Di Xu, Zhang Guofeng, Zhang Yaqin, Takeda Kazuyo, Rivera Rosado Leslie A, Zhang Baolin
Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, MD, United States.
Oncotarget. 2013 Sep;4(9):1349-64. doi: 10.18632/oncotarget.1174.
TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. We have previously shown that deficiency of DR4 and DR5 on the surface membrane is a critical mechanism of cancer cell resistance to the recombinant human TRAIL and its receptor agonistic antibodies, which are being evaluated clinically for treating cancers. In certain cancer cells, DR4 and DR5 were found to be mislocalized in intracellular compartments yet to be characterized. Here, we report a novel role of autophagy in the regulation of dynamics of TRAIL death receptors. We first assessed basal levels of autophagosomes in a panel of 11 breast cancer cell lines using complementary approaches (LC3 immunoblotting, RFP-LC3 fluorescence microscopy, and electron microscopy). We found high levels of basal autophagosomes in TRAIL resistant breast cancer cell lines (e.g. BT474 and AU565) and relevant mouse xenograft models under nutrition-rich conditions. Notably, DR4 and DR5 co-localized with LC3-II in the autophagosomes of TRAIL-resistant cells. Disruption of basal autophagosomes successfully restored the surface expression of the death receptors which was accompanied by sensitization of TRAIL-resistant cells to TRAIL induced apoptosis. By contrast, TRAIL-sensitive cell lines (MDA-MB-231) are characterized by high levels of surface DR4/DR5 and an absence of basal autophagosomes. Inhibition of lysosomal activity induced an accumulation of autophagosomes and a decrease in surface DR4 and DR5, and the cells became less sensitive to TRAIL-induced apoptosis. These findings demonstrate a novel role for the basal autophagosomes in the regulation of TRAIL death receptors. Further studies are warranted to explore the possibility of using autophagosome markers such as LC3-II/LC3-I ratios for prediction of tumor resistance to TRAIL related therapies. The results also provide a rationale for future non-clinical and clinical studies testing TRAIL agonists in combination with agents that directly inhibit autophagosome assembly.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）通过靶细胞表面表达的死亡受体（DRs）4和/或5诱导细胞凋亡。我们之前已经表明，细胞膜表面DR4和DR5的缺失是癌细胞对重组人TRAIL及其受体激动性抗体产生抗性的关键机制，这些抗体正在进行癌症治疗的临床评估。在某些癌细胞中，发现DR4和DR5定位错误于尚未明确特征的细胞内区室中。在此，我们报告自噬在调节TRAIL死亡受体动态方面的新作用。我们首先使用互补方法（LC3免疫印迹、RFP-LC3荧光显微镜和电子显微镜）评估了一组11种乳腺癌细胞系中的自噬体基础水平。我们发现在营养丰富的条件下，TRAIL抗性乳腺癌细胞系（如BT474和AU565）及相关小鼠异种移植模型中存在高水平的基础自噬体。值得注意的是，在TRAIL抗性细胞的自噬体中，DR4和DR5与LC3-II共定位。基础自噬体的破坏成功恢复了死亡受体的表面表达，同时TRAIL抗性细胞对TRAIL诱导的凋亡变得敏感。相比之下，TRAIL敏感细胞系（MDA-MB-231）的特征是高水平的表面DR4/DR5以及不存在基础自噬体。溶酶体活性的抑制导致自噬体积累以及表面DR4和DR5减少，并且细胞对TRAIL诱导的凋亡变得不那么敏感。这些发现证明了基础自噬体在调节TRAIL死亡受体方面的新作用。有必要进一步研究探索使用自噬体标志物（如LC3-II/LC3-I比率）预测肿瘤对TRAIL相关疗法抗性的可能性。这些结果也为未来非临床和临床研究测试TRAIL激动剂与直接抑制自噬体组装的药物联合使用提供了理论依据。
