Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Leipziger Straße 44, 39120, Magdeburg, Germany.
Systems-Oriented Immunology and Inflammation Research Group, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124, Braunschweig, Germany.
Cell Death Dis. 2019 May 16;10(6):384. doi: 10.1038/s41419-019-1609-y.
Clear cell renal cell carcinoma (ccRCC) is the most-prominent tumor type of kidney cancers. Resistance of renal cell carcinoma (RCC) against tumor therapy is often owing to apoptosis resistance, e.g., by overexpression of anti-apoptotic proteins. However, little is known about the role of the apoptosis inhibitor c-FLIP and its potential impact on death receptor-induced apoptosis in ccRCC cells. In this study, we demonstrate that c-FLIP is crucial for resistance against CD95L-induced apoptosis in four ccRCC cell lines. Strikingly, downregulation of c-FLIP expression by short hairpin RNA (shRNA)interference led to spontaneous caspase activation and apoptotic cell death. Of note, knockdown of all c-FLIP splice variants was required to induce apoptosis. Stimulation of ccRCC cells with CD95L induced NF-κB and MAP kinase survival pathways as revealed by phosphorylation of RelA/p65 and Erk1/2. Interestingly, CD95L surface expression was high in all cell lines analyzed, and CD95 but not TNF-R1 clustered at cell contact sites. Downstream of CD95, inhibition of the NF-κB pathway led to spontaneous cell death. Surprisingly, knockdown experiments revealed that c-FLIP inhibits NF-κB activation in the context of CD95 signaling. Thus, c-FLIP inhibits apoptosis and dampens NF-κB downstream of CD95 but allows NF-κB activation to a level sufficient for ccRCC cell survival. In summary, we demonstrate a complex CD95-FLIP-NF-κB-signaling circuit, in which CD95-CD95L interactions mediate a paracrine survival signal in ccRCC cells with c-FLIP and NF-κB both being required for inhibiting cell death and ensuring survival. Our findings might lead to novel therapeutic approaches of RCC by circumventing apoptosis resistance.
透明细胞肾细胞癌 (ccRCC) 是肾脏肿瘤中最主要的肿瘤类型。肾细胞癌 (RCC) 对肿瘤治疗的耐药性通常归因于细胞凋亡抵抗，例如通过抗凋亡蛋白的过度表达。然而，关于凋亡抑制剂 c-FLIP 的作用及其对 ccRCC 细胞死亡受体诱导凋亡的潜在影响知之甚少。在这项研究中，我们证明 c-FLIP 对于四种 ccRCC 细胞系中 CD95L 诱导的凋亡抵抗至关重要。引人注目的是，短发夹 RNA (shRNA) 干扰下调 c-FLIP 表达导致自发的半胱天冬酶激活和凋亡细胞死亡。值得注意的是，需要敲低所有 c-FLIP 剪接变体才能诱导凋亡。用 CD95L 刺激 ccRCC 细胞，如 RelA/p65 和 Erk1/2 的磷酸化所揭示的，诱导 NF-κB 和 MAP 激酶存活途径。有趣的是，在分析的所有细胞系中，CD95L 表面表达都很高，而 CD95 而不是 TNF-R1 聚集在细胞接触部位。CD95 下游，NF-κB 途径的抑制导致自发细胞死亡。令人惊讶的是，敲低实验表明 c-FLIP 在 CD95 信号转导中抑制 NF-κB 的激活。因此，c-FLIP 抑制凋亡并抑制 NF-κB 下游的 CD95，但允许 NF-κB 激活到足以维持 ccRCC 细胞存活的水平。总之，我们证明了一个复杂的 CD95-FLIP-NF-κB 信号通路，其中 CD95-CD95L 相互作用介导 ccRCC 细胞中的旁分泌存活信号，c-FLIP 和 NF-κB 都需要抑制细胞死亡并确保存活。我们的发现可能会通过绕过细胞凋亡抵抗为 RCC 带来新的治疗方法。
