Mps1 kinase regulates tumor cell viability via its novel role in mitochondria

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells.Massive chromosome missegregation induces cell death as observed by Theodor Boveri in the early 1900s.¹ However, the underlying mechanism remains elusive. The spindle assembly checkpoint (SAC) is a dominant machine monitoring chromosomal segregation during mitosis by delaying the onset of anaphase until all chromosomes are properly captured by microtubules. The SAC consists of kinetochore association sensors, including Mps1 (monopolar spindle 1), Bub1 (budding uninhibited by benzimidazole 1 homolog) and Aurora B; a signaling transducer termed the mitotic checkpoint complex (MCC), including CDC20 (cell division cycle 20), BubR1 (Bub1-related kinase), Bub3 (budding uninhibited by benzimidazole 3 homolog) and Mad2 (mitotic arrest deficient-like 2); and an effector APC/C (anaphase-promoting complex/cyclosome) that is inhibited by MCC in response to an active SAC.² Loss of SAC by inactivation of checkpoint sensors or signaling transducers elicits massive chromosome missegregation, induces severe gain or loss of chromosomes and eventually causes cell death.^{3, 4, 5, 6} Meanwhile, a weakened SAC due to the haploinsufficiency of the checkpoint proteins Mad1, Mad2, Bub1, BubR1 and CENP-E (centromere protein E) does not cause cell death but facilitates tumorigenesis.^{7, 8, 9, 10, 11} These studies suggest that the fate of these cells is dependent on their respective degree of SAC deficiency. Notably, in these studies SAC proteins were constitutively disturbed, raising the possibility that other signaling pathways could be affected as SAC proteins have functions beyond SAC regulation.^{12, 13, 14}Mps1 is an essential component of SAC that senses SAC signal by promoting MCC formation via kinetochore recruitment of Mad2, CENP-E and Knl1 (kinetochore-null protein 1).^{15, 16, 17, 18, 19} Recent studies show that Mps1 can discriminate between on or off SAC signaling by binding to NDC80c via the motif that associates microtubules.^{20, 21} Following SAC, Mps1 is involved in regulating chromosome alignment by phosphorylating Borealin, a component of chromosomal passenger complex (CPC).^{22, 23} In addition, Mps1 plays multiple roles beyond mitosis, including centrosome duplication, cytokinesis, ciliogenesis and DNA damage response.^{18, 24, 25, 26, 27, 28} Mps1 is indispensable for cell survival as loss of Mps1 function by specific siRNA or Mps1 kinase inhibitors causes significant cell death; it has been proposed that Mps1 regulates this process through its roles in SAC.^{29, 30, 31}Mps1 kinase is overexpressed in a variety of tumor types.^{32, 33, 34, 35} In breast cancer, high levels of Mps1 correlate with tumor grades; reducing Mps1 level induces massive apoptosis but allows a selective survival of tumor cells with less aneuploidy.³² Our recent results in colon cancer cells showed that overexpression of Mps1 facilitate the survival of tumor cells with higher aneuploidy by decreasing SAC threshold.³⁵ To further uncover the roles of high levels of Mps1 in tumorigenesis, we examined Mps1 levels in various stages of colon cancer tissues and found that Mps1 level peaks in tissues at stage II, at which stage tumor cells encounter various survival stresses, including genome instability. Aneuploid colon cancer cell lines bear higher levels of Mps1 than diploid cell lines and the amount of Mps1 required for cell survival is far more than that of maintaining SAC, suggesting that other functions of Mps1 are also employed to maintain cell viability. Short-term inhibition of Mps1 activity in mitosis with inhibitors at a dose of more than SAC depletion is sufficient to cause dividing cell death and increase mitochondrial fragmentation simultaneously. Finally, we found that Mps1 can regulate the release of cytochrome c by associating with mitochondrial protein VDAC1 (voltage-dependent anion channel 1). Based on these findings, we postulated that high levels of Mps1 contribute to survival of aneuploid cancer cells via its roles in SAC and mitochondria.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

Implication of different domains of the Leishmania major metacaspase in cell death and autophagy

