Cristina Muñoz‐Pinedo

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.

The endoplasmic reticulum is an organelle with multiple functions. The synthesis of transmembrane proteins and proteins that are to be secreted occurs in this organelle. Many conditions that impose stress on cells, including hypoxia, starvation, infections and changes in secretory needs, challenge the folding capacity of the cell and promote endoplasmic reticulum stress. The cellular response involves the activation of sensors that transduce signaling cascades with the aim of restoring homeostasis. This is known as the unfolded protein response, which also intersects with the integrated stress response that reduces protein synthesis through inactivation of the initiation factor eIF2α. Central to the unfolded protein response are the sensors PERK, IRE1 and ATF6, as well as other signaling nodes such as c-Jun N-terminal kinase 1 (JNK) and the downstream transcription factors XBP1, ATF4 and CHOP. These proteins aim to restore homeostasis, but they can also induce cell death, which has been shown to occur by necroptosis and, more commonly, through the regulation of Bcl-2 family proteins (Bim, Noxa and Puma) that leads to mitochondrial apoptosis. In addition, endoplasmic reticulum stress and proteotoxic stress have been shown to induce TRAIL receptors and activation of caspase-8. Endoplasmic reticulum stress is a common feature in the pathology of numerous diseases because it plays a role in neurodegeneration, stroke, cancer, metabolic diseases and inflammation. Understanding how cells react to endoplasmic reticulum stress can accelerate discovery of drugs against these diseases.

The endoplasmic reticulum ( ER ) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one‐third of cellular proteins, and is thus inextricably linked to the maintenance of cellular homeostasis and the fine balance between health and disease. Specific ER stress signalling pathways, collectively known as the unfolded protein response ( UPR ), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand and the UPR initially aims to restore ER homeostasis and normal cellular functions. However, if this fails, then the UPR triggers cell death. In this review, we provide a UPR signalling‐centric view of ER functions, from the ER 's discovery to the latest advancements in the understanding of ER and UPR biology. Our review provides a synthesis of intracellular ER signalling revolving around proteostasis and the UPR , its impact on other organelles and cellular behaviour, its multifaceted and dynamic response to stress and its role in physiology, before finally exploring the potential exploitation of this knowledge to tackle unresolved biological questions and address unmet biomedical needs. Thus, we provide an integrated and global view of existing literature on ER signalling pathways and their use for therapeutic purposes.

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis.

Cellular metabolism influences life and death decisions. An emerging theme in cancer biology is that metabolic regulation is intricately linked to cancer progression. In part, this is due to the fact that proliferation is tightly regulated by availability of nutrients. Mitogenic signals promote nutrient uptake and synthesis of DNA, RNA, proteins and lipids. Therefore, it seems straight-forward that oncogenes, that often promote proliferation, also promote metabolic changes. In this review we summarize our current understanding of how ‘metabolic transformation’ is linked to oncogenic transformation, and why inhibition of metabolism may prove a cancer′s ‘Achilles’ heel’. On one hand, mutation of metabolic enzymes and metabolic stress sensors confers synthetic lethality with inhibitors of metabolism. On the other hand, hyperactivation of oncogenic pathways makes tumors more susceptible to metabolic inhibition. Conversely, an adequate nutrient supply and active metabolism regulates Bcl-2 family proteins and inhibits susceptibility to apoptosis. Here, we provide an overview of the metabolic pathways that represent anti-cancer targets and the cell death pathways engaged by metabolic inhibitors. Additionally, we will detail the similarities between metabolism of cancer cells and metabolism of proliferating cells.

The release of mitochondrial intermembrane space proteins to the cytosol is a key event during apoptosis. We used in situ fluorescent labeling of proteins tagged with a short tetracysteine-containing sequence to follow the release of Smac, Omi, adenylate kinase-2, cytochrome c , and apoptosis-inducing factor (AIF) during apoptosis and compared the release with that of cytochrome c tagged with GFP in individual cells observed over time. We observed a caspase-independent, simultaneous release of cytochrome c , Smac, Omi, and adenylate kinase-2. Although AIF release also was caspase-independent and commenced with that of the other proteins, it proceeded much more slowly and incompletely from mitochondria, perhaps because of a requirement for a secondary event. These results suggest that these proteins are released through the same mitochondrial pore and that apoptosis may not be regulated through a selective release of individual mitochondrial proteins. The timing and extent of AIF release makes it unlikely that it is involved in the induction of apoptosis, either upstream or downstream of mitochondrial outer membrane permeabilization.

