Necroptosis in cancer: An angel or a demon?

Abstract

In the past few decades, apoptosis has been regarded as the only form of programmed cell death. However, the traditional view has been challenged by the identification of several forms of regulated necrosis, including necroptosis. Necroptosis is typified by a necrotic cell death morphology and is controlled by RIP1, RIP3, and mixed lineage kinase domain–like protein. The physiological role of necroptosis is to serve as a “fail-safe” form of cell death for cells that fail to undergo apoptosis during embryonic development and disease defense. Currently, established studies have indicated that necroptosis is involved in cancer initiation and progression. Although elevated necroptosis contributes to cancer cell death, extensive cell death also increases the risk of proliferation and metastasis of the surviving cells by inducing the generation reactive oxygen species, activation of inflammation, and suppression of the immune response. Thus, questions regarding the overall impact of necroptosis on cancer remain open. In this review, we introduce the basic knowledge regarding necroptosis, summarize its dual effects on cancer progression, and analyze its advantages and disadvantages in clinical applications.

Introduction

Cell death is a fundamental pathological process that is involved in multiple diseases, including inflammatory ailments and cancer. Cell death processes have traditionally been divided into the following two types: necrosis and apoptosis. Necrosis was previously viewed as an accidental form of cell death and regarded as an unregulated passive process, while apoptosis was viewed as cell suicide and regarded as the only form of programmed cell death.^1–3 However, this view has been altered by recent studies that have identified several forms of regulated necrosis,⁴ including phosphoribosyl pyrophosphate (PPRP)-1-mediated necrotic death,⁵ p53-mitochondrial function–mediated necrosis,⁶ pyroptosis,⁷ ferroptosis,⁸ and necroptosis.⁹ Thus, caspase-dependent apoptosis is not the only form of programmed cell death.

Necroptosis has recently gained increasing attention as a novel but well-studied form of programmed cell death. Degterev et al.,⁹ who originally coined the term “necroptosis,” showed that necroptosis is characterized by a necrotic cell death morphology and by activation of autophagy. This form of programmed cell death can occur when the cells suffer from severe stress or are treated with chemotherapy or inflammatory factors.^10–13 Cancer is a malignant disease that is associated with aberrant cell death regulation. Recent studies have shown that necroptosis plays complex roles that are both positive and negative in the development of cancer. In this review, we summarize the current studies surrounding necroptosis and its roles in cancer progression and therapy.

Necroptosis: a novel form of programmed cell death

The discovery of necroptosis

Several investigations provided evidence for necroptosis long before the term was proposed. The first discovery of necroptosis can be tracked to the 1988 observation that tumor necrosis factor (TNF) triggers necrotic death in multiple cell types.¹⁴ For a long time, it was believed that this type of TNF-triggered cell death was apoptosis. In 1998, Vercammen et al.¹⁵ reported that TNF induced the rapid death of murine L929 fibrosarcoma cells in a necrotic manner when cellular caspases were repressed by inhibitors. Around the same time, Kawahara et al.¹⁶ observed that Fas-associated protein with death domain (FADD)-mediated cell death was independent of caspase-8 activation. These studies suggested that the apoptosis-related molecules regulated a novel form of cell death that was different from apoptosis and more similar to necrosis. This novel finding motivated investigators to explore the potential mechanism behind it. A landmark study of necroptosis was published in 2000 when RIP1 kinase was identified as the critical regulator of necrotic death in caspase-inhibited cells.¹⁷ In 2005, Degterev et al.⁹ called this Fas/TNF receptor (TNFR) receptor family-triggered, nonapoptotic form of cell death “necroptosis” and invented a small molecule, necrostain-1, that blocked necroptosis. A subsequent study proved that necrostain-1 inhibited RIP1 kinase.¹⁸ In 2009, three independent studies proved that RIP3 was required for necroptosis and was the downstream target of RIP1.^19–21 In 2012, mixed lineage kinase domain–like protein (MLKL) was identified as a necroptosis executor, which functioned after interacting with and being phosphorylated by RIP3 at its threonine 357 and serine 358 residues.²² The discovery of MLKL enabled a rough mapping of the necroptosis regulatory pathway.