Metacaspases (MCAs) are cysteine peptidases expressed in plants, fungi and protozoa, with a caspase-like histidine–cysteine catalytic dyad, but differing from caspases, for example, in their substrate specificity. The role of MCAs is subject to debate: roles in cell cycle control, in cell death or even in cell survival have been suggested. In this study, using a Leishmania major MCA-deficient strain, we showed that L. major MCA (LmjMCA) not only had a role similar to caspases in cell death but also in autophagy and this through different domains. Upon cell death induction by miltefosine or H₂O₂, LmjMCA is processed, releasing the catalytic domain, which activated substrates via its catalytic dyad His/Cys and a proline-rich C-terminal domain. The C-terminal domain interacted with proteins, notably proteins involved in stress regulation, such as the MAP kinase LmaMPK7 or programmed cell death like the calpain-like cysteine peptidase. We also showed a new role of LmjMCA in autophagy, acting on or upstream of ATG8, involving Lmjmca gene overexpression and interaction of the C-terminal domain of LmjMCA with itself and other proteins. These results allowed us to propose two models, showing the role of LmjMCA in the cell death and also in the autophagy pathway, implicating different protein domains.Apoptosis is, in most cases, associated with and depends on the activation of cys-dependent peptidases, named caspases.^{1, 2} Once activated, initiator caspases induce a proteolytic cascade via the activation of effector caspases that ultimately cleave numerous substrates, thereby causing the typical morphological features of apoptosis.^{3, 4} Despite their essential role in apoptosis, caspases are also involved in non-apoptotic events, including inflammation, cell proliferation, cell differentiation⁵ and the cell survival process autophagy, a major catabolic process in eukaryotic cells that allows cells to survive nutrient starvation due to engulfment of a portion of the cytoplasm by a specific membrane, delivery to lysosomes or vacuoles and digestion by hydrolytic enzymes.^{6, 7, 8, 9, 10} Plants, fungi and protozoa are devoid of caspases but express metacaspases (MCAs).¹¹MCAs are cysteine peptidases of the clan CD, family 14, with a caspase-like histidine–cysteine catalytic dyad.^{12, 13} However, besides their distant similarity to caspases,¹⁴ MCAs prefer arginine/lysine in the P1 position, whereas caspases prefer aspartic residues.^{15, 16} The role of MCAs in cell death is still enigmatic. For example, in the yeast Saccharomyces cerevisiae, YCA1 has a role in cell death,^{17, 18} whereas, although only partly dependent on its conserved catalytic cysteine, it also facilitates the removal of unfolded proteins, prolonging cellular life span.¹⁹ Similarly, some metacaspases have roles, outside of death, in stress acclimation pathways, as in Aspergillus fumigatus²⁰ or in the unicellular planctonic organisms diatoms.^{21, 22} In Arabidopsis thaliana, AtMC1 is a positive regulator of cell death and a survival factor for aging plants,²³ whereas AtMC2 negatively regulates cell death.²⁴ Trypanosoma brucei TbMCA2, TbMCA3 and TbMCA5 and Leishmania major MCA are involved in cell cycle regulation.^{25, 26}Leishmania are parasitic protozoa responsible for the neglected tropical disease leishmaniasis, transmitted to humans by the bite of the sand fly. In the insect, parasites proliferate as free-living flagellated forms called procyclic promastigotes within the midgut before differentiating into virulent metacyclic promastigotes and migrating to the proboscis.^{27, 28} In the mammalian host, promastigotes are taken up by macrophages and transform into amastigotes. Under a variety of stress stimuli, apoptosis-like morphological and biochemical features have been described in Leishmania, among which are cell shrinkage, chromatin condensation, DNA fragmentation or mitochondrial depolarization.^{29, 30, 31, 32, 33, 34, 35, 36, 37, 38} Despite the evidence of morphological and biochemical markers of cell death in dying Leishmania, very little is known about the cell death pathway and the implicated executioner proteins. Indeed, essential proteins involved in mammalian apoptosis, death receptors, small pro- and anti-apoptotic molecules and caspases, are apparently not encoded in the genome of Leishmania³⁹ and the role of Leishmania MCA in cell death is still controversial, certain authors suggesting a role as a negative regulator of intracellular amastigote proliferation, instead of having a caspase-like role in the execution of cell death.⁴⁰LmjMCA contains different domains: an N-terminal domain with a Mitochondrion Localization Signal (MLS),⁴¹ a caspase-like catalytic domain and a C-terminal proline-rich domain.⁴¹ On the basis of this domain structure, LmjMCA can be classified among the type I metacaspases,¹⁶ a subclass more generally defined in higher plants and characterized by the presence of an N-terminal prodomain and a short linker between the large and small subunits, as initiator caspases in metazoans.¹¹ Upon induction of cell death by heat shock, H₂O₂ or drugs like miltefosine or curcumin, LmjMCA is processed and the catalytic domain is released,⁴¹ liberating the C-terminal domain. It was therefore interesting to investigate the functional roles of the different domains.In this report, we studied the role of L. major MCA (LmjMCA), using an MCA-deficient strain and overexpressing independently the catalytic and the C-terminal domains. The results confirmed that MCA was not essential to L. major survival. In contrast, LmjMCA processing, releasing its catalytic and C-terminal domains, induced cell death in L. major, whereas the overexpression of Lmjmca gene triggered autophagy after interaction of the C-terminal domain with itself and with other proteins, acting on or upstream of the autophagic protein ATG8.     

Live imaging the phagocytic activity of inner ear supporting cells in response to hair cell death

