Identification And Mechanism Study Of Genes That Regulate Breast Cancer Metastasis

Objective:To explore the effect and mechanism of Tetratricopeptide Repeat Domain 17(TTC17)on breast cancer metastasis.Methods:CRISPR/Cas9 whole genome knockout library was used for in vitro screening to find candidate genes relating to breast cancer metastasis.Meanwhile,using the exome sequencing results of breast cancer tissues combined with breast cancer data of TCGA database for data analysis,64 genes related to breast cancer metastasis were screened.After that,we constructed a 64-gene CRISPR/Cas9 customized library and screened it via lung metastasis mouse model.Both in vivo and in vitro screening suggested that TTC17 had the potential to affect breast cancer metastasis.Then the KM-Plotter,UALCAN and TCGA data platforms were used to analyze the expression of TTC 17 in breast cancer.Therefore,in vivo and in vitro experiments were conducted to verify the role of TTC 1 7 in regulating breast cancer metastasis.The function experiments included cell invasion,wound healing and clone formation experiments.Animal models included 4T1 orthotopic breast cancer mouse model and MDA-MB-231 lung metastasis mouse model.To explore the mechanism that TTC17 regulates breast cancer metastasis,RNA-seq was performed on TTC17 knockout/control breast cancer cells.Then,we used molecular biology experiments and related pathway inhibitor to verify the mechanism.In addition,in order to find approved anti-cancer drugs that can reverse the malignant phenotype caused by the decreased expression of TTC 17.we screened 313 approved anti-cancer drugs in vitro.At the same time,immunohistochemical staining of breast cancer clinical cohort tissues was used to analyze the relationship between TTC 17 expression and breast cancer metastasis.Results:We used CRISPR/Cas9 knockout in vivo and in vitro screening,combined with data analysis of breast cancer tissue exon sequencing results and TCGA breast cancer data,confirming that TTC 17 has the potential function of regulating breast cancer metastasis.Then through the data analysis of TCGA and CPTAC breast cancer data,we found that the expression of TTC 17 was down-regulated in breast cancer tissues and TTC 17 expression level showed a decrease in expression level as the tumor progressed.KMPLOT analysis showed that patients with low expression of TTC 17 had significantly shorter relapse-free survival.Experiment results showed that the knockout or knockdown of TTC 17 significantly enhances the invasion and migration ability of breast cancer cells;TTC17 overexpression inhibited cell invasion,healing and clonal formation in breast cancer cells.The orthotopic breast cancer model and lung metastasis model also show that the decrease of TTC17 expression can significantly promote the lung metastasis ability of breast cancer.KEGG enrichment analysis of differentially expressed genes between TTC17 knockout and control breast cancer cells showed that TTC17 regulated genes were mainly enriched in RAP1 pathway and TGF-β pathway.Meanwhile,we used cBioportal to find that TTC17 is significantly negatively correlated with the expression of genes in RAP1 signaling pathway.Through Western blot we found that the protein levels of RAP1A and CDC42(key molecules of the RAP1 pathway)in TTC17 knockdown breast cancer cell lines were significantly increased.In addition,Inhibiting the activity of CDC42 can reverse the increased migration ability of breast cancer cells caused by TTC17 knockdown.On the side,we found that TTC17 knockdown significantly reduced TGF-β2 protein level via Western blot.Drug library screening results showed that Pirfenidone,Paclitaxel,Palbociclib,Rapamycin,etc.had different drug inhibition in TTC17 knockdown and control cell lines.Interestingly,Rapamycin has the function of inhibiting CDC42 activity and Pirfenidone is an inhibitor of TGF-β pathway.The above further verified that TTC17 can regulate RAP1 and TGF-β signaling pathway.Meanwhile,Immunohistochemical staining results showed that the expression of TTC17 in breast cancer tissues with metastasis was lower than breast cancer tissues without metastasis.Conclusion:TTC17 can regulate the invasion and metastasis of breast cancer.Patients with low TTC17 expression had poor prognosis.The RAP1/CDC42 signaling pathway and TGF-β signaling pathway are potential pathways by which TTC17 regulated breast cancer metastasis.This study provides new potential target for the treatment of patients with breast cancer metastasis,as well as new potential target for diagnosis and prognosis.Objective To explore the effect of bromodomain and WD repeat domain containing 3(BRWD3)on lymph node metastasis in breast cancer and its mechanism.Methods Firstly,in vitro cell invasion experiments were used to explore the effect of BRWD3 on the invasion phenotype of breast cancer cell lines.And using mouse lymph node metastasis model and lung metastasis model,we investigate the role of BRWD3 in regulating breast cancer lymph node metastasis and lung metastasis in BALB/c nude mice.Secondly,The BRWD3 co-expressed genes were searched through cBioPortal databases to analyze the biological functions and pathways of BRWD3,constructed a BRWD3 molecular regulatory network,which is examined with Western blot analysis partly.At last,the public breast cancer dataset and the KM-Plotter analysis platform was used to analyze the expression of BRWD3 in breast cancer and the relationship between the expression of BRWD3 and breast cancer prognosis.Results In vitro experiments show that knockdown of BRWD3 can significantly promote the invasion of MDA-MB-231 and inhibit cell proliferation.The mouse lymph node metastasis model showed that knockdown of BRWD3 could significantly promote lymph node metastasis of breast cancer.However,the mouse lung metastasis model showed that BRWD3 did not affect the mouse lung metastasis ability.Furthermore,analysis of the co-expressed genes of BRWD3 revealed that functional clustering results showed that they are mainly involved in gene expression regulation,DNA damage repair,chromosome organization and modification,ubiquitination,etc.Meanwhile,KEGG enrichment analysis showed that it was involved in signaling pathways such as ubiquitination,oxidative phosphorylation,MAPK,etc.In addition,via Western blot experiment,it was found that knockdown of BRWD3 can promote the phosphorylation of ERK1/2.Moreover,BRWD3 expression in breast cancer with lymph node metastasis is significantly lower than in patients without lymph node metastasis.Further,the survival analysis in KM-Plotter found that the prognosis of patients with low expression of BRWD3 was poor,which was significantly lower than that of patients with high expression of BRWD3.Conclusions BRWD3 can regulate breast cancer invasion in vitro and lymph node metastasis in vivo.Afterwards,the prognosis of patients with low expression of BRWD3 is poor.Meanwhile,Ubiquitination,oxidative phosphorylation,MAPK pathway,etc.were the possible regulation pathways of BRWD3,which provide a new theoretical basis for the research and application of molecular markers related to breast cancer lymph node metastasis.