Necroptosis pathways in tumorigenesis

Abstract

Necroptosis is a caspase-independent form of programmed cell death executed by the receptor interacting protein kinase 1 (RIPK1)-RIPK3-mixed lineage kinase domain-like protein (MLKL) signaling cascade, deregulation of which can cause various human diseases including cancer. Escape from programmed cell death is a hallmark of cancer, leading to uncontrolled growth and drug resistance. Therefore, it is crucial to further understand whether necroptosis plays a key role in therapeutic resistance. In this review, we summarize the recent findings of the link between necroptosis and cancer, and discuss that targeting necroptosis is a new strategy to overcome apoptosis resistance in tumor therapy.

1. Regulation and mechanisms of necroptosis

Apoptosis is the best described programmed cell death [1] induced by various intrinsic and extrinsic factors and is mediated by the activation of caspases. Several types of regulated cell death (RCD), beyond apoptosis, have been characterized, such as pyroptosis, ferroptosis, necroptosis, and cuprotosis [2,3]. Pyroptosis, ferroptosis, and necroptosis are categorized as regulated necrosis. Pyroptosis is an inflammation-induced and highly immunogenic RCD [2]. Ferroptosis is an iron-induced RCD characterized by lipid peroxidation and iron dependence, showing promise of potential antitumor treatment [4,5]. On the other hand, cuprotosis is a recently discovered form of intracellular cupper-dependent RCD driven by copper-induced mitochondrial stress [3].

Necroptosis is best-studied regulated necrotic cell death, which is activated in a caspase-independent manner and characterized as a lytic form of cell death [6–8]. Necroptosis is accompanied by the release of damage-associated molecular patterns (DAMPs) and cytokines, thereby triggering proinflammatory responses. On the other hand, apoptosis is a non-lytic form of cell death in which cellular contents are kept inside apoptotic bodies and thus causes no inflammatory effects. Necroptosis is induced in apoptosis-deficient cells. Therefore, it is regarded as a backup cellular defense mechanism. Necroptosis exerts essential physiological function in development and tissue homeostasis such as antiviral defense and tissue injury repair. Dysregulated necroptosis leads to various pathological conditions, such as chronic intestinal inflammation [9,10], inflammatory skin disease [11,12], non-alcoholic steatohepatitis (NASH) [13,14], multiple sclerosis (MS) [15], and amyotrophic lateral sclerosis (ALS) [16]. In addition, recent findings have revealed a tight correlation between necroptosis and cancer, which has attracted increasing attention. Therefore, understanding the precise molecular mechanism of necroptosis is essential to design possible therapeutic intervention strategies for various necroptosis-mediated human diseases.

Necroptosis is induced by the ligands of the death receptor family, such as tumor necrosis factor (TNF)-α, Fas Ligand (FasL), and TNF-related apoptosis-inducing ligand (TRAIL), by binding to their cognate receptors when caspases are blocked by chemical inhibitors or genetic knockout [17] (Fig. 1). TNF/TNF receptor 1 (TNFR1)-mediated necroptosis is the most well-studied caspase-independent necrosis [6,18,19]. Mechanistically, TNF-α binding to TNFR1 induces the formation of a complex called the TNFR1 signaling complex–complex I, where RIPK1 is ubiquitinated by upstream E3 ligases, including cellular inhibitor of apoptosis (cIAP) 1/2 and linear ubiquitin chain assembly complex (LUBAC) [20–24]. The main downstream pathways of the complex I are NF-κB and mitogen-activated protein kinases (MAPKs), which promote cellular survival [25]. RIPK1 will be deubiquitinated by cylindromatosis protein (CYLD) and A20 when cIAP1/2 is degraded by the small-molecule, second mitochondria-derived activator of caspase (SMAC) mimetics [26–29]. The deubiquitination of RIPK1 triggers RIPK1 dissociation from the complex I and subsequently promotes the formation of complex IIa (RIPK1, FAS-associated death domain protein (FADD), caspase-8, etc.) [30,31], which can cause the activation of caspase-8 and induction of apoptosis. Biologically, activated caspase 8 cleaves RIPK1 and RIPK3 to block the formation of complex IIb (caspase-8, FADD, RIPK1, RIPK3, and MLKL) and the induction of necroptosis. When caspases are blocked by inhibitors (e.g., zVAD) or by the genetic deletion, which prevents the cleavage of the key necroptosis regulators RIPK1 and RIPK3, complex IIb or necrosome will be formed in a RIPK1-kinase-dependent manner [32,33]. In the necrosome, RIPK1 binds to RIPK3 through their respective RIP homotypic interaction motif (RHIM) domains to activate RIPK3, which in turn mediates the phosphorylation and activation of the downstream substrate, MLKL. Subsequently, oligomerization of MLKL disrupts the integrity of plasma membranes and causes cell death [34,35] (Table 1).

