Role Of CCR4 In Colorectal Cancer Metastasis And The Underlying Mechnaisms

Objectives:To investigated the effects of CCR4 on cell invasion and metastasis,as well as the underlying molecular mechanisms.Methods:The expression of CCR4 in colorectal cancer tissues was detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR),western blot and immunohistochemistry analysis.Several clinical and pathologic factors were compared with CCR4 expression.The migratory and invasive abilities of CRC cells were examined by wound healing and transwell assays.The molecular mechanisms responsible for CCR4-mediated cells invasion were explored by a Tumor Metastasis PCR Array and immnoblotting.We further treated CRC cells with different concentrations of rh-TNF-?.CHIP and luciferase reporter assays were used to detect binding site of NF-?B in the CCR4 promoter region.Results:CCR4 expression levels were significantly increased in CRC samples compared to paired normal tissues from 16 patients.In a retrospective cohort,a significant association was observed between the CCR4 negative group and positive group in tumor size,invasion depth,lymphatic metastasis,and TNM stage.Kaplan-Meier survival curves showed a strong correlation between CCR4 level and poorer 5-year overall survival and disease-free survival.Wound healing and transwell assays showed that CCR4 promoted invasive abilities of CRC cells.The results form liver metastasis model showed that CCR4 played a positive role in metastasis of CRC cells.A Tumor Metastasis PCR Array was then performed,MMP13 was selected for further investigation because it has the largest up-regulated alteration.After treatment with si-MMP13 and CL82198,the increased capacities induced by CCR4 overexpression were dramatically abolished.MMP13 expression was then analyzed in the TMA containing 116 paired CRC tissues.A positive correlation between CCR4 expression and MMP13 was observed.We father demonstrated that the effect of CCR4 was dependent on the activation of ERK/NF-?B/MMP13 pathway.TNF-a incubation increased the expression of CCR4 in a dose-dependent manner.CHIP and luciferase reporter assays also detected binding site of NF-kB in the CCR4 promoter region upon TNF-a stimulation.Conclusion?Chemokine receptor CCR4 facilitates metastasis of CRC cells by activating ERK/NF-?B/MMP13 pathway.Overexpression of CCR4 is correlated with poor survival of CRC patients.Moreover,CCR4 could be induced by the cytokine TNF-a in NF-?B-dependent manner.