Mechanism Of USP37 In The Regulation Of Breast Cancer Stemness,EMT And Cisplatin Sensitivity Via Hedgehog Pathway

Breast cancer is the deadliest form of carcinoma affecting women,with approximately 1.7 million cases diagnosed worldwide in 2012.Although there are effective treatments against some types of breast carcinoma,such as the subtype with abnormal overexpression of the HER2/Neu oncogene,the majority of patients diagnosed with breast cancer still remain incurable.The existence of breast cancer stem cells(BCSCs)is deemed to be the main reason for tumor recurrence,metastasis and therapeutic resistance due to their self-renewal and differentiation abilities.CD44+ ?CD24-or aldehyde dehydrogenase1(ALDH1)phenotypes is the efficient measure to identify BCSCs from breast cancer populations.Therefore,it is necessary to seek more critical biomarkers within the distinct molecular subtypes for isolation and identification the CSC subpopulation,and that biomarkers can be acquired significantly implication for comprehending the breast cancer stem cells biology mechanism.Growing evidence suggests that the activation of epithelial-mesenchymal transition(EMT)process is closely related to cancer stem-like cells characteristics.EMT describes the process that epithelial cells detached from their neighbors and transfer to other sites of the tissue via dissolving basement membrane and passing through the extra-cellular matrix.EMT could facilitate the generation of cancer stem cells from more differentiated cancer cells.Moreover,studies suggested that breast cancer stem-like cells express markers associated with an EMT.Whereas,EMT is considered to be closely controlled,the whole process is difficult to understand because of the changement of main factors and the occurrence of complex events.The process is considered to be controlled by a series of signaling pathways including Notch,Wnt/?-catenin and Hedgehog et al.Hedgehog pathway is responsibility for the maintenance of the stem cell and EMT in contributing to the evolution of breast cancer.Ubiquitination describes a highly conservative and reversible modification process aiming at protein degradation,which is involved in nearly all aspects of cell biology.Deubiquitinating enzymes(DUBs)can prevent ubiquitin from degrading target proteins.Deregulated DUBs expressions are frequently associated with tumorigenesis,such as cell self-renewal,apoptosis and EMT.It has been confirmed that DUBs are essential for regulation of stem cell-related markers and controlling the various steps of metastatic progression,including invasion,dissemination and eventual metastatic tumor to distant organs.Ubiquitin specific peptidase 37(USP37),a novel DUB,is a member of ubiquitin-specific processing proteases family.Its function is initially identified as a potent regulator of cell cycle through accelerating the G1/S transition,in which USP37 is expressed at the highest level.Pan et al.reported that the high expression of USP37 gene was detected in lung cancer patients and it promoted cell viability and the Warburg effect by deubiquitinating and stabilizing pluripotent stem factor c-Myc protein.Above these advances,it was implicated that USP37 gene may be associated with stemness of cells in cancer progression.However,the biology function of USP37 in the regulation of breast cancer stem cells and EMT process remains unexplored.This study focused on the effect of USP37 on breast cancer stemness.Finally,the relationship between USP37 and Hedgehog signaling pathway were further studied.Part ?Expression of USP37 in breast cancer tissues and breast cancer stem cellsObjective: To investigate the expression of USP37 in breast cancer tissues and breast cancer cells,and its relationship with breast cancer types,prognosis and related signaling pathways.Methods: The expression of USP37 in breast cancer tissues and adjacent normal tissues was detected by IHC assay.Western blotting assay was also used to detect USP37 in breast cancer cell lines.Secondly,the distribution of USP37 expression in breast cancer and the correlation between USP37 expression and the overall survival rate were detected by The Cancer Genome Atlas(TCGA)database.Gene set enrichment analysis(GSEA)was utilized to evaluate potential mechanism of USP37 in breast cancer.To further investigate the expression of USP37 in breast cancer stem cells,we selected breast cancer stem cells by MACS and detected the m RNA expression of USP37 in breast cancer stem cells by real-time quantitative PCR assay and cellular immunofluorescence assay.At the same time,the differential expression of USP37 in adherent cells and corresponding cell spheres was detected by western blotting.Results: Bioinformatics analysis demonstrated that USP37 gene was elevated in breast cancer tissues and its overexpression was strongly correlated with the increased mortality rate.GSEA analysis showed that USP37 expression was positively associated