| Objective Androgen receptor(AR)is highly expressed in human epithelial growth factor receptor 2(HER2)-positive breast cancer(BC),about 60% of which express AR,and has a poor prognosis.Erb B3 binding protein 1(EBP1)is a key factor regulating cell cycle and can promote cell differentiation.It has been reported that EBP1 can directly regulate AR-related genes in prostate cancer,but the specific mechanism of them in HER2-positive BC is still unclear.The purpose of this study was to investigate the role and the relevant mechanism of EBP1-AR pathway in HER2-positve BC.Methods 1.Firstly,140 cases of HER2-positive and 142 cases of HER2-negative invasive breast cancer paraffin tissue samples were randomly selected to perform immunohistochemistry staining.Statistical software SPSS24.0 was used to analyze the relationship between AR,EBP1 expression and some clinical pathological factors by Chi-squared tests.Spearman correlation method and Kaplan-Meier analyses were used to analyze the correlation between AR and EBP1 and the survival analysis separately.2.Then 3 cases of matched HER2-positive freshcancer tissues and adjacent normal tissues were randomly selected from Breast Cancer Pathology and Research Laboratory Department,Tianjin Medical University Cancer Hospital.The tissue proteins were extracted.The protein expression level of EBP1 was detected by Western blot.8 cases of HER2-positive freshcancer tissues and adjacent normal tissues were randomly selected and extracted tissue RNA,real-time quantitative PCR(RT-qPCR)method to detect the expression level of EBP1 nucleic acid.3.In HER2-positive breast cancer cell lines SK-BR-3,MDA-MB-453,and HCC1954,the basic expression of EBP1 was detected by Western blot,and then EBP1-over-expressing and EBP1-shRNA-expressing plasmids were constructed respectively.SK-BR-3 and MDA-MB-453 were transfected with EBP1-overexpressing and EBP1-shRNA-expressing plasmids separately to analyze the AR and HER2 expression by RT-qPCR and Western blot.Wound-healing test,Transwell assay,CCK8 and plate clone formation assay were performed to measure cell migration,invasion and proliferation ability.4.Co-immunoprecipitation(Co-IP)assay was performed to verify the formation of EBP1 and AR protein molecular complexes,and the dual luciferase reporter gene was performed to detect the regulation of EBP1 on AR.5.To explore the role of AR in HER2-positive breast cancer,SK-BR-3 cells were transfected AR-expressing and AR-shRNA-expressing plasmids respectively.Western blot and RT-qPCR analysis were performed to detect the protein and the nucleic acid level expression of EBP1 and HER2.In order to further verify the role of EBP1 between AR and HER2,one group was transfected with AR-shRNA-expressing plasmid,and the other group was transfected with AR-shRNA-expressing and EBP1-expressing plasmid simultaneously in SK-BR-3 cells.Wound-healing test,Transwell assay,CCK8 and plate clone formation assay were performed to measure cell migration,invasion and proliferation ability.6.To determine the effect of AR on the interaction of EBP1 and HER2 in the HER2-positive breast cancer cell line HCC1954,one group was transfected with EBP1-expressing plasmid,one group was transfected with EBP1-expressing and AR-expressing plasmid at the same time,and the third group was transfected with EBP1-expressing and AR-shRNA-expressing plasmid at the same time.RT-qPCR analysis was performed to determine the HER2 expression in these three subgroup.7.HER2-positive breast cancer cells SK-BR-3,which were stably transfected with EBP1-shRNA-expressing plasmid,AR-shRNA-expressing/EBP1-expressing plasmid and the corresponding unloaded plasmid,were planted in double immunodeficiency mice by 5 nude mice per group.After 48 days of culture,the tumor tissues were removed,and the incidence of transplanted tumors,tumor size,and liver and lung metastases conditions were observed.The tumors were stained with HE and immunohistochemical staining.The liver and lung tissues were stained with HE to observe whether the occurrence of metastasis.Results 1.In HER2-positive and HER2-negative invasive breast cancer paraffin tissue samples,EBP1 was expressed in cytoplasm and AR was expressed in nucleus.Among HER2-positive invasive breast cancer paraffin tissue samples,the high expression rate of EBP1 was 47.9%(67 / 140),the low expression rate of EBP1 was 52.1%(73/140).EBP1 expression was related to histological grade(P <0.001),AR expression(P <0.001),and lymph node metastasis(P = 0.013).In HER2-negative invasive breast cancer paraffin tissue samples,the high expression rate of EBP1 was 62.0%(88/142),and the low expression rate of EBP1 was 38.0%(54/142).The expression of EBP1 was related to the expression of PR(P = 0.002),not histological grade,AR expression or lymph node metastasis.In HER2-positive breast cancer cases,the positive rate of AR is 60%(84/140),the negative rate of AR is 40%(56/140),the AR expression was related to histological grade(P = 0.001),lymph node metastasis(P = 0.042)and the expression of EBP1(P <0.001).The expression of AR was negatively correlated with the expression of EBP1(r =-0.802,P <0.001)by Spearman analysis.In HER2-positive breast cancer,patients with high expression of EBP1 have a better prognosis than those with EBP1-low expression,and patients with AR-positive expression have a worse prognosis than patients with AR-negative expression.The disease-free survival and overall survival of breast cancer patients with AR-positive/ EBP1-low expression were shorter than those of other groups(AR-positive/EBP1 high-expression group,AR-negative/EBP1-low expression group,and AR-negative/EBP1-high expression group).Co-expression of AR and EBP1 is an independent prognostic factor both in disease-free survival(DFS)(P = 0.015)and overall survival(OS)(P = 