Fig. 1.

Canonical TNF/TNFR1 dependent Necroptosis Signaling Cascades. Upon TNF-α stimulation, the activated TNF receptor (TNFR) interacts with TNFR1-associated death domain protein (TRADD), TRAFs, and RIPK1 and recruits cIAP1 and cIAP2 to form a plasma membrane-associated complex, resulting in RIPK1 polyubiquitination. Inhibition of cIAPs (by SMAC or SMAC mimetics) leads to deubiquitination of RIPK1 and dissociation of RIPK1 from the complex. RIPK1 then binds to FADD and caspase-8 to form a complex called complex IIa, which activates caspase-8 and leads to apoptosis induction. When caspase-8 activity is blocked, RIPK1 binds to RIPK3 to form a complex IIb or necrosome in a RIPK1-kinase-dependent manner. Then, RIPK3 undergoes auto-phosphorylated and subsequent activation, allowing RIPK3 to recruit and phosphorylate MLKL. The translocation of phosphorylated MLKL to the plasma membrane promotes necroptosis by disrupting plasma and intracellular membrane integrity. KD: kinase domain, DD: death domain.

Table 1.

Necroptotic factors and their roles in necroptosis.

Necroptotic factors	Key roles in necroptosis	Antagonists
RIPK1	Being deubiquitinated under apoptosis-deficient conditions, interacts with RIPK3 to form necrosome	Necrostatin-1 (Nec-1)	[6]
RIPK3	Forms necrosome with RIPK1 and phosphorylates MLKL	GSK843, GSK872	[36]
MLKL	Being phosphorylated by RIPK3, oligomerizes and translocates to plasma membrane to induce membrane permeabilization and necroptosis	Necrosulfonamide (NSA)	[37]
cIAP1/2	Polyubiquitinates RIPK1 via K63-linkage to induce NF-κB signaling	SMAC mimetics	[38]
LUBAC	Polyubiquitinates RIPK1via M1-linkage to induce NF-κB signaling
CYLD	Deubiquitinates RIPK1 to promote complex II formation
A20	Deubiquitinates RIPK1 to promote complex II formation

In addition to these death-receptor ligand-induced necroptosis, lipopolysaccharides (LPS)–toll-like receptor 4 (TLR4) or poly(I:C)–TLR3 treatment with the inhibition of caspases by zVAD-fmk can also induce TIR domain-containing adaptor-inducing interferon-β (TRIF)-mediated necroptosis [39,40] (Fig. 2). TRIF triggers the activation of RIPK3 by the direct RHIM-RHIM interaction to initiate necroptosis [41,42]. On the other hand, z-DNA/RNA binding protein-1 (ZBP1), which was suggested as a cytoplasmic DNA sensor capable of inducing type I interferons (IFNs), and nuclear factor-κB (NF-κB) activation, has been shown to induce RIPK3-mediated necroptosis independently of RIPK1 in response to double-stranded DNA [43–47]. Additionally, interferon has been reported to induce RIPK1/RIPK3-mediated necrosis when caspase-8 or FADD was absent [48,49].

Fig. 2.

Other stimuli that are capable of inducing necroptosis. FASL, TRAIL, LPS, dsRNA (such as poly (I:C)), and interferon γ (IFNγ), can stimulate their respective receptors to activate TRIF, RIPK1, and subsequent binding to RIPK3 via their RHIM domain to activate RIPK3. Viral infection directly activates RIPK3 through ZBP1/DAI (DNA-dependent activator of interferon regulatory factors) binding to RIPK3. Activated RIPK3 then phosphorylates MLKL and causes the oligomerization of MLKL, membrane insertion of MLKL oligomers, and disruption of plasma and intracellular membrane integrity to induce necroptosis.

