Complex roles of necroptosis in cancer

Complex roles of necroptosis in cancer 程序性坏死在癌症中的复杂作用 1 2 3 4 first-author 1 2 3 4 1 2 3 4 1 2 3 4 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:hesudan2018@163.com †E-mail:hesudan2018@163.com hesudan2018@163.com Cyrus Tang Hematology Center and Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou 215123, China Cyrus Tang Hematology Center and Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou 215123, China Key Laboratory of Stem Cells and Biomedical Materials of Jiangsu Province and Chinese Ministry of Science and Technology, Soochow University, Suzhou 215123, China Key Laboratory of Stem Cells and Biomedical Materials of Jiangsu Province and Chinese Ministry of Science and Technology, Soochow University, Suzhou 215123, China Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medial College, Beijing 100005, China Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medial College, Beijing 100005, China Suzhou Institute of Systems Medicine, Suzhou 215123, China Suzhou Institute of Systems Medicine, Suzhou 215123, China Necroptosis is a tightly regulated form of necrosis that requires the activation of receptor-interacting protein (RIP) kinases RIPK1 and RIPK3, as well as the RIPK3 substrate mixed lineage kinase domain-like protein (MLKL). Because of membrane rupture, necroptotic cells release damage-associated molecular patterns (DAMPs) that evoke immune responses. Necroptosis is emerging as an important cellular response in the modulation of cancer initiation, progression, and metastasis. Necroptosis of cancer cells is considered to be an immunogenic cell death capable of activating anti-tumor immunity. Necroptosis also participates in the promotion of myeloid cell-induced adaptive immune suppression and thus contributes to oncogenesis. In addition, necroptosis of endothelial cells and tumor cells is conducive to tumor metastasis. In this review, we summarize the current knowledge of the complex role of necroptosis in cancer and discuss the potential of targeting necroptosis components for cancer therapies. 细胞程序性坏死是一种受到严格调控的细胞坏死形式,其发生依赖于受体相互作用蛋白(RIP)激酶RIPK1和RIPK3以及RIPK3底物混合谱系激酶域样蛋白(MLKL)的活化.由于细胞膜破裂,发生程序性坏死的细胞会释放损伤相关分子模式(DAMPs)分子,进而引发炎症反应.越来越多的研究表明细胞程序性坏死参与调控癌症的发生、发展和转移.肿瘤细胞程序性坏死被认为是一种具有激活抗肿瘤免疫功能的免疫原性细胞死亡,从而抑制肿瘤生长.细胞程序性坏死也被发现能增强髓系细胞诱导的适应性免疫抑制,进而促进肿瘤发生.此外,内皮细胞和肿瘤细胞的程序性坏死会促进肿瘤细胞的转移.在这篇综述中,我们总结了细胞程序性坏死信号通路及其在癌症中的复杂作用机制,并讨论了靶向程序性坏死调控蛋白在癌症治疗中的作用. Cell death Necroptosis Cancer Mixed lineage kinase domain-like protein (MLKL) Receptor-interacting protein (RIP) kinase 细胞死亡 细胞程序性坏死 癌症 混合谱系激酶域样蛋白(MLKL) 受体相互作用蛋白(RIP)激酶 Introduction 1 Cell death is an essential aspect of development and tissue homeostasis in multicellular organisms. Apoptosis is a well-defined form of programmed cell death characterized by cell shrinkage, DNA condensation, and the formation of apoptotic bodies that are surrounded by an intact plasma membrane (Kerr et al., 1972 1972 ). Apoptosis is mediated by a family of cystine proteases termed caspases; the activity of these caspases is initiated by apoptotic stimuli via the activation of the death receptor pathway (“extrinsic pathway”) or the mitochondrial pathway (“intrinsic pathway”). In contrast, necrosis has traditionally been viewed as an unregulated form of cell death characterized by morphological features including cell swelling and cell membrane rupture. More recently, however, a regulated form of necrosis known as necroptosis (also “programmed necrosis”) has been experimentally confirmed in detail. Necroptosis is controlled by receptor-interacting protein (RIP) kinases RIPK1, RIPK3, and mixed lineage kinase domain-like protein (MLKL) (Holler et al., 2000 2000 ; Cho et al., 2009 2009 ; He et al., 2009 2009 ; Zhang et al., 2009 2009 ; Sun et al., 2012 2012 ; Zhao et al., 2012 2012 ). Necroptosis was first considered as a backup form of cell death that can function when apoptosis is inhibited in response to the activation of death receptors (He and Wang, 2018 2018 ; Yuan et al., 2019 2019 ), but it is now well established that necroptosis can be initiated by various stimuli, including the ligands of death receptors, Toll-like receptors (TLRs), interferon (IFN) receptors, and certain pathogens (Fig. 1 1 ). Necroptosis can be caused by a variety of stimuli, including the ligands of the death receptor, TLRs, IFN receptors, and certain pathogens. TNF-α-activated TNFR1 forms Complex I by recruiting RIPK1, TRADD, cIAP1/2, and TRAF2. In Complex I, RIPK1 is ubiquitinated by cIAP1/2 and eventually activates NF-κB signaling pathway. In the presence of Smac mimetic, which leads to the degradation of cIAPs, RIPK1 is disassociated from Complex I and deubiquitinated by CYLD, followed by interacting with FADD and procaspase-8 to form a cytosolic protein complex (Complex II). This complex activates caspase-8 and eventually leads to apoptosis. When caspase-8 activity is inhibited, RIPK1 interacts with RIPK3 to form a protein complex (necrosome) via their respective RHIM domains, leading to activation of both RIPK1 and RIPK3. Activated RIPK3 further phosphorylates MLKL, which forms oligomerization and translocates to the plasma membrane for the execution of necroptosis. The necroptotic cells finally release cellular contents including DAMPs, which act as endogenous danger signals to elicit inflammatory responses. The RHIM-dependent interaction between RIPK3 and RIPK1 is also required for IFNα/β/γ-induced necroptosis. Furthermore, RIPK3 can be activated through interacting with other RHIM-containing proteins such as TRIF, DAI, and the viral protein ICP6. In TLR3 or TLR4-induced necroptosis, TRIF is recruited to RIPK3 through their