Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, CSIC-Universidad de Sevilla-Universidad Pablo de Olavide, Avda Américo Vespucio 24, 41092, Sevilla, Spain.
University of Stuttgart, Institute of Cell Biology and Immunology, Stuttgart, Germany.
Cell Death Dis. 2022 Feb 3;13(2):111. doi: 10.1038/s41419-022-04574-6.
Protein misfolding or unfolding and the resulting endoplasmic reticulum (ER) stress frequently occur in highly proliferative tumors. How tumor cells escape cell death by apoptosis after chronic ER stress remains poorly understood. We have investigated in both two-dimensional (2D) cultures and multicellular tumor spheroids (MCTSs) the role of caspase-8 inhibitor cFLIP as a regulator of the balance between apoptosis and survival in colon cancer cells undergoing ER stress. We report that downregulation of cFLIP proteins levels is an early event upon treatment of 2D cultures of colon cancer cells with ER stress inducers, preceding TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) upregulation, caspase-8 activation, and apoptosis. Maintaining high cFLIP levels during ER stress by ectopic expression of cFLIP markedly inhibits ER stress-induced caspase-8 activation and apoptosis. Conversely, cFLIP knockdown by RNA interference significantly accelerates caspase-8 activation and apoptosis upon ER stress. Despite activation of the proapoptotic PERK branch of the unfolded protein response (UPR) and upregulation of TRAIL-R2, MCTSs are markedly more resistant to ER stress than 2D cultures of tumor cells. Resistance of MCTSs to ER stress-induced apoptosis correlates with sustained cFLIP expression. Interestingly, resistance to ER stress-induced apoptosis is abolished in MCTSs generated from cFLIP knockdown tumor cells. Overall, our results suggest that controlling cFLIP levels in tumors is an adaptive strategy to prevent tumor cell's demise in the unfavorable conditions of the tumor microenvironment.
蛋白质错误折叠或展开以及由此导致的内质网（ER）应激经常发生在高度增殖的肿瘤中。肿瘤细胞在慢性 ER 应激后如何避免细胞凋亡仍然知之甚少。我们在二维（2D）培养物和多细胞肿瘤球体（MCTS）中研究了半胱天冬酶-8 抑制剂 cFLIP 作为调节发生 ER 应激的结肠癌细胞中凋亡与存活平衡的作用。我们报告说，下调 cFLIP 蛋白水平是结肠癌细胞 2D 培养物在用 ER 应激诱导剂处理时的早期事件，早于 TNF 相关凋亡诱导配体受体 2（TRAIL-R2）上调、半胱天冬酶-8 激活和凋亡。通过异位表达 cFLIP 在 ER 应激期间维持高 cFLIP 水平可显著抑制 ER 应激诱导的半胱天冬酶-8 激活和凋亡。相反，通过 RNA 干扰敲低 cFLIP 可显著加速 ER 应激时半胱天冬酶-8 的激活和凋亡。尽管未折叠蛋白反应（UPR）的促凋亡 PERK 分支被激活并且 TRAIL-R2 上调，但 MCTS 对 ER 应激的抵抗力明显强于肿瘤细胞的 2D 培养物。MCTS 对 ER 应激诱导的凋亡的抗性与持续的 cFLIP 表达相关。有趣的是，在源自 cFLIP 敲低肿瘤细胞的 MCTS 中，ER 应激诱导的凋亡抗性被消除。总体而言，我们的结果表明，控制肿瘤中的 cFLIP 水平是一种适应性策略，可以防止肿瘤细胞在肿瘤微环境的不利条件下死亡。
