Direct Regulation Of NCK1 By STAT3 Promotes Metastasis And Angiogenesis Of Coloretal Cancer

Backgroud and ObjectiveIt has been reported that NCK1 is involved in cytoskeleton remodeling and could enhance the ability of cell movement.We considered that NCK1 might be related to tumorigenesis.The purpose of this study was to detect the expression and function of NCK1 in colorectal cancer(CRC),and to explore the molecular mechanism of its abnormal expression and function in CRC.MethodsWe determined the expression level of NCK1 in CRC tissues with quantitative real-time(qRT-PCR),western blot and immunohistochemistry(IHC),and then studied its clinical significance;The functional effects of NCK1 in CRC cell lines were performed using a loss-of-function or gain-of-function approach;The relationship between STAT3 and NCK1 was analyzed by gene expression analyses in 42 pairs of CRC samples using spearman correlation;the binding site between STAT3 and NCK1 was confirmed using dual luciferase assay and chromatin immunoprecipitation(ChIP)assay in CRC cells;western blot analysis was performed to elucidating the phosphorylation status of PAK1 protein and ERK;The relationships among NCK1,PAK1 and PYK2 were detected by confocal immunofluorescence and immunoprecipitation,and the PYK2-siRNA was transfected into the NCK1-upregulated CRC to detect the effect of PYK2 on the phosphorylation of PAK1 induced by NCK1.Finally,the NCK1 interference lentiviral vector was transfected into CRC cells overexpressing STAT3,to explore the possible mechanism of STAT3-NCK1-PAK1-ERK regulating axis in the development of colorectal cancer.Result:The average expression level of NCK1 was significantly up-regulated in CRC specimens compared with their normal counterparts,and the NCK1 expression level in CRC was positively correlated with serosal invasion,lymph metastasis,and tumor-node-metastasis(TNM)classification while reversely correlated to differentiation;Gain-of-function and loss-of-function studies showed that ectopic expression of NCK1 enhanced metastasis and angiogenesis in CRC cells;Spearman correlation analyses showed that STAT3 and NCK1 were positively related in expression in 42 pairs of colorectal cancer tissues(r=0.739,P=0.000);Promoter activation and binding analyses denoted that STAT3 promoted the NCK1 expression by directly acting on the NCK1 promoter.Ectopic expression of NCK1 significantly induced the phosphorylation of PAK1 and ERK,treatment with the p-PAK1 inhibitor could abrogate the activities of p-PAK1 and p-ERK by overexpression of NCK1.Therefore,the phosphorylation of PAK1 was involved in the activation of the ERK pathway induced by NCK1;Reciprocal Co-immunoprecipitation assay showed that NCK1 pull down PAK1 as well as PYK2 reciprocally in the SW480 cells;The depletion of PYK2 could reduce the phosphorylation of PAK1 and ERK compared to the negative control.All these evidences suggested that the activating the PAK1/ERK signaling pathway by NCK1 might be dependent on the interaction of PYK2.The overexpression of STAT3 significantly facilitated the phosphorylation of PAK1 and ERK in RKO cells while the knockdown of NCK1 partially reduced the expression of p-PAK1 and p-ERK in the RKO/STAT3 cells.These findings suggest a novel STAT3 to NCK1 to PAK1/ERK signaling mechanism potentially critical for CRC metastasis and angiogenesis.Conclusion:Increased expression of NCK1 in colorectal cancer tissues was positively correlated with the progression of colorectal cancer.Under the transcriptional regulation of STAT3,NCK1 could activate the ERK pathway by induces the phosphorylation of PAK1,which is ultimately reflected the role of NCK1 in the metastasis and angiogenesis of colorectal cancer.