RHIM domains. During infection of MCMV or influenza A virus, DAI activates RIPK3 to induce necroptosis. In the mouse cells infected with HSV-1/2, binding of ICP6/10 to mouse RIPK3 leads to the activation of RIPK3 and necroptosis. However, in the human natural host cells, ICP6/10 also can interact with human RIPK3, and this association results in the inactivation of RIPK3. Necroptosis can be inhibited by Nec-1 (RIPK1 inhibitor), GSK872 (RIPK3 inhibitor), and NSA (human MLKL inhibitor). Casp: caspase; cIAP1/2: cellular inhibitor of apoptosis 1/2; cFLIP: cellular Fas-associated DD-like interleukin 1β-converting enzyme-inhibitory protein; CYLD: cylindromatosis; DAI: DNA-dependent activator of interferon-regulatory factor; DAMPs: damage-associated molecular patterns; dsRNA: double-stranded RNA; FasL: Fas ligand; FADD: Fas-associating protein with a novel death domain; HSV: herpes simplex virus; IFN: interferon; IFNR: IFN receptor; ICP6: ribonucleotide reductase large subunit; LPS: lipopolysaccharide; MCMV: murine cytomegalovirus; MLKL: mixed lineage kinase domain-like protein; Nec-1: necrostatin-1; NEMO: NF-κB essential modifier; NF-κB: nuclear factor-κB; NSA: necrosulfonamide; RIPK: receptor-interacting protein (RIP) kinase; RHIM: RIP homotypic interaction motif; TLR: Toll-like receptor; TNF-α: tumor necrosis factor-α; TNFR1: TNF receptor 1; TRADD: TNFR1-associated death domain protein; TRAIL: TNF-related apoptosis-inducing ligand; TRAILR: TRAIL receptor; TRAF2: TNFR-associated factor 2; TRIF: Toll/interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β Cancer is a malignant disease associated with abnormal cell proliferation and cell death. It has been established that cancer cells can evade apoptotic responses. Therefore, induction of necroptosis could be an alternative strategy for killing cancer cells. However, increasing evidence is accumulating that many cancer cells dampen necroptosis via epigenetic silencing of RIPK3 (Koo et al., 2015 2015 ; Yang et al., 2017 2017 ). In contrast to apoptosis, necroptosis results in the release of damage-associated molecular patterns (DAMPs), which further activate immune responses. The inflammatory implications of necroptosis enhance the immunogenicity of dying cancer cells and thereby increase anti-tumor immunity (Yatim et al., 2015 2015 ; Aaes et al., 2016 2016 ; Yang et al., 2016 2016 ). In addition to its suppressive role for tumorigenesis, necroptosis has been implicated in oncogenesis and in cancer metastasis. Viewed collectively, it is thus clear that necroptosis functions in complex ways in cancer initiation and development. This review focuses on recent advances in the understanding of the necroptosis machinery and its role in cancer initiation, development, metastasis, and treatment. Discovery of necroptosis as a form of regulated necrosis 1 The tumor necrosis factor (TNF) superfamily of cytokines, including TNF-α, TNF-related apoptosis-inducing ligand (TRAIL), and FasL/CD95L, is classic ligands for the activation of death receptors. Ligation of a death receptor (e.g. TNF receptor 1 (TNFR1)) by its ligand (e.g. TNF-α) triggers the extrinsic pathway of apoptosis. After the initial discovery that TNF-α is able to induce necrosis in certain cell lines in addition to apoptosis (Laster et al., 1988 1988 ), it was later found that TNF-induced necrosis is aggravated by inhibition of caspases in the presence of the chemical caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (zVAD-fmk) and by the expression of a viral inhibitor of caspase-8, cytokine response modifier A (CrmA) (Vercammen et al., 1998 1998 ). Furthermore, more evidence suggests that apoptosis induced by TNF, TRAIL, and FasL/CD95L can be switched to necrosis when caspase activity is defective or inhibited. In 2000, the protein kinase RIPK1 was identified as an essential regulator for necrotic cell death induced by TNF-α, TRAIL, or FasL/CD95L in a kinase-activity-dependent manner (Holler et al., 2000 2000 ). It thus became clear that death receptor-initiated necrosis is tightly regulated by the kinase activity of RIPK1. This form of death receptor-initiated RIPK1-dependent necrosis was referred to as “programmed necrosis” (Chan et al., 2003 2003 ). In 2005, the term necroptosis was introduced to describe this regulated necrosis when necrostatin-1 (Nec-1) was identified as a specific chemical inhibitor of TNF-induced necrosis (Degterev et al., 2005 2005 ). Nec-1 was further found to inhibit the kinase activity of RIPK1 (Degterev et al., 2005 2005 ), and this inhibitor has been used extensively for the detection of necroptosis and for the evaluation of the in vivo basic biological significance of necroptosis (Yuan et al., 2019 2019 ). In 2009, the kinase RIPK3 was found to regulate necroptosis (Cho et al., 2009 2009 ; He et al., 2009 2009 ; Zhang et al., 2009 2009 ). RIPK3 can associate with RIPK1 to form a protein complex (termed a necrosome) through the RIP homotypic interaction motif (RHIM) domains of both proteins (Sun et al., 2002 2002 ). Formation of the necrosome activates RIPK3, and this process leads to the phosphorylation of RIPK3. In 2012, MLKL was found to be a downstream substrate of RIPK3 and its phosphorylation by RIPK3 was confirmed to be essential for necroptosis execution (Sun et al., 2012 2012 ; Zhao et al., 2012 2012 ). RIPK3-mediated phosphorylation of MLKL activates MLKL, leading to MLKL oligomerization and membrane translocation for the execution of necroptosis (Cai et al., 2014 2014 ; Chen et al., 2014 2014 ; Wang HY et al., 2014 2014 ). Activation and execution of necroptosis 1 TNF-induced necroptosis is the best characterized pathway of