Nrip2, A Novel Regulator Of The Non-canonical Wnt Pathway, Promotes The Self-renewal Of Colorectal Cancer Initiating Cells

Aim:Colorectal cancer is one of the most common malignant tumors, accounting for the second cause of death in the world. In recent years, although great progess has been achieved to improve the survival rates of them in the chemical therapy, the prognosis of the patients with advanced colorectal cancer is still depressed, with total mortality of40%. To elucidate the molecular mechanism of colorectal cancer is the premise and basis for the treatment of colorectal cancer. Recent studies have indicated that colorectal cancer initiating cells(CCICs) are involved in development, invasion, metastasis and drug resistance of colon cancer. The Wnt pathway palys an important role in the self renewal of CCICs, while the mechanism remains unknown. The purpose of this study is to demonstrate the biological effect of nuclear receptor interacting protein2(Nrip2) on colon cancers, which was screened and identified, and was related to the activation of Wnt pathway in CCICs; detect its role in the self renewal of CCICs; identify its key target protein; and look for the potential targets of Nrip2effect on CCICs.Methods:Nrip2related to the activation of the Wnt pathway was screened through constructing cDNA retroviral library of colospheres from SW620cells. To explore the expression of Nrip2in colon cancer, immunohistochemistry and mRNA fluorescence in situ hybridization were used in colon cancer tissue, and RT-qPCR was used in colospheres enriched from colon cancer cells and clinical samples. To evaluate the effect of Nrip2on self renewal of CCICs, the colospheres formation ability, the expression of iPS and EMT factors, and the tumorigenesis in vivo were observed. To investigate the above mechanism, the target protein of Nrip2, Frizzledl(Fzdl) was screened by nano LC-MS/MS sequencing followed by the analysis of binding site between Nrip2and Fzd1. Using co-immunoprecipitation and Western blot, retinoic acid-related orphan receptor beta (Rorβ) was screened and identified as a key target of Nrip2regulating the noncanonical Wnt pathway, which effect on the colospheres formation ability, the expression EMT factors, and the tumorigenesis in vivo were observed. HMG-box transcription factor1(HBP1) was screened as a core target of Rorp by gene chip. The effect of Nrip2enzyme activity on CCICs was observed with treatment of PepstatinA on colon cancer cells.Results:The overexpression of Nrip2related to the activation of Wnt pathway was observed by constructing cDNA retroviral library of colospheres enrich from SW620. The overespression of Nrip2was verified in colon cancer cells, stem cells, and tissues. Overexpression of Nrip2promotes self renewal of CCICs. We found that Nrip2could bind to Fzdl, while not in combination with β-catenin. Nrip2binded to Rorβ to induce its degradation, which was screened by co-immunoprecipitation. The enzyme inactivity can prevent degradation of Rorβ, promoting it from translocating into the nucleus. Rorβ could inhibit the self renewal of CCICs by inhibiting the Wnt pathway. HBP1was screened as a key target of Rorβ by gene chip, which could bind to the TCF/LEF4to block the transcription of down stream target molecules. Inactivation of Nrip2inhibited the self renewal of CCICs.Conclusion:We found that Nrip2and Rorβ expressed in normal colon tissue and colon cancer cells and tissues for the first time. Nrip2, with over-expression in colon cancer stem cells, could combine with Rorβ depending on its enzyme activity, degrade Rorp, inhibit the transcription of HBP1, and promote the targets expression of Wnt pathway, which was defined as a novel non canonical Wnt pathway-Nrip2/Rorβ/HBPl pathway. The enzyme activity of Nrip2is a potential target to block the self renewal of colon cancer stem cells.