The molecular mechanism of necroptosis

The TNF family of cytokines, including TNF-α, FASL, and TRAIL, are well-studied stimuli that trigger necroptosis.²³ We will focus on TNF-α here. The TNF-α combination with TNFR1 activates TNFR1 and induces the receptor trimerization. The activated receptors then interact with RIP1 through TNFR1-associated death domain protein (TRADD), and they recruit cIAP1/2 and TNFR-associated factor 2 (TRAF2) to form a complex at the plasma membrane. In this complex, the cIAP1/2 E3 ubiquitin ligases induce ubiquitination of RIP1, while TRAF2 mediates the ubiquitination of RIP1.²⁴ Ubiquitination of RIP1 generates binding sites for TAB2/3 (the adaptor protein that mediates the activation of transforming growth factor beta–activated kinase 1 (TAK1)) and NEMO (the regulatory subunit of the IκB kinase (IKK) complex), which leads to further recruitment and activation of TAK1 and IKKα/β. TAK1 activates the IKK complex by phosphorylating it, which results in the phosphorylation and subsequent degradation of IκBα. Nuclear factor kappa B (NF-κB) localizes to the nucleus due to IκBα degradation and promotes cell survival by regulating the expression of multiple genes.^24,25 In addition, the IKKα/IKKβ complex protects cells from RIPK1-dependent death by phosphorylating RIPK1; this is independent of its role in NF-κB activation.²⁶ However, when cIAPs are degraded or antagonized, RIP1 ubiquitination is blocked, and deubiquitination of RIP1 is induced by two deubiquitinases, cylindromatosis (CYLD) and A20,^27,28 which results in the dissociation of RIP1 from the plasma membrane and a functional conversion of RIP1 from promoting survival to promoting cell death by apoptosis or necroptosis.^29,30 Cytoplasmic RIP1 binds to FADD and recruits procaspase-8 to form complex II in the cytoplasm. Complex II might already form in cells that do not undergo cell death, but the cells survive because complex II harbors the caspase-8 inhibitor.³¹ Once caspase-8 is activated in this complex, it induces apoptosis through the caspase pathway; the activated caspase-8 simultaneously cleaves RIP1 and blocks the functions of this kinase.^32,33 However, once caspase-8 activity is inhibited by specific conditions, the RIP1-mediated death model will transition from apoptosis to necroptosis. During this process, an amyloid-like signaling complex, known as the necrosome, is formed. In the necrosome, RIP1 functions as the kinase that interacts with the RIP3 downstream molecular regulator through their common homotypic interaction motifs (RHIM) to promote phosphorylation of RIP3 at serine 227.^19,34 The MLKL pseudo kinase is the downstream executioner of RIP3-regulated necroptosis.²² Generally, MLKL exists in the cytoplasm as a monomer. The activated RIP3 binds to MLKL through its C-terminal kinase domain and induces MLKL phosphorylation at threonine 357 and serine 358.²² Phosphorylation leads to the oligomerization of MLKL and membrane translocation of the MLKL oligomers.^35,36 MLKL oligomers subsequently disrupt membrane integrity and increase membrane permeabilization through nonspecific pore formation or by interacting with the TRPM7 calcium channel protein to promote calcium and sodium influx,^37,38 and cell death is induced. The activated MLKL also translocates to the intracellular membrane and increases organelle permeabilization.³⁵ Recently, Xia et al.³⁹ reported that MLKL forms transmembrane cation channels, which are permeable to Mg²⁺, Na⁺, and K⁺ but not to Ca²⁺. This triggers MLKL-induced membrane depolarization and cell death. In addition to the TNF family, other cytokines, including interferon (IFN), can trigger necroptosis through the RIP1–RIP3 complex.⁴⁰