with cell growth and metastasis while negatively related to cell apoptosis in the TCGA breast cancer samples.USP37 expression was elevated in breast cancer tissues and breast cancer cell lines.Moreover,we also detected that USP37 was overexpressed in breast cancer stem cells.IHC assay revealed that USP37 expression was mainly localized in the cytoplasm and was occasionally detected in the nucleus.The results of the H scores showed that the expression level of USP37 in breast cancer tissues was significantly higher than that in the corresponding adjacent tissues.The expression of USP37 in breast cancer cell lines was significantly higher than that in mammary epithelial cells MCF-10 A.The protein expression level of USP37 in tumor spheres was significantly higher than that of its corresponding adherent cells(P<0.05).The expression of USP37 in the CD44+/CD24-cell population was significantly higher than that in the non-CD44+/CD24-cell population by real-time quantitative PCR assay and cellular immunofluorescence assay.At the same time,the m RNA level of ALDH1 was significantly elevated in the CD44+/CD24-cell population.Discussions: 1.The expression of USP37 in breast cancer tissues is significantly higher than that in normal tissues adjacent tissues,and its high expression is closely related to the poor prognosis of breast cancer patients,suggesting that USP37 is cancerous in breast cancer.2.By GSEA analysis,high expression of USP37 is closely related to cell proliferation,metastasis and cell anti-apoptosis.3.Compared with the normal tissues,the protein expression of USP37 in breast cancer tissues was significantly increased.USP37 is highly expressed in breast cancer stem cells,suggesting that USP37 may be involved in improving the characteristics of breast cancer stem cells.Part ?The effect of USP37 on breast cancer stemness in vitroObjective: To investigate whether USP37 is involved in the regulation of breast cancer cell stemness,epithelial-mesenchymal transition and cisplatin sensitivity in vitro.The effect of USP37 on Hedgehog signaling pathway and Bcl-2/Bax is further investigated.Methods: In this study,we changed USP37 expression in breast cancer cell lines MCF-7 and MDA-MB-231 by transient transfection or lentiviral transfection.The effect of USP37 protein on breast cancer cell stemness were detected by sphere formation assay,wound healing assay,transwell assay and CCK-8 assay.Related markers in EMT,cancer stemness and cell apoptosis were examined by western blotting.Results: Silenced USP37 could inhibit the abilitiy of sphere formation in breast cancer cells.The number and size of cell spheres in the sh USP37#2 group were significantly lower than those in the sh Scramble group.Down-regulation of USP37 in breast cancer cells significantly inhibited stem cell markers(ALDH1 and OCT4)and Hedgehog signaling pathways(Smo and Gli-1)(P < 0.05).Wound healing assay and transwell assay showed that knockdown of USP37 cells significantly reduced the migration and invasion of breast cancer cells(P<0.05).After 48 hours of transfection of breast cancer cells by USP37 si RNA,USP37 was successfully knocked down in MCF-7 and MDA-MB-231 as shown by western blotting or q RT-PCR.Silicend USP37 significantly reduced the protein expression levels of Snail1,N-cadherin and Vemintin,and increased E-cadherin.Additionally,the results of immunofluorescence assay were consistent with the results of western blotting assay.The cell survival rate of sh USP37#2 group was significantly lower than that of sh Scramble group with cisplatin treatment(P<0.05).As shown in the colony formation experiment,the number of colonies formed by sh USP37#2 cells was significantly lower than that of the sh Scramble group at different concentrations of cisplatin.The protein levels of Bax and Cleaved caspase9 in sh USP37#2,cisplatin-treated and sh USP37#2+cisplatin groups were increased by western blotting assay.At the same time,the expression level of Bcl-2 protein in sh USP37#2+ cisplatin group showed the lowest expression level;on the contrary,the protein level of Bax and Cleaved caspase9 was the highest.Up-regulation of USP37 in breast cancer cells MCF-7 can increase the ability of breast cancer cells to form spheres.The number and size of cell spheres in upregulated USP37 group were significantly higher than those in CTL group.Up-regulation of USP37 in MCF-7 cells significantly promoted the expression of protein levels in stem cell markers(ALDH1 and OCT4)and Hedgehog signaling pathways(Smo and Gli-1)(P<0.05).The cell invasion and migration ability of the USP37 group was significantly higher than that of the CTL group(*P<0.05,**P<0.01).Overexpression of USP37 inhibits the protein expression levels of Snail1,N-cadherin and Vemintin,and the protein expression level of E-cadherin is elevated.The trendency of cellular immunofluorescence was consistent with the results of western blotting.The CCK-8 cytotoxicity assay