Hearing loss and balance disorders affect millions of people worldwide. Sensory transduction in the inner ear requires both mechanosensory hair cells (HCs) and surrounding glia-like supporting cells (SCs). HCs are susceptible to death from aging, noise overexposure, and treatment with therapeutic drugs that have ototoxic side effects; these ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic drug cisplatin. Although both classes of drugs are known to kill HCs, their effects on SCs are less well understood. Recent data indicate that SCs sense and respond to HC stress, and that their responses can influence HC death, survival, and phagocytosis. These responses to HC stress and death are critical to the health of the inner ear. Here we have used live confocal imaging of the adult mouse utricle, to examine the SC responses to HC death caused by aminoglycosides or cisplatin. Our data indicate that when HCs are killed by aminoglycosides, SCs efficiently remove HC corpses from the sensory epithelium in a process that includes constricting the apical portion of the HC after loss of membrane integrity. SCs then form a phagosome, which can completely engulf the remaining HC body, a phenomenon not previously reported in mammals. In contrast, cisplatin treatment results in accumulation of dead HCs in the sensory epithelium, accompanied by an increase in SC death. The surviving SCs constrict fewer HCs and display impaired phagocytosis. These data are supported by in vivo experiments, in which cochlear SCs show reduced capacity for scar formation in cisplatin-treated mice compared with those treated with aminoglycosides. Together, these data point to a broader defect in the ability of the cisplatin-treated SCs, to preserve tissue health in the mature mammalian inner ear.Hearing loss affects more than 360 million people worldwide and is often irreversible.¹ Mechanosensory hair cells (HCs), the receptor cells of hearing and balance, are not regenerated in the adult mammal and their death results in permanent hearing loss.^{2, 3} HCs are surrounded by glia-like supporting cells (SCs) that are necessary for HC survival and function (reviewed in Monzack et al.).⁴ SCs perform many functions, including providing critical trophic factors, preventing excitotoxicity, and mediating regeneration in those systems (non-mammalian vertebrates) capable of replacing lost HCs.^{5, 6, 7, 8, 9, 10, 11} When HCs die, SCs also preserve the integrity and function of the remaining tissue by forming scars and clearing dead HCs.^{2, 12, 13, 14, 15, 16, 17} Maintaining a fluid barrier at the surface of the sensory epithelium after damage is necessary to preserve the electro-chemical gradient that drives HC depolarization and therefore sensory transduction after the onset of hearing (reviewed in Wangemann).¹⁸Several major stressors cause HC death,^{19, 20, 21, 22} including aging, noise trauma, and exposure to therapeutic drugs with ototoxic side effects. When a HC is killed by noise or aminoglycoside antibiotics, surrounding SCs form a filamentous actin (F-actin) cable that constricts the HC at its apex.^{2, 12, 13, 14, 15, 16, 17} This process separates the apical portion of the cell, including the stereocilia bundle, from the HC body and preserves a sealed reticular lamina.²³ In the chick utricle, following the apical constriction of dead HCs, the SCs engulf and phagocytose the remaining HC corpse.¹⁵ Additional data from the chick indicate that the ototoxic drug cisplatin impairs some SC functions, including regeneration of HCs or clearance of HC debris.²⁴ We hypothesized that SCs would have significant phagocytic activity in the mature mammalian inner ear, and that cisplatin would impair this activity. To examine these dynamic processes, we live-imaged SC phagocytic activity in the adult mouse utricle and compared the SC responses with HC stress and death caused by aminoglycosides versus cisplatin.     

Artesunate induces necrotic cell death in schwannoma cells

Established as a potent anti-malaria medicine, artemisinin-based drugs have been suggested to have anti-tumour activity in some cancers. Although the mechanism is poorly understood, it has been suggested that artemisinin induces apoptotic cell death. Here, we show that the artemisinin analogue artesunate (ART) effectively induces cell death in RT4 schwannoma cells and human primary schwannoma cells. Interestingly, our data indicate for first time that the cell death induced by ART is largely dependent on necroptosis. ART appears to inhibit autophagy, which may also contribute to the cell death. Our data in human schwannoma cells show that ART can be combined with the autophagy inhibitor chloroquine (CQ) to potentiate the cell death. Thus, this study suggests that artemisinin-based drugs may be used in certain tumours where cells are necroptosis competent, and the drugs may act in synergy with apoptosis inducers or autophagy inhibitors to enhance their anti-tumour activity.Artemisinin, a sesquiterpene lactone isolated from the Chinese herb Artemisia annua L., has profound activity against malaria.¹ Artemisinin contains an endoperoxide moiety that reacts with iron to produce toxic reactive oxygen species (ROS). When malaria parasite (Plasmodia) consumes iron-rich haemoglobin within its acidic food vacuole in erythrocytes, the exposure of artemisinin to haem-derived iron results in lethal ROS production that exerts fatal toxicity to the parasite.² Therefore, artemisinin, its water-soluble derivative artesunate (ART) and other analogues are potent in killing malarial parasites.^1,3Cancer cells contain substantial free iron, resulting from their higher-rate iron uptake via transferrin receptors compared with normal cells. Therefore, artemisinin-based drugs such as ART possess selective toxicity to cancer cells.^{4, 5, 6} Importantly, the pharmacokinetics and tolerance of ART as an anti-malarial drug have been well documented, with clinical studies showing excellent safety. Collectively, these properties make artemisinin-based compounds attractive drug candidates for cancer chemotherapy. Artemisinin and ART have been shown to induce cell death in multiple cancer cells, including colon, breast, ovarian, prostate,⁷ pancreatic⁸ and leukaemia⁹ cancer cells. Preliminary in vivo experiments also indicate the therapeutic potential for these drugs as anti-cancer treatments. In animal models, artemisinin or ART has shown promising results in Kaposi Sarcoma,¹⁰ pancreatic cancer¹¹ and hepatoma,¹² while compassionate use of ART in uveal melanoma patients fortifies standard chemotherapy potential for the patients.¹³ Currently, ART is on clinical trial for breast cancer treatment (ClinicalTrials.gov ID: {"type":"clinical-trial","attrs":{"text":"NCT00764036","term_id":"NCT00764036"}}NCT00764036).Programmed cell death (PCD) is one of the critical terminal paths for the cells of metazoans. Among PCD, apoptosis has been well studied and it is known that caspase activation is essential in this process.¹⁴ In addition to apoptosis, necroptosis is another form of PCD. The RIP1-RIP3 complex highlights the signals that regulate necroptosis.^{15, 16, 17} Artemisinin derivatives, mostly ART, have been suggested to lead to apoptosis via ROS production in cancer cells. Efforts have been focused on ROS-mediated mitochondrial apoptosis,^9,18,19 and DNA damage²⁰ in cancer cells. Recent data suggest that artemisinin and its derivatives may induce cell death or inhibit proliferation through diverse mechanisms in different cell types. Artemisinin or its analogues were shown to inhibit cell proliferation in multiple cancer cells by regulating cell-cycle arrest^{21, 22, 23} or inducing apoptosis.^24,25 Nevertheless, the detailed molecular mechanisms underlying artemisinin or ART-induced cell death are poorly understood, thus need to be further addressed.Neurofibromatosis 2 (NF2) is caused by the loss of NF2 gene encoding Merlin protein. NF2 gene mutations cause the low grade tumour syndrome, composed of schwannomas, meningiomas and ependymomas.²⁶ All spontaneous schwannomas, the majority of meningiomas and a third of ependymomas are caused by NF2 gene mutations. Notably, approximately 10% of intracranial tumours are schwannomas.²⁷ Interestingly, NF2 gene mutations are also found in a variety of cancers, including breast cancer and mesothelioma.^{28, 29, 30} The low grade tumours caused by NF2 gene mutations do not respond well to current cancer drugs and therapy is restricted to surgery and radiosurgery.²⁶ Therefore, there is a need for drug treatment of the diseases. Here, we show that ART sufficiently induced schwannoma cell death in both RT4 cell line and human primary cells. Importantly, we show, for the first time, that ART-induced cell death is largely dependent on necroptosis. Our data suggest that ART has great potential in schwannoma chemotherapy, especially when used in synergy with an apoptosis-inducing drug and/or an autophagy-inhibitory drug.     