Within this complex regulatory network, the ubiquitination status of RIP1 is regarded as a deciding factor toward its role in promoting cell death versus cell survival.⁴¹ CYLD and A20, two important RIP1 deubiquitinases, have been well recognized for promoting cell death by inducing the release of RIP1 from the cell membrane, which limits NF-κB activation. However, a recent study found that CYLD and A20 exert opposing effects on the stability of the LUBAC-generated linear ubiquitin (M1) in the TNFR1-associated signaling complex.⁴² CYLD cleaves M1 chains and promotes cell death, whereas A20 protects M1 chains from cleavage and consequently inhibits cell death. Thus, the interplay between LUBAC, linear ubiquitination, A20, and CYLD is important for regulating cell death. In addition, RIP1 is not essential for necroptosis, though it plays an important regulatory role during this process. Several stimuli can trigger necroptosis by directly activating RIP3. Lipopolysaccharides (LPS) or double-stranded RNAs (dsRNA) can interact with Toll-like receptors 3 and 4 (TLR3/4) to directly activate RIP3 through TRIF (TIR-domain-containing adapter-inducing interferon-β), a RHIM-containing protein.^43,44 During virus-induced necroptosis, a DNA-dependent activator of interferon-regulatory factors (DAI) interacts with RIP3 through its RHIM, which mimics the RIP1/3 complex and results in cell death.⁴⁵ Chemically enforced RIP3 oligomerization can also cause necroptosis to bypass RIP1 and occur in a RHIM domain-independent manner.⁴⁶ Multiple stimuli can trigger necroptosis through RIP1-dependent and/or -independent mechanisms (Figure 1), but to date, RIP3 and MLKL are regarded as irreplaceable downstream regulators of necroptosis.

Figure 1.

The mechanism of necroptosis. TNF, FASL, and TRAIL bind to their corresponding receptors, which results in receptor trimerization. The activated receptors then interact with RIP1 and induce RIP1 dissociation from the plasma membrane. RIP1 functions as a kinase that interacts with RIP3 and promotes RIP3 phosphorylation, which then binds to MLKL and induces MLKL phosphorylation. Phosphorylation promotes MLKL oligomerization and membrane translocation of the MLKL oligomers. The MLKL oligomers disrupt the membrane integrity, increase membrane permeabilization through nonspecific pore formation or through interactions with calcium channels to promote calcium and sodium influx, and induce cell death. IFN, LPS, viral infections, and several anti-cancer drugs can trigger necroptosis in RIP1-independent models.

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Physiological function

As with apoptosis, the goal of necroptosis is to eliminate unnecessary or abnormal cells from the body. This is important for embryonic development and disease defenses. Caspase-8 is a key determinant of apoptosis and necroptosis. The protease activity of caspase-8 is required to activate apoptosis and suppress necroptosis.³³ Necroptosis can be stimulated when cells are treated with zVAD, a pan-caspase inhibitor,⁴⁷ because an inactivated caspase-8 cannot cleave the two key regulators of necroptosis, RIP1 and RIP3.^32,33,48 However, the zVAD treatment also drives autocrine production of TNF, which is an important necroptosis trigger.⁴⁹ Moreover, Chen et al.⁵⁰ indicated that necroptosis eliminates cells that fail to die through apoptosis. These studies imply that necroptosis serves as a “fail-safe” form of cell death for cells that fail to undergo apoptosis. In this way, necroptosis functions as a supplemental death model for apoptosis.

Pathological characteristics and necroptosis detection

The morphological features of necroptosis are nearly identical to those of necrosis.⁵¹ A cell that undergoes necroptosis loses its plasma membrane integrity, which results in increased permeabilization. Thus, the cell exhibits cytoplasmic swelling and vacuolization. The organelles in the plasma, including the mitochondria, also swell. Furthermore, the permeabilized plasma membrane and subsequent leakage of the intracellular contents cause necroptosis to trigger immune and inflammatory responses in neighboring tissues.^52–54 Unlike apoptosis, nuclear condensation and fragmentation are absent during necroptosis. In addition, the necroptotic process consumes less energy when adenosine triphosphate (ATP) is depleted.