Tumors show an increased rate of glucose uptake and utilization. For this reason, glucose analogs are used to visualize tumors by the positron emission tomography technique, and inhibitors of glycolytic metabolism are being tested in clinical trials. Upregulation of glycolysis confers several advantages to tumor cells: it promotes tumor growth and has also been shown to interfere with cell death at multiple levels. Enforcement of glycolysis inhibits apoptosis induced by cytokine deprivation. Conversely, antiglycolytic agents enhance cell death induced by radio- and chemotherapy. Synergistic effects are likely due to regulation of the apoptotic machinery, as glucose regulates activation and levels of proapoptotic BH3-only proteins such as Bim, Bad, Puma and Noxa, as well as the antiapoptotic Bcl-2 family of proteins. Moreover, inhibition of glucose metabolism sensitizes cells to death ligands. Glucose deprivation and antiglycolytic drugs induce tumor cell death, which can proceed through necrosis or through mitochondrial or caspase-8-mediated apoptosis. We will discuss how oncogenic pathways involved in metabolic stress signaling, such as p53, AMPK (adenosine monophosphate-activated protein kinase) and Akt/mTOR (mammalian target of rapamycin), influence sensitivity to inhibition of glucose metabolism. Finally, we will analyze the rationale for the use of antiglycolytic inhibitors in the clinic, either as single agents or as a part of combination therapies.

Similar to most if not all pro-apoptotic members of the Bcl-2 family, Bid (and its truncated product t-Bid) triggers cell death via mitochondrial membrane permeabilization (MMP). This effect can be monitored in intact cells, upon microinjection of recombinant Bid protein into the cytoplasm, as well as in purified mitochondria, upon addition of Bid protein. Here we show that Bid-induced MMP can be inhibited, both in cells and in the cell-free system, by three pharmacological inhibitors of the permeability transition pore complex (PTPC), namely cyclosporin A, N-methyl-4-Val-cyclosporin A, and bongkrekic acid (a ligand of the adenine nucleotide translocase, ANT, one of the PTPC components). Bid effects on synthetic membranes were studied either in proteoliposomes or in synthetic bilayers subjected to electrophysiological measurements. Full length Bid preferentially permeabilizes membranes and induces the formation of large conductance channels at neutral pH, when added to liposomes or bilayers containing both purified ANT and Bax, yet has no or little effect combined with ANT or Bax alone. t-Bid acts on membranes containing ANT alone with the same efficiency as on those containing both ANT and Bax. These results suggest that the proapoptotic effects of Bid are mediated, at least in part, by its functional interaction with ANT, one of the major components of PTPC.

During apoptosis, mitochondrial outer membrane permeabilization (MOMP) is often a point-of-no-return; death can proceed even if caspase activation is disrupted. However, under certain conditions, resistance to MOMP-dependent, caspase-independent cell death is observed. Mitochondrial recovery represents a key process in this survival. Live cell imaging revealed that during apoptosis not all mitochondria in a cell necessarily undergo MOMP. This incomplete MOMP (iMOMP) was observed in response to various stimuli and in different cell types regardless of caspase activity. Importantly, the presence of intact mitochondria correlated with cellular recovery following MOMP, provided that caspase activity was blocked. Such intact mitochondria underwent MOMP in response to treatment of cells with the Bcl-2 antagonist ABT-737, suggesting that the resistance of these mitochondria to MOMP lies at the point of Bax or Bak activation. Thus, iMOMP provides a critical source of intact mitochondria that permits cellular survival following MOMP.

Most cancer cells exhibit increased glycolysis for generation of their energy supply. This specificity could be used to preferentially kill these cells. In this study, we identified the signaling pathway initiated by glycolysis inhibition that results in sensitization to death receptor (DR)-induced apoptosis. We showed, in several human cancer cell lines (such as Jurkat, HeLa, U937), that glucose removal or the use of nonmetabolizable form of glucose (2-deoxyglucose) dramatically enhances apoptosis induced by Fas or by tumor necrosis factor-related apoptosis-inducing ligand. This sensitization is controlled through the adenosine monophosphate (AMP)-activated protein kinase (AMPK), which is the central energy-sensing system of the cell. We established the fact that AMPK is activated upon glycolysis block resulting in mammalian target of rapamycin (mTOR) inhibition leading to Mcl-1 decrease, but no other Bcl-2 anti-apoptotic members. Interestingly, we determined that, upon glycolysis inhibition, the AMPK–mTOR pathway controlled Mcl-1 levels neither through transcriptional nor through posttranslational mechanism but rather by controlling its translation. Therefore, our results show a novel mechanism for the sensitization to DR-induced apoptosis linking glucose metabolism to Mcl-1 downexpression. In addition, this study provides a rationale for the combined use of DR ligands with AMPK activators or mTOR inhibitors in the treatment of human cancers.