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death'' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death'' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.Defining life and death is more problematic than one would guess. In 1838, the work of several scientists including Matthias Jakob Schleiden, Theodor Schwann and Rudolf Carl Virchow culminated in the so-called ‘cell theory'', postulating that: (1) all living organisms are composed of one or more cells; (2) the cell is the basic unit of life; and (3) all cells arise from pre-existing, living cells.¹ Only a few decades later (in 1885), Walter Flemming described for the first time some of the morphologic features that have been largely (but often inappropriately) used to define apoptosis throughout the past four decades.^{2, 3, 4}A corollary of the cell theory is that viruses do not constitute bona fide living organisms.⁵ However, the discovery that the giant Acanthamoeba polyphaga mimivirus can itself be infected by other viral species has casted doubts on this point.^{6, 7, 8} Thus, the features that underlie the distinction between a living and an inert entity remain a matter of debate. Along similar lines, defining the transition between an organism''s life and death is complex, even when the organism under consideration is the basic unit of life, a cell. From a conceptual standpoint, cell death can obviously be defined as the permanent degeneration of vital cellular functions. Pragmatically speaking, however, the precise boundary between a reversible alteration in homeostasis and an irreversible loss of cellular activities appears to be virtually impossible to identify. To circumvent this issue, the Nomenclature Committee on Cell Death (NCCD) previously proposed three criteria for the identification of dead cells: (1) the permanent loss of the barrier function of the plasma membrane; (2) the breakdown of cells into discrete fragments, which are commonly referred to as apoptotic bodies; or (3) the engulfment of cells by professional phagocytes or other cells endowed with phagocytic activity.^{9, 10, 11}However, the fact that a cell is engulfed by another via phagocytosis does not imply that the cell-containing phagosome fuses with a lysosome and that the phagosomal cargo is degraded by lysosomal hydrolases.^{12, 13, 14} Indeed, it has been reported that engulfed cells can be released from phagosomes as they preserve their viability, at least under some circumstances.¹⁵ Thus, the NCCD recommends here to consider as dead only cells that either exhibit irreversible plasma membrane permeabilization or have undergone complete fragmentation. A compendium of techniques that can be used to quantify these two markers of end-stage cell death in vitro and in vivo goes beyond the scope of this review and can be found in several recent articles.^{16, 17, 18, 19, 20, 21, 22, 23, 24, 25}Importantly, cell death instances can be operationally classified into two broad, mutually exclusive categories: ‘accidental'' and ‘regulated''. Accidental cell death (ACD) is caused by severe insults, including physical (e.g., elevated temperatures or high pressures), chemical (e.g., potent detergents or extreme variations in pH) and mechanical (e.g., shearing) stimuli, is virtually immediate and is insensitive to pharmacologic or genetic interventions of any kind. The NCCD thinks that this reflects the structural disassembly of cells exposed to very harsh physicochemical conditions, which does not involve a specific molecular machinery. Although ACD can occur in vivo, for instance as a result of burns or traumatic injuries, it cannot be prevented or modulated and hence does not constitute a direct target for therapeutic interventions.^{23, 26, 27, 28} Nonetheless, cells exposed to extreme physicochemical or mechanical insults die while releasing elevated amounts of damage-associated molecular patterns (DAMPs), that is, endogenous molecules with immunomodulatory (and sometimes cytotoxic) activity. Some DAMPs can indeed propagate an unwarranted cytotoxic response (directly or upon the involvement of innate immune effectors) that promotes the demise of local cells surviving the primary insult.^{16, 19, 29, 30, 31} Intercepting DAMPs or blocking DAMP-ignited signaling pathways may mediate beneficial effects in a wide array of diseases involving accidental (as well as regulated) instances of cell death.^{19, 32}At odds with its accidental counterpart, regulated cell death (RCD) involves a genetically encoded molecular machinery.^{9, 33} Thus, the course of RCD can be altered by means of pharmacologic and/or genetic interventions targeting the key components of such a machinery. Moreover, RCD often occurs in a relatively delayed manner and is initiated in the context of adaptive responses that (unsuccessfully) attempt to restore cellular homeostasis.^{34, 35, 36, 37, 38} Depending on the initiating stimulus, such responses can preferentially involve an organelle, such as the reticular unfolded protein response, or operate at a cell-wide level, such as macroautophagy (hereafter referred to as autophagy).^{39, 40, 41, 42, 43, 44} Thus, while ACD is completely unpreventable, RCD can be modulated (at least to some extent, see below) not only by inhibiting the transduction of lethal signals but also by improving the capacity of cells to mount adaptive responses to stress.^{45, 46, 47, 48, 49, 50} Importantly, RCD occurs not only as a consequence of microenvironmental perturbations but also in the context of (post-)embryonic development, tissue homeostasis and immune responses.^{51, 52, 53, 54} Such completely physiologic instances of RCD are generally referred to as ‘programmed cell death'' (PCD) (Figure 1).^{9, 33}Open in a separate windowFigure 1Types of cell death. Cells exposed to extreme physical, chemical or mechanical stimuli succumb in a completely uncontrollable manner, reflecting the immediate loss of structural integrity. We refer to such instances of cellular demise with the term ‘accidental cell death'' (ACD). Alternatively, cell death can be initiated by a genetically encoded machinery. The course of such ‘regulated cell death'' (RCD) variants can be influenced, at least to some extent, by specific pharmacologic or genetic interventions. The term ‘programmed cell death'' (PCD) is used to indicate RCD instances that occur as part of a developmental program or to preserve physiologic adult tissue homeostasisFor the purpose of this discussion, it is useful to keep in mind the distinction that is currently made between the initiation of RCD and its execution. The term execution is generally used to indicate the ensemble of biochemical processes that truly cause the cellular demise. Conversely, initiation is commonly used to refer to the signal transduction events that activate executioner mechanisms. Thus, the activation of caspase-8 (CASP8) in the course of FAS ligand (FASL)-triggered apoptosis is widely considered as an initiator mechanism, whereas the consequent activation of caspase-3 (CASP3) is categorized as an executioner mechanism (see below).^{51, 55, 56, 57}Here, the NCCD formulates a set of recommendations to discriminate between essential and accessory aspects of RCD, that is, between those that etiologically mediate its occurrence and those that change its kinetics or morphologic and biochemical manifestations.     

