Necroptosis: an alternative cell death program defending against cancer

Abstract

One of the hallmarks of cancer is resistance to programmed cell death, which maintains the survival of cells en route to oncogenic transformation and underlies therapeutic resistance. Recent studies demonstrate that programmed cell death is not confined to caspase-dependent apoptosis, but includes necroptosis, a form of necrotic death governed by Receptor-Interacting Protein 1 (RIP1), RIP3, and Mixed Lineage Kinase Domain-Like (MLKL) protein. Necroptosis serves as a critical cell-killing mechanism in response to severe stress and blocked apoptosis, and can be induced by inflammatory cytokines or chemotherapeutic drugs. Genetic or epigenetic alterations of necroptosis regulators such as RIP3 and cylindromatosis (CYLD), are frequently found in human tumors. Unlike apoptosis, necroptosis elicits a more robust immune response that may function as a defensive mechanism by eliminating tumor-causing mutations and viruses. Furthermore, several classes of anticancer agents currently under clinical development, such as SMAC and BH3 mimetics, can promote necroptosis in addition to apoptosis. A more complete understanding of the interplay among necroptosis, apoptosis, and other cell death modalities is critical for developing new therapeutic strategies to enhance killing of tumor cells.

Keywords: Cancer; MLKL; Necroptosis; RIP1; RIP3.

Figure 1. Morphological features of necroptosis and apoptosis in cancer cells

HT29 colon cancer cells treated with an anticancer drug for 48 hours were analyzed by transmission electron microscopy. The cell undergoing necroptosis shows plasma membrane rupture and permeabilization, compared to the intact plasma membrane with blebbing in the apoptotic cell (red arrowheads). The necroptotic cell exhibits cytoplasm swelling and vacuolization, which are absent in the apoptotic cell (green arrowheads). The necroptotic cell has swelled mitochondria, in contrast to those in the apoptotic cells (yellow arrowheads). The necroptotic cell also lacks condensed and fragmented nuclei seen in the apoptotic cell (blue arrowheads).

Figure 2. Necroptosis regulators and pathways

Upon TNF-α stimulation, the activated TNF receptor (TNFR) interacts with RIP1 and recruits cIAP1 and cIAP2 to form a plasma membrane associated complex, resulting in RIP1 polyubiquitination (Ubs). Inhibition of cIAPs (by SMAC or SMAC mimetics) leads to deubiquitination of RIP1 by CYLD and dissociation of RIP1. RIP1 then binds to FADD and procaspase-8 to form a complex, which activates caspase-8 (Casp-8) and leads to apoptosis induction. If caspase-8 activity is inhibited, RIP1 binds to RIP3 to form necrosome and promotes RIP3 auto-phosphorylation and subsequent activation, allowing RIP3 to recruit and phosphorylate MLKL. This results in oligomerization of MLKL, membrane insertion of MLKL oligomers, disruption of plasma and intracellular membrane integrity, and necroptotic death. Other necroptotic stimuli, including FASL, TRAIL, LPS, dsRNA (such as poly (I:C)) and interferon γ (IFNγ), can stimulate their respective receptors to activate RIP1 and/or RIP3 to promote necroptosis. Viral infection directly activates RIP3 through DAI. Anticancer agents and genotoxic stress can also induce RIP1/RIP3-dependent necroptosis. Several inhibitors of necroptosis have been developed, including the RIP1 inhibitor necrostatin-1 (Nec-1), the RIP3 inhibitors GSK843 and GSK872, and the MLKL inhibitor necrosulfonamide (NSA).