Abstract Cancer cells are markedly different from normal cells with regards to how their metabolic pathways are used to fuel cellular growth and survival. Two basic metabolites that exemplify these differences through increased uptake and altered metabolic usage are glucose and glutamine. These molecules can be catabolized to manufacture many of the building blocks required for active cell growth and proliferation. The alterations in the metabolic pathways necessary to sustain this growth have been linked to therapeutic resistance, a trait that is correlated with poor patient outcomes. By targeting the metabolic pathways that import, catabolize, and synthesize essential cellular components, drug-resistant cancer cells can often be resensitized to anticancer treatments. The specificity and efficacy of agents directed at the unique aspects of cancer metabolism are expected to be high; and may, when in used in combination with more traditional therapeutics, present a pathway to surmount resistance within tumors that no longer respond to current forms of treatment. Cancer Res; 73(9); 2709–17. ©2013 AACR.

Metabolic stress occurs frequently in tumors and in normal tissues undergoing transient ischemia. Nutrient deprivation triggers, among many potential cell death-inducing pathways, an endoplasmic reticulum (ER) stress response with the induction of the integrated stress response transcription factor ATF4. However, how this results in cell death remains unknown. Here we show that glucose deprivation triggered ER stress and induced the unfolded protein response transcription factors ATF4 and CHOP. This was associated with the nontranscriptional accumulation of TRAIL receptor 1 (TRAIL-R1) (DR4) and with the ATF4-mediated, CHOP-independent induction of TRAIL-R2 (DR5), suggesting that cell death in this context may involve death receptor signaling. Consistent with this, the ablation of TRAIL-R1, TRAIL-R2, FADD, Bid, and caspase-8 attenuated cell death, although the downregulation of TRAIL did not, suggesting ligand-independent activation of TRAIL receptors. These data indicate that stress triggered by glucose deprivation promotes the ATF4-dependent upregulation of TRAIL-R2/DR5 and TRAIL receptor-mediated cell death.

Cell proliferation requires the coordination of multiple signaling pathways as well as the provision of metabolic substrates. Nutrients are required to generate such building blocks and their form of utilization differs to significant extents between malignant tissues and their nontransformed counterparts. Thus, oncogenes and tumor suppressor genes regulate the proliferation of cancer cells also by controlling their metabolism. Here, we discuss the central anabolic functions of the signaling pathways emanating from mammalian target of rapamycin, MYC, and hypoxia-inducible factor-1. Moreover, we analyze how oncogenic proteins like phosphoinositide-3-kinase, AKT, and RAS, tumor suppressors such as phosphatase and tensin homolog, retinoblastoma, and p53, as well as other factors associated with the proliferation or survival of cancer cells, such as NF-κB, regulate cellular metabolism.

ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased DDIT3 and TRIB3 mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer.Abbreviations: 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ9-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response.

Cellular starvation is typically a consequence of tissue injury that disrupts the local blood supply but can also occur where cell populations outgrow the local vasculature, as observed in solid tumors. Cells react to nutrient deprivation by adapting their metabolism, or, if starvation is prolonged, it can result in cell death. Cell starvation also triggers adaptive responses, like angiogenesis, that promote tissue reorganization and repair, but other adaptive responses and their mediators are still poorly characterized. To explore this issue, we analyzed secretomes from glucose-deprived cells, which revealed up-regulation of multiple cytokines and chemokines, including IL-6 and IL-8, in response to starvation stress. Starvation-induced cytokines were cell type-dependent, and they were also released from primary epithelial cells. Most cytokines were up-regulated in a manner dependent on NF-κB and the transcription factor of the integrated stress response ATF4, which bound directly to the IL-8 promoter. Furthermore, glutamine deprivation, as well as the antimetabolic drugs 2-deoxyglucose and metformin, also promoted the release of IL-6 and IL-8. Finally, some of the factors released from starved cells induced chemotaxis of B cells, macrophages, and neutrophils, suggesting that nutrient deprivation in the tumor environment can serve as an initiator of tumor inflammation.

