ER-?36,a Novel Variant Of ER-?,Madiated Autophagy Induced By Tamoxifen In Glioma Cells

Glioblastomas are the most common and aggressive primary brain tumors in adults,with uncontrolled proliferation,invasiveness and resistance to conventional and novel therapeutic approaches.Treatment of malignant brain tumors is one of the toughest challenges in the field of medical sciences.Despite recent advances in surgery and radiation therapy,prognosis for high-grade gliomas is uniformly fatal.Thus,new and effective treatments are required,particularly to overcome the drug resistance in these tumors.Tamoxifen(TAM),was first used as drug for clinical treatment in 1969 in England,and was widely used in the treatment of ER+ breast cancers and Endometrial Carcinoma.TAM is a selective estrogen receptor modulator(SERM)with mixed agonist/antagonist activities that has been used widely as an effective treatment of all stages of estrogen receptor(ER)-positive breast cancer.Recently,TAM has been shown to inhibit glioma cell proliferation and induce cell apoptosis in vitro.First report of a continuous treatment of glioma patients with recurrent tumours with high doses of TAM described a response rate of 30%.Understanding the mechanism of TAM-induced apoptosis of malignant glioma cells would help in determining the basis for differential sensitivity to TAM and possibility of modifying the response to TAM treatment.ER-?36,a novel variant of ER-? with a molecular weight of 36 kDa,was firstly identified and clonedd by Zhao-Yi Wang.ER-?36 was also proposed to transduce membrane-initiated estrogen signaling.The expression of ER-?36 was correlated with clinical phenotypes and endocrine therapy responses of patients with various cancers,particularly breast cancer.The present review surveys updated knowledge on ER-?36 biology,non-nuclear receptor functions,its role in estrogen-dependent tumors and its action in human breast cancer diagnosis and treatment,especially emphasize on endocrine resistance.Our previous results revealed that ER-?36 was expressed in primary culture neurons,glial cells and glioblastoma cells,for the first time a regulatory role of ER-?36 in TAM resistent in glioblastoma cells.Autophagy is a physiological process of cellular mechanism which recycles its large molecules and endogenous in the cytoplasm,in order to generate energy and metabolic precursors in order to prolong survival.Preclinical studies have also revealed a role of autophagy in resistance.While treatment of glioblastoma includes radiotherapy and chemotherapy,generally,glioblastomas are resistant to the current therapeutics due to the hyperactivation of the PI3K-Akt-mTOR survival pathway by excessive stimulation via autophagy.Whereas,the function of autophagy is not always clear.So,autophagy was involved in resistance to TAM in gliomas? Whether ER-?36 rugulated autophagy which was induced by TAM?To investigate the effect of tamoxifen on autophagy in glioma cells with different expression of ER-?36,we employed methods including MTT assays for the survival of U87-MG glioblastoma cells with ER-?36 knockdown after tamoxifen treatment.Quantitative real-time PCR for evaluating its predictive value for tamoxifen treatment in GBM.In this study,we have exmined whether ER-?36 could promote tamoxifen resistance in glioma cells.Results show that:(1)Treatment with TAM could inhibit the cell viability of glioblastoma cells,it's a dose-dependent and time-dependent manner.But U251 and U87-MG cells had different sensibilities.(2)TAM induced autophagy and expression of autophagy associated genes in U87-MG cells.5?M TAM increased LC3II/LC3 I,acid hydrolases,Beclin-1,ATG-5 and ATG-12,decreased the expression of p62.(3)ER-?36 was expressed in glioma cells,but significantly higher in U87-MG cells.TAM induced expression of ER-?36 mRNA in glioma cells.(4)Established ER-?36 knockdown stabilized cells(U87-36KD).Knock down ER-?36 increased sensitivity of the U87-MG cells to TAM and decreaced autophagy;Overespression of ER-?36 decreased sensitivity of the U251 cells to TAM and induced autophagy.(5)Tamoxifen-resistant U251 cells were screened.These cells had a six fold higher ER-?36 mRNA expression,showed elevated basal autophagy levels,and could be significantly resensitized to tamoxifen by knockdown ER-?36.(6)ER-?36 activated PI3K/Akt/mTOR signaling pathway was closely associated with autophagy induced by TAM in glioma cells.Inhibit the pathway could increase cells sensitivity to TAM,reduce autophagy.Conclusions: Together,these results indicated that ER-?36 induced autophagy in glioma cells.The process was regulated by PI3K/Akt/mTOR signaling pathway.Activation of the pathway may be the basis of glioma cells resistant to TAM.