Al-Lamki R S, Lu W, Manalo P, Wang J, Warren A Y, Tolkovsky A M, Pober J S, Bradley J R
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
Department of Pathology, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
Cell Death Dis. 2016 Jun 30;7(6):e2287. doi: 10.1038/cddis.2016.184.
We previously reported that renal clear cell carcinoma cells (RCC) express both tumor necrosis factor receptor (TNFR)-1 and -2, but that, in organ culture, a TNF mutein that only engages TNFR1, but not TNFR2, causes extensive cell death. Some RCC died by apoptosis based on detection of cleaved caspase 3 in a minority TUNEL-positive cells but the mechanism of death in the remaining cells was unexplained. Here, we underpin the mechanism of TNFR1-induced cell death in the majority of TUNEL-positive RCC cells, and show that they die by necroptosis. Malignant cells in high-grade tumors displayed threefold to four fold higher expression of both receptor-interacting protein kinase (RIPK)1 and RIPK3 compared with non-tumor kidney tubular epithelium and low-grade tumors, but expression of both enzymes was induced in lower grade tumors in organ culture in response to TNFR1 stimulation. Furthermore, TNFR1 activation induced significant MLKL(Ser358) and Drp1(Ser616) phosphorylation, physical interactions in RCC between RIPK1-RIPK3 and RIPK3-phospho-MLKL(Ser358), and coincidence of phospho-MLKL(ser358) and phospho-Drp1(Ser616) at mitochondria in TUNEL-positive RCC. A caspase inhibitor only partially reduced the extent of cell death following TNFR1 engagement in RCC cells, whereas three inhibitors, each targeting a different step in the necroptotic pathway, were much more protective. Combined inhibition of caspases and necroptosis provided additive protection, implying that different subsets of cells respond differently to TNF-α, the majority dying by necroptosis. We conclude that most high-grade RCC cells express increased amounts of RIPK1 and RIPK3 and are poised to undergo necroptosis in response to TNFR1 signaling.
我们之前报道过，肾透明细胞癌细胞（RCC）同时表达肿瘤坏死因子受体（TNFR）-1和-2，但在器官培养中，一种仅与TNFR1结合而不与TNFR2结合的TNF突变体可导致大量细胞死亡。基于在少数TUNEL阳性细胞中检测到裂解的半胱天冬酶3，一些RCC细胞通过凋亡死亡，但其余细胞的死亡机制尚不清楚。在此，我们阐明了TNFR1诱导大多数TUNEL阳性RCC细胞死亡的机制，并表明它们通过坏死性凋亡死亡。与非肿瘤肾小管上皮和低级别肿瘤相比，高级别肿瘤中的恶性细胞显示受体相互作用蛋白激酶（RIPK）1和RIPK3的表达高出三到四倍，但在器官培养中，低级别肿瘤中这两种酶的表达在TNFR1刺激下会被诱导。此外，TNFR1激活诱导了显著的混合谱系激酶结构域样蛋白（MLKL）（Ser358）和动力相关蛋白1（Drp1）（Ser616）磷酸化，RCC中RIPK1-RIPK之间以及RIPK3-磷酸化-MLKL（Ser358）之间发生物理相互作用，并且在TUNEL阳性RCC的线粒体中磷酸化-MLKL（ser358）和磷酸化-Drp1（Ser616）同时出现。半胱天冬酶抑制剂仅部分降低了RCC细胞中TNFR1结合后细胞死亡的程度，而三种分别靶向坏死性凋亡途径中不同步骤的抑制剂则具有更强的保护作用。对半胱天冬酶和坏死性凋亡的联合抑制提供了累加性保护，这意味着不同的细胞亚群对TNF-α的反应不同，大多数细胞通过坏死性凋亡死亡。我们得出结论，大多数高级别RCC细胞表达增加的RIPK1和RIPK3，并准备好在TNFR1信号传导时发生坏死性凋亡。