Native cytochrome c (cyt c ) has a compact tertiary structure with a hexacoordinated heme iron and functions in electron transport in mitochondria and apoptosis in the cytoplasm. However, the possibility that protein modifications confer additional functions to cyt c has not been explored. Disruption of methionine 80 (M80)-Fe ligation of cyt c under nitrative stress has been reported. To model this alteration and determine if it confers new properties to cyt c , a cyt c mutant (M80A) was constitutively expressed in cells. M80A-cyt c has increased peroxidase activity and is spontaneously released from mitochondria, translocating to the cytoplasm and nucleus in the absence of apoptosis. Moreover, M80A models endogenously nitrated cyt c because nitration of WT-cyt c is associated with its translocation to the cytoplasm and nucleus. Further, M80A cyt c may up-regulate protective responses to nitrative stress. Our findings raise the possibility that endogenous protein modifications that disrupt the M80-Fe ligation (such as tyrosine nitration) stimulate nuclear translocation and confer new functions to cyt c in nonapoptotic cells.

Programmed Cell Death is essential for the life cycle of many organisms. Cell death in multicellular organisms can occur as a consequence of massive damage (necrosis) or in a controlled form, through engagement of diverse biochemical programs. The best well known form of programmed cell death is apoptosis. Apoptosis occurs in animals as a consequence of a variety of stimuli including stress and social signals and it plays essential roles in morphogenesis and immune defense. The machinery of apoptosis is well conserved among animals and it is composed of caspases (the proteases which execute cell death), adapter proteins (caspase activators), Bcl-2 family proteins and Inhibitor of Apoptosis Proteins (IAPs). We will describe in this chapter the main apoptotic pathways in animals: the extrinsic (death receptor-mediated), the intrinsic/mitochondrial and the Granzyme B pathway. Other forms of non-apoptotic Programmed Cell Death which occur in animals will also be discussed. We will summarize the current knowledge about apoptotic-like and other forms of cell death in other organisms such as plants and protists. Additionally, we will discuss the hypothesis that apoptosis originated as part of a host defense mechanism. We will explore the similarities between the protein complexes which mediate apoptosis (apoptosomes) and complexes involved in immunity: inflammasomes. Additional functions of apoptotic proteins related to immune function will be summarized, in an effort to explore the evolutionary origins of cell death.

Alveolar and embryonal rhabdomyosarcomas are childhood tumors that do not respond well to current chemotherapies. Here, we report that the glycolytic inhibitor 2-deoxyglucose (2-DG) can efficiently promote cell death in alveolar, but not embryonal, rhabdomyosarcoma cell lines. Notably, 2-DG also induced cell differentiation accompanied by downregulation of PAX3/FOXO1a, the chromosome translocation-encoded fusion protein that is a central oncogenic driver in this disease. Cell death triggered by 2-DG was associated with its ability to activate Bax and Bak. Overexpression of the antiapoptotic Bcl-2 homologues Bcl-x(L) and Mcl-1 prevented apoptosis, indicating that cell death proceeds through the mitochondrial pathway. Mechanistic investigations indicated that Mcl-1 downregulation and Noxa upregulation were critical for 2-DG-induced apoptosis. In addition, 2-DG promoted eIF2α phosphorylation and inactivation of the mTOR pathway. Mcl-1 loss and cell death were prevented by downregulation of the endoplasmic reticulum (ER) stress-induced protein ATF4 and by incubating cells in the presence of mannose, which reverted 2-DG-induced ER stress but not ATP depletion. Thus, energetic stresses created by 2-DG were not the primary cause of cell death. Together, our findings suggest that glycolysis inhibitors such as 2-DG may be highly effective in treating alveolar rhabdomyosarcoma and that Noxa could offer a prognostic marker to monitor the efficacy of such agents.

A common process associated with oxidative stress and severe mitochondrial impairment is the opening of the mitochondrial permeability transition pore, as described in many neurodegenerative diseases. Thus, inhibition of mitochondrial permeability transition pore opening represents a potential target for inhibiting mitochondrial-driven cell death. Among the mitochondrial permeability transition pore components, cyclophilin D is the most studied and has been found increased under pathological conditions. Here, we have used in vitro and in vivo models of X-linked adrenoleukodystrophy to investigate the relationship between the mitochondrial permeability transition pore opening and redox homeostasis. X-linked adrenoleukodystrophy is a neurodegenerative condition caused by loss of function of the peroxisomal ABCD1 transporter, in which oxidative stress plays a pivotal role. In this study, we provide evidence of impaired mitochondrial metabolism in a peroxisomal disease, as fibroblasts in patients with X-linked adrenoleukodystrophy cannot survive when forced to rely on mitochondrial energy production, i.e. on incubation in galactose. Oxidative stress induced under galactose conditions leads to mitochondrial damage in the form of mitochondrial inner membrane potential dissipation, ATP drop and necrotic cell death, together with increased levels of oxidative modifications in cyclophilin D protein. Moreover, we show increased expression levels of cyclophilin D in the affected zones of brains in patients with adrenomyeloneuropathy, in spinal cord of a mouse model of X-linked adrenoleukodystrophy (Abcd1-null mice) and in fibroblasts from patients with X-linked adrenoleukodystrophy. Notably, treatment with antioxidants rescues mitochondrial damage markers in fibroblasts from patients with X-linked adrenoleukodystrophy, including cyclophilin D oxidative modifications, and reverses cyclophilin D induction in vitro and in vivo. These findings provide mechanistic insight into the beneficial effects of antioxidants in neurodegenerative and non-neurodegenerative cyclophilin D-dependent disorders.

