Abstract：
Current treatment options for triple-negative breast cancer (TNBC) are limited to toxic drug combinations of low efficacy. We recently identified an aryl-substituted fatty acid analogue, termed CTU, that effectively killed TNBC cells in vitro and in mouse xenograft models in vivo without producing toxicity. However, there was a residual cell population that survived treatment. The present study evaluated the mechanisms that underlie survival and renewal in CTU-treated MDA-MB-231 TNBC cells. RNA-seq profiling identified several pro-inflammatory signaling pathways that were activated in treated cells. Increased expression of cyclooxygenase-2 and the cytokines IL-6, IL-8 and GM-CSF was confirmed by real-time RT-PCR, ELISA and Western blot analysis. Increased self-renewal was confirmed using the non-adherent, in vitro colony-forming mammosphere assay. Neutralizing antibodies to IL-6, IL-8 and GM-CSF, as well as cyclooxygenase-2 inhibition suppressed the self-renewal of MDA-MB-231 cells post-CTU treatment. IPA network analysis identified major NF-κB and XBP1 gene networks that were activated by CTU; chemical inhibitors of these pathways and esiRNA knock-down decreased the production of pro-inflammatory mediators. NF-κB and XBP1 signaling was in turn activated by the endoplasmic reticulum (ER)-stress sensor inositol-requiring enzyme 1 (IRE1), which mediates the unfolded protein response. Co-treatment with an inhibitor of IRE1 kinase and RNase activities, decreased phospho-NF-κB and XBP1s expression and the production of pro-inflammatory mediators. Further, IRE1 inhibition also enhanced apoptotic cell death and prevented the activation of self-renewal by CTU. Taken together, the present findings indicate that the IRE1 ER-stress pathway is activated by the anti-cancer lipid analogue CTU, which then activates secondary self-renewal in TNBC cells.
摘要：
三阴性乳腺癌(TNBC)的当前治疗选择限于低功效的毒性药物组合。我们最近确定了一种芳基取代的脂肪酸类似物，称为CTU，在体外和体内小鼠异种移植模型中有效杀死TNBC细胞而不产生毒性。然而,有残留的细胞群体在治疗中存活。本研究评估了CTU处理的MDA-MB-231TNBC细胞中存活和更新的基础机制。RNA-seq谱分析鉴定了在处理的细胞中被激活的几种促炎信号传导途径。实时RT-PCR证实了环氧合酶-2和细胞因子IL-6，IL-8和GM-CSF的表达增加，ELISA和蛋白质印迹分析。使用非粘附剂证实了自我更新的增加，体外集落形成乳腺球试验。中和IL-6，IL-8和GM-CSF的抗体，以及环氧合酶-2抑制抑制CTU处理后MDA-MB-231细胞的自我更新。IPA网络分析确定了由CTU激活的主要NF-κB和XBP1基因网络;这些途径的化学抑制剂和esiRNA敲低降低了促炎介质的产生。NF-κB和XBP1信号依次被内质网（ER）-应激传感器肌醇要求酶1（IRE1）激活，介导展开的蛋白质反应。与IRE1激酶和RNase活性抑制剂共同治疗，降低磷酸化NF-κB和XBP1s的表达和促炎介质的产生。Further,IRE1抑制还增强了凋亡性细胞死亡，并阻止了CTU对自我更新的激活。一起来看,本研究结果表明，IRE1ER应激途径被抗癌脂质类似物CTU激活，然后激活TNBC细胞的二次自我更新。