Currently, the primary method for confirming necroptosis involves detection of the key molecules that control necroptosis (i.e., RIP1, RIP3, and MLKL). First, increases in RIP1, RIP3, and MLKL at the messenger RNA (mRNA) and protein levels are potential indicators of increased necroptosis.⁵⁵ Second, the phosphorylated RIP1, RIP3, and MLKL proteins are in their activated forms during necroptosis. Thus, determining the activation states of these molecules by assessing their phosphorylation states by immunoblotting or immunostaining is one way to demonstrate necroptosis.^35,56 Finally, necroptosis can be detected by interfering with the expression or function of RIP1, RIP3, and MLKL by small interfering RNA (siRNA) knockdown or with small-molecule inhibitors.^22,44,57,58 Recently, Wallberg et al.⁵⁹ established an effective method to detect necroptosis by fluorescence-activated cell sorting (FACS). The following three dyes were used to label the cells: 4′,6-diamidino-2-phenylindole (DAPI) to stain dead cells; Annexin V to stain apoptotic cells; tetramethylrhodamine methyl ester (TMRM), a cell-permeable fluorescent dye that is sequestered by active mitochondria, to label live cells. Thus, the combination of Annexin V/DAPI/TMRM can label the entire cell population and distinguish living cells (TMRM+/Annexin V^FITC−/DAPI−) from dying or dead cells (apoptosis: TMRM−/Annexin V^FITC+/DAPI−; necrosis: TMRM−/Annexin V^FITC+/DAPI+).

The role of necroptosis in cancer: a double-edged sword

Cancer is a disease that is closely related to cell death, and resistance to cell death is a hallmark of cancer. As one of multiple forms of cell death, necroptosis has been recognized for its critical roles in defending against cancer. Numerous studies have suggested that necroptosis suppresses the initiation and progression of cancer and facilitates its therapy. The anti-tumor effects of necroptosis have been recently reviewed.⁵⁰ However, emerging data have revealed that necroptosis can promote cancer progression, which suggests that necroptosis is a double-edged sword in cancer progression.

Necroptotic defense against cancer

Like other forms of programmed cell death, necroptosis plays important roles in maintaining homeostasis and toward preventing diseases, particularly tumor formation. Accumulating data suggest that necroptosis functions against cancer by inhibiting its initiation, growth, and metastasis. In addition, necroptosis is a mechanism by which chemotherapeutic compounds can eliminate cancer cells.

First, dysregulation of necroptosis contributes to tumor initiation and growth. Numerous studies have shown that key necroptosis regulators are dysregulated due to genetic or epigenetic alterations during tumor progression; this results in tumor cell survival. For example, RIP3 is significantly reduced in most cases of acute myeloid leukemia (AML), which results in decreases in apoptosis/necroptosis and the promotion of NF-κB-mediated survival.⁶⁰ Likewise, another study shows that the genetic loss of RIP3 promotes the development of AML by enhancing the accumulation of leukemia-initiating cells in mice, and the link between RIP3 suppression and the blockage of cell death has been validated in primary AML patient cohorts.⁶¹ RIP1 and RIP3 are also significantly decreased in colon cancer tissues compared to adjacent normal colon tissues, and their decreases impair the cancer cells response to necroptosis triggers.⁶² Similarly, downregulation of RIP3 and CYLD causes chronic lymphocytic leukemia (CLL) cells to become resistant to necroptosis that is stimulated by TNF-α in combination with zVAD.⁶³ In addition, RIP3 is reportedly reduced in breast cancer tissues due to genomic methylation, which promotes tumor growth and development.⁶⁴ MiR-155-5p overexpression inhibits necroptosis by targeting RIP1, which results in cell proliferation.⁶⁵ In addition, downregulation of the key executor of necroptosis, MLKL, in pancreatic adenocarcinomas and ovarian cancers is associated with decreased patient survival.^66,67 Other necroptosis-related molecules are dysregulated in cancer, including the IAPs, which function as inhibitors of necroptosis.⁶⁸