Article13 May 2020Open Access Source DataTransparent process Tumors defective in homologous recombination rely on oxidative metabolism: relevance to treatments with PARP inhibitors Álvaro Lahiguera Álvaro Lahiguera Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Petra Hyroššová Petra Hyroššová Departament de Ciències Fisiològiques, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Agnès Figueras Agnès Figueras Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Diana Garzón Diana Garzón Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Roger Moreno Roger Moreno Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Vanessa Soto-Cerrato Vanessa Soto-Cerrato Departament de Patologia i Terapèutica Experimental, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Iain McNeish Iain McNeish Department of Surgery and Cancer, Imperial College, London, UK Search for more papers by this author Violeta Serra Violeta Serra orcid.org/0000-0001-6620-1065 Experimental Therapeutics Group, Vall d'Hebron Institute of Oncology, Barcelona, Spain CIBERONC, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Conxi Lazaro Conxi Lazaro Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERONC, Instituto de Salud Carlos III, Madrid, Spain Hereditary Cancer Program, Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Pilar Barretina Pilar Barretina Medical Oncology Department, Institut Català d'Oncologia, IDIBGI, Girona, Spain Search for more papers by this author Joan Brunet Joan Brunet CIBERONC, Instituto de Salud Carlos III, Madrid, Spain Hereditary Cancer Program, Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Hereditary Cancer Program, Institut Català d'Oncologia, IDIBGI, Girona, Spain Medical Sciences Department, School of Medicine, University of Girona, Girona, Spain Search for more papers by this author Javier Menéndez Javier Menéndez Program against Cancer Therapeutic Resistance (ProCURE), Metabolism and Cancer Group, Catalan Institute of Oncology, Girona, Spain Girona Biomedical Research Institute (IDIBGI), Girona, Spain Search for more papers by this author Xavier Matias-Guiu Xavier Matias-Guiu Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERONC, Instituto de Salud Carlos III, Madrid, Spain Servei d'Anatomia Patològica, Hospital Universitari de Bellvitge, L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author August Vidal August Vidal Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Departament de Patologia i Terapèutica Experimental, Universitat de Barcelona, Barcelona, Spain Servei d'Anatomia Patològica, Hospital Universitari de Bellvitge, L'Hospitalet de Llobregat, Barcelona, Spain Xenopat, Carrer de la Feixa Llarga S/N, L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Alberto Villanueva Alberto Villanueva Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Xenopat, Carrer de la Feixa Llarga S/N, L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Barbie Taylor-Harding Barbie Taylor-Harding Womens Cancer Program, Cedars-Sinai Medical Center, Los Angeles, CA, USA Search for more papers by this author Hisashi Tanaka Hisashi Tanaka Womens Cancer Program, Cedars-Sinai Medical Center, Los Angeles, CA, USA Search for more papers by this author Sandra Orsulic Sandra Orsulic David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Alexandra Junza Alexandra Junza Metabolomics Platform, Department of Electronic Engineering (DEEEA), Universitat Rovira i Virgili, Tarragona, Spain Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain Search for more papers by this author Oscar Yanes Oscar Yanes Metabolomics Platform, Department of Electronic Engineering (DEEEA), Universitat Rovira i Virgili, Tarragona, Spain Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain Search for more papers by this author Cristina Muñoz-Pinedo Cristina Muñoz-Pinedo Cell Death Regulation Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Luís Palomero Luís Palomero Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Miquel Àngel Pujana Miquel Àngel Pujana Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author José Carlos Perales José Carlos Perales Departament de Ciències Fisiològiques, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Francesc Viñals Corresponding Author Francesc Viñals [email protected] [email protected] orcid.org/0000-0002-9918-6751 Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Departament de Ciències Fisiològiques, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Álvaro Lahiguera Álvaro Lahiguera Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Petra Hyroššová Petra Hyroššová Departament de Ciències Fisiològiques, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Agnès Figueras Agnès Figueras Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Diana Garzón Diana Garzón Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Roger Moreno Roger