The Molecular Mechanism Of SND1 Promoting TGFβ Signal Transduction And Breast Cancer Metastasis

Objectives:Breast cancer is one of the malignant diseases that endanger the health of women in the world.Distant metastasis of advanced breast cancer is responsible for more than 90% of all breast cancer-related deaths.Breast cancer metastasis is a complex cell-biological process which involves local invasion of primary cancer cells and their dissemination to distant organ sites and metastatic colonization.TGFβ signaling pathway can regulate the tumor invasion and migration via promoting epithelial–mesenchymal transition(EMT)and regulating the expression of series of genes involved in tumor invasion and migration.SND1(Staphylococcal nuclease domain-containing 1)protein,which is a conserved and muti-functional protein,has a role in the regulation of gene expression as a transcriptional co-activator and implicates in breast cancer related processes.In the previous studies,we found that SND1 could bind the promoter of transcription factors and regulators in TGFβ signaling pathway,which suggested that SND1 is a potential regulator in the TGFβ signaling pathway.However,the correlation between SND1,TGFβ signaling pathway and breast cancer metastasis is still unknown.The aim of this study is to explore the molecular mechanism of SND1 involved in breast cancer metastasis by regulating TGFβ signal transduction.Methods:1.Immunohistochemistry of breast cancer samples with or without lymph nodes metastasis and in vivo metastasis assay were performed to analyze the correlation between SND1 and breast cancer metastasis.The Kaplan–Meier method was used to analyze the correlation between SND1 and breast cancer prognosis.2.Genome-wide Chromatin Immunoprecipitation(Ch IP)-chip was performed to delineate the transcriptional targets of SND1.TCGA breast cancer database was used to find the gene co-expressed with SND1.The two dates were combined to analysis and predict SND1 downstream target genes.3.Western Blot(WB),Polymease Chain Reaction(PCR)and dual luciferase assay were performed to investigate the physiological function of SND1 in regulating the expression of SMAD2,SMAD3,SMAD4 genes.Pathway Commons 2 software was used to analysis Smad downstream genes which were regulated by SND1.The analysis was further verified by q PCR.4.MEME online software was used to identify the SND1-binding sites in the promoter regions of SMAD2,SMAD3,SMAD4 genes.Ch IP and Electrophoretic Mobility Shift Assay(EMSA)were performed to confirm whether SND1 could bind to the promoter regions of SMAD2,SMAD3,SMAD4 genes.5.The molecular mechanism of SND1 regulation of SMAD2,SMAD3,SMAD4 gene transcription was analyzed by Co-Immunoprecipitation(Co IP)and Ch IP.6.GST-pulldown assay,Realtime PCR,WB,Ch IP and dual luciferase assay were performed to identify the functional domain of SND1 in regulating the expression of SMAD2,SMAD3,SMAD4 genes.7.MCF-7-con(con)and MCF-7-SND1-sh-1(SND1-sh-1)cells were treated with 4 ng/m L TGFβ1.WB,immunofluorescence(IF)and dual luciferase assay were performed to investigate the influence of different SND1 protein levels on TGFβ signal transduction.A TGFβ1 signal response reporter assay was performed to investigate the influence of different SND1 protein levels on TGFβ receptor activity.Wound-healing and Transwell invasion assays were performed to investigate the influence of different SND1 protein levels on TGFβ1 promotion of migration and invasion.Results:1.SND1 expression is correlated with metastasis and poor prognosis in human breast cancer.2.Screening SMAD2,SMAD3 and SMAD4 as target genes of SND1 protein by analyzing the Ch IP-chip and TCGA datas.3.SND1 promotes the expression of SMAD2,SMAD3,SMAD4 and their downstream genes in breast cancer cells.4.SND1 promotes gene transcriptional activation of SMAD2,SMAD3,SMAD4 by binding to conserved motifs in the promoter region.5.SND1 interacts with histone acetyltransferase GCN5 and recruits GCN5 to each promoters of SMAD2,SMAD3,SMAD4.The recruitment of GCN5 to SMAD2,SMAD3,SMAD4 promoters would sequentially enhance histone acetylation and gene transcriptional.6.The tudor-containing TD domain of SND1 is the GCN5-bingding domain and plays a major role in promoting SMAD2,SMAD3,SMAD4 genes transcriptional activation.7.After TGFβ1 treatment,SND1 knockdown dose not affect the activation of TGFβ receptor but decreases the phosphorylation level of Smad2 and Smad3 and attenuates the TGFβ1 respond transcription.Conclusions:1.SND1 can bind to SMAD2,SMAD3,SMAD4 genes promoter conserved motifs and recruit GCN5 to acetylate H3K9 and that through this activity,SND1 promotes SMAD2,SMAD3,SMAD4 genes transcription.The tudor-containing TD domain of SND1 plays a major role in this process.2.SND1 can affect the signal transduction of TGFβ signaling pathway by regulating the expression of TGFβ downstream Smad proteins and further affects the breast cancer metastasis phenotype via TGFβ signaling pathway.