USP12 Promotes Breast Cancer Metastasis By Maintaining The Stability Of Midkine Protein

Breast cancer is the most common female malignant tumor in the world,and it is a serious threat to women’s health.However,the metastasis of breast cancer in the later stage is the main cause of death in breast cancer patients.But so far,our basic theoretical research on metastatic breast cancer is still insufficient,and there is a lack of clinically effective detection targets and therapeutic drugs.Metastatic breast cancer is still a major worldwide problem in the tumor community.More and more studies have shown that deubiquitinating enzymes(DUBs)maintain the stability of many important proteins during tumor metastasis through deubiquitinating enzymes,play important biological functions,and participate in tumor metastasis.However,the specific functions and mechanisms of deubiquitinating enzymes in breast cancer metastasis are still unclear.Therefore,in this study,we studied the function and mechanism of deubiquitinating enzyme in the process of breast cancer metastasis,aiming to provide a new theoretical basis and therapeutic target for the molecular diagnosis and treatment of breast cancer metastasis.In this study,we screened 74 DUBs molecules related to the Distant Metastasis-Free Survival(DMFS)of breast cancer through the Kaplan-Meier plotter database.Through statistical analysis,we found that a total of 11 DUBs molecules were significantly associated with breast cancer DMFS(p<0.05).Subsequently,we used the mouse orthotopic transfer model to screen and verify the candidate DUBs molecules in vivo experiments.We found that knocking down USP12(Ubiquitin-specific protease 12)among these candidate DUBs molecules has the most significant inhibitory effect on the formation of nodule formation in mice with orthotopic breast cancer lung metastasis,and overexpression of USP12 can promote the formation of nodule formation in mice with orthotopic breast cancer lung metastasis.This suggests that USP12 may be a DUBs molecule that plays an important role in the process of breast cancer metastasis.However,when we used metastatic breast cancer MDA-MB-231 cells for in vitro Transwell metastasis experiments,we found that no matter overexpression or knockdown of USP12 did not affect the migration and invasion ability of breast cancer MDA-MB-231 cells.However,when we used ELISA(Enzyme linked immunosorbent assay)to detect the enriched supernatants of breast cancer MDA-MB-231 cells and MCF7 cells,we found that the breast cancer MDA-MB after overexpression of USP12,the levels of angiogenesis-related factors VEGF-A(Vascular endothelial growth factor-A)and VEGF-C(Vascular endothelial growth factor-C)in the supernatant of MDA-MB-231 cells and MCF7 cells increased,while knocking down USP12 was the opposite.In addition,we found through angiogenesis-related in vitro experiments HUVEC migration experiments and tube formation experiments that the enriched breast cancer MDA-MB-231 cells and MCF7 cell supernatants after overexpression of USP12 can promote endothelial cell angiogenesis.On the contrary,using the supernatant of breast cancer cells after knockdown of USP12 has an inhibitory effect on the occurrence of angiogenesis in vitro.In order to find the downstream substrates of USP12,we overexpressed USP12 and Control in HEK293T cells,using iTRAQ(isobaric tags for relative and absolute quantification)technology to detect differentially expressed genes between the two groups.We found 36 differentially expressed genes,of which 21 genes were up-regulated and 15 genes were down-regulated.We use MDK(Midkine)protein as a candidate substrate for USP12 deubiquitination enzyme.We overexpressed USP12 in HEK293T cells,and found that the protein level of MDK increased by protein immunoblotting,but the mRNA level of MDK did not change.We found the interaction between USP12 and MDK through IP(immunoprecipitation)and pull down experiments.Through cell immunofluorescence experiments,it was found that USP12 and MDK proteins co-localized in the cytoplasm of breast cancer MDA-MB-231 cells and MCF7 cells,which further demonstrated the interaction between MDK and USP12.Ubiquitination experiments and CHX(Cycloheximide)inhibition experiments found that USP12 can reduce the ubiquitin level on MDK protein through deubiquitination enzyme action,thereby increasing MDK protein in HEK293T cells and breast cancer MDA-MB-231 cells and Stability in MCF7 cells.We use the enriched breast cancer cell supernatant after knocking down USP12 to inhibit the angiogenesis of endothelial cells in vitro,but when we overexpress MDK at the same time,this inhibitory effect disappears.Through in vivo experiments,we found that knockdown USP12 inhibited the occurrence of breast cancer lung metastasis in mice,but at the same time,this inhibitory effect disappeared after overexpression of MDK.This shows that USP12 promotes breast cancer angiogenesis and participates in metastasis by maintaining the stability of MDK protein.Finally,we found in clinical tissue samples of breast cancer that compared with adjacent tissues,USP12 and MDK proteins are expressed at high levels in breast cancer tissues,and their protein expression levels are positively correlated.Finally,we found through database analysis that patients with high expression of USP12 and MDK often have a worse prognosis.In summary,our study reveals for the first time the function and mechanism of the USP12-MDK axis in the process of breast cancer metastasis,which USP12 maintains the stability of MDK protein through the action of deubiquitinating enzymes,and promotes angiogenesis and participates in breast cancer.Transfer.This study is expected to provide new ideas and new targets for basic research and clinical diagnosis and treatment of metastatic breast cancer.