2. Downregulation in the core necroptotic factors in cancer

It has been reported that the key mediators of necroptosis, including RIPK3, MLKL, and CYLD, are downregulated in different cancer cells (Table 2). CYLD is a deubiquitinating enzyme identified as a key positive regulator of TNF/TNFR1-mediated necroptosis [26,27,50]. IKKβ-dependent CYLD phosphorylation at Ser568 following TNFR1 stimulation potentiates its deubiquitinase (DUB) activity towards K-63 linked ubiquitin chain on RIPK1 [51]. CYLD was originally demonstrated as a tumor suppressor gene, which was reported to be mutated in Familial Cylindromatosis [52]. These mutations have been found in a variety of human cancers and can lead to resistance to chemotherapy. Lymphoid enhancer-binding factor 1 (LEF1), a downstream effector of the Wnt/β-catenin pathway, is likely responsible for repressing CYLD at the transcriptional levels [53]. Moreover, Snail was found to negatively regulate the transcription levels of CYLD, which promotes tumor progression in malignant melanoma [54]. It was also found that the expression of CYLD was decreased in the colon and hepatocellular carcinoma cells and tumor samples [55].

Table 2.

Downregulations and mutations of necroptotic factors in cancer.

Cancer	Necroptotic factors downregulated in cancer
Acute myeloid leukemia	RIPK3	[64]
Chronic lymphocytic leukemia	CYLD	[53]
Melanoma	RIPK3, CYLD	[54] [65]
Breast cancer	RIPK3	[66]
Colorectal cancer	RIPK3, MLKL, CYLD	[55] [56] [67]
Head and neck squamous cell carcinoma	RIPK1	[68]
Gastric cancer	MLKL	[69]
Ovarian cancer	MLKL	[70]
Mutation	Expected effect on function
RIPK3 V458M	Identified in RHIM domain: Loss-of-function	[60]
MLKL L291P	Identified in pseudokinase domain: Loss-of-function	[63]

It has been well demonstrated that DNA methylation plays a key role in the silence of the expression of RIPK3. Notably, RIPK3 expression is often silenced in cancer cells in a methylation-dependent mechanism in many cancer cell lines, which may explain why cancer cells can also escape from the caspase-independent necroptosis [56–58]. Consistent with this finding, treatment with the hypomethylating agent decitabine (5-AD) or knockdown of DNMT1 (DNA cytosine-5-methyltransferases) restored RIPK3 expression in multiple cell lines, thereby promoting sensitivity to necroptosis inducers in a RIPK3-dependent manner [58]. There is growing evidence suggesting that RIPK3 silencing in tumor cells is selected during the process of tumor progression, and RIPK3 down-regulation confers cancer cells to chemotherapeutic resistance in cancers [58,59]. Thus, reactivation of RIPK3 expression in cancer cells by using hypomethylating agents might provide an opportunity for triggering chemotherapy-induced cell death. Furthermore, somatic V458M mutations, which resides within RIPK3 RHIM domain, is likely a loss-of-function mutation based on the biochemical analysis showing that V458 is a critical residue for RHIM-mediated protein interaction [60].

The MLKL expression levels were decreased in several cancer cells, including pancreatic adenocarcinoma and primary ovarian cancers, and the downregulation of MLKL was correlated with decreased patient survival [61]. In addition, in human tumor progression, mutations in F398I and L291P have been identified in the pseudokinase domain of MLKL [62]. Ectopic expression of MLKL L291P mutant, but not F398I, facilitates the cells resistant to TNF/TNFR1-mediated necroptosis, suggesting that the L291P is a loss-of-function mutation [63] (Table 2).

3. Necroptosis in tumorigenesis

It has been reported that several necroptosis-related factors exert their function in tumorigenesis of a range of cancers. Reactive oxygen species (ROS) production and cytochrome c release accompanied with mitochondrial dysfunction was induced by TNFα via RIPK1 [71]. Tumor cell necroptosis was induced by Tag7-Hsp70 cytotoxic complex through permeabilization of lysosomes and mitochondria [72]. In prostate adenocarcinoma cell line PC-3, ROS-mediated necroptosis was induced by biogenic selenium nanoparticles through TNF activation, independent of RIPK3 and MLKL, regulated by RIPK1 [73]. In prostate cancer cells under lactic acidosis, necroptosis was induced through mitochondrial dysfunction with the cell communication network factor 1 (CCN1) [74]. In human colon cancer cells, RIPKs collaborated with ROS during necroptosis, which was promoted by z-VAD and cobalt chloride, a reagent inducing hypoxia-inducible factor-1α (HIF1α) expression and mimicking the hypoxic microenvironment of tumor tissue [75]. In human gastric cancer cells, caspase-independent necroptosis was induced by HUHS1015, a newly synthesized naftopidil analog, in association with the accumulation of apoptosis-inducing factor-homologous mitochondrion-associated inducer of death (AMID) in the nucleus [76]. In addition, p53 mutation, SMAC mimetics, and other factors were reported to be involved in the necroptosis of tumors. In colorectal cancer, necroptosis was rare, and p53 mutation might result in autophagy upregulation [77]. Necroptosis was promoted by SIRT3, inhibiting the growth of human small-cell lung cancer cells, and the expression of SIRT3 could regulate the stability of mutant p53 by controlling ubiquitination-mediated proteasomal degradation of the protein [78]. In necroptosis of colon cancer cells, AMP-activated protein kinase (AMPK) played an inhibitory role with p53 null mutation under nutrient starvation [79]. In apoptosis-resistant cancer cells, necroptosis was triggered by IFNγ synergizing with SMAC mimetics [80]. Caspase inhibition combined with SMAC analog, LCL161, induced a necroptosis effect on human breast cancer drug-resistant cells [81,82]. RIPK3 expresses in mouse models of colorectal cancer and a subset of human colorectal cancer cells appears to be the deciding factor of cancer cell susceptibility to SMAC mimetic-induced necroptosis [59].

In many types of cancers, necroptosis is found to be actively involved. In lung cancer, necroptosis was triggered by Betanodavirus B2 protein [83]. Low-level expression of necroptosis factors, such as RIPK3 and PELI1, combined with high-level expression of the DNA damage response factor p53, served as an important indicator in predicting the survival of stage I non-small-cell lung cancer (NSCLC) patients with the squamous cell carcinoma subtype [84]. In colorectal cancer cells, PFK-15 induced genome instability and necroptosis were induced by PFK-15, of which cytotoxicity and genotoxicity were attenuated by deprivation of necroptosis. In this way, a more intimate relationship among PFKFB3, necroptosis, and genome instability, could be revealed, which awaits further in-depth studies [85].

In colon cancer cells, activation of hepatocyte growth factor (HGF) gene was caused by genomic instability, which promoted cancer cell resistance to necroptosis [86]. Besides, adipoRon suppressed tumor growth of pancreatic cancer by inducing necroptosis [87]. In the early stages of prostate cancer, necroptosis was more activated via induced RIPK3 expression, while repressed during the final stages of tumor progression. Moreover, the repression of RIPK3 is related to the increase of both PSA levels and tumor volume, which represents the tumor progression in the final stages [88]. In prostate cancer cells, apoptosis and necroptosis were differentially facilitated by reticulocalbin-1 down-regulation [89]. On the other hand, sensitivity of renal cancer cells to cisplatin-induced necroptosis was regulated by miR-124, targeting the calpain small subunit 1 (Capn4)-CCR4-NOT transcription complex subunit 3 (CNOT3) axis [90]. In low-grade serous, but not serous borderline ovarian tumor cells, necroptosis-like cell death was induced by CD40 [91].

