NgBR Induces The Paclitaxel Resistance Of Estrogen Receptor Positive Breast Cancer Via Glycolysis

Backgrounds:Although chemotherapy is the main therapy and can improve the overall survival and prognosis of breast cancer,chemoresistance remains a major obstacle in clinical.In particular,most breast cancer is estrogen receptor(ER)positive,but it responds less well to chemotherapy than ER negative breast cancer.Therefore,it is particularly important to find a new target for improving chemoresistance of ER positive breast cancer.The activated glycolysis in ER positive breast cancer cells not only fulfills the needs of rapid growth and proliferation through enhancing glucose uptake and promoting glycolysis but also induces chemoresistance through promoting oncogene signaling pathways and protective autophagy.Our previous study found that Nogo B receptor(NgBR)regulated p53 and survivin expression,inhibited apoptosis,and induced chemoresistance through regulating transcription of estrogen receptor alpha(ER?)in ER positive breast cancer.A previous study on mechanism of NgBRregulated paclitaxel resistance in ER positive breast cancer focused on oncogene signaling pathways,the effects of NgBR on glycolysis in ER positive breast cancer has not been reported.Objectives:Our study focused on glycolysis to explore the molecular mechanism of NgBR inducing paclitaxel resistance and provided a new idea and direction for improving chemoresistance of ER positive breast cancer.Methods:(1)The study used CCK-8 and colony formation assays to convince the palitaxel resistance of paclitaxel-resistance ER positive breast cancer(MCF-7/PTX)cells by comparing with paclitaxel-sensitive ER positive breast cancer(MCF-7)cells.Then RT-PCR and Western Blot of the two cell lines were used to detect the expressions of NgBR and glycolysis-related proteins in MCF-7/PTX cells.(2)To vertify the significances of NgBR and glycolysis-related proteins in MCF-7/PTX cells,MCF-7/PTX cells were transfected NgBR si RNA and/or plasmids of glycolysis-related proteins,treated with estrogen and/or high concentration of paclitaxel,then detected with CCK-8,colony formation,and cell apoptosis assays.(3)To vertify the regulation of NgBR on hypoxia inducible factor-1 alpha(HIF-1?)which was the upstream protein of glycolysis,MCF-7/PTX cells were transfected HIF-1? si RNA and/or NgBR plasmid,treated with estrogen,then detected with CCK-8,lactate production,adenosine triphosphate(ATP)production assays,and Western Blot.(4)To further explore the regulatory mechanism of NgBR on HIF-1?,MCF-7/PTX cells were transfected with NgBR plasmid,treated with estrogen and/or high concentration of paclitaxel,then detected by RT-PCR,Western Blot,nuclear protein extraction,and immunofluorescence.MCF-7/PTX cells were transfected with NgBR plasmid and/or treated with estrogen,then detected by chromatin immunoprecipitation assay.(5)Breast tissue samples from 59 ER positive breast cancer patients before and after neoadjuvant chemotherapy were collected and stained with HIF-1? and/or NgBR immunohistochemistry and performed correlation analysis.Results:(1)Expressions and significance of NgBR and glycolysis-related proteins in MCF-7/PTX cells.Our study confirmed the paclitaxel resistance of MCF-7/PTX cells and found that NgBR and glycolysis-related proteins expression were increased in MCF-7/PTX cells by comparing with MCF-7 cells,which suggested that glycolysis-related proteins might have connection with paclitaxel resistance of ER positive breast cancer.Estrogen treatment could not only promote cell survival rate and colony formation ability and inhibit cell apoptosis,but also counteract inhibition of high concentration of paclitaxel treatment on MCF-7/PTX cells,which suggested that estrogen treatment might exert a protective effect on MCF-7/PTX cells by regulating glycolysis.In addition,NgBR knockdown could inhibit cell survival rate and colony formation ability and promote cell apoptosis of MCF-7/PTX cells,while glycolysisrelated proteins overexpresssion could partially attenuate the inhibition of NgBR knockdown on MCF-7/PTX cells,which suggested that NgBR might induce paclitaxel resistance by regulating glycolysis-related proteins expression in MCF-7/PTX cells.