Nogo-B Receptor Increases The Resistance To Tamoxifen In Estrogen Receptor Positive Breast Cancer Cells

Backgrounds: Breast cancer is the most common cancer in females around the world,and the incidence is increasing year by year.About 75% of the cases are estrogen receptor alpha(ERα)positive breast cancer.These patients undergo adjuvant endocrine therapy to increase disease free survival(DFS)and overall survival(OS)after surgery.According to the National Comprehensive Cancer Network(NCCN)guideline,invasive breast cancer patients who are ERα positive or progesterone receptor(PR)positive are eligible for tamoxifen for five to ten years.Although the efficacy of tamoxifen is obvious,one third of the ERα positive breast cancer patients gained resistance to tamoxifen during or after tamoxifen treatment.The underlying mechanisms of developing resistance,especially acquired resistance,to tamoxifen are complex and numerous,including decreased ERα expression downregulated,mutation of ERα,ligand-independent activation of ERα or its co-activators through phosphorylation,the change of energy consumption during metabolism,autophagy or abnormal DNA damage-repair in tumor cells.Nogo isoforms,including Nogo-A,-B and-C,are members of a reticulon protein superfamily.Nogo-B is mainly expressed in peripheral tissues.Ng BR was identified as a Nogo-B receptor specific for the amino terminus of Nogo-B(Am Nogo-B).Ng BR is expressed in many tissues or organs such as liver,brain,kidney and breast,and it is necessary for angiogenesis,cell apoptosis and resistance to chemotherapy in hepatocellular carcinoma cells.Our recent findings demonstrated that Ng BR binds farnesylated Ras and recruits Ras to the plasma membrane,which is a critical step required for receptor tyrosine kinases(RTKs)-mediated activation of Ras signaling in cancer cells and tumorigenesis.The level of Ng BR is also up-regulated in breast cancer cells and is in accordance with the level of survivin.A proteomic study also showed that Ng BR is essential for promoting epithelial-mesenchymal transition(EMT)in breast cancer cells.And previous study has shown that EMT is also involved in tamoxifen resistance.However,the role of Ng BR in tamoxifen resistance of ERα positive breast cancer is still unknown.Objectives: In this study,we found the expression of Ng BR is increased in tamoxifen resistant ERα positive breast cancer cells.Mechanistically,our data demonstrated the role of Ng BR in EGF-induced phosphorylation of ERα and the change of sensitivity to tamoxifen.Methods: 1、Colony formation assay and CCK-8 assay were used to compare the sensitivity to tamoxifen between MCF-7,T47 D and their cultured tamoxifen resistant cells.Western blot results were used to demonstrate the difference of important tumor makers.2、Ng BR was knocked down by using small interfering RNA(si RNA)to detect the change of sensitivity to tamoxifen.Flow cytometry and colony formation assay were used to detect cell apoptosis and cell viability.3、P53 was knocked down by si RNA to compare the change of Ng BR,survivin and the effects of silencing p53 or survivin on tamoxifen resistance.4、The data of breast cancer patients was collected from GEO database,and was used to analyze RNA level of NUS1 and TP53.5、Western blot was used to detect the effects of Ng BR on EGF pathway.The function of Ng BR through Ras was confirmed using immunoprecipitation(IP)assay.6、Immunohistochemistry(IHC)was used to confirm the relationship between Ng BR,Nogo-B and survivin in 22 breast cancer tissues.7、The survival information of breast cancer patients was collected from kmplot website and was used to analyze the effects of Ng BR on prognostics.Results: 1、According to the results of colony formation assay and CCK-8 assay,MCF-7-Tam R and T47D-Tam R cells were more resistant to tamoxifen than MCF-7 and T47 D cells.The levels of Ng BR m RNA and protein were higher in MCF-7-Tam R and T47 DTam R cells.The expression of survivin was higher,while the levels of p53 and ERα are lower in MCF-7-Tam R and T47D-Tam R cells.2、Through flow cytometry,trypan blue staining and colony formation assay,we confirmed that MCF-7-Tam R and T47D-Tam R restored the sensitivity to tamoxifen after Ng BR knockdown.3、Ng BR depletion in MCF-7-Tam R cells increased the expression of p53,but decreased the amount of survivin at the protein level.Knockdown of p53 increased the resistance of MCF-7 cells to 4-OHT,while survivin knockdown restored the sensitivity of MCF-7-Tam R cells to 4-OHT.The results of T47 D and T47D-Tam R are similar to those of MCF-7 and MCF-7-Tam R cells.4、The data from GEO database of 60 ERα positive breast cancer patients indicated a negative correlation between NUS1 and TP53.5、EGF treatment increased the phosphorylation of AKT,ERK and MDM2 in MCF-7-Tam R cells treated with control non-silencing(NS)si RNA.Ng BR knockdown attenuated EGF-stimulated phosphorylation of AKT,ERK and MDM2.IP result indicated that EGF stimulation increases the association of Ng BR with activated Ras.6、The result of IHC indicated that cases that highly expressed Ng BR also highly expressed survivin.In cases that negatively or weakly expressed Ng BR,the expression of survivin was also low.7、Kaplan–Meier analysis revealed that high expression of Ng BR was associated with poor Relapse Free Survival(RFS)of ERα positive breast cancer patients(n=755)and of patients receiving endocrine therapy(n=335)Conclusion: 1、The expression of Ng BR is increased in tamoxifen resistant ERα positive breast cancer cells;2、Ng BR knockdown increases the protein levels of p53 and decreases survivin;3、Ng BR knockdown with si RNA attenuates EGF-induced phosphorylation of ERα and restores the sensitivity to tamoxifen in ERα positive breast cancer cells;4、Ng BR expression is associated with poor survival of ERα positive breast cancer patients.