The plant metacaspase AtMC1 in pathogen-triggered programmed cell death and aging: functional linkage with autophagy

Autophagy is a major nutrient recycling mechanism in plants. However, its functional connection with programmed cell death (PCD) is a topic of active debate and remains not well understood. Our previous studies established the plant metacaspase AtMC1 as a positive regulator of pathogen-triggered PCD. Here, we explored the linkage between plant autophagy and AtMC1 function in the context of pathogen-triggered PCD and aging. We observed that autophagy acts as a positive regulator of pathogen-triggered PCD in a parallel pathway to AtMC1. In addition, we unveiled an additional, pro-survival homeostatic function of AtMC1 in aging plants that acts in parallel to a similar pro-survival function of autophagy. This novel pro-survival role of AtMC1 may be functionally related to its prodomain-mediated aggregate localization and potential clearance, in agreement with recent findings using the single budding yeast metacaspase YCA1. We propose a unifying model whereby autophagy and AtMC1 are part of parallel pathways, both positively regulating HR cell death in young plants, when these functions are not masked by the cumulative stresses of aging, and negatively regulating senescence in older plants.An emerging theme in cell death research is that cellular processes thought to be regulated by linear signaling pathways are, in fact, complex. Autophagy, initially considered merely a nutrient recycling mechanism necessary for cellular homeostasis, was recently shown to regulate cell death, mechanistically interacting with components that control apoptosis. Deficient autophagy can result in apoptosis^{1, 2, 3} and autophagy hyper-activation can also lead to programmed cell death (PCD).⁴ In addition, the pro-survival function of autophagy is mediated by apoptosis inhibition and apoptosis mediates autophagy, although this cross-regulation is not fully understood.⁵In plants, autophagy can also have both pro-survival and pro-death functions. Autophagy-deficient plants exhibit accelerated senescence,^{6, 7, 8} starvation-induced chlorosis,^{6, 7, 9} hypersensitivity to oxidative stress¹⁰ and endoplasmic reticulum stress.¹¹ Further, autophagy-deficient plants cannot limit the spread of cell death after infection with tissue-destructive microbial infections.^{12, 13} The plant phytohormone salicylic acid (SA) mediates most of these phenotypes.⁸ Autophagy has an essential, pro-survival role in situations where there is an increasing load of damaged proteins and organelles that need to be eliminated, that is, during aging or stress. Autophagy has an opposing, pro-death role during developmentally regulated cell death^{14, 15} or during the pathogen-triggered hypersensitive response PCD (hereafter, HR) that occurs locally at the site of attempted pathogen attack.^{16, 17} The dual pro-death/pro-survival functions of plant autophagy remain a topic of active debate.Also under scrutiny are possible novel functions of caspases and caspase-like proteins as central regulators of pro-survival processes. Caspases were originally defined as executioners of PCD in animals, but increasing evidence indicates that several caspases have non-apoptotic regulatory roles in cellular differentiation, motility and in the mammalian immune system.^{18, 19, 20}Yeast, protozoa and plants do not have canonical caspases, despite the occurrence of morphologically heterogeneous PCDs.²¹ More than a decade ago, distant caspase homologs termed metacaspases were identified in these organisms using structural homology searches.²² Metacaspases were classified into type I or type II metacaspases based on the presence or absence of an N-terminal prodomain, reminiscent of the classification in animals into initiator/inflammatory or executioner caspases, respectively. Despite the architectural analogy between caspases and metacaspases, differences in their structure, function, activation and mode of action exist.^{23, 24, 25}Metacaspases mediate PCD in yeast,^{26, 27, 28, 29, 30, 31} leishmania,^{32, 33} trypanosoma³⁴ and plants.²⁴ We demonstrated that two type I metacaspases, AtMC1 and AtMC2, antagonistically regulate HR in Arabidopsis thaliana.³⁵ Our work showed that AtMC1 is a positive regulator of HR and that this function is mediated by its catalytic activity and negatively regulated by the AtMC1 N-terminal prodomain. AtMC2 antagonizes AtMC1-mediated HR.Besides AtMC2, new examples of metacaspases with a pro-life/non-PCD role are emerging. Protozoan metacaspases are involved in cell cycle dynamics^{34, 36, 37, 38} and cell proliferation.³⁹ The yeast metacaspase Yca1 alters cell cycle dynamics⁴⁰ and interestingly, is required for clearance of insoluble protein aggregates, thus contributing to yeast fitness.⁴¹Here, we explore the linkage between plant autophagy and AtMC1 function in the context of pathogen-triggered HR and aging. Our data support a model wherein autophagy and AtMC1 are part of parallel pathways, both positively regulating HR cell death in young plants and negatively regulating senescence in older plants.     

HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death

Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90''s previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3.Necroptosis is an inflammatory, caspase-independent form of regulated cell death characterised by loss of cellular membrane integrity and release of cytoplasmic contents.¹ It is believed to have evolved as a defence mechanism against viruses;^{2, 3} however, there is increasing evidence that deregulated necroptosis has a role in the pathogenesis of a range of inflammatory, autoimmune and neurodegenerative diseases.^{4, 5, 6, 7, 8} Reduced capacity to undergo necroptosis has been correlated to increased aggressiveness of cancers;^{9, 10} and therapeutic initiation of necroptosis is currently being investigated as a cancer therapy.^{11, 12} Additionally, there is emerging evidence that the necroptotic signalling pathway has a general role in the modulation of inflammation.^{13, 14, 15, 16, 17} As such, unravelling the molecular events governing necroptosis, and potential avenues for therapeutic intervention, is of enormous interest.Necroptosis is initiated through activation of death receptors, such as Tumour Necrosis Factor Receptor 1 (TNFR1), or through microbial activation of pattern recognition receptors, such as Toll-like receptors or intracellular viral DNA sensors.^{3, 18, 19, 20} Receptor ligation initiates a signalling cascade, whereby Receptor Interacting Protein Kinase (RIPK)-3 oligomerises and is phosphorylated, a process known to be regulated by association with other effectors, such as the protein kinase RIPK1, TIR-domain-containing adapter-inducing IFN-β (TRIF), or DNA-dependent activator of IFN regulatory factors (DAI), via their RIP Homotypic Interaction Motifs (RHIMs).^{2, 21, 22} Once activated, RIPK3 phosphorylates the pseudokinase domain of Mixed Lineage Kinase domain-Like (MLKL), the most downstream known obligate effector of the necroptotic signalling pathway, to induce its activation.^{23, 24} MLKL phosphorylation is thought to trigger a molecular switch,^{25, 26, 27} leading to the unleashing of the N-terminal executioner four-helix bundle (4HB) domain,²⁸ MLKL oligomerisation and translocation to cellular membranes where cell death occurs via an incompletely-understood mechanism.^{28, 29, 30}Molecular chaperones have an integral role in modulating both the structure and function of proteins. One such chaperone is heat-shock protein 90 (HSP90), which interacts with a diverse group of protein ‘clients'', the largest group comprising the kinases and pseudokinases, with 50% of the human kinome estimated to interact with HSP90.³¹ These interactions are dependent on the recognition of the kinase or pseudokinase domain by the HSP90 co-chaperone Cdc37, which enables HSP90 to confer protein stabilisation, assist in late-stage folding and conformational modifications, and mediate intracellular transport.^{32, 33, 34, 35}It has already been demonstrated that the necroptotic pathway is subject to modulation by HSP90. RIPK1 is well established as an HSP90 client protein, with a number of studies finding HSP90 inhibition affects both the stability and function of RIPK1 and promotes an apoptotic phenotype.^{36, 37, 38, 39, 40, 41} More recently, RIPK3 was also identified as an HSP90 client.^{2, 42, 43} Surprisingly, HSP90 inhibition did not markedly impact RIPK3 abundance or stability, but rather was essential for RIPK3''s necroptotic functions, such as phosphorylation of MLKL.⁴² However, whether MLKL itself is a client of HSP90 has not been investigated.In this study, using a phenotypic screen for small-molecule inhibitors of MLKL-driven cell death, we identified HSP90 as a modulator of necroptosis that functions on, or downstream of, the terminal effector, MLKL. HSP90 inhibition did not markedly reduce levels of MLKL in human U937 or mouse dermal fibroblasts, suggesting instead that HSP90 has an active role in governing MLKL-mediated cell death. This idea is supported by our finding that cell death driven by the S345D activated mutant of MLKL in Ripk3-deficient fibroblasts in the absence of necroptotic stimuli was suppressed by three distinct chemical classes of HSP90 inhibitor, but MLKL abundance was not impacted by HSP90 inhibition. Although our data indicate that MLKL binds HSP90 weakly or transiently, HSP90 activity was essential for the assembly of MLKL into high molecular weight complexes and the membrane translocation known to precede cell death. These findings suggest an expanded role for HSP90 in regulating necroptosis, and further our understanding of the mechanisms controlling MLKL-mediated cell death.     