Moreno Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Vanessa Soto-Cerrato Vanessa Soto-Cerrato Departament de Patologia i Terapèutica Experimental, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Iain McNeish Iain McNeish Department of Surgery and Cancer, Imperial College, London, UK Search for more papers by this author Violeta Serra Violeta Serra orcid.org/0000-0001-6620-1065 Experimental Therapeutics Group, Vall d'Hebron Institute of Oncology, Barcelona, Spain CIBERONC, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Conxi Lazaro Conxi Lazaro Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERONC, Instituto de Salud Carlos III, Madrid, Spain Hereditary Cancer Program, Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Pilar Barretina Pilar Barretina Medical Oncology Department, Institut Català d'Oncologia, IDIBGI, Girona, Spain Search for more papers by this author Joan Brunet Joan Brunet CIBERONC, Instituto de Salud Carlos III, Madrid, Spain Hereditary Cancer Program, Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Hereditary Cancer Program, Institut Català d'Oncologia, IDIBGI, Girona, Spain Medical Sciences Department, School of Medicine, University of Girona, Girona, Spain Search for more papers by this author Javier Menéndez Javier Menéndez Program against Cancer Therapeutic Resistance (ProCURE), Metabolism and Cancer Group, Catalan Institute of Oncology, Girona, Spain Girona Biomedical Research Institute (IDIBGI), Girona, Spain Search for more papers by this author Xavier Matias-Guiu Xavier Matias-Guiu Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERONC, Instituto de Salud Carlos III, Madrid, Spain Servei d'Anatomia Patològica, Hospital Universitari de Bellvitge, L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author August Vidal August Vidal Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Departament de Patologia i Terapèutica Experimental, Universitat de Barcelona, Barcelona, Spain Servei d'Anatomia Patològica, Hospital Universitari de Bellvitge, L'Hospitalet de Llobregat, Barcelona, Spain Xenopat, Carrer de la Feixa Llarga S/N, L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Alberto Villanueva Alberto Villanueva Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Xenopat, Carrer de la Feixa Llarga S/N, L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Barbie Taylor-Harding Barbie Taylor-Harding Womens Cancer Program, Cedars-Sinai Medical Center, Los Angeles, CA, USA Search for more papers by this author Hisashi Tanaka Hisashi Tanaka Womens Cancer Program, Cedars-Sinai Medical Center, Los Angeles, CA, USA Search for more papers by this author Sandra Orsulic Sandra Orsulic David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Alexandra Junza Alexandra Junza Metabolomics Platform, Department of Electronic Engineering (DEEEA), Universitat Rovira i Virgili, Tarragona, Spain Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain Search for more papers by this author Oscar Yanes Oscar Yanes Metabolomics Platform, Department of Electronic Engineering (DEEEA), Universitat Rovira i Virgili, Tarragona, Spain Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain Search for more papers by this author Cristina Muñoz-Pinedo Cristina Muñoz-Pinedo Cell Death Regulation Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Luís Palomero Luís Palomero Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author Miquel Àngel Pujana Miquel Àngel Pujana Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Search for more papers by this author José Carlos Perales José Carlos Perales Departament de Ciències Fisiològiques, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Francesc Viñals Corresponding Author Francesc Viñals [email protected] [email protected] orcid.org/0000-0002-9918-6751 Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain Departament de Ciències Fisiològiques, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Author Information Álvaro Lahiguera1,2, Petra Hyroššová3, Agnès Figueras1,2, Diana Garzón1,2, Roger Moreno1,2, Vanessa Soto-Cerrato4, Iain McNeish5, Violeta Serra6,7, Conxi Lazaro2,7,8, Pilar Barretina9, Joan Brunet7,8,10,11, Javier Menéndez12,13, Xavier Matias-Guiu2,7,14, August Vidal2,4,14,15, Alberto Villanueva1,2,15, Barbie Taylor-Harding16, Hisashi Tanaka16, Sandra Orsulic17, Alexandra Junza18,19, Oscar Yanes18,19, Cristina Muñoz-Pinedo20, Luís Palomero1,2, Miquel Àngel Pujana1,2, José Carlos Perales3 and Francesc Viñals *,*,1,2,3 1Program Against Cancer Therapeutic Resistance (ProCURE), Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain 2Oncobell Program, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain 3Departament de Ciències Fisiològiques, Universitat de Barcelona, Barcelona, Spain 4Departament de Patologia i Terapèutica Experimental, Universitat de Barcelona, Barcelona, Spain 5Department of Surgery and