Mechanism Study On SIPA1 Facilitating Macrophage Recruitment During Breast Cancer Metastasis

Metastasis is the main cause of death in cancer patients,and the changes of tumour microenvironment（TME）is a critical factor leading to metastasis.It has been found that there are many kinds of immune cells infiltrating in the microenvironment of breast cancer,especially tumour-associated macrophages（TAMs）and regulatory T cells.Among these cells,TAMs have been known to associate with tumour angiogenesis,invasion and metastasis.Extracellular vesicles（EVs）derived from breast cancer cells are critical media involved in the communication between tumour cells and TAMs in the TME,which promote cancer cell invasion and metastasis.Signal-induced proliferation-associated 1（SIPA1）protein is a trigger of metastasis in breast cancer.However,it is unclear whether SIPA1promotes metastasis by regulating macrophage infiltration in the TME.The aim of this study was to investigate how breast cancer cells expressing SIPA1 recruit macrophages to promote metastasis,and to clarify the role of SIPA1 in regulating TME.The main results are concluded as follow.（1）Firstly,the correlation between the expression of SIPA1 and infiltration of macrophages in the tumour microenvironment was elucidated.Immunofluorescence staining of tissue microarray from breast cancer patients were analysed and the results revealed that there was a positive correlation between SIPA1 average fluorescence intensity（AFI）and the ratio of CD68-positived macrophages.Analysis of the breast invasive carcinoma（BRCA）data from the TCGA database also illustrated that SIPA1 expression levels were positively correlated with macrophage abundance.Furthermore,the number of macrophages in the SIPA1-high expressed metastatic MDA-MB-231 cell xenograft tumours was significantly higher than that in SIPA1 knockdown MDA-MB-231 cell（231/si）xenograft tumours.These results suggest that SIPA1 may regulate macrophage infiltration in breast cancer tissues.（2）Secondly,we investigated that how SIPA1 recruited macrophages in breast cancer tissue.THP1 macrophages were treated with EVs derived from breast cancer cell lines with knockdown or overexpression of SIPA1.The results showed that EVs derived from low SIPA1-expression breast cancer cells significantly recruited less migrated macrophages.In contrast,EVs derived from MCF7 cells overexpressing SIPA1（MCF7/SIPA1）significantly recruited more migrated macrophages than that from MCF7.Moreover,the MDA-MB-231cell orthotopic tumours were further treated with tumour cell-derived EVs,and the results showed that both of the number of macrophages in orthotopic tumours and metastatic tumour nodules on the surface of lung tissues in mice treated with MDA-MB-231-derived EVs（231-EVs）were higher than those with 231/si-derived EVs（231/si-EVs）or PBS.These results suggest that high SIPA1-expressed breast cancer cells recruit macrophages and promote lung metastasis by modulating tumour cell-derived EVs.（3）Then,the key SIPA1-regulated molecules were screened in EVs.Proteomics analysis of 231-EVs and 231/si-EVs and screened 158 candidate molecules that only exist in 231-EVs.Subsequently,the 158 candidate molecules were enriched by gene ontology（GO）,and four biological processes related to cell migration were screened out,among which the consensus molecule MYH9 was picked out.The chromatin immunoprecipitation,luciferase reporter gene,and Western blotting assays revealed that SIPA1 interacted with the promoter region of MYH9（the gene encoding myosin-9）,and upregulated MYH9transcription,increasing myosin-9 levels in cells and EVs.These results suggest that SIPA1in breast cancer cells can regulate protein composition in EVs and positively regulate MYH9expression in cells.（4）Finally,we validated whether myosin-9 in EVs regulates macrophage infiltration and the relationship between MYH9 expression and prognosis in breast cancer patients.Myosin-9 levels in tumour-derived EVs were decreased or increased by knocking down or overexpressing of MYH9 in breast cancer cells.Myosin-9-enriched EVs significantly promoted macrophage migration,while downregulation of MYH9 expression in cancer cells or use of blebbistatin（a myosin-9 inhibitor）reduced myosin-9 content or function in EVs and significantly reduced macrophage migration.Further investigation the TCGA-BRCA data showed that both the expression of SIPA1 and MYH9 were positively correlated with the expression of three typical markers of TAMs（CD86,CSF1R,and CCR2）,respectively.When analysing survival data from breast cancer patients in Kaplan-Meier website,we found that higher MYH9 expression was associated with shorter relapse-free survival in breast cancer patients in the SIPA1^highexpression group.These results indicate that myosin-9 in tumour-derived EVs promotes macrophage infiltration,suggesting that TAMs infiltration may be abundant in breast cancer patients with both SIPA1 and MYH9 high expression,leading to poor prognosis.In conclusion,SIPA1 targets the MYH9 promoter,upregulates its transcription in breast cancer cells,and increases myosin-9 content in tumour cell-derived EVs.Myosin-9-enriched EVs enhance macrophage infiltration and promote breast cancer metastasis.Therefore,myosin-9 in breast cancer cell-derived EVs could be a potential marker of breast cancer metastasis.Inhibition of myosin-9 in cells or EVs may be a new approach for the treatment of breast cancer metastasis.