0.002).2.In three randomly selected paired HER2-positive freshcancer tissues and adjacent tissues,Western blot results showed that EBP1 was lowly expressed in tumor tissues while highly expressed in adjacent normal tissues.In 8 randomly selected paired HER2-positive freshcancer tissues and adjacent tissues,RT-qPCR assay results also showed that EBP1 was lowly expressed in tumor tissues and highly expressed in adjacent normal tissues.3.Western Blot results showed that SK-BR-3,HCC1954,and MDA-MB-453 breast cancer cell lines’ phenotypes were HER2 positive,AR positive,and low expression of EBP1.Immunofluorescence showed AR nuclear expression and EBP1 plasma expression.Then SK-BR-3 and MDA-MB-453 cells with low EBP1 expression were infected with the EBP1-expressing plasmid lentivirus to upregulate the expression of EBP1.The EBP1 plasmid empty template lentivirus was used as a control group.Western blot and RT-qPCR results showed that AR and HER2 decreased in the EBP1-expressing group compared with the transfected empty-loaded plasmid group both in protein and nucleic acid levels.Wound healing,Transwell,CCK8 and plate clone formation assay results showed that EBP1-expressing could inhibit the migration,invasion and proliferative ability of HER2-positive breast cancer cells.Compared with the transfected empty plasmid group,the EBP1-shRNA-expressing plasmid group showed that the protein and nucleic acid expression levels of AR and HER2 increased.Meanwhile,wound healing,Transwell,CCK8 and plate clone formation assay results showed that EBP1-shRNA-expressing plasmid group could promote the migration,invasion and proliferation ability of HER2-positive breast cancer cells.4.The co-IP experimental results show that AR and EBP1 proteins could form protein complexes.The results of the double luciferase report experiment show that in the 293-T cell lines,one group was simultaneously transferred into the AR full-length promoter plasmid and EBP1 overexpression plasmid,and the other group was simultaneously transferred into the AR full-length promoter mutant plasmid and EBP1 over-expressed plasmids,and the other group was transfected into unloaded plasmids which served as controls.The ratio of the firefly luciferase reaction intensity to the internal reference Renilla luciferase reaction ratio was measured,and the ratios in the three groups had statistically significance.5.SK-BR-3 cells were transfected with AR-expressing and AR-shRNA-expressing lentiviral vector plasmids respectively to evaluate the EBP1 and HER2 expression.Western blot and RT-qPCR results showed that HER2 increased after AR overexpression at the protein and nucleic acid level,while HER2 decreased after AR low-expression at the protein and nucleic acid level.There was no significant change in EBP1 expression between AR-expressing and AR-shRNA-expressing subgroup.Then AR-shRNA-expressing/EBP1-expressing lentiviral vector plasmid was stably transfected into SK-BR-3 cells.Western blot and RT-qPCR results showed that compared with AR-shRNA-expressing subgroup,HER2 expression level further decrease in the AR-shRNA-expressing/EBP1-expressing subgroup.Wound healing,Transwell,CCK8 and plate clone formation assay results also showed that migration,invasion,and proliferation ability were further reduced in the AR-shRNA-expressing/EBP1-expressing subgroup.6.RT-qPCR results showed that compared with the EBP1-expressing subgroup and AR-expressing/ EBP1-expressing subgroup,the expression level of HER2 was the lowest in EBP1-expressing/ AR sh-RNA-expressing subgroup.AR plays a role in the regulation of HER2 by EBP1.7.Breast cancer cell lines SK-BR-3 from three different treatment subgroups(EBP1-shRNA-expressing plasmid,AR-shRNA-expressing/EBP1-expressing plasmid and the corresponding unloaded plasmid)was injected into double immunodeficient mice by 5 nude mice per group.The tumor size and growth situation of the three subgroups were measured every two days.After 48 days of culture,the tumor tissues were removed,and the incidence of transplanted tumors,tumor size,and liver and lung metastases conditions were observed.The tumors were stained with HE and immunohistochemical staining.The liver and lung tissues were stained with HE to observe whether the occurrence of metastasis.The nude mice in the EBP1 shRNA-expressing plasmid subgroup had the largest tumor volume,the nude mice in the AR shRNA-expressing plasmid / EBP1 expressing plasmid subgroup had the smallest tumor volume,and the immunohistochemical results of Ki67 showed that Ki67 expression was the lowest in the AR shRNA-expressing plasmid / EBP1 expressing plasmid subgroup.Ki67 expression was the highest in the EBP1 shRNA-expressing plasmid subgroup.Lung metastasis occurred in the EBP1 shRNA-expressing plasmid subgroup,while no lung metastasis was seen in the other subgroups.The results of this experiment indicated that down-regulating the expression of EBP1 could promote the ability of tumor growth,proliferation and metastasis,and up-regulating EBP1 while down-regulating AR expression could inhibit the ability of tumor growth,proliferation and metastasis.Conclusions Our findings provide that the expression of EBP1 and AR in HER2-positive breast cancer tissues has a negative correlation between each other.Co-expression of EBP1 and AR is an independent prognostic factor for disease-free survival and overall survival.In HE2-positive breast cancer cell lines,the expression of EBP1 affects the expression of AR and HER2,overexpression and lowexpression of EBP1 can affect the migration,invasion and proliferation ability of these cell lines and AR also plays an important role in the regulation of HER2 by EBP1.These findings will provide important evidence for treating AR and EBP1 as targets for patients with HER2-positive breast cancer. |