Cancer, Imperial College, London, UK 6Experimental Therapeutics Group, Vall d'Hebron Institute of Oncology, Barcelona, Spain 7CIBERONC, Instituto de Salud Carlos III, Madrid, Spain 8Hereditary Cancer Program, Institut Català d'Oncologia, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain 9Medical Oncology Department, Institut Català d'Oncologia, IDIBGI, Girona, Spain 10Hereditary Cancer Program, Institut Català d'Oncologia, IDIBGI, Girona, Spain 11Medical Sciences Department, School of Medicine, University of Girona, Girona, Spain 12Program against Cancer Therapeutic Resistance (ProCURE), Metabolism and Cancer Group, Catalan Institute of Oncology, Girona, Spain 13Girona Biomedical Research Institute (IDIBGI), Girona, Spain 14Servei d'Anatomia Patològica, Hospital Universitari de Bellvitge, L'Hospitalet de Llobregat, Barcelona, Spain 15Xenopat, Carrer de la Feixa Llarga S/N, L'Hospitalet de Llobregat, Barcelona, Spain 16Womens Cancer Program, Cedars-Sinai Medical Center, Los Angeles, CA, USA 17David Geffen School of Medicine at UCLA, Los Angeles, CA, USA 18Metabolomics Platform, Department of Electronic Engineering (DEEEA), Universitat Rovira i Virgili, Tarragona, Spain 19Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain 20Cell Death Regulation Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain *Corresponding author. Tel: +34 93 2607344; E-mails: [email protected]; [email protected] EMBO Mol Med (2020)12:e11217https://doi.org/10.15252/emmm.201911217 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mitochondrial metabolism and the generation of reactive oxygen species (ROS) contribute to the acquisition of DNA mutations and genomic instability in cancer. How genomic instability influences the metabolic capacity of cancer cells is nevertheless poorly understood. Here, we show that homologous recombination-defective (HRD) cancers rely on oxidative metabolism to supply NAD+ and ATP for poly(ADP-ribose) polymerase (PARP)-dependent DNA repair mechanisms. Studies in breast and ovarian cancer HRD models depict a metabolic shift that includes enhanced expression of the oxidative phosphorylation (OXPHOS) pathway and its key components and a decline in the glycolytic Warburg phenotype. Hence, HRD cells are more sensitive to metformin and NAD+ concentration changes. On the other hand, shifting from an OXPHOS to a highly glycolytic metabolism interferes with the sensitivity to PARP inhibitors (PARPi) in these HRD cells. This feature is associated with a weak response to PARP inhibition in patient-derived xenografts, emerging as a new mechanism to determine PARPi sensitivity. This study shows a mechanistic link between two major cancer hallmarks, which in turn suggests novel possibilities for specifically treating HRD cancers with OXPHOS inhibitors. Synopsis Homologous recombination-defective (HRD) cancers need high levels of NAD+ and ATP for alternative PARP-dependent DNA repair. HRD cancer cells undergo a characteristic metabolic shift that include enhanced OXPHOS, opening new opportunities for treatment with OXPHOS inhibitors like metformin. Studies in different cancer BRCA-mutated models depict a metabolic shift that includes enhanced expression of the oxidative phosphorylation (OXPHOS) pathway and a decline in the glycolytic Warburg phenotype. HRD cancers rely on oxidative metabolism to supply NAD+ and ATP for Poly (ADP-ribose) polymerase (PARP)-dependent DNA repair mechanisms. In consequence HRD tumors are more sensitive to OXPHOS inhibitors, such as metformin, and NAD+ concentration changes. Moreover, shifting from an OXPHOS to a highly glycolytic metabolism interferes with the sensitivity to PARP inhibitors (PARPi) in these HRD cells. The paper explained Problem Metabolism and the generation of reactive oxygen species (ROS) contribute to the acquisition of DNA mutations and genomic instability in cancer. But how genomic instability influences the metabolic capacity of cancer cells is nevertheless poorly understood. Results Here, we show that homologous recombination-defective (HRD) and in general genomic instable cancers rely on oxidative metabolism to supply key metabolites important for PARP-dependent DNA repair mechanisms. Studies in breast and ovarian cancer HRD models depict a metabolic shift that includes repression of the glycolytic phenotype and enhanced expression of the oxidative phosphorylation (OXPHOS) pathway and its key components. In consequence, HRD cells are more sensitive to inhibitors of OXPHOS, as metformin. On the other hand, shifting from an OXPHOS to a highly glycolytic metabolism interferes with the sensitivity to PARP inhibitors in these HRD cells, emerging as a new mechanism that determines PARP inhibitor sensitivity. Impact This study shows a mechanistic link between two major cancer hallmarks, which in turn suggests novel possibilities for specifically