Nogo-B Receptor Increases The Resistance Of Estrogen Receptor Positive Breast Cancer To Paclitaxel

Objective:Intrinsic or acquired chemoresistance is a hurdle in oncology.Only 7%-16%of estrogen receptor a(ERa)positive breast cancer cases achieve a pathological complete response(pCR)after neo-adjuvant chemotherapy.Nogo-B receptor(NgBR)is a cell surface receptor that binds farnesylated Ras and promotes Ras translocation to the plasma membrane.The purpose of this study was to investigate whether NgBR expression is involved in promoting ERa positive breast cancer cell resistance to paclitaxel.Findings from this study implicate a novel therapeutic target for treating ERa positive breast cancer in neo-adjuvant/adjuvant chemotherapy.Method:1.To examine the roles of NgBR in regulating the response of ERa positive breast cancer to neoadjuvant chemotherapy,we randomly collected breast tissue samples from 38 ER-positive breast cancer cases and performed immunostaining to examine NgBR and Nogo-B expression levels in breast tumor sections before and after neo-adjuvant chemotherapy.Combined the pathologic response with NgBR/Nogo-B expression to analyze the correlation between them.2.To determine the contribution of NgBR to the E2 induced resistance of ERa positive breast cancer to paclitaxel,we knocked down NgBR in T47D/MCF-7 cells,CCK-8 experiment was used to test cell viability,clonogenic survival assay was used determined the capability of in vitro tumorigenesis,AO/EB staining and Annexin V-FITC/PI staining-based flow cytometry approach were used to determine cell apoptosis.3.To determine the molecular mechanism by which NgBR increases ERa-mediated paclitaxel resistance,we examined NgBR-mediated downstream signaling pathways(such as phos-Akt,phos-ERK and phos-ERa)and the expression of apoptosis regulators,such as survivin and p53.4.T47D and MCF-7 cells were treated with E2 after knockdown NgBR,ChIP-PCR assay was performed to determine how NgBR is involved in ERa-mediated gene transcription.5.To determine the contribution of NgBR to the EGF induced resistance of ERa positive breast cancer to paclitaxel,we knocked down NgBR in T47D cells,CCK-8 experiment was used to test cell viability,clonogenic survival assay was used determined the capability of in vitro tumorigenesis,AO/EB staining and Annexin V-FITC/PI staining-based flow cytometry approach were used to determine cell apoptosis.6.To determine the molecular mechanism by which NgBR increases EGF induced ERa-mediated paclitaxel resistance.we examined NgBR-mediated downstream signaling pathways(such as phos-Akt,phos-ERK and phos-ERα)and the expression of apoptosis regulators,such as survivin.7.Real time RT-PCR was used to determine the mRNA level of NgBR and survivin induced by E2 or EGF.Result:Here,we demonstrate NgBR as a potential therapeutic target for ERa positive breast cancer patients to attenuate paclitaxel resistance.Initially,Thirty-two cases did not demonstrate or partially demonstrated a response to neoadjuvant chemotherapy.Among these 32 cases,22 cases show increased or unchanged NgBR expression after chemotherapy and 26 cases showing increased or unchanged Nogo-B expression after chemotherapy.We further examined the association of NgBR expression with the outcome of ERα positive breast cancer patients.NgBR(NUS1)mRNA expression data were retrieved from a gene-expression profiling dataset.Subsequent Kaplan-Meier analysis revealed a significantly reduced Recurrence Free Survival for patients with high NgBR-expression in tumors as compared to patients with low NgBR-expression in tumors.Next,we demonstrated that knockdown NgBR of T47D and MCF-7 human ERa positive breast cancer cells can reduce E2 or EGF induced cell viability,NgBR knockdown further decreases viability of T47D cells treated with E2 and paclitaxel;clonogenic capability of NgBR knockdown cells was significantly decreased under the condition of paclitaxel treatment alone or along with E2/EGF stimulation;the results of AO/EB staining and flow cytometry showed that E2/EGF treatment attenuated paclitaxel-induced apoptosis of T47D cells,but NgBR knockdown restores the sensitivity of T47D cells to paclitaxel by showing increased apoptosis.To determine the molecular mechanism by which NgBR increases ERa-mediated paclitaxel resistance,Western Blot was used to examine NgBR-mediated downstream signaling pathways and altered the expression of apoptosis regulators.NgBR knockdown enhanced paclitaxel-induced cell apoptosis by modulating expression of p53 and survivin in ERa positive breast cancer cells via NgBR-mediated PI3K/AKT and MAPK/ERK signaling pathways.NgBR knockdown attenuated either E2 or EGF stimulated phosphorylation of ERa at Serine 118 residue.What’s more,E2/EGF treatment can increase the protein and mRNA level of NgBR and survivin.Finally,the ChIP-PCR assay further demonstrated that NgBR knockdown decreased ERa binding to the estrogen response element(ERE)of the ERα target gene and increased the binding of p53 to the promoter region of survivin to attenuate survivin transcription.Conclusions:In this study,we demonstrated that NgBR is highly expressed in ERα positive breast cancer residue tumor that survived after neo-adjuvant chemotherapy.NgBR knockdown could increase p53 expression,decrease survivn expression by enhancing the binding of p53 to the promoter region of survivin.Consequently,the results of cell viability and apoptosis assays clearly demonstrate that NgBR knockdown attenuates the E2-induced resistance to paclitaxel.In addition,NgBR knockdown attenuates the EGF-induced resistance to paclitaxel by decreasing EGF-stimulated phosphorylation of Akt,ERK,ERa and EGF-induced expression of survivin.Our study elucidated the important roles of NgBR in promoting the acquired resistance of ERa positive breast cancer to paclitaxel.