4. Necroptosis promotes inflammation and cancer metastasis

Necroptotic cells could directly induce inflammation by releasing massive damage-associated molecular patterns (DAMPs) into the tissue microenvironment, including interleukin-1 family cytokines, nucleic acids and ribonucleoproteins, histones and high mobility group box 1 protein (HMGB) family members, and ATP [56] (Fig. 3). DAMPs are usually detected by pattern recognition receptors that activate immune responses by inducing the expression of cytokines and chemokines [92, 93]. Metastasis is thought to be an important cause of cancer patients’ mortality. Immune cells and secreted cytokines, chemokines, and growth factors collectively create an inflammatory microenvironment that promotes the invasive and metastatic ability of cancer cells. Necroptosis may facilitate tumor cell metastasis by promoting inflammation. To this end, TNF α has been shown to play a key role in tumor development. It has been demonstrated that elevated expression of TNF-α was elevated in many malignant tumors, which is associated with poor prognosis [94]. As such, inactivation of RIPK1 with the specific inhibitor Nec-1 has been shown to alleviate inflammation and colitis-associated tumorigenesis in a mouse model of the disease [95].

Fig. 3.

Role of Necroptosis in Cancer.

Tumor metastasis largely depends on the capacity to escape from the bloodstream by passing the endothelial barrier. Tumor cell extravasation through endothelium is a key step in the process of metastasis. Tumor cells were shown to induce necroptosis of endothelial cells by activating death receptor 6 (DR6) to promote tumor cell extravasation and metastasis. Treatment of mice with the RIPK1 inhibitor, Nec-1, or genetically knockout of RIPK3 or MLKL in endothelial cells reduced tumor-cell-induced endothelial necroptosis, tumor cell extravasation, and metastasis [96]. Thus, targeting RIPK1 kinase-dependent necroptotic signaling pathways might be promising anti-metastatic therapies.

Necroptotic cells release damage-associated molecular patterns (DAMPS) to promote inflammation, which leads to cancer growth and metastasis. However, necroptosis can also activate naïve CD8⁺ T cells for immune defense against cancer. Moreover, tumor cells can induce endothelial cell necroptosis to promote metastasis. On the other hand, many cancer cells can escape from necroptosis by downregulating RIPK3 and MLKL.

5. Necroptosis and mouse cancer model

Mouse models have been used as an efficient tool to evaluate the critical function of necroptosis in regulating tumorigenesis and metastasis. The breast cancer orthotopic mouse model demonstrated that CRISPR-Cas9-mediated MLKL silencing in Mvt-1 mouse mammary tumor cells decreased necroptosis in core tumor necrotic regions, leading to the conversion of necroptosis into apoptotic cell death mode in the tumor. While MLKL KO in the Mvt-1 cells had little effect on tumor growth, the WT Mvt-1-derived tumors were highly metastatic to the lung compared with MLKL-KO cells, suggesting the role of necroptosis in increasing lung metastatic potential of breast cancer cells [97]. In the context of AML, the murine bone marrow transplantation model demonstrated that deletion of RIPK3 or MLKL in myeloid cells in combination with FMS-like tyrosine kinase 3 (FLT3) mutation induces leukemogenesis, confirming the tumor suppressor role of the RIPK3/MLKL signaling [64]. Caspase-8-deficient colorectal cancer (CRC) model mouse displays high sensitivity to SMAC mimetics, which was reversed by RIPK3 deletion, suggesting that RIPK3 may be a critical determinant factor of SMAC mimetic therapy in CRC patients [59]. In the investigation of liver cancer lineage commitment, hepatocyte-specific MLKL knockout mice demonstrate that necroptosis microenvironment promotes transformed hepatocytes into intrahepatic cholangiocarcinoma (ICC), while apoptotic cell surrounded environment facilitates the hepatocytes to develop hepatocellular carcinoma (HCC) [98].

6. Necroptosis and cancer immunity

The activation of naïve cytotoxic CD8⁺ T cells is critical for immunity against most tumors. Necroptosis has been shown to activate specific types of immune cells and adaptive immunity. Necroptotic cells can provide both antigens and inflammatory stimuli for dendritic cells (DCs), which in turn activate CD8⁺ T cells through a process called antigen cross-priming [99,100] (Fig. 3). Activation of the NF-κB pathway can provide both antigen and immune stimulation, thereby supporting DC-mediated cross-priming of CD8⁺ T cells. Cross-priming of CD8⁺ T cells depends on the RIPK1 scaffold protein function and NF-κB-mediated transcription activity. It has been demonstrated that RIPK3, the key executor of necroptosis, can regulate cytokine expression to mediate the natural killer T (NKT) cell function, and genetic knockout of RIPK3 led to decreased immune responses to metastatic tumor cells by the NKT cells [101]. These findings strongly suggest that necroptosis might play a critical role in anti-tumor immunity by activating CD8⁺ T cells or NKT cells.

