that CHIP directly regulated the stability of CSC related protein CD166 in protein level.Rescue assays were designed by suppressing the expression level of CHIP in HN13-shCD166 cells and by reducing the expression level of CD166 in UMSCC12-shCHIP cells.Consequently,we managed to increase CD166 expression level in HN13-shCD166 cells by knocking down CHIP expression.Compared with HN13-Scrambled cells,the decreased colony and microsphere forming abilities of HN13-shCD166 cells were obviously enhanced with two pairs of siRNA sequences targeting CHIP.In UMSCC12-shCHIP cells,its increased CD166 expression was reduced by two pairs of siRNA sequences targeting CD166.UMSCC12-shCHIP cells presented a higher CD166 expression and CSC properties than UMSCC12-Scrambled cell,and knocking down CD166 expression in UMSCC12-shCHIP significantly decreased its colony and microsphere formation.These data strengthen the point that CHIP-CD166 axis plays important roles in regulating CSC properties of HNCs.Subsequently,we identified that CHIP directly regulates the stability of CD166 protein through the ubiquitin proteasome system in a HSP70/HSP90 dependent manner.In the clinical samples and in carcinogen-induced mouse tongue cancers,a significant negative correlation was observed between the expression levels of CHIP and CD166.The above data indicated an important role of CHIP-CD166-proteasome axis in the initiation and development of oral squamous cancer.In order to evaluate the translation potential of CD166 in cancer areas,a series of meta-analyses were conducted.Based on the current clinical studies,total CD166 expression was observed to be correlated to cancer risk,membrane CD166 expression correlated to nodal metastasis,and cytoplasmic CD166 expression correlated to TNM stage and disease-free survival.Membrane CD166,cytoplasmic CD166 and soluble CD166 expression showed great potential to be used as a panel of biomarkers for cancer overall survival prediction.Conclusion:Our findings demonstrated that CHIP-CD166-proteasome axis participates in regulating CSC properties in oral squamous cancer,suggesting that the regulation of CD166 by CHIP could provide new options for diagnosing and treating in the patients with oral squamous cancer.