C9orf140,A Novel Axin1-interacting Protein,mediates The Negative Feedback Loop Of Wnt/β-catenin Signaling

Wnt（wingless-type MMTV integration site family）signaling pathway is highly conserved in organisms,which is named after the Wnt ligands that activate this signaling pathway.Wnt signaling pathway plays an important role in embryonic development,and is closely related to tumorigenesis and tumor development.Generally,the Wnt signaling pathway mainly refers to the canonical Wnt signaling pathway which is mediated byβ-catenin,namely the Wnt/β-catenin signaling pathway.The activation of Wnt/β-catenin signaling pathway depends on whether the key factorβ-catenin in the cytoplasm can accumulate and enter the nucleus to regulate the downstream target genes.The target genes of Wnt/β-catenin signaling pathway are mainly related to cell proliferation,stem cell maintenance and tumorigenesis.The key regulatory steps involved in the Wnt/β-catenin signaling pathway are mediated by theβ-catenin destruction complex in the cytoplasm.The destruction complex consists of the central scaffolding protein Axin and otherβ-catenin binding proteins and protein kinases,ensuring precise regulation of the phosphorylation,ubiquitination and proteasomal degradation ofβ-catenin.Wnt/β-catenin signaling activity is maintained in homeostasis by an expanding list of molecular determinants.However,the molecular components and the regulatory mechanisms involved remain to be elucidated.C9orf140 is one of the differentially expressed genes during carcinogenesis which was isolated and characterized in recent years,and more and more experimental evidence showed that C9orf140 expression was detected in a variety of tumor cell lines and tumor tissues,including gastric cancer,colorectal cancer,hepatocellular carcinoma,renal cell carcinoma,glioma,and breast cancer.C9orf140 may be closely related to tumorigenesis and its specific expression may contribute to tumor cell proliferation,migration and invasion.Moreover,the expression of C9orf140 is cell cycle-dependent,which is higher in the G₁ and M phases than in the S and G₂ phases,and highest in the M phase.The disorder of cell cycle is one of the most distinctive features of tumorigenesis.C9orf140 may be involved in the regulation of cell cycle,and thus play a role in tumorigenesis.The present results show that C9orf140 may be oncogenic and a potential diagnostic marker in clinical oncology and a molecular target of drug therapy.However,there are few reports on C9orf140-interacted proteins or signaling pathways that C9orf140 involved in.The relationship between C9orf140-related signaling pathways and its biological functions needs to be further studied.Here,we identified C9orf140 as a novel Axin1-interacting protein by tandem-affinity purification and mass spectrometry,and we confirmed their interaction by immunoprecipitation,pull-down and immunofluorescence assays in vitro.We further showed that C9orf140 is a negative regulator of Wnt/β-catenin signaling in cultured cells.It functions upstream ofβ-catenin,outcompetes PP2A for binding to Axin1,influences the balance between phosphorylation and de-phosphorylation ofβ-catenin,and ultimately compromises Wnt3A-inducedβ-catenin accumulation.Interestingly,Wnt induced C9orf140expression viaβ-catenin.We propose that C9orf140 mediates a negative feedback loop of Wnt/β-catenin signaling by interacting with Axin1.Our results provide new insights into the role of C9orf140 from the perspective of protein interaction and signaling pathways.We identified C9orf140 as a novel Axin1-interacting protein a negative regulator of Wnt/β-catenin signaling.These results advance the current understanding of the exquisite control of Wnt/β-catenin signaling cascade,and provide evidence of the new role of C9orf140.