Mechanistic Study On The Role Of Bcl-3 In Canonical Wnt Signaling And The Stemness Maintenance Of Colorectal Cancer Cells

Objectives:Colorectal cancer is a vital threat for human lives since the global morbidity and fatality rates rank 3rd among all cancers in recent years.The tumor stem cells within colorectal cancer play key roles in the occurrence,development and drug tolerance of this malignant tumor.Canonical Wnt signaling is one of the key regulators that maintains colorectal cancer stem cells.Our aim is to determine whether Bcl-3 participates in Wnt signaling mediated tumor stem cell maintenance in colorectal cancer.Besides,our results revealed that BCL-3 stabilized Smad3 protein and regulate lung metastasis of breast cancer in cultured cells and mouse model with spontaneous lung metastasis of breast cancer.In this study,we further designed experiments to understand the mechanism how TGF?signaling is regulated by Bcl-3.Methods:Several colorectal cancer cell lines were used to determine whether and how Bcl-3 regulated canonical Wnt signaling.We performed Western Blot,Q-PCR,CoIP,ChIP and FACS analysis to examine how Bcl-3 regulated Wnt signaling at protein and transcription levels as well as the effect of Bcl-3 on the growth and drug resistance of colorectal cancer stem cells?CSC?.Apc^min mice were employed as a model to observe the differences of intestinal adenoma growth between Apc^minin and Bcl-3^-/-Apc^min mice.Meanwhile,through MMTV-PyMT mouse model,our results suggested that Bcl-3 is critical for the lung metastasis of breast cancer.We also revealed that Bcl-3 interacts with Smad3and RBX1 by Co-IP.Results:We showed that Bcl-3 translocated into the nucleus upon Wnt3a stimulation and stabilized CBP,which in turn increased the acetylation of lysine49 of?-catenin and further enhanced the binding of?-catenin to the promoter regions of target genes.Bcl-3 knockdown decreased the number of colorectal cancer stem cells and in vitro oncosphere growth ability of colorectal cancer stem cells while increased their sensitivity to chemotherapeutic drugs.By contrast,Bcl-3overexpression increased the numbers of colorectal CSCs and decreased the sensitivity to chemotherapeutic drugs.There was no significant difference in the numbers of adenomas between Apc^minin and Bcl-3^-/-Apc^min mice.However,the expression of CSC related genes in adenoma from Bcl-3^-/-Apc^min mice was significantly lower than that in the control group.The lung metastasis of primary breast cancer cell are significantly decreased and overexpression of Smad3 partially rescued the decreased metastasis.We further identified the binding site between Bcl-3 and Smad3.Conclusion:Upon the activation of canonical Wnt/?-catenin signaling initiated by Wnt3a the phosphorylation of Bcl-3 by GSK-3 was decreased,resulting in inhibition of proteasome-dependent Bcl-3 degradation.Bcl-3 translocated into the nucleus to form a complex with CBP and?-catenin.This transcriptional complex bounded to the promoter region of Wnt target genes and initiated target genes expression.Bcl-3 depletion elevated the CBP degradation and diminished the acetylation of lysine 49?-catenin,in turn weakening the interaction between?-catenin and the promoter region,consequently decreasing the transcription process of Wnt target genes.Bcl-3 is required for the maintainace of the number and proliferation of colorectal cancer stem cell.Furthermore,our results showed that Bcl-3 promote the drug tolerance suggesting that Bcl-3 is a putative target for colorectal cancer.Bcl-3 also showed a higher expression level breast cancer than normal tissue.Meanwhile,Bcl-3 interfere affects the interaction between E3-ligase RBX1 and Smad3 and thus affected the protein level of Smad3.