Tumor necrosis factor receptor‐2 signaling pathways promote survival of cancer stem‐like CD133(+) cells in clear cell renal carcinoma

Clear cell renal cell carcinoma (ccRCC) contains cancer stem‐like cells (CSCs) that express CD133 (ccRCC‐CD133(+)). CSCs are rarely in cell cycle and, as nonproliferating cells, resist most chemotherapeutic agents. Previously, we reported that tumor necrosis factor receptor‐2 (TNFR2) signaling promotes the cell cycle entry of ccRCC‐CD133(+)CSCs, rendering them susceptible to cell‐cycle‐dependent chemotherapeutics. Here, we describe a TNFR2‐activated signaling pathway in ccRCC‐(CD133+)CSCs that is required for cell survival. Wild‐type (wt)TNF or R2TNF but not R1TNF (TNF muteins that selectively bind to TNFR2 and TNFR1) induces phosphorylation of signal transducer and activator of transcription 3 (STAT3) on serine(727) but not tyrosine(705), resulting in pSTAT3(Ser727) translocation to and colocalization with TNFR2 in mitochondria. R2TNF signaling activates a kinase cascade involving the phosphorylation of VEGFR2, PI‐3K, Akt, and mTORC. Inhibition of any of the kinases or siRNA knockdown of TNFR2 or STAT3 promotes cell death associated with mitochondrial morphological changes, cytochrome c release, generation of reactive oxygen species, and TUNEL(+)cells expressing phosphorylated mixed lineage kinase‐like (MLKL). Pretreatment with necrostatin‐1 is more protective than z‐VAD.fmk, suggesting that most death is necroptotic and TNFR2 signaling promotes cell survival by preventing mitochondrial‐mediated necroptosis. These data suggest that a TNFR2 selective agonist may offer a potential therapeutic strategy for ccRCC.