(2)The regulation of NgBR on HIF-1?Estrogen treatment could significantly promote NgBR,HIF-1?,glucose transporter 1(GLUT1),hexokinases(HK2),and lactate dehydrogenase(LDHA)expression,enhance lactate and ATP production and cell survival rate of MCF-7/PTX cells,which indicated that estrogen treatment might regulate glycolysis by promoting NgBR and HIF-1? expression and estrogen relied on ER? to exert regulatory function,which suggested that ER? might have a regulatory function on HIF-1?.Furthermore,HIF-1? knockdown did not change NgBR expression,but could significantly inhibit GLUT1,HK2,and LDHA expression,decrease lactate and ATP production and cell survival rate of MCF-7/PTX cells;NgBR overexpression could significantly promote HIF-1? expression and reverse inhibition of HIF-1?knockdown.This indicated that the effects of NgBR on glycolysis production and cell survival rate were achieved by regulating HIF-1? expression,and then affecting GLUT1,HK2,and LDHA expression.(3)The regulatory mechanism of NgBR on HIF-1?Estrogen,high concentration of paclitaxel treatment,and NgBR overexpression could promote HIF-1? expression and reduce sensativity of MCF-7/PTX cells to paclitaxel by promoting NF-?B transcription,phosphorylation,and nuclear transferation.This indicated that estrogen treatment could regulate HIF-1?expression through activating nuclear factor kappa B subunit(NF-?B)signaling pathway,thereby maintaining the paclitaxel resistance of MCF-7/PTX cells.High concentration of paclitaxel treatment also could regulate HIF-1? expression through activating NF-?B signaling pathway,thereby resisting the drug toxicity of high concentration of paclitaxel.NgBR also could regulate HIF-1? expression via NF-?B signaling pathway.In addition,both estrogen treatment and NgBR overexpression could promote ER? binding to the estrogen response element(ERE)region of HIF-1? gene and indicated that NgBR could regulate HIF-1? expression by promoting HIF-1?transcription through ER?.In particular,estrogen treatment and NgBR overexpression could also directly promote ER? binding to the ERE region of GLUT1 gene,which indicated that NgBR could directly regulate GLUT1 expression through ER?.Both estrogen treatment and NgBR overexpression could also significantly enhance HIF-1? binding to the hypoxia-responsive element(HRE)regions of GLUT1,HK2,and LDHA genes,which indicated that estrogen treatment could further enhance the transcription of HIF-1? on GLUT1,HK2,and LDHA genes and promote GLUT1,HK2,and LDHA expression.This suggested that the regulation of ER? on HIF-1? transcription.In addition,NgBR could also promote GLUT1,HK2,and LDHA expression through regulating HIF-1? transcription.(4)Correlation analysis of NgBR,HIF-1? expression and the effects of chemotherapy in ER positive breast cancer patientsPatients with positive HIF-1? expression appeared to be less sensitive to neoadjuvant chemotherapy with paclitaxel compared with patients with negative HIF-1? expression.ER positive breast cancer patients with high NgBR expression also had high HIF-1? expression,which indicated that the expression of NgBR and HIF-1? was correlated.Conclusions:1.NgBR and glycolysis-related proteins were involved in paclitaxel resistance of ER positive breast cancer.2.NgBR regulated glycolysis via HIF-1? expression in paclitaxel-resistance ER positive breast cancer cells,and the correlation analysis of pathological samples from ER positive breast cancer patients confirmed that the expression of NgBR and HIF-1? was correlated.3.NgBR overexpression promoted HIF-1? expression,enhanced glycolysis,and induced paclitaxel resistance through ER?/HIF-1? and NF-?B/HIF-1? signaling pathways in ER positive breast cancer cells.4.NgBR overexpression promoted GLUT1 expression,enhanced glucose uptake,promoted glycolysis,and induced paclitaxel resistance through ER?/GLUT1 and HIF-1?/GLUT1 signaling pathways in ER positive breast cancer cells.5.NgBR knockdown restored paclitaxel sensitivity of ER positive breast cancer cells through inhibiting glycolysis.