Nuclear ULK1 promotes cell death in response to oxidative stress through PARP1

Reactive oxygen species (ROS) may cause cellular damage and oxidative stress-induced cell death. Autophagy, an evolutionarily conserved intracellular catabolic process, is executed by autophagy (ATG) proteins, including the autophagy initiation kinase Unc-51-like kinase (ULK1)/ATG1. Although autophagy has been implicated to have both cytoprotective and cytotoxic roles in the response to ROS, the role of individual ATG proteins, including ULK1, remains poorly characterized. In this study, we demonstrate that ULK1 sensitizes cells to necrotic cell death induced by hydrogen peroxide (H₂O₂). Moreover, we demonstrate that ULK1 localizes to the nucleus and regulates the activity of the DNA damage repair protein poly (ADP-ribose) polymerase 1 (PARP1) in a kinase-dependent manner. By enhancing PARP1 activity, ULK1 contributes to ATP depletion and death of H₂O₂-treated cells. Our study provides the first evidence of an autophagy-independent prodeath role for nuclear ULK1 in response to ROS-induced damage. On the basis of our data, we propose that the subcellular distribution of ULK1 has an important role in deciding whether a cell lives or dies on exposure to adverse environmental or intracellular conditions.Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide (H₂O₂), are formed by the incomplete reduction of oxygen during oxidative phosphorylation and other enzymatic processes. ROS are signaling molecules that regulate cell proliferation, differentiation, and survival.^{1, 2, 3} Accumulation of ROS (i.e., oxidative stress) on exposure to xenobiotic agents or environmental toxins can cause cellular damage and death via apoptotic or nonapoptotic pathways.^{4, 5, 6} Oxidative stress-induced cellular damage and death have been implicated in aging, ischemia-reperfusion injury, inflammation, and the pathogenesis of diseases (e.g., neurodegeneration and cancer).⁷ Oxidative stress also contributes to the antitumor effects of many chemotherapeutic drugs, including camptothecin^{8, 9} and selenite.^{10, 11}Autophagy, an evolutionarily conserved intracellular catabolic process, involves lysosome-dependent degradation of superfluous and damaged cytosolic organelles and proteins.¹² It is typically upregulated under conditions of perceived stress and in response to cellular damage. The consequence of autophagy activation – whether cytoprotective or cytotoxic – appears to depend on the cell type and the nature and extent of stress. Although most studies indicate a cytoprotective role for autophagy, some evidence suggests that it contributes to cell death in response to oxidative stress.^{13, 14, 15, 16, 17} Studies have also indicated that autophagy may be suppressed in response to oxidative stress, thereby sensitizing certain cells to apoptosis.^{18, 19} Unc-51-like kinase/autophagy 1 (ULK1/ATG1) is a mammalian serine–threonine kinase that regulates flux through the autophagy pathway by activating the VPS34 PI(3) kinase complex and facilitating ATG9-dependent membrane recycling.²⁰ Results from two studies suggest that ULK1 expression is altered in response to oxidative stress, and that the corresponding effects on autophagy contribute to cell death.^{18, 21}For example, p53-mediated upregulation of ULK1 and increase in autophagy promote cell death in osteosarcoma cells exposed to sublethal doses of camptothecin,²¹ yet mutant p53-mediated suppression of ULK1 impairs autophagic flux and promotes apoptosis in selenite-treated NB4 cells.¹⁸ Here we investigated the role of ULK1 in cells exposed to H₂O₂.     

Dysferlin regulates cell membrane repair by facilitating injury-triggered acid sphingomyelinase secretion