De novo necroptosis created an inflammatory environment mediating tumor susceptibility to immune checkpoint inhibitors [102]. In vivo, Poly(I:C)-induced, TLR3/RIPK3-dependent necroptosis supported immune effector-mediated tumor elimination [103]. In mouse tumor cells, necroptosis was mediated by P. aeruginosa, inducing long-lasting systemic anti-tumor immunity [104]. In human leukocyte antigen (HLA)-negative tumor cells, necroptosis was induced by FasL on the surface of Tag7 (PGRP-S)-activated lymphocytes with the involvement of lysosomes and mitochondria [105]. In adoptively transferred T cells, necroptosis was induced by blocking TCR restimulation, improving tumor control [106].

Tumor cell TLR3 could directly induce necroptosis by poly(I:C), independent of immune effector-mediated tumor shrinkage [107]. In the gut epithelium, the TSC complex subunit 1 (TSC1)/ mechanistic target of rapamycin (mTOR) pathway stood for a metabolic and innate immune checkpoint for intestinal dysfunction and inflammation, and mTOR hyperactivation triggered by western diet or Tsc1 ablation led to epithelium necroptosis [108]. In esophageal squamous cell carcinoma, necroptosis represented as an independent prognostic factor, and it correlated with tumor-infiltrating lymphocytes [109]. In colorectal cancer, oxaliplatin resistance was promoted in part by tumor-associated macrophages via methyltransferase 3, N6-Adenosine-Methyltransferase Complex Catalytic Subunit (METTL3)-mediated m(6)A of TNF receptor-associated factor 5 (TRAF5) and necroptosis [110]. Prostate cancer progression was promoted by SIRT3 and SIRT6 through inhibiting necroptosis-mediated innate immune response [111]. Poor treatment outcome of human papillomavirus positive cervical cancer was predicted by low necroptosis process through decreasing tumor-associated macrophages M1 polarization [112]. A favorable immune cell signature and programmed death-ligand 1 expression in cholangiocarcinoma was correlated with necroptosis [113].

7. Necroptosis and antitumor therapies

Recently, several anti-cancer agents relevant to necroptosis have been reported. Shikonin is widely recognized to play a part in the effect of necroptosis on tumors. Fe(III)-shikonin supramolecular nanomedicine played a role in combined therapy of tumors via ferroptosis and necroptosis [114]. In pancreatic cancer, necroptosis was induced by shikonin, regulating the expression of RIPK1/RIPK3 and synergizing the activity of gemcitabine [115]. Shikonin reduces the growth of docetaxel-resistant prostate cancer cells mainly through inducing necroptosis [116]. In bladder cancer, cisplatin resistance was largely overcome by shikonin, in part through inducing necroptosis [117]. Moreover, in estrogen receptor (ER) positive breast cancer, shikonin has been reported to induce necroptosis and apoptosis [118]. On the other hand, epidermal growth factor receptor (EGFR)-expressing cancer cells were sensitized by amino acid starvation culture condition to gefitinib-mediated cytotoxicity, inducing atypical necroptosis [119].

In multiple cancers, several agents exert an anti-cancer function. In human lung cancer cells, necroptosis was induced by 11-Methoxytabersonine with Autophagy via AMPK/mTOR and c-Jun N-terminal kinase (JNK) signaling pathways [120]. In non-small cell lung cancer, necroptosis was suppressed via RIPK3 promoter methylation, increasing cancer resistance to chemotherapy [121]. 2-Methoxy-6-Acetyl-7-Methyljuglone inhibited lung cancer by inducing necroptosis by targeting RIPK1 [122]. In human non-small cell lung cancer, necroptosis could be triggered by Deoxypodophyllotoxin [123]. In lung cancer cell line A549, cisplatin-triggered cell death was contributed by MLKL–phosphatidylinositol transfer protein alpha (PITPα) signaling-mediated necroptosis [124]. In gastric cancer cells, RIPK1-mediated necroptosis and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation were induced by Astaxanthin [125]. Furthermore, colorectal cancer growth and metastasis were suppressed by resibufogenin through necroptosis [126]. In addition, in colon cancer cells, necroptosis was induced by dimethyl fumarate [127]. In breast cancer cells, Alysicarpus vaginalis ethyl acetate fraction (AVEAF) exerted anticancer activities associated with ROS-mediated mitochondrial-mediated intrinsic pathway of apoptosis and necroptosis [128]. Bcl-2-associated death promoter (BAD) sensitized breast cancer cells to docetaxel with increased mitotic arrest and necroptosis [129].