Second, in addition to inhibiting cancer cell survival and proliferation, necroptosis prevents metastasis. Fu et al.⁶⁹ found that shikonin inhibits cancer cell metastasis by inducing RIP1/3 expression and promoting necroptosis. RIP1/3 may promote anti-metastatic outcomes by regulating oxidative stress to kill metastatic tumor cells;⁷⁰ RIP3 promotes reactive oxygen species (ROS) production, and high ROS levels inhibit cancer cell metastasis.^19,71 Third, necroptosis inhibits tumor progression by triggering the activation of immunity against tumor. For example, PolyIC, a viral dsRNA analog, induces colon cancer cell necroptosis and promotes tumor elimination by activating dendritic cell (DC)-induced anti-tumor immunity.⁷² Schmidt et al.⁷³ demonstrated that interleukin-1α (IL-1α), which is released from PolyIC-induced necroptotic cancer cells, activates DCs, and the DCs then function as anti-tumor agents by producing cytotoxic cytokine IL-12 or by activating CD8+T cells.⁷⁴ In addition, RIP3 is also involved in natural killer (NK) T cell-mediated immune responses. A crucial role for NK T cell-mediated activation of the RIP3–PGAM5–NFAT/Drp1 signaling pathway has been demonstrated, and the responses of NK T cells to metastatic tumor cells are reduced in RIP3(−/−) mice.⁷⁵

Finally, several therapeutic reagents eliminate cancer cells using the necroptosis mechanism. Theoretically, DNA-damaging agents should be ineffective toward inducing cell apoptosis due to the p53 losses or mutations that are typical of most cancers. However, DNA-damaging compounds are effective in cancer chemotherapy. Several DNA-damaging agent–based chemotherapeutic drugs have recently been shown to kill cancer cells by inducing necroptosis through the RIP1/RIP3/MLKL pathway.^50,76,77 For example, Taxol, etoposide, and 5-fluorouracil (5-FU) induce necroptosis by activating RIP1 in multiple cancer cell types,^78–80 whereas shikonin induces necroptosis in cancer cells through ROS production and RIP1/RIP3 activation.¹¹ In addition to DNA-damaging agents, various compounds have also been recently proven to promote necroptosis in specific tumor cells. For example, TRAIL induces necroptosis in cancer cells by suppressing caspases^13,20 and by activating RIP1/RIP3.⁸¹ Several kinase inhibitors, including staurosporine and sorafenib, also induce RIP1/MLKL-dependent necroptosis.^73,82 In addition, RIP3 silencing causes chemoresistance to 5-FU, cisplatin, camptothecin, and etoposide in colon and breast cancer cells,⁶⁴ whereas restoring the key regulators of necroptosis improves the cancer cell sensitivity to chemotherapeutics.⁸³ These data suggest that necroptosis plays positive roles in cancer therapy.

Promotion of cancer progression by necroptosis

It is easy to understand and accept the viewpoint that necroptosis provides a defense against cancer. However, emerging research suggests that cell death promotes cancer progression, and several recent studies have shown that necroptosis is also involved in the progression of specific, though not all, cancer types.