necroptosis. Activation of TNFR1 by TNF-α is known to activate nuclear factor-κB (NF-κB) signaling, apoptosis, and necroptosis. Upon TNF-α ligation, TNFR1 is activated by inducing trimer formation. Activated TNFR1 receptors form a membrane signaling complex (called Complex I) by recruiting RIPK1, TNFR1-associated death domain protein (TRADD), cellular inhibitors of apoptosis (cIAPs), and TNFR-associated factor 2 (TRAF2) (Micheau and Tschopp, 2003 2003 ). In this complex, the E3 ubiquitin ligases, cIAP1 and cIAP2, induce RIPK1 ubiquitination (Bertrand et al., 2008 2008 ; Haas et al., 2009 2009 ). RIPK1 further generates binding sites for NF-κB essential modifier (NEMO), which leads to further recruitment and activation of IκB kinase (IKK) α/β and promotes activation of the NF-κB signaling pathway (Gerlach et al., 2011 2011 ; O'Donnell et al., 2012 2012 ). Smac (Diabo) is a pro-apoptotic protein that can interact with inhibitor of apoptosis proteins (IAPs) to relieve their suppression of apoptosis. In the presence of Smac mimetic (a chemical compound that mimics Smac and induces the degradation of cIAPs), RIPK1 is deubiquitinated by cylindromatosis (CYLD) or A20 (Wertz et al., 2004 2004 ; Wright et al., 2007 2007 ; Hitomi et al., 2008 2008 ). RIPK1 is disassociated from Complex I and binds to Fas-associating protein with a novel death domain (FADD) and procaspase-8 to form Complex II in the cytoplasm (Micheau and Tschopp, 2003 2003 ). The precursor caspase-8 was cleaved into the active caspase-8, which further cleaves the downstream casepase-3/7, eventually leading to apoptosis. When caspase-8 activation is inhibited, RIPK1 interacts with RIPK3 to form a protein complex (necrosome) via their RHIM domains (Declercq et al., 2009 2009 ), leading to activation and phosphorylation of both RIPK1 (human RIPK1 at Ser14/15 and Ser161/Ser166, and mouse RIPK1 at Lys45/Lys115) (Degterev et al., 2008 2008 ; McQuade et al., 2013 2013 ) and RIPK3 (human RIPK3 at Ser227 and mouse RIPK3 at Thr231/Ser232) (Sun et al., 2012 2012 ; Chen et al., 2013 2013 ). Activated RIPK3 further phosphorylates the substrate MLKL, which is a pseudokinase. RIPK3-mediated phosphorylation of MLKL (human MLKL at Thr357/Ser358 and mouse MLKL at Ser345/Ser347) (Sun et al., 2012 2012 ; Tanzer et al., 2015 2015 ; Rodriguez et al., 2016 2016 ) induces a conformational change of MLKL and leads to the activation of MLKL, which further forms oligomerization and translocates to the plasma membrane for the execution of necroptosis (Cai et al., 2014 2014 ; Chen et al., 2014 2014 ; Wang X et al., 2014 2014 ). It has been shown that MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis (Liu et al., 2017 2017 ). Dovey et al. ( 2018 2018 ) identified inositol phosphate (IP) kinases inositol polyphosphate multikinase (IPMK) and inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) as critical mediators of MLKL oligomerization and membrane localization. The highly phosphorylated IP molecule produced by these IP kinases, IP6, displaces the MLKL auto-inhibitory region, suggesting the requirement of a specific IP code in coordinating MLKL conformational changes to unleash necroptosis. Prior to membrane disruption, the activation of MLKL triggers the formation of plasma membrane bubbles, which display exposed phosphatidylserine (Gong et al., 2017 2017 ; Yoon et al., 2017 2017 ; Zargarian et al., 2017 2017 ). Endosomal sorting complex required for transport-III (ESCRT-III) components are required for the formation of these membrane bubbles and for the release of phosphor-MLKL-containing vesicles (Gong et al., 2017 2017 ; Yoon et al., 2017 2017 ). Inhibition of ESCRT components increased the sensitivity of cells to necroptosis. These results suggest that the ESCRT-III machinery provides protection against MLKL-mediated membrane damage by actively shedding broken membrane blebs. The necroptotic cells finally release cellular contents including DAMPs, which act as endogenous danger signals to elicit inflammatory responses. Activation of RIPK3 is essential for the induction of necroptosis. The RHIM-dependent interaction of RIPK3 and RIPK1 is also required for IFN-induced necroptosis (Thapa et al., 2013 2013 ) (Fig. 1 1 ). In addition to interacting with RIPK1, RIPK3 can be activated by binding to other RHIM-containing proteins, including Toll/interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β (TRIF), DNA-dependent activator of interferon-regulatory factor (DAI, also known as ZBP1 or DLM1), and the viral protein ribonucleotide reductase large subunit (ICP6) (Wang X et al., 2014 2014 ; Guo et al., 2015 2015 ; Huang et al., 2015 2015 ) (Fig. 1 1 ). TLR3 and TLR4 are pathogen recognition receptors that can activate NF-κB activation and interferon regulatory factor 3 (IRF3)-mediated production of type I IFNs via the adaptor protein TRIF (Yamamoto et al., 2003 2003 ). TLR3 can be activated by double-stranded RNA (dsRNA) molecules such as poly(I:C), while TLR4 recognizes the lipopolysaccharide (LPS), a component of Gram-negative bacteria. In TLR3-or TLR4-induced necroptosis, TRIF is recruited to RIPK3 via their respective RHIM domains (He et al., 2011 2011 ; Najjar et al., 2016 2016 ). Blockage of RIPK1 kinase activity by Nec-1 inhibits TLR4-induced macrophage necroptosis, while RIPK1 is not required for TLR3-induced necroptosis of mouse fibroblasts (He et al., 2011 2011 ; Kaiser et al., 2013 2013 ), suggesting that RIPK3 can be activated by TRIF. In response to infection of a mutant murine cytomegalovirus (MCMV) which lacks the viral RIHM-containing protein viral inhibitor of RIP activation (vIRA) or influenza A virus, DAI activates RIPK3 and