Necroptosis: A new form of cell death and its importance in targeting cancer therapy resistance

By Hung-His (Chelsea) Chen

One of the biggest challenges faced by cancer patients is resistance to therapy. Therefore, cancer research aims to develop novel therapeutic interventions that bypass apoptosis. Necroptosis, an alternative programmed cell death pathway, has become a popular area of research as a potential cancer therapy to combat apoptosis resistant cancer cells. 

Apoptosis is one of the most well-defined, programmed cell death  (PCD) pathways. Apoptosis is the body’s defence against danger and cancer development. Unfortunately, evasion of apoptosis is third hallmarks of cancer, leading to tumourgenesis and therapy resistance. Until recently, apoptosis was believed to be the only form of PCD, until the discovery of necroptosis, which is also known as programmed necrosis. Although necroptosis is a form of programmed cell death, it leads to the upregulation of many pro-inflammatory cytokines such as IL-6, 8, and 18 –  this is usually not seen in apoptosis. 

The most well-defined necroptotic pathway is the Tumour Necrosis Factor alpha (TNFα)-TNFR1 signalling pathway. Upon stimulation by TNFα, complex I, consisting of cellular Inhibitors of Apoptosis (cIAP), Receptor Intreacting Protein Kinase 1 (RIPK1), Tumour Necrosis Factor Receptor Type 1-Associated Death Domain Protein (TRADD), and Tumour Necrosis Associated Factor (TRAF)2/5. This pathway leads to cell-survival through the NF-kB and MAPK pathway to signal the upregulation of pro-inflammatory cytokines (Pasparakis et al., 2015). Destabilisation of Complex I leads to Complex IIa formation, consisting of TRADD, Fas-Associated Protein with Death Domain (FADD), caspase-8 and signals for apoptosis independent of RIPK1 (Pasparakis et al., 2015). Inhibition of cIAP by BV6 drives Complex IIb formation, consisting of RIPK1, RIPK3, FADD and caspase-8. However, the presence of caspase-8 inhibits the activity of RIPK1 and RIPK3, and the pathway leads to apoptosis as well. Finally, inhibition of caspase-8 by pan-caspase inhibitor, BV6, drives Complex III formation, leading to necroptosis (Sun et al., 2012). Upon TNFa stimulation, RIPK1 gets ubiquitinated and phosphorylated. Phosphorylated RIPK1 interacts with RIPK3 via their RIP Homolytic Interaction Motif (RHIM) domain and phosphorylates RIPK3 (Kaiser et al., 2013). Phosphorylated RIPK3 recruits and phosphorylates MLKL, where the latter then oligomerises and gets translocated to the plasma membrane to cause membrane rupture.  

The pathway can also be stimulated by bacterial endotoxins through toll-like receptors mediated signalling pathways. In the LPS-TLR4 signalling pathway, TRIF binds to RIPK1 and RIPK3 by their RHIM domain to stimulate the phosphorylation and oligomerisation of MLKL, leading to necroptosis (Kaiser et al., 2013). 

Immunosurveillance is the ability of immune cells to identify and eliminate foreign invaders. Although there is no evidence suggesting the role of necroptotic signalling in immune cell activation, there has been evidence suggesting RIPK3’s role in regulating Natural Killer T-cell (NKT)’s anti-tumour response (Gong et al., 2019). Werthmöller et al. has reported the use of zVAD to induce necroptosis during therapy increases  DC and CD8+ T cell infiltration, hence reducing tumour growth (Werthmöller et al., 2015). Additionally, its pro-inflammatory nature meant the release of cytokines like IL-1a to activate dendritic cells to secrete IL-12, which plays a crucial role in anti-tumour immunity (Gong et al., 2019). 

Interestingly, it is with this same pro-inflammatory nature that promotes tumourigenesis. The extensive pro-inflammatory consequences of necroptosis have been reported to encourage cancer metastasis and suppress the immune system (Gong et al., 2019). The pro-inflammatory immune cells engaged upon the stimulation of necroptosis enhances metastasis, angiogenesis, and cell proliferation; all of these contribute to a worse prognosis (Gong et al., 2019). Furthermore, IL-1a, that’s supposedly beneficial to host tumour defence as aforementioned, can directly trigger neighbouring cells to undergo cell proliferation and neoplastic transformation (Gong et al., 2019). Moreover, extensive inflammatory microenvironment can also trigger the release of ROS, leading to DNA damage and hence genome instability and tumorigenesis (Gong et al., 2019). 

Additionally, RIPK3 are reported to have different effects on different cancer types. There is a decrease in RIPK3 expression in breast cancer, acute myeloid leukaemia, as well as head and neck squamous cell carcinoma, which has been reported to have worsened prognosis and accelerated tumour growth and enhanced tumour survival (Gong et al., 2019). On the other hand, necroptotic component RIPK1 has been found to be at increased levels in glioblastoma and lung cancer cells – it being reported to have worsened prognosis and promoted oncogenesis respectively (Gong et al., 2019). 

Although targeting cancer therapies focused on necroptosis sounds promising, due to its ability to stimulate the immune system, more research has to be done to ensure the therapies benefit not harm the host. For the therapy to be effective, the inflammatory responses should be controlled and limited to ensure the therapy does not harm organs. 

References:

Gong, Y., Fan, Z., Luo, G., Yang, C., Huang, Q., Fan, K., Cheng, H., Jin, K., Ni, Q., Yu, X. & Liu, C. 2019, “The role of necroptosis in cancer biology and therapy”, Molecular cancer, vol. 18, no. 1, pp. 100.

Kaiser, W.J., Sridharan, H., Huang, C., Mandal, P., Upton, J.W., Gough, P.J., Sehon, C.A., Marquis, R.W., Bertin, J. & Mocarski, E.S. 2013, “Toll-like Receptor 3-mediated Necrosis via TRIF, RIP3, and MLKL”, The Journal of biological chemistry, vol. 288, no. 43, pp. 31268-31279.

Pasparakis, M. & Vandenabeele, P. 2015, “Necroptosis and its role in inflammation”, Nature (London), vol. 517, no. 7534, pp. 311-320.

Sun, L., Wang, H., Wang, Z., He, S., Chen, S., Liao, D., Wang, L., Yan, J., Liu, W., Lei, X. & Wang, X. 2012, “Mixed Lineage Kinase Domain-like Protein Mediates Necrosis Signaling Downstream of 

RIP3 Kinase”, Cell (Cambridge), vol. 148, no. 1-2, pp. 213-227.

Werthmöller, N., Frey, B., Wunderlich, R., Fietkau, R. & Gaipl, U.S. 2015, “Modulation of radiochemoimmunotherapy-induced B16 melanoma cell death by the pan-caspase inhibitor zVAD-fmk induces anti-tumor immunity in a HMGB1-, nucleotide- and T-cell-dependent manner”, Cell death & disease, vol. 6, no. 5, pp. e1761.

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