Abstract
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.
Highlights
Renal cell carcinoma (RCC) accounts for 3.8% of malignancies of the adult and over 90% of kidney tumors[1]
The apoptosis inhibitor c-FLIP was expressed by all four cell lines analyzed, with c-FLIPL expression being higher than expression of the short splice variants c-FLIPS and c-FLIPR in clearCa-2 and -3 cell lines (Fig. 1b)
We focused on the role of c-FLIP-mediated CD95L resistance in RCC
Summary
Renal cell carcinoma (RCC) accounts for 3.8% of malignancies of the adult and over 90% of kidney tumors[1]. RCC is a cancer of the proximal renal tubular epithelium and the collecting tubular epithelium and comprises a variety of malignancies that differ in histology and on the molecular level[1,2,3]. One particular type of RCC, the clear cell RCC (ccRCC), often exhibits mutations in the von Hippel-Lindau (VHL) tumor suppressor gene and in PBRM1, a component of the PBAF chromatin-remodeling complex[4]. Another main characteristic of RCC is the Resistance to apoptosis is a hallmark of cancer cells and can lead to tumor formation[7]. The protein Fas-associated protein with death domain (FADD) binds to CD95 and acts as recruitment platform
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