USP1 Promots Breast Cancer Cell Metastasis And Its Possible Mechanisms

Background and Objective: The main cause of death in patients with breast cancer is distant metastasis.Deubiquitinating enzymes(DUBs)regulate stability,cellular localization and activity of certain proteins by deconjugating ubiquitin or ubiquitin chain from them,thereby influence a variety of biological processes,such as cell cycle,DNA damage repair,chromatin remodeling.Deregulation of DUBs plays an important role in tumorigenesis and tumor progression.Our preliminary screening experiments showed that USP1 knockdown can significantly inhibit the migration of breast cancer cells.The aim of this study is to explore whether and how USP1 regulates the metastatic activity of breast cancer cells.Method: MCF7 were transfected with si RNAs targeting different DUBs to identify DUBs that impact breast cancer cell migration by cell migration assay.Breast cancer cells were lentivirally transduced with USP1 or the sh RNA targeting USP1.Cell proliferation,migration and invasion,in vivo tumor metastasis ability was analyzed by MTT,clone formation,in vitro migration and invasion assays,as well as orthotropic tumor model.The enzyme-dependent activity of USP1 in regulation of tumor cell metastasis was analyzed by a selective USP1 inhibitor and a USP1 de-ubiquitinating enzyme-inactive mutant generated by site-directed mutagenesis.The downstream events of USP1 were delineated through RNA sequencing,KEGG annotation and IPA analysis,and the results were verified by real-time RT-PCR.The expression pattern of USP1 and Snail in human or mouse breast tumor cells was examined by western blot,real-time RT-PCR and IHC analyses.Data mining based on published databases was performed to analyze the prognostic significance of USP1 and Snail expression as well as the correlation of their abundances in human breast carcinomas.Results: In human breast carcinoma tissues,USP1 protein levels in breast cancer tissue and lymph node metastases were significantly higher than that in nontumor breast cells.Neither Knockdown nor overexpression of USP1 influence proliferation of MCF7,MDA-MB-231 and 4T1 cells.Knockdown of USP1 inhibited breast cancer cell migration and invasion,whereas overexpression of USP1 promoted cell migration and invasion.In orthotropic 4T1 breast tumor model,USP1 significantly promoted formation of metastatic foci in lung without influencing the primary tumor formation.Notably,the USP1 mutant deficient in de-ubiquitinating enzyme activity failed to promote migration of MCF7 cells,and the USP1 inhibitor ML323 had inhibitory effects on tumor cell migration,indicating that deubiquitinating activity was essential for USP1-mediated regulation of cell migration.We further analyzed the alterations of gene profile in MCF7 cells with USP1 knockdown by RNAseq.Pathway enrichment analysis with DAVID showed that the transcript levels of genes involved in focal adhesion were downregulated in cells with USP1 knockdown relative to control cells.In addition,IPA analysis for probing enriched function demonstrated that the factors related to cell movement of breast cancer cells were transcriptionally down-regulated by USP1 inhibition.Importantly,RNAseq results also showed that USP1 inhibition in tumor cells resulted in decreases in expression of Snail and its downstream genes.Furthermore,USP1 overexpression upregulated Snail expression at both m RNA and protein levels.Silencing Snail in USP1-overexpressing breast cells significantly counteracted USP1-mediated enhancement of migration.By data mining of published databases,we found a significantly positive correlation between USP1 and Snail expression,and that USP1 overexpression was associated poor prognosis and the presence of metastasis in breast cancer.Both high m RNA expression of USP1 and Snail are correlated with a poor survival prognosis.Conclusions: USP1 promotes metastatic breast tumor growth but does not prevent primary tumor.Upregulated Snail activity is involved in the metastasis–promoting function of USP1 in breast cancer cells.Targeting USP1 may be a potential strategy to manipulate breast cancer metastasis.