Emerging research on the promotion of cancer progression by necroptosis

First, several clinical studies have suggested that increased cell death is associated with a high degree of malignancy and low survival rate. For example, a high apoptotic index (AI), which is usually defined as the number of apoptotic cells per square millimeter of tumor tissue, is correlated with malignant cellular features (i.e., high histological grade, high nuclear grade, and comedo-type necrosis). It indicates cell proliferation (high mitotic index and Ki67 immuno-labeling) and invasiveness and is linked to unfavorable disease outcomes in breast cancer.^84,85 In colon cancer, a high AI is also associated with malignant cellular features.⁸⁵ Conversely, increase in Bcl-2, an apoptosis inhibiting factor, is associated with a good cancer prognosis. For example, Sinicrope et al.⁸⁵ found that Bcl-2 expression is the most important predictor of overall survival, and low or absent Bcl-2 expression is a significant prognostic indicator of a poor survival outcome in node-negative colon cancer. Similarly, Poincloux et al.⁸⁶ also found that although the extent of Bcl-2 expression in tumor cells is negatively associated with slower local tumor growth, the loss of Bcl-2 expression is correlated with an increased rate of relapse in stage II colon cancers. Researchers have also demonstrated that Bcl-2 expression is a predictive marker for favorable prognoses in breast cancer.^87–89 Callagy et al. have shown that breast cancer patients with high Bcl-2 expression levels have better survival rates, particularly within the first 5 years of the patient’s diagnosis. Moreover, they have revealed Bcl-2 as an independent predictor of breast cancer outcomes.⁸⁷ From a five-study analysis of 11,212 women with early-stage breast cancers, Dawson et al.⁸⁸ also concluded that Bcl-2 is a favorable prognostic marker in breast cancer. In addition, an assessment of 103 breast cancer cases by Zhang et al. showed that the clinical pathologic features of centrally necrotizing carcinomas are mostly characterized as basal-like. The centrally necrotizing carcinomas have more aggressive biological patterns, including high proliferative activities and high rates of recurrence and metastasis, which suggests that extensive cell death results in high fecundity and stimulates the proliferation and metastasis of surviving cells.^90,91

Second, several researchers have indicated that cell death from therapy leads to a poorer cancer prognosis. For example, antagonists of IAP, an inhibitor of apoptosis, are used as anti-cancer agents that induce apoptosis in clinical treatments. However, Yang et al.⁹² recently found that IAP antagonists increase tumor growth and metastasis in the bone by activating NF-κB-inducing kinase (NIK) with a subsequent enhancement of alternative NF-κB signaling in osteoclasts; this causes osteolysis and provides a favorable microenvironment for cancer growth and metastasis. In addition, anti-angiogenesis reagents kill cancer cells by reducing the blood supply. However, Ebos et al.⁹³ have reported that several vascular endothelial growth factor receptor (VEGFR)/platelet-derived growth factor receptor (PDGFR) kinase inhibitors could accelerate metastatic tumor growth and decrease overall survival in mice that received these therapeutic reagents, though the anti-tumor benefits of this reagent class were observed in orthotopically transplanted tumors. Interestingly, tanshinone IIA, a vasoactive reagent, has been used to evaluate the effects of inducing vascular normalization on cancer growth and metastasis in an animal model that had undergone palliative resections of hepatocellular carcinomas. The authors demonstrate that tanshinone IIA improves blood circulation and inhibits cancer metastasis in vivo by upregulating VEGFR1/PDGFR, though it did not eliminate the original tumor.⁹⁴ Another recent study shows that increased tumor angiogenesis and co-administration of low-dose Cilengitide also extends survival in murine and human cancer models in vivo.⁹⁵

Finally, emerging research supports the idea that necroptosis promotes cancer progression. For example, Liu et al. reported that knockouts of RIP1, RIP3, or MLKL, the key regulators of necroptosis, in cancer cells significantly attenuate their proliferation in vitro and reduce their ability to form tumors in vivo. Moreover, in a xenograft model, tumor growth is significantly delayed after treatments with necrosulfonamide, a chemical inhibitor of necroptosis.⁹⁶ Furthermore, the same study showed that higher expression of phosphorylated MLKL is a poor prognostic marker of human esophageal and colon cancers.⁹⁶ Recently, Seifert et al.⁹⁷ reported that necroptosis promotes pancreatic carcinogenesis. They found that RIP1 and RIP3 are highly expressed in pancreatic ductal adenocarcinomas and that inhibition of RIP1 or deletion of RIP3 prevents pancreatic carcinoma progression in vivo.⁹⁷ In addition, Strilic and colleagues found that tumor cell–induced endothelial cell necroptosis promotes cancer metastasis.⁹⁸ Tumor cell extravasation is a critical step for cancer metastasis, and this process is believed to resemble leukocyte transendothelial migration. However, the interaction patterns of tumor and endothelial cells and the mechanisms that regulate the interaction process are unclear. By treating endothelial cells with a chemical inhibitor of RIP1 (necrostatin-1) or by specifically deleting RIP3, Strilic et al. found that endothelial cell necroptosis is reduced with a concomitant decrease in tumor cell extravasation and metastasis. However, the loss of caspase-8 in the endothelial cells promotes these processes. These results suggest that tumor cells induce endothelial cell necroptosis, which contributes to cancer cell metastasis.⁹⁸ Another study demonstrates that this process is mediated by the interaction between amyloid precursor proteins on tumor cells and death receptor 6 on endothelial cells.⁹⁸ These results suggest that necroptosis can promote tumor growth and that necroptosis regulatory genes may be promising targets for cancer therapies. In summary, cell death (by necroptosis) can increase the risk of cancer progression.

The possible mechanisms behind the promotion of cancer progression by necroptosis

Although the mechanisms that underlie the promotion of cancer progression by necroptosis are not well known, it is believed that the major mechanisms by which necroptosis promotes the proliferation and metastasis of living cancer cells involve the induction of inflammation and ROS and/or the suppression of tumor immunity.

First, necroptosis may promote tumor progression by activating inflammation. The increased permeability of the cellular membrane results in a release of damage-associated molecular patterns (DAMPs) from necroptotic cells (i.e., the high-mobility group box 1 (HMGB1) protein) that trigger inflammation.^52,53 Tumor-associated chronic inflammation may stimulate tumor proliferation and invasion by activating inflammatory cells to produce multiple growth factors.^99–102 For example, it has been extensively observed that the macrophage density in cancer tissues is correlated with a poor prognosis, and tumor associated macrophages are reportedly required for the migration and invasion of multiple cancer cell types in response to highly expressed colony stimulating factor 1 (CSF-1), IL-3, epidermal growth factor (EGF), and CCL-2.^103,104 In addition to the inflammation that is induced by dead cells, the key regulators of necroptosis promote cell death–independent inflammation.¹⁰⁵ Najjar and colleagues have reported that the activation of RIP1 and RIP3 in LPS-stimulated macrophages promotes sustained activation of cFos, Erk, and NF-κB, which are required for the expression of inflammatory cytokines. This process requires the TRIF adaptor molecule, but it is independent of the cell death functions of RIP1 and RIP3.¹⁰⁵ This study suggests that crosstalk between necroptosis and inflammation is complex. Recently, the association between necrosome components or necroptosis and inflammation has been extensively reviewed elsewhere.^106,107

Second, necroptosis may promote tumor progression by inducing ROS production. It is well known that elevated ROS is associated with genomic instability,^108,109 whereas genomic instability plays an important role in malignant transformation and cancer progression through the accumulation of multiple mutations.^110,111 Numerous studies have indicated that ROS induces normal cell transformation into tumor cells and promotes cell growth and invasion of the transformed cells.¹¹² For example, hexavalent chromium, a well-known human carcinogen, promotes the malignant transformation of human bronchial epithelial cells by inducing ROS generation, whereas inhibiting ROS overproduction suppresses malignant transformation and tumor formation.¹¹³ Both endogenous and exogenous ROS promote cell proliferation in multiple cancers by activating the PI3K/protein kinase B (PKB) and c-jun n-terminal kinase (JNK) signaling pathways.^114–116 H₂O₂ promotes colorectal cancer cell invasion by upregulating matrix metalloproteinase (MMP-7) via JNK/C-Jun and extracellular signal–regulated kinase (ERK)/C-Fos activation in an activator protein 1 (AP-1)-dependent manner.¹¹⁷ Cell death–induced inflammation produces ROS. For example, during chronic inflammation, activated macrophages generate and release ROS¹¹⁸ and secrete cytokines, including TNF; TNF stimulates ROS production through a Rac-cytosolic phospholipase A2-leukotriene B4 cascade.¹¹⁹ In addition, necroptosis itself can directly induce ROS production. For example, Zhang and colleagues have reported that RIP3 enhances the production of ROS by increasing energy metabolism in NIH 3T3 cells.²¹ Recently, Yu et al.¹²⁰ confirmed that RIP3 promotes ROS production in cancer cells, which is one mechanism by which neoalbaconol (NA), an isolate of Albatrellus confluens, induces necroptosis in cancer cells. Several recent studies have observed that necroptosis accompanies increased ROS production in multiple cancer cell types.^121–123

Finally, necroptosis may promote cancer progression by inhibiting tumor immunity. Recently, Seifert and colleagues have reported that necroptosis promotes pancreatic cancer progression by repressing tumor immunity. They show that necroptosis in pancreatic cancer tissues induces the expression of CXCL1, a chemokine attractant, that blocks the infiltration of immune cells into tumor tissues, which subsequently promotes pancreatic cancer progression.⁹⁷

Taken together, the net effect of necroptosis on cancer progression remains unclear. On the one hand, necroptosis inhibits tumor initiation and progression by killing the tumor cells; on the other hand, the dead cells release numerous materials or induce inflammatory reactions that contribute to cell growth and metastasis.^124–126 Thus, applications of novel necroptosis-based strategies for cancer treatments should be cautiously approached.

Perspective

The basic characteristics, biological functions, and molecular mechanisms of necroptosis have been reviewed here. As a novel form of programmed cell death, necroptosis has a strong potential for applications in cancer prognoses and treatments. However, multiple questions remain that need further explorations. First, specific markers for the detection of necroptosis are nonexistent; a determination of necroptosis currently depends on the combined use of multiple methods. Necroptosis is relatively complex. Moreover, several in vivo methods have limitations, including FACS. Thus, a search for specific necroptosis markers will significantly improve the necroptosis detection efficiency and overcome the limitations of in vivo necroptosis detection methods. Second, current studies have shown that RIP3 and MLKL are the central molecules in necroptosis regulation. However, it is unknown whether there is a crosstalk between the RIP–MLKL pathway and other signaling pathways and whether there are other molecules that control necroptosis in addition to RIP and MLKL. Exploring these questions will improve the understanding of the necroptosis mechanism. Third, suppression of apoptosis can promote necroptosis. However, little is known about the association between necroptosis and other forms of programmed cell death. Elucidating the different conditions that favor the different forms of programmed cell death will be important for understanding tumor development and for choosing reasonable therapeutic approaches to induce cell death. Lastly, the role of necroptosis in tumor progression remains unclear. Although multiple studies support the anti-tumor functions of necroptosis, increasing evidence suggests that necroptosis also promotes cancer progression. However, mechanistic studies of the promotion of cancer progression by necroptosis are in their infancy. Moreover, it is unclear whether there is a regulatory mechanism that controls a functional shift by necroptosis within the different stages of cancer progression. Therefore, additional mechanistic studies of the role of necroptosis in regulating cancer progression are expected.

In conclusion, necroptosis is a novel form of programmed cell death, and emerging studies suggest that necroptosis plays crucial functions in tumor formation and progression. However, it is too early to conclude whether necroptosis is a friend or foe to human cancer.

X.L., Y.H., and X.L. designed and outlined the work. T.W., Y.J., W.Y., L.Z., and X.J. consulted the literature and completed the manuscript. T.W., Y.J., and W.Y. contributed equally to this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the National Natural Science Foundation of China (Grant No. 81302061 to T.W. and Grant Nos 81401961 and 81641101 to X.L.), Postdoctoral Scientific Research Development Fund of Heilongjiang Province (Grant No. LBH-Q14104 to X.L. and Grant No. LBH-Q15082 to T.W.), Natural Science Foundation of Heilongjiang Province (Grant No. H2016006 to X.L.), and Wu-Lian-De Youth Science Foundation of Harbin Medical University (Grant No. WLD-QN1411 to X.L.).