NDRG1 Suppresses Cancer Cell Proliferation Via Modulating P21 Phosphorylation In A P53-independent Manner

This study is to investigate whether the phosphorylation of p21 by N-myc downstream-regulated gene 1(NDRG1)affects its stabilityin a P53-independent manner in cancer cells.NDRG1,is reported to act as a tumorsuppressor in colorectal,prostate,breast and pancreatic cancer,which could inhibit tumor cell migration,invasion,especially tumor metastasis.In a serious of long-term researches of NDRG1 demonstrated that the over-expression of NDRG1 in colorectal cancer,prostate cancer,breast cancer,and pancreatic cancer tissues could inhibit invasion and migration of tumor.Hence,NDRG1 has been classified as a metastasis suppressor rather than a proliferation inhibitor for a long time.A recent foreign research report indicated that NDRG1 could regulate the expression of p21,which was a tumor proliferation inhibitor.In the study,it revealed that NDRG1 could suppress tumor proliferation through up-regulating p21.However,the underlying mechanism of how NDRG1 modulates in detail p21,of which one signal pathway has rarely been explored.We revealed that the stability of p21 wasregulated by a varity of protein kinase in the process of post-translational modification,which could promote or inhibit the proliferation of tumor cells.Therefore,the current study is focused on the mechanism of phosphorylation of p21 and the function of NDRG1.First,we applied the immunohistochemstry assay to investigate the relative relationship of NDRGl protein expression in tumor tissue comparing with normal colorectal tissue in colorectal cancer patients between the proliferation and the prognosis of patients.NDRGl protein expression was significantly decreased in tumor tissue comparing to that in paired normal colorectal tissue was firstly showed in the assay.Further we analyzed the NDRG1 protein expression in tumor size.It revealed that the tumor size was bigger than 3cm,the number of negative NDRG1 was far smaller than the number of positive NDRG1.Hence,it indicated that NDRG1 had a function of inhibiting proliferation in cancer cells.In addition,we also analyzed that NDRG 1 was interacted to the prognosis of colorectal cancer patients.The patients with NDRG1 positive expression had significantly better overall survival and cancer-free survival comparing to those with NDRG1 negative expression.We builded up the stable NDRG1 over-expression and knocking-down models in colorectal and prostate cancer cell lines by transfecting through the retrovirus.Then we demonstrated precious phenomenon that NDRG1 suppressed cancer cell proliferation by immunofluorescence,CCK-8 assay and plat colony formation assay in those well-established tumor cell lines in a p53-independent manner.Morever,western blotting was used to analyze the relative relationship existed between NDRG1 and p21 phosphorylation at Thr145.And co-immunoprecipitation was used to further reveal that NDRG1 phosphorylated p21 at Thr145 through a protein –protein interaction.At last,we designed a BALB/c nude mouse xenograft model in the animal experiment center of Ruijin Hospital.The results revealed again that NDRG1 inhibited p53-independent cancer cells via regulating pThr145-p21.This study indicated that NDRG1,a tumor inhibitor factor,was able to inhibit proliferation of tumor cells in a p53-independent manner through regulating pThr145-p21.It expended the function of NDRG1 in anti-tumor field.This indicated that NDRG1 could be regarded as a prognostic biomarker for colorectal cancer and prostate cancer.Moreover,it helped to invest the novel a novel targeting therapy for human solid tuor proliferation.