Cisplatin-induced necroptosis was also found to be mediated by RIPK1, which could govern proliferation through G2/M checkpoint progression [130]. In androgen‑dependent prostate cancer cells, RIPK1‑dependent necroptosis was induced by Ophiopogonin D’ (OPD’) [131]. Moreover, tumor cells could be sensitized for TRAIL-induced necroptosis by a clinically approved anti-leukemia drug, homoharringtonine [132].

In addition to drug therapy, other tumor therapeutic methods can also be applied to exert necroptosis. Through the RIPK1/RIPK3/MLKL/JNK/interleukin-8 (IL-8) pathway, tumor repopulation after radiotherapy was regulated by necroptosis. Necroptosis was triggered by ROS accumulation and Ca²⁺ overload, partly through RIPK1/RIPK3/MLKL/JNK/IL-8 pathway. Tumor repopulation after radiotherapy was regulated by necroptosis [133]. Necroptosis triggered by ROS accumulation and Ca²⁺ overload partly explained the inflammatory responses and anti-cancer effects associated with 1 Hz, 100 mT extremely low frequency-magnetic fields (ELF-MF) [134]. A potent anti-tumor effect was induced by stereotactic body radiation combined with oncolytic vaccinia virus, triggering tumor cell necroptosis and DAMPs [135]. Photothermal/photodynamic therapy of ovarian cancer was guided by CuS-MnS(2) nano-flowers for magnetic resonance imaging via necroptosis [136]. Non-small cell lung cancer cell killing was enhanced by ablative hypofractionated radiation therapy through preferential stimulation of necroptosis [137]. In keeping with these results, response of human colon cancer cell lines to supra-normal temperatures was associated with autophagy, apoptosis, and necroptosis [138]. In ovarian cancer, ceramide nanoliposomes were found to be necroptosis-inducing and MLKL-dependent chemotherapeutic reagents [139]. In breast cancer cells, apoptosis resistance was overcome by carbon nanodots for on-demand chemophotothermal therapy combination to elicit necroptosis [140]. Immunogenic necroptosis played a role in the anti-tumor photodynamic action of BAM-SiPc, a silicon(IV) phthalocyanine-based photosensitizer [141].

8. Concluding remarks

Accumulating evidence suggests that certain cancer cells can undergo necroptosis under some physical or chemical stimuli, and such triggering necroptosis could be a promising alternative strategy for killing cancer cells that fail to die by apoptosis. Moreover, important biological issues remain as follows: (i) Most studies on cancer and necroptosis were using cell-based assays in vitro, and thus more in vivo studies need to be performed to check the efficacy of necroptosis on killing cancer cells. (ii) Dissecting the functional role of necroptosis and underlying mechanism in tumor development and cancer therapy will be important for taking advantage of necroptosis-based anti-cancer therapies. (iii) More physiological and pathological systems need to be defined to study necroptosis. So far, in the necroptosis field, most of the studies were relatively artificial as caspase inhibition, TNFα treatment, or gene knockouts are needed to induce necroptosis. (iv) Cancer immunotherapy has been proved successful. It is fascinating to further investigate if induction of necroptosis can increase the efficacy of cancer immunotherapies. It will be crucial to know the specific roles regarding which DAMPs and other signaling molecules are released during necroptosis on the immune response and cancer immune surveillance and tumor promotion. In summary, RIPK1/RIPK3/MLKL-dependent necroptosis as a new form of cell death would be further explored to develop new anti-cancer therapies to overcome the resistance to proapoptotic chemotherapeutic agents.

Data Availability

No data was used for the research described in the article.