Abstract
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 MLKLSer358 and Drp1Ser616 phosphorylation, physical interactions in RCC between RIPK1-RIPK3 and RIPK3-phospho-MLKLSer358, and coincidence of phospho-MLKLser358 and phospho-Drp1Ser616 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.
Highlights
Renal clear cell carcinoma (RCC) is resistant to chemotherapy and 5-year survival rates of metastatic disease are only 5–15%.1 Many anticancer agents act via induction of apoptosis, and apoptotic deficiency may be a cause of chemoresistance
Tumor necrosis factor-α (TNF) can induce necroptosis, via TNF receptor 1 (TNFR1), through a pathway involving TNFR-associated protein with a death domain (TRADD), receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3).2,6–10 Signaling through tumor necrosis factor receptor 2 (TNFR2), which is expressed on RCC cells, does not generally induce cell death, but can potentiate TNFR1mediated programmed necrosis via TNFR1.11 Activation of caspase 8 generally inhibits death by necroptosis so that necroptosis is more evident when caspase activation is inhibited or in cells lacking Fasassociated protein with a death domain (FADD) or caspase 8.12,13 In necroptosis, RIPK1 is recruited to TRADD via a death domain (DD) located in its C terminus[14,15] and RIPK1 recruits
We previously reported that ligation of TNFR1 promotes cell death in malignant tubular epithelial cells in RCC, which we had attributed to apoptosis based on detection of cleaved caspase 3 in some of the TUNEL-positive cells.[27]
Summary
Renal clear cell carcinoma (RCC) is resistant to chemotherapy and 5-year survival rates of metastatic disease are only 5–15%.1 Many anticancer agents act via induction of apoptosis, and apoptotic deficiency may be a cause of chemoresistance. Recent studies have identified a caspase-independent form of programmed cell death, termed necroptosis,[2] that may provide an alternative pathway for tumor killing.[3,4] Tumor necrosis factor-α (TNF), acting through TNF receptor 1 (TNFR1), induces apoptosis of many different types of malignant cells through caspase 8 activation, which is mediated through the assembly of a signaling complex involving TNFR-associated protein with a death domain (TRADD), receptor-interacting protein kinase 1.
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