treating HRD cancers with OXPHOS inhibitors. Introduction Genomic instability and deregulation of cellular energetics are two of the hallmarks of cancer cells, as re-defined by Hanahan and Weinberg (2011). Genomic instability arises when mechanisms that protect genomes from alterations in DNA are overcome. Consequently, as mutation rates increase, cells can adapt better to changes in the tumor environment, response to therapies, etc. An increase in genomic instability could be caused by alterations in the DNA damage response (DDR), including the mechanisms responsible for (i) detecting DNA damage, (ii) DNA repair, and/or (iii) detoxification (Jackson & Bartek, 2009). The DNA repair machinery corrects, among other things, double- and single-strand DNA breaks (DSBs and SSBs, respectively). DSBs are repaired by processes such as homologous recombination (HR) and non-homologous end joining (NHEJ), and SSBs are repaired by base excision repair (BER) and nucleotide excision repair (NER) mechanisms. BRCA1 and BRCA2 proteins play a key role in HR, and mutations in these genes cause defective HR and increased genomic instability (Roy et al, 2011). Poly(ADP-ribose) polymerase (PARP) proteins are DNA damage sensors because they can bind to breaks and nicks in DNA and transduce signals to the DNA repair machinery by attaching branched poly(ADP-ribose) (PAR) chains to various proteins (Krishnakumar & Kraus, 2010; Gupte et al, 2017; Lord & Ashworth, 2017). Cancer cells with defective HR depend on alternative DNA repair pathways, such as BER and NHEJ, both of which are promoted by PARP enzymes. This is the basis of the clinical utility of PARP inhibitors in HR-deficient cases (Lord & Ashworth, 2017). Tumoral cells must maintain a constant supply of nutrients and energy to sustain the growth and cell division that characterize cancer (Boroughs & DeBerardinis, 2015). In order to do so, a large proportion of tumoral cells switch their metabolism to aerobic glycolysis (the Warburg effect), a preference for increased glycolysis regardless of the presence of oxygen (Hsu & Sabatini, 2008; Vander Heiden et al, 2009; Ward & Thompson, 2012). Thus, glycolytic intermediates expand to cope with biosynthetic pathways, such as those involved in the synthesis of nucleotides and amino acids (Hsu & Sabatini, 2008; Vander Heiden et al, 2009; Ward & Thompson, 2012; Hay, 2016). However, a preference for aerobic glycolysis does not always involve reduced respiratory capacity. In fact, tumoral cells depend on a continuing supply of TCA cycle intermediates, such as those serving as direct precursors of lipids, and those providing carbons and cofactors in the nucleotide and amino acid synthesis pathways (Vyas et al, 2016). Thus, depending on the tumoral cells’ intrinsic capacity (oncogene profile, metabolic program, proliferative state, etc), the characteristics of the tumor environment (e.g., with or without hypoxia, and metabolic substrates produced by the tumoral stroma), and the effects of the cancer treatment (e.g., presence of drugs and ROS generated by the treatment), the metabolism of cancer cells can change and readapt. Examples of this are cancer cells subjected to chronically low in vitro glucose levels, which increase oxidative phosphorylation (OXPHOS) to maintain growth (Birsoy et al, 2014), chemotherapy-resistant breast tumoral cells, which have a higher mitochondrial oxidative metabolic rate than do sensitive cells (Janzer et al, 2014), and antiangiogenic treatment in breast and lung cancer models that induce a shift from a largely glycolytic metabolism to a more oxidative one (Navarro et al, 2016). The capacity to detect and repair DNA damage and the metabolic characteristics of a cell are coordinated (Berkers et al, 2013; Imai & Guarente, 2014; Shimizu et al, 2014). Different repair mechanisms rely on specific metabolites, which may, in turn, limit the capacity of cancer cells to cope with different chemotherapies. PARP enzymes directly depend on the cellular metabolic state, particularly on NAD+ levels, for their functions (Krishnakumar & Kraus, 2010; Gupte et al, 2017); hence, a direct role for PARP proteins linking DNA damage and metabolic capacities in different situations has been proposed (Bai et al, 2007; Sakamaki et al, 2009; Brace et al, 2016). Acute DNA damage inhibits glutamine metabolism to control cell cycle arrest through SIRT4, a family of proteins that also depend on NAD+ for their activity (Jeong et al, 2013). Meanwhile, DDR upregulates the pentose phosphate pathway and glycolysis to promote nucleotide synthesis and an open global chromatin structure (Cosentino et al, 2011; Liu et al, 2015; Efimova et al, 2016). However, the metabolic requirements of BRCA-dependent HR DNA repair mechanisms remain unknown. In this study, analyses of cancer cell models, patient-derived xenografts (PDXs), