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TNFR1 is a crucial regulator of NF‐ĸB‐mediated proinflammatory cell survival responses and programmed cell death (PCD). Deregulation of TNFα‐ and TNFR1‐controlled NF‐ĸB signaling underlies major diseases, like cancer, inflammation, and autoimmune diseases. Therefore, although being routinely used, antagonists of TNFα might also affect TNFR2‐mediated processes, so that alternative approaches to directly antagonize TNFR1 are beneficial. Here, we apply quantitative single‐molecule localization microscopy (SMLM) of TNFR1 in physiologic cellular settings to validate and characterize TNFR1 inhibitory substances, exemplified by the recently described TNFR1 antagonist zafirlukast. Treatment of TNFR1‐mEos2 reconstituted TNFR1/2 knockout mouse embryonic fibroblasts (MEFs) with zafirlukast inhibited both ligand‐independent preligand assembly domain (PLAD)‐mediated TNFR1 dimerization as well as TNFα‐induced TNFR1 oligomerization. In addition, zafirlukast‐mediated inhibition of TNFR1 clustering was accompanied by deregulation of acute and prolonged NF‐ĸB signaling in reconstituted TNFR1‐mEos2 MEFs and human cervical carcinoma cells. These findings reveal the necessity of PLAD‐mediated, ligand‐independent TNFR1 dimerization for NF‐ĸB activation, highlight the PLAD as central regulator of TNFα‐induced TNFR1 oligomerization, and demonstrate that TNFR1‐mEos2 MEFs can be used to investigate TNFR1‐antagonizing compounds employing single‐molecule quantification and functional NF‐ĸB assays at physiologic conditions.
Mutations in the Von Hippel–Lindau (VHL) gene are the driving force in many forms of clear cell renal cell carcinoma (ccRCC) and promote hypoxia-inducible factor (HIF)-dependent tumor proliferation, metastasis and angiogenesis. Despite the progress that has already been made, ccRCC generally remain resistant to conventional therapies and ccRCC patients suffer from metastasis and acquired resistance, highlighting the need for novel therapeutic options. Cysteinyl leukotriene receptor 1 (CysLTR1) antagonists, like zafirlukast, are administered in bronchial asthma to control eicosanoid signaling. Intriguingly, long-term use of zafirlukast decreases cancer risk and leukotriene receptor antagonists inhibit tumor growth, but the mechanisms still remain unexplored. Therefore, we aim to understand the mechanisms of zafirlukast-mediated cell death in ccRCC cells. We show that zafirlukast induces VHL-dependent and TNFα-independent non-apoptotic and non-necroptotic cell death in ccRCC cells. Cell death triggered by zafirlukast could be rescued with antioxidants and the PARP-1 inhibitor Olaparib, and additionally relies on HIF-2α. Finally, MG-132-mediated proteasome inhibition sensitized VHL wild-type cells to zafirlukast-induced cell death and inhibition of HIF-2α rescued zafirlukast- and MG-132-triggered cell death. Together, these results highlight the importance of VHL, HIF and proteasomal degradation in zafirlukast-induced oxidative cell death with potentially novel therapeutic implications for ccRCC.
Men and women differ substantially regarding height, weight, and body fat. Interestingly, previous work detecting genetic effects for waist-to-hip ratio, to assess body fat distribution, has found that many of these showed sex-differences. However, systematic searches for sex-differences in genetic effects have not yet been conducted. Therefore, we undertook a genome-wide search for sexually dimorphic genetic effects for anthropometric traits including 133,723 individuals in a large meta-analysis and followed promising variants in further 137,052 individuals, including a total of 94 studies. We identified seven loci with significant sex-difference including four previously established (near GRB14/COBLL1, LYPLAL1/SLC30A10, VEGFA, ADAMTS9) and three novel anthropometric trait loci (near MAP3K1, HSD17B4, PPARG), all of which were significant in women, but not in men. Of interest is that sex-difference was only observed for waist phenotypes, but not for height or body-mass-index. We found no evidence for sex-differences with opposite effect direction for men and women. The PPARG locus is of specific interest due to its link to diabetes genetics and therapy. Our findings demonstrate the importance of investigating sex differences, which may lead to a better understanding of disease mechanisms with a potential relevance to treatment options.