他莫昔芬(tamoxifen，TAM)作为雌激素受体阳性(estrogen receptor，ER+)乳腺癌的一线化疗药物使大多数患者受益，但原发性和继发性耐药问题严重影响临床治疗效果。深入研究ER+乳腺癌TAM耐药机制，改善治疗效果是当前亟待解决的问题。抑癌因子NDRG2(N-myc downstream regulated gene 2，NDRG2)在肿瘤发生发展中发挥重要作用，但是否参与ER+乳腺癌TAM耐药尚不清楚。本研究旨在探明NDRG2在ER+乳腺癌TAM耐药中发挥的作用和机制。通过RT-PCR与免疫印迹分析对比TAM敏感型和耐药型ER+乳腺癌细胞发现，NDRG2的mRNA转录水平和蛋白质翻译水平在TAM耐药细胞中表达显著下调，且与耐药能力负相关(P＜0。001);CCK-8细胞毒性实验和软琼脂克隆形成实验证实，在耐药细胞中过表达NDRG2可显著降低TAM药物半抑制浓度IC5。和软琼脂克隆形成率(P＜0。001)，逆转耐药表型。分子机制上，X-box结合蛋白1(X-box bind-ing protein 1，XBP1)mRNA 剪切实验与内质网相关降解(endoplasmic-reticulum associated degradation，ERAD)报告蛋白的结果显示，过表达NDRG2可增强耐药细胞中剪切型XBP1s mRNA转录与ERAD报告蛋白CD3e-YFP表达(P＜0。001)，引发耐药细胞内质网强应激反应;免疫印迹检测结果显示，过表达NDRG2可显著提高耐药细胞中内质网应激感受器肌醇需要激酶1α(inositol requiring enzyme 1，IRE 1α)的磷酸化水平及其下游因子，例如内质网EIP辅助因子(endoplasmic reticulum-lo-calized DnaJ 4，ERdj4)、PKR 蛋白激酶的细胞抑制剂(cellular Inhibitor of the PKR protein kinase，P58IPK)、α 甘露糖苷酶样应激蛋白(er degradation enhancing α mannosidase likeprotein，EDEM)和蛋白质二硫键异构酶家族 A 成员 5(protein disulfide isomerase family a member 5，PDIA5)的表达水平(P＜0。001)。小鼠异种移植瘤研究进一步证实，在耐药细胞中过表达NDRG2可增强TAM治疗效果，显著抑制耐药移植瘤生长(P＜0。001)。以上研究结果表明，通过提高耐药细胞中NDRG2表达，增强TAM治疗引发的内质网强烈应激，可逆转ER+乳腺癌细胞耐药性，改善TAM治疗效果。研究结果为解决ER+乳腺癌TAM耐药问题提供了新的思路和有价值的潜在药物靶点。
NDRG2 Activates Endoplasmic Reticulum Stress via IRE1α-XBP1 to Reverse Tamoxifen Resistance in ER+Breast Cancer
Tamoxifen(TAM)has been widely used for the treatment of ER+breast cancer.However,the inevitable emergence of resistance to tamoxifen obstructs the successful treatment of this cancer.The tumor suppressor gene N-myc downstream-regulated gene 2(NDRG2)plays a significant role in the de-velopment of ER+breast cancer.However,it is unclear whether NDRG2 participates in mediating TAM resistance in ER+breast cancer.Here,we investigate the expression of NDRG2 mRNA and protein in TAM-sensitive and TAM-resistant ER+breast cancer cells.The results of immunoblotting experiments re-vealed a negative correlation between NDRG2 expression and TAM resistance ability in ER+breast cancer cells(P＜0.001).CCK-8 cell viability assays and soft agar colony formation assays showed that NDRG2 overexpression in TAM resistant cells significantly reduced the TAM IC50 value and the soft agar colony formation rate(P＜0.001).For the mechanism,the ERAD reporter protein assays showed that NDRG2 overexpression upregulated the expression of the ERAD reporter protein CD3ε-YFP and increased the lev-els of spliced XBP1s mRNA,leading to severe endoplasmic reticulum stress in TAM resistant cells(P＜0.001).Immunoblot analysis confirmed that overexpression of NDRG2 significantly increased the level of phosphorylation of the endoplasmic reticulum stress sensor IRE 1α and the expression levels of its down-stream protein factors,including ERdj4,P58IPK,EDEM and PDIA5(P＜0.001).The in vivo xenograft tumor experiments in mice further verified that NDRG2 overexpression significantly inhibited the growth of resistant tumors,which enhanced the therapeutic effect of TAM(P＜0.001).These findings indicate that increasing NDRG2 expression and triggering severe endoplasmic reticulum stress upon TAM treatment can reverse the resistance of ER+breast cancer cells to TAM and inhibits the growth of ER+breast canc-er tumors.Our results provide valuable new insights and potential targets for improving the clinical man-agement of TAM-resistance and prognosis in ER+breast cancer.
NETL
NSTL
万方数据