was performed in the 2-16 ?g/ml cisplatin concentration range,and the cell survival rate of the overexpressed USP37 group was significantly higher than that of the CTL group(P<0.05).We observed the upregulation of Bax and cleaved caspase-9 and downregulation of Bcl-2 in MCF-7 cells and MDA-MB-231 cells after transfection with USP37 sh RNA and/ or treatment with cisplatin for 48 h.Conclusion:1.Downregulation of USP37 can inhibit the stemness of breast cancer cells.Conversely,overexpression of USP37 can enhance the stemness of breast cancer cells.2.Knockdown of USP37 expression hampered cell migration,invasion and EMT in breast cancer cells.Overexpression of USP37 can promote cell migration,invasion and EMT in breast cancer cells.3.Knockdown of USP37 can induce cell apoptosis and enhance the sensitivity of breast cancer cells breast cancer cells to cisplatin.Conversely,overexpression of USP37 can increase the drug resistance of breast cancer cells to cisplatin.Part ?The mechanism of USP37 on stemness of breast cancer cells in vitroObjection: To investigate the effect of USP37 on the stemness and epithelial-mesenchymal transition of breast cancer cells regulated by Hedgehog signaling pathway.Explore the interaction between USP37 and Gli-1 and the effect of USP37 on Gli-1.Methods: The effect of Hedgehog agonist(Purmorphamin)on USP37 and Hedgehog signaling pathway was detected by western blotting.Immunofluorescence assay,Transwell assay,and sphere formation assay were used to investigate whether USP37 regulates breast cancer cells stemness and epithelial-mesenchymal transition through Hh pathway.MG132,CHX and co-immunoprecipitation experiments were used to study whether USP37 binds to Gli-1 protein and stabilizes its protein activity.Results: The protein levels of Smo and Gli-1 were significantly up-regulated at 24 h and 48 h after PM treatment,and the protein expression of USP37 was significantly increased.Western blotting assay showed that PM promoted Hedgehog signaling pathway,stemness and EMT in breast cancer cells.However,after knockdown USP37 with 0.5 ?M PM treatment,Hedgehog signaling pathway and the ability of stemness and epithelial-mesenchymal transition in breast cancer cells were inhibited.Immunofluorescence assay indicated the same trendency as the results of western blotting.The results of co-immunoprecipitation indicated that there was an interaction between endogenous USP37 and Gli-1.Immunofluorescence assay showed the co-localization of USP37-Flag and Gli-1.MG132 experiments showed that the inhibition of Gli-1 by the downregulation of USP37 was alleviated after treatment with MG132 for 4 hours.The protein expression level of Gli-1 in the silencing USP37 group was significantly lower than that in the control group after adding CHX at the concentration of 50 ?g/ml for 20 minutes in MCF-7 and MDA-MB-231 cells.However,Gli-1 protein in MCF-7 cells with USP37 overexpession was significantly higher than that in the control group after treatment with CHX at the concentration of 50 ?g/ml for 90 minutes.Conclusion: 1.Purmorphamine can activate Hedgehog signaling pathway and up-regulate USP37 protein expression.It indicates that USP37 is involved in the Hedgehog signaling pathway.2.Purmorphamine can promote the ability of stemness,epithelial mesenchymal transition,sphere formation and cell invasion in breast cancer cells.3.Down-regulation of USP37 can inhibit the stemness,epithelial-mesenchymal transition and invasion of breast cancer cells by blocking Hedgehog pathway.Part ?USP37 knockdown inhibits tumorigenicity and increases sensitivity to cisplatin in vivoObjective: To investigate the inhibition of silienced USP37 on tumorigenicity and the sensitivity to cisplatin in vivo.Methods: MCF-7 cells were infected with sh Scramble or sh USP37#2.The treated breast cancer cell lines were inoculated into nude mice.During the process of cell growth,the growth curve of the tumor was made.The nude mice were treated with cisplatin to observe the tumor formation.Finally,the tumor weights were measured.Hedgehog/Bcl-2 signaling pathway was detected by IHC assay or western blotting assay.Results: We constructed the breast cancer cell line with stably down-regulated USP37.The volume and weight of tumors formed by sh Scramble group were significantly higher than those of sh USP37#2 group(P<0.05).The tumor volume and weight of sh USP37#2 with cisplatin treatment group were significantly lower than those of cisplatin treatment group/sh USP37#2 group(P<0.05).The protein expression levels of Smoothened,Gli-1,ALDH1,OCT4 and Bcl-2 in sh USP37#2 with cisplatin treatment group showed the lowest expression trendency(P<0.05).Conclusion: 1.Knockdown of USP37 inhibits the tumorigenicity of breast cancer in vivo.2.Knockdown of USP37 can inhibit the breast cancer stemness and Bcl-2 by repressing Hedgehog signaling pathway,resulting in enhancing the sensitivity of breast cancer cells to cisplatin.