necroptosis of host cells independently of both RIPK1 and TRIF (Upton et al., 2010 2010 , 2012 2012 ; Kuriakose et al., 2016 2016 ; Najjar et al., 2016 2016 ; Nogusa et al., 2016 2016 ; Thapa et al., 2016 2016 ). In mouse cells infected with human herpes simplex virus (HSV)-1, mouse RIPK3 recruits the viral protein ICP6 and activates necroptosis. Together, these studies have established RIPK3 as a central molecular node for receiving necroptosis-initiating signals from multiple pathways via RIHM-dependent interactions. Interestingly, RIPK3 can also be inactivated via RIHM-mediated interaction with the viral protein ICP6 in the natural host human cells (Huang et al., 2015 2015 ; Yu et al., 2016 2016 ). Necroptosis in tumor suppression 1 Defective necroptosis response in many cancer cells 2 Cell death of tumor cells is generally viewed as beneficial to cancer prevention and therapy, and evasion of apoptosis is considered a critical hallmark of cancer cells. It is therefore not surprising that cancer cells often have defects in their necroptosis responses. Downregulation of RIPK3 was observed in multiple types of human cancer samples, including breast cancers (Koo et al., 2015 2015 ), colorectal cancers (Feng et al., 2015 2015 ), and acute myeloid leukemia (AML) cells (Nugues et al., 2014 2014 ). Indeed, RIPK3 was found to be silenced in a variety of cancer cells. Treatment with a hypomethylating agent or knockdown of DNA methyltransferase 1 (DNMT1) or UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) promoted hypomethylation of the Ripk3 promoter, leading to the induction of RIPK3 expression in RIPK3-null cancer cells (Koo et al., 2015 2015 ; Yang et al., 2017 2017 ). These results suggest that defects in RIPK3 expression in cancer cells may result from methylation of the Ripk3 promoter. Restored expression of RIPK3 by hypomethylating agents promoted the sensitivity to chemotherapeutics (Koo et al., 2015 2015 ). Hypomethylating agents have been used for clinical treatment of myelodysplastic syndrome and AML. Therefore, induction of RIPK3 expression by hypomethylating agents may provide an opportunity for the development of anti-cancer therapies for treating RIPK3-null cancer cells. Moreover, it has been shown that low RIPK3 expression is associated with poor clinical outcomes in human colorectal cancer and that overexpression of RIPK3 can attenuate the migration and invasion of colorectal cancer cells (Feng et al., 2015 2015 ). The ectopic expression of RIPK3 in RIPK3-null cancer cells inhibited tumor growth (Koo et al., 2015 2015 ; Yang et al., 2017 2017 ). These results suggest that the deletion or downregulation of RIPK3 in tumor cells favors cell survival and tumorigenesis. In addition, low expression of MLKL was shown to be associated with poor prognosis in patients with early-stage resected pancreatic adenocarcinoma (Colbert et al., 2013 2013 ), ovarian cancer (He et al., 2013 2013 ), and cervical cancer (Ruan et al., 2015 2015 ); the mechanism leading to the downregulation of MLKL in these cancer samples has not been explored. These studies suggest that core necroptosis components RIPK3 and MLKL may serve as tumor suppressors. Activation of necroptosis as a mechanism to overcome apoptosis resistance 2 Induction of cancer cell death is an important strategy for killing cancer cells. Cancer cells typically develop resistance to apoptosis via defective caspase activity owing to gene mutations or silencing. Since necroptosis tends to occur in the absence of caspase activation, it is conceivable that necroptosis is an alternative mode of cell death to overcome apoptosis resistance. This idea is strongly supported by the discovery that necroptosis is activated in caspase-8-deficient colorectal cancer cells in response to Smac mimetic and that necroptosis results in significant tumor regression in both a hereditary and a xenograft mouse model (He et al., 2017 2017 ) (Fig. 2 2 ). The Smac mimetic birinapant, in combination with the clinical caspase-8 inhibitor emricasan/IDN-6556, was shown to effectively trigger necroptosis of acute myelogenous leukemia (AML) cells, and this was found to prolong the survival of mice bearing MLL-AF9 or MLL-ENL birinapant-resistant AML cells (Brumatti et al., 2016 2016 ). Birinapant has been also shown to induce RIPK1-dependent apoptosis and necroptosis in patient-derived acute lymphoblastic leukemia (ALL) cells and to exhibit an anti-tumor effect on Smac mimetic-sensitive ALL in vivo (McComb et al., 2016 2016 ). Another Smac mimetic (BV6) was able to activate TNF-dependent necroptosis in patient-derived AML cells, although these cells were resistant to apoptosis (Safferthal et al., 2017 2017 ). Similarly, BV6 could trigger necroptosis in pancreatic carcinoma cells when caspase activation was blocked (Hannes et al., 2016 2016 ). Necroptosis can be induced for killing certain tumor cells by some chemotherapy drugs. Moreover, necroptosis of tumor cells acts as ICD, which is characterized by the release of ATP and DAMPs (e.g. HMGB1) from dying cells. This ICD promotes DC maturation and CD8 + T cell anti-tumor immunity. MTX: mitoxantrone; ICD: immunogenic cell death; ATP: adenosine triphosphate; DAMPs: damage-associated molecular patterns; HMGB1: high mobility group box 1; DC: dendritic cell Additionally, the plant natural product shikonin was shown to induce cell death of MCF-7 that could be blocked by Nec-1 (Han et al., 2007 2007 ) (Fig. 2 2 ). Further studies have shown that shikonin upregulates the expression levels of RIPK1 and RIPK3 and induces the formation of the RIPK1/RIPK3 necrosome complex in multiple cancer cells (Fu et al., 2013 2013 ; Park et al., 2013 2013 ; Lu et al., 2017 2017 ). A reactive oxygen species (ROS) scavenger was shown to attenuate shikonin-induced necrosome formation (Park et al., 2013 2013 ; Lu et al., 2017 2017 ). Treatment of shikonin repressed tumor growth of both primary and metastatic osteosarcomas in vivo and resulted in increased tumor necrosis and elevated levels of RIPK1 and RIPK3 in primary tumor tissues (Fu et al., 2013 2013 ) (Table 1 1 ). Therefore, triggering necroptosis of tumor cells may represent an attractive new strategy for the treatment of cancer, including apoptosis-resistant cancer. LLC1: Lewis lung carcinoma line 1; Nec-1: necrostatin-1; AML: acute myeloid leukemia; TNF: tumor necrosis factor; IFN: interferon; NSA: necrosulfonamide; MTX: mitoxantrone; PDA: pancreatic ductal adenocarcinoma; RIPK: receptor-interacting protein (RIP) kinase; TAMs: tumor-associated macrophages; PD-1: checkpoint receptor; ICOS: co-stimulatory ligand Cancer model Small molecule Major finding Reference Osteosarcoma Shikonin Shikonin increased tumor necrosis and repressed tumor growth of both primary and metastatic osteosarcomas in vivo Fu et al., 2013 2013 2 Lung metastasis models of B16 and LLC1 Nec-1 Nec-1 reduced tumor cell-induced endothelial necroptosis and inhibited tumor cell extravasation and metastasis Strilic et al., 2016 2016 5-(2,3-Dihydro-1 H -indol-5-yl)-7 H -pyrrolo[2,3- d ] pyrimidin-4-amine (RIPK1 inhibitor) This compound reduced the metastasis of B16 tumor cells in vivo Li et al., 2018 2018 2 AML BV6 BV6 activated TNF-dependent necroptosis in patient-derived AML blasts Safferthal et al., 2017 2017 Nec-1 Nec-1 in combination with IFN-γ significantly enhanced human primary AML cell differentiation and attenuated the clonogenic capacity Xin et al., 2017 2017 Breast cancer NSA NSA inhibited xenograft tumor growth of MDA-MB-231 in nude mice Liu et al., 2016 2016 2 Colorectal cancer Smac mimetic Smac mimetic promoted the necroptosis of caspase-8-deficient colorectal cancer cells and inhibited the development of MC38 and Apc Min/+ mouse colorectal cancer He et al., 2017 2017 GSK872 GSK872 significantly reduced tumors in the spontaneous intestinal tumor model (Apc Min/+ mice) Jayakumar and Bothwell, 2019 2019 EL4, TC-1 MTX Cancer cell necroptosis induced by MTX exhibited the features of immunogenic cell death (ICD) and activated anticancer immune responses Yang et al., 2016 2016 PDA GSK'547 (RIPK1 inhibitor) Inhibition of RIPK1 by GSK'547 induced immunogenic programming of TAMs, leading to activation of adaptive immune responses and tumor suppression. GSK'547 synergizes with PD-1 and ICOS-based Immunotherapy Wang et al., 2018 2018 Tumor suppressive activity of necroptosis through control of apoptosis 2 Strong in vivo evidence for RIPK3-mediated tumor suppression has been provided by the finding that RIPK3 deletion enhances the development of hepatic tumors in a hepatocellular carcinoma (HCC) model induced by the liver parenchymal cell (LPC)-specific deletion of transforming growth factor-β (TGF-β)-activated kinase 1 ( Tak1 ) (Vucur et al., 2013 2013 ). Hepatocyte cell death is commonly involved in the progression of liver fibrosis and the HCC that is caused by chronic liver disease. LPC-specific TAK1 knockout (TAK1 LPC-KO ) mice display severe hepatic inflammation as well as spontaneous LPC apoptosis and necrosis (Bettermann et al., 2010 2010 ). Compared to TAK1 LPC-KO and TAK1/Casp-8 LPC-KO (combined ablations of TAK1 and caspase-8 in LPC) mice, TAK1 LPC-KO /RIPK3 −/− (ablation of TAK1 in LPC together with a full knockout of Rip3 ) mice exhibited significantly higher inflammatory responses in the liver, suggesting that LPC apoptosis rather than necroptosis contributes to hepatic inflammation (Vucur et al., 2013 2013 ). Loss of RIPK3 caused increased apoptosis and proliferation of TAK1-deficient LPC, leading to increased inflammation and carcinogenesis in TAK1 LPC-KO /RIPK3 −/− mice (Vucur et al., 2013 2013 ). Therefore, manipulation of hepatic apoptosis and necroptosis signaling might represent a new strategy for the prevention and/or treatment of HCC. Recently, it has been shown that the expression of Ripk3 was higher in intrahepatic cholangiocarcinoma (ICC) than in HCC (Seehawer et al., 2018 2018 ). The necroptosis-dependent hepatic cytokine microenvironment leads to ICC development, while suppression of necroptosis shifted cell death towards apoptosis and switched ICC to HCC (Seehawer et al., 2018 2018 ). These results suggest that the tumor microenvironment determines lineage commitment in liver tumorigenesis. Tumor suppressive activity of necroptosis through promotion of cell death and differentiation 2 The tumor suppressive activity of necroptosis is also supported by studies in AML. AML is maintained by a rare population of leukemia-initiating cells (LICs) characterized by inhibition of differentiation and cell death. It has been shown that RIP3 expression is significantly reduced in CD34 + cells from AML patients compared with that in CD34 + cells from healthy donors (Nugues et al., 2014 2014 ). Re-expression of RIPK3 in RIP3-null leukemia cell lines induced apoptosis in a smaller proportion of cells, and also activated necroptosis in the presence of the caspase inhibitor, while expression of a kinase dead form of RIPK3 (RIPK3-D161N) in mouse DA1-3b leukemia cells triggered massive apoptosis which was shown to depend on NF-κB activity (Nugues et al., 2014 2014 ). The induced expression of RIPK3-D161N but not wild-type RIPK3 prolonged the survival of mice after injection of leukemia cells (Nugues et al., 2014 2014 ). These results suggest that a decrease in RIPK3 expression in AML cells may contribute to the suppression of both necroptosis and apoptosis. Moreover, RIPK3 was found to be expressed at reduced levels in samples from patients with FMS-like tyrosine kinase 3 (FLT3)-mutated AML (Höckendorf et al., 2016 2016 ) (Fig. 3 3 ). Mice lacking RIPK3 showed enhanced survival of FLT3-internal tandem duplication (FLT3-ITD)-transformed hematopoietic stem and progenitor cells (HSPCs) and increased leukemogenesis. Similarly, MLKL-deficient mice accelerated the onset of AML (Höckendorf et al., 2016 2016 ). It has been suggested that TNFR-mediated RIPK1/RIPK3-dependent cell death restricts the accumulation of malignant progenitors. In addition, RIPK3-mediated inflammasome activation promoted differentiation of LICs by eliciting interleukin-1β (IL-1β) signaling (Höckendorf et al., 2016 2016 ). These results suggest that RIPK3 has tumor-suppressive function(s) in FLT3-mutated AML subtypes through the promotion of necroptosis and the differentiation of LICs. In the FLT3-ITD-induced AML model, RIPK3 promotes necroptosis of LIC and stimulates LIC differentiation by activating the inflammasome to release IL-1β. In the MA9-AML cells, inhibition of RIPK1 or RIPK3 sensitizes cells to IFN-γ-induced differentiation and reduces clonogenic capacity. AML: acute myeloid leukemia; FLT3: FMS-like tyrosine kinase 3; ITD: internal tandem duplication; LIC: leukemia-initiating cell; IL-1β: interleukin-1β; MA9: Mll-AF9; IFN: interferon; RIPK: receptor-interacting protein (RIP) kinase Necroptosis in anti-tumor immunity 2 Immunogenic cell death (ICD) can activate host immune responses, especially to malignant tumor cells, to produce strong and persistent anti-cancer immunity (Casares et al., 2005 2005 ). Therefore, the necroptosis of cancer cells is becoming increasingly important because of its roles in immunogenicity and in activating the immune system. Studies have shown that induction of cancer cell necroptosis triggers the release of DAMPs to induce dendritic cell (DC) maturation (Yatim et al., 2015 2015 ; Aaes et al., 2016 2016 ) (Fig. 2 2 ). It was observed that release of inflammatory mediators such as DAMPs by dying cells is not enough to cause CD8 + T cell cross-priming. Rather, robust cross-priming was found to require RIPK1 signaling and NF-κB-induced intracellular transcription of dying cells (Yatim et al., 2015 2015 ). Moreover, vaccination with these necroptotic cancer cells induced cross-priming of T cells and the production of IFN-γ in response to tumor antigen stimulation, and this prevented tumor growth, thus demonstrating the immunogenic nature of necroptotic cancer cells and its association with anti-tumor immunity (Aaes et al., 2016 2016 ). Additionally, chemotherapy agents like mitoxantrone (MTX) can reinstate anticancer immunosurveillance by inducing the ICD of tumor cells. Cancer cells undergoing necroptosis induced by MTX exhibited biochemical hallmarks of ICD including calreticulin (CRT) exposure and the release of adenosine triphosphate (ATP) and high mobility group box (HMGB). Deletion of RIPK3 or MLKL in cancer cells repressed chemotherapy-induced ICD and anticancer immune responses in vivo (Yang et al., 2016 2016 ). These results suggest that necroptosis can contribute to ICD signaling and tumor immunogenicity. Additionally, it was found that RIPK3 reduction in colorectal cancer was associated with accumulation of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (Yan et al., 2018 2018 ). Reduction of RIPK3 in MDSCs led to upregulation of NF-κB p65 and cyclooxygenase-2 (COX-2) which catalyzed the synthesis of prostaglandin E 2 (PGE 2 ). Addition of PGE 2 inhibited RIPK3 expression, but enhanced the expression of p65 and COX-2 in MDSCs (Yan et al., 2018 2018 ). Blockage of COX-2 or PGE 2 receptors by chemical inhibitors reversed the downregulated RIPK3 and the immunosuppressive activity of MDSCs, and reduced the tumorigenesis of colorectal cancer (Yan et al., 2018 2018 ). These results suggest that the RIPK3-PGE 2 circuit regulates the infiltration and activity of MDSCs and colorectal cancer carcinogenesis, and thus may be a potential target for the treatment of colorectal cancer. Necroptosis in tumorigenesis 1 Necroptosis in macrophage-mediated immune suppression 2 Although it is believed that the induction of necroptosis in cancer cells contributes to the death of cancer cells and the activation of anti-tumor immunity, necroptosis has been reported to promote tumorigenesis. Chronic inflammation has been recognized as one of the hallmarks of cancer and is known to be involved in tumor progression (Coussens and Werb, 2002 2002 ). Given the fact that necroptosis induces the release of DAMPs and inflammatory responses, it is conceivable that necroptosis may stimulate tumor growth. Higher expression levels of RIPK1 and RIPK3 were observed in human pancreatic ductal adenocarcinoma (PDA) than in the surrounding normal pancreas tissue (Seifert et al., 2016 2016 ) (Fig. 4a 4a ). Deficiency of RIPK3 in mice or inhibition of RIPK1 protected against oncogenic progression of Kras G12D - induced PDA in mice (Seifert et al., 2016 2016 ). RIPK1/RIPK3 signaling-induced chemokine (C-X-C motif) ligand 1 (CXCL1) expression and ligation of Mincle by SAP130 were shown to promote macrophage-induced adaptive immune suppression and therefore accelerated oncogenesis (Seifert et al., 2016 2016 ), suggesting that inhibition of RIPK1 or RIPK3 could be beneficial for treating PDA. It is noteworthy that RIPK3-deficient mice were not protected against B16 melanoma or subcutaneously implanted cancer cells with mutant Kras and p53 , suggesting that the pro-tumorigenic action of RIPK3 occurs in a cell-type and context-dependent manner. Additionally, it has been shown that RIPK1 was highly expressed in tumor-associated macrophages in the PDA model and suppression of RIPK1 by a selective inhibitor GSK547-induced STAT1-dependent immunogenic reprogramming of tumor-associated macrophages (TAMs), leading to the activation of adaptive immune responses and tumor suppression (Wang et al., 2018 2018 ) (Table 1 1 ). Inhibition of RIPK1 synergized with checkpoint receptor (PD-1) and co-stimulatory ligand (ICOS)-based immunotherapies (Wang et al., 2018 2018 ) (Table 1 1 ). Unexpectedly, treatment of the RIPK1 inhibitor provided additive protection in RIPK3-deficient mice, suggesting that the tumor-promoting effects of RIPK1 in PDA are independent of RIPK3. (a) In the PDA model, necroptosis of pancreatic epithelial cells induces CXCL1 expression and ligation of Mincle by SAP130, leading to accumulation of MDSCs and suppression of adaptive immunity. (b) In the spontaneous intestinal tumor model (Apc Min/+ mice), the RIPK3 kinase inhibitor GSK872 significantly reduces tumor growth in the small intestine by inhibiting the accumulation of I-MDSC, which mediates expansion of IL-17-producing T cells. CXCL1: chemokine (C-X-C motif) ligand 1; DC: dendritic cell; MDSC: myeloid-derived suppressor cell; I-MDSC: intermediate MDSC; TAM: tumor-associated macrophage; IL-17: interleukin-17; RIPK: receptor-interacting protein (RIP) kinase Recently, it has been reported that inhibition of RIPK3 kinase activity by the RIPK3 kinase inhibitor small molecule GSK872 significantly reduced tumors in the small intestine of a spontaneous intestinal tumor model (Apc Min/+ mice) (Jayakumar and Bothwell, 2019 2019 ) (Fig. 4b 4b , Table 1 1 ). Intermediate MDSCs (I-MDSCs) were significantly decreased in the GSK872-treated Apc Min/+ mice, while monocytic MDSCs (M-MDSCs) and macrophages were moderately reduced. Injection of I-MDSC enhanced tumor growth and increased the number of CD4 + IL-17 + cells in a mouse MC38 colon cancer model; in contrast, these phenotypes were suppressed by treatment with GSK872 (Jayakumar and Bothwell, 2019 2019 ). Although these results suggest a role for RIPK3 in the promotion of intestinal tumors by regulating I-MDSCs-mediated expansion of IL-17-producing T cells, further studies will be required to dissect any necroptosis-dependent or necroptosis-independent functions of RIPK3 in the tumorigenesis of Apc Min/+ mice, for example by using genetic deletion of necroptosis components. Other mechanisms for necroptosis-mediated tumor promotion 2 There was also evidence showing that deletion of RIPK1, RIPK3, or MLKL in human breast cancer MDA-MB-231 cells repressed xenograft tumor growth in nude mice (Liu et al., 2016 2016 ). Similarly, deletion of RIPK3 in human 4T1 breast cancer reduced tumor growth in syngeneic Balb/C mice (Liu et al., 2016 2016 ). Moreover, the MLKL inhibitor necrosulfonamide (NSA) inhibited growth of MDA-MB-231 xenograft tumors (Liu et al., 2016 2016 ) (Table 1 1 ). Although previous studies have shown that necroptosis promotes cancer cell death and therefore benefits cancer treatment, the results of Liu et al. ( 2016 2016 ) suggest the apparent tumor-promoting properties of necroptosis components in these cancer cells. p65/RelA phosphorylation, a marker indicating NF-κB activation, was reduced in RIPK3 knockout cells, while p65/RelA phosphorylation was not changed in RIPK1 or MLKL knockout cells. It is believed that aberrant activation of NF-κB is observed in most cancers and increases tumor proliferation and tumor-related inflammation. Further investigations are needed to confirm the impact of NF-κB signaling on RIPK3-mediated tumor progression and to delineate any mechanism(s) underlying tumorigenesis as driven by necroptosis components. In addition, it has been shown that RIPK3 is more highly expressed in the M4 and M5 subtypes of AML (Xin et al., 2017 2017 ) (Fig. 3 3 ). Knockout of RIPK1 or RIPK3 in MA9-AML cells sensitized cells to IFN-γ-induced differentiation and repressed leukemia development (Xin et al., 2017 2017 ). Treatment with the RIPK1 inhibitor Nec-1 combined with IFN-γ significantly enhanced human primary AML cell differentiation and attenuated clonogenic capacity, suggesting that blockage of RIPK1/RIPK3 signaling may be a new approach for treatment of several subtypes of AML in combination with IFN-γ (Xin et al., 2017 2017 ) (Table 1 1 ). However, such a negative role for RIPK1/RIPK3 in AML cell differentiation is inconsistent with the previous observation that RIPK3 signaling promotes LIC cell death and differentiation in FLT3-IDT-induced AML (Höckendorf et al., 2016 2016 ). The role of RIPK1/RIPK3 signaling in AML pathogenesis thus appears to vary among the different subtypes of AML, so the therapeutic potential of strategies targeting necroptosis must be carefully evaluated in relevant mouse models and in large and diverse patient cohorts. Necroptosis in the promotion of metastasis 1 Metastasis is the major cause of cancer-related death and is understood to be a complex process resulting in the spread of cancer cells to distant parts of the body through the circulatory system. It has been shown that in vitro co-culture of tumor cells with endothelial cells induces endothelial cell death via RIPK1-RIPK3-MLKL-dependent necroptosis (Strilic et al., 2016 2016 ). In lung metastasis models of B16 and LLC1 in vivo, endothelium-specific caspase 8 deficiency could inhibit apoptosis and further promote necroptosis, which eventually aggravated lung metastasis (Strilic et al., 2016 2016 ). Endothelial cell-specific deletion of RIPK3, the complete loss of MLKL, or necrosatin-1 treatment was each shown to reduce tumor cell-induced endothelial necroptosis as well as tumor cell extravasation and metastasis. This endothelial necroptosis was induced via the activation of death receptor 6 (DR6) in endothelial cells upon exposure to amyloid precursor protein (APP) in tumor cells. These results suggest a role for necroptosis in the disruption of vascular endothelial barriers for promoting metastasis (Strilic et al., 2016 2016 ) (Fig. 5a 5a ). Recently, it has been reported that endothelial TGF-β-activated kinase 1 (TAK1) acts as a negative regulator of endothelial necroptosis and metastasis (Yang et al., 2019 2019 ). TAK1 deletion resulted in increased expression of RIPK3 in endothelial cells and enhanced both necroptosis of endothelial cells and metastasis of tumor cells (Yang et al., 2019 2019 ). Deficiency of RIPK3 effectively inhibited tumor metastasis in epithelium-specific TAK1 knockout mice (Yang et al., 2019 2019 ). These results support the idea that sustained TAK1 signaling is required for controlling the low levels of necroptosis components in endothelial cells. (a) Necroptosis of endothelial cells is induced via the activation of DR6 in endothelial cells upon exposure to APP in tumor cells. This process destroys vascular endothelial barriers to promote metastasis. (b) Activation of RIPK1/RIPK3 signaling promotes VEGF-induced vascular permeability to mediate tumor cell extravasation independent of necroptosis. (c) Necroptosis of tumor cells promotes tumor metastasis. APP: amyloid precursor protein; DR6: death receptor 6; RIPK: receptor-interacting protein (RIP) kinase; VEGF: vascular endothelial growth factor; VEGFR: VEGF receptor Nevertheless, there is also evidence showing that loss of RIPK3 kinase activity or loss of MLKL had no significant effect on metastasis, although the complete loss of RIPK3 or loss of RIPK1 kinase activity was confirmed to suppress tumor cell extravasation and metastasis (Hänggi et al., 2017 2017 ). Cell death in primary endothelial cells upon co-incubation of tumor cells was shown to be independent of RIPK3 or MLKL. It was found that RIPK1 kinase activity and RIPK3 promote vascular endothelial growth factor (VEGF)-dependent activation of p38/HSP27 mitogen-activated protein (MAP) kinase signaling axis to facilitate vascular permeability and vessel sprouting (Hänggi et al., 2017 2017 ) (Fig. 5b 5b ). These results suggest a necroptosis-independent function for RIPK1/RIPK3 in vascular permeability that may contribute to tumor metastasis. Although further studies are needed to elucidate the precise molecular mechanism through which RIPK1 and RIPK3 may mediate tumor cell extravasation, these observations reveal that RIPK1 and RIPK3 can be viewed as potential targets for developing therapeutic interventions against metastasis. Indeed, studies showed that blockage of RIPK1 kinase activity by Nec-1 or a different RIPK1 inhibitor significantly reduced tumor metastasis in vivo (Strilic et al., 2016 2016 ; Li et al., 2018 2018 ) (Table 1 1 ). In addition to endothelial cell necroptosis, necroptosis of tumor cells was recently found to promote tumor metastasis (Jiao et al., 2018 2018 ). The phosphorylation of MLKL, the marker of necroptosis activation, was detected in areas of tumor necrosis in both human breast cancer samples and mouse breast cancer models, including a spontaneous breast cancer model (MMTV-PyMT mice) and syngeneic breast cancer model (Jiao et al., 2018 2018 ). Deletion of MLKL in mouse MVT-1 breast cancer cells greatly reduced breast cancer metastasis to the lung (Jiao et al., 2018 2018 ). These results provide strong evidence suggesting that tumor cell necroptosis occurs during tumor development and participates in the pathogenesis of cancer metastasis (Fig. 5c 5c ). Concluding remarks 1 Necroptosis is now recognized as a major form of regulated cell death. It is mediated by RIPK1/RIPK3 activation, followed by MLKL phosphorylation, oligomerization, and membrane translocation, and finally membrane disruption. Necroptosis is becoming increasingly attractive as a field of study given its implications in the pathogenesis of inflammatory diseases, degenerative diseases, and cancer. As a highly inflammatory form of regulated cell death, necroptosis has very complicated roles in cancer initiation, development, and metastasis. Although the necroptosis field has developed rapidly in recent years, it also faces many challenges. For example, it remains unclear how the action of MLKL links to membrane leakage. Given that RIPK3 expression is restricted in most tumor cells, we do not yet understand the action of RIPK3 in other cells including immune cells and stromal cells in the tumor microenvironment. RIP kinase necroptosis components are now understood to mount inflammatory responses by causing necroptosis or directly regulating inflammatory signaling pathways, but it is not yet known if RIPK1 and/or RIPK3 activate the necroptosis pathway or/and the immune responses to affect tumorigenesis. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1900160 Project supported by the National Natural Science Foundation of China (Nos. 31671436, 31600133, 31771533, and 31830051), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Natural Science Foundation of Jiangsu Province (No. BK20160314), the Fok Ying Tung Education Foundation for Young Teachers (No. 151020), and the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (Nos. 2017NL31002 and 2017NL31004) 2019-03-29 2019-04-22 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2019 2019 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 05 20