Dysferlin deficiency compromises the repair of injured muscle, but the underlying cellular mechanism remains elusive. To study this phenomenon, we have developed mouse and human myoblast models for dysferlinopathy. These dysferlinopathic myoblasts undergo normal differentiation but have a deficit in their ability to repair focal injury to their cell membrane. Imaging cells undergoing repair showed that dysferlin-deficit decreased the number of lysosomes present at the cell membrane, resulting in a delay and reduction in injury-triggered lysosomal exocytosis. We find repair of injured cells does not involve formation of intracellular membrane patch through lysosome–lysosome fusion; instead, individual lysosomes fuse with the injured cell membrane, releasing acid sphingomyelinase (ASM). ASM secretion was reduced in injured dysferlinopathic cells, and acute treatment with sphingomyelinase restored the repair ability of dysferlinopathic myoblasts and myofibers. Our results provide the mechanism for dysferlin-mediated repair of skeletal muscle sarcolemma and identify ASM as a potential therapy for dysferlinopathy.Dysferlinopathy is a progressive muscle wasting disease, which is classified as limb-girdle muscular dystrophy type 2B (LGMD2B) or Miyoshi muscular dystrophy 1, based on its muscle involvement.^{1, 2} Dysferlin deficit leads to altered vesicle formation and trafficking,^{3, 4} poor repair of injured cell membranes,^{5, 6} and increased muscle inflammation.^{7, 8} Dysferlin contains C2 domains that are found in Ca²⁺-dependent membrane fusion proteins such as synaptotagmins.⁹ Thus, dysferlin is thought to regulate muscle function by regulating vesicle trafficking and fusion.^{10, 11, 12, 13} Dysferlin deficiency has also been implicated in conflicting reports regarding the fusion ability of dysferlinopathic myoblasts.^{4, 14, 15, 16} With such diverse roles for dysferlin, the mechanism through which dysferlin deficiency results in muscle pathology is unresolved. As skeletal muscle-specific re-expression of dysferlin rescues all dysferlinopathic pathologies,^{17, 18} myofiber repair has been suggested to be the unifying deficit underlying muscle pathology in dysferlinopathy.¹⁹ Repair of injured cell membranes requires subcellular compartments, which in mammalian cells include lysosomes,¹¹ enlargeosomes,²⁰ caveolae,²¹ dysferlin-containing vesicles,⁵ and mitochondria.²²Cells from muscular dystrophy patients that have normal dysferlin expression exhibit normal lysosome and enlargeosome exocytosis.²³ However, dysferlinopathic muscle cells exhibit enlarged LAMP2-positive lysosomes, reduced fusion of early endosomes, altered expression of proteins regulating late endosome/lysosome fusion, and reduced injury-triggered cell-surface levels of LAMP1.^{4, 11, 12} In non-muscle cells, lack of dysferlin reduces lysosomal exocytosis.²⁴ These findings implicate lysosomes in dysferlin-mediated muscle cell membrane repair. In one model for lysosome-mediated cell membrane repair, Ca²⁺ triggers vesicle–vesicle fusion near the site of injury, forming ‘membrane patch'', which fuses to repair the wounded cell membrane.^{25, 26, 27, 28} In another model, lysosome exocytosis following cell membrane injury by pore-forming toxins leads to secretion of the lysosomal enzyme acid sphingomyelinase (ASM), which causes endocytosis of pores in the damaged cell membranes.^{21, 29, 30} Both these models have been suggested to be involved in the repair of injured muscle cells.^{21, 28}To examine the muscle cell pathology in dysferlinopathy, we have developed dysferlinopathic mouse and human models. Use of these models shows that a lack of dysferlin does not alter myogenic differentiation but causes poor repair of even undifferentiated muscle cells. We show that dysferlin is required for tethering lysosomes to the cell membrane. Fewer lysosomes at the cell membrane in dysferlinopathic cells results in slow and reduced lysosome exocytosis following injury. This reduction in exocytosis reduces injury-triggered ASM secretion, which is responsible for the poor repair of dysferlinopathic muscle cells. Extracellular sphingomyelinase (SM) fully rescues the repair deficit in dysferlinopathic cells and mouse myofibers, offering a potential drug-based therapy for dysferlinopathy.     

Thrombospondin-1 signaling through CD47 inhibits cell cycle progression and induces senescence in endothelial cells

CD47 signaling in endothelial cells has been shown to suppress angiogenesis, but little is known about the link between CD47 and endothelial senescence. Herein, we demonstrate that the thrombospondin-1 (TSP1)-CD47 signaling pathway is a major mechanism for driving endothelial cell senescence. CD47 deficiency in endothelial cells significantly improved their angiogenic function and attenuated their replicative senescence. Lack of CD47 also suppresses activation of cell cycle inhibitors and upregulates the expression of cell cycle promoters, leading to increased cell cycle progression. Furthermore, TSP1 significantly accelerates replicative senescence and associated cell cycle arrest in a CD47-dependent manner. These findings demonstrate that TSP1-CD47 signaling is an important mechanism driving endothelial cell senescence. Thus, TSP1 and CD47 provide attractive molecular targets for treatment of aging-associated cardiovascular dysfunction and diseases involving endothelial dysregulation.Endothelial cell (EC) senescence is accompanied with vascular dysfunction, including arterial stiffening and remodeling,¹ impaired angiogenesis,^{2, 3} reduced endothelial repair capability and increased incidence of cardiovascular disease.^{4, 5, 6} Cellular senescence can occur in vivo or in vitro in response to various stressors,^{7, 8, 9, 10} leading to suppression of cell proliferation. EC senescence has been reported to contribute to the pathogenesis of age-associated vascular diseases, such as atherosclerosis.¹¹ Thus, further understanding the mechanisms of EC senescence may help to identify effective targets for antisenescence therapy and treatment aging-associated cardiovascular disorders.Previous studies have shown that the secreted matricellular protein thrombospondin-1 (TSP1) is as potent inhibitor of angiogenesis¹² and its antiangiogenic activity is mediated by its receptors, CD36^{13, 14} and CD47.^{15, 16} CD47 is a ubiquitously expressed transmembrane protein that serves as a ligand for signal regulatory protein-α and is a signaling receptor of TSP1. The TSP1-CD47 pathway has an important role in several fundamental cellular functions, including proliferation, apoptosis, inflammation and atherosclerotic response.¹⁷ Ligation of CD47 by TSP1 has been shown to inhibit nitric oxide (NO)/cGMP signaling in vascular cells, leading to suppression of angiogenic responses.¹⁶ Recently, it was reported that lack of CD47 expression in ECs may enable these cells to spontaneously gain characteristics of embryonic stem cells.¹⁸ However, the potential role of CD47 in regulation of EC senescence has not been well explored. The present study was initiated to determine the role and mechanisms of TSP1-CD47 signaling pathway in regulating cell cycle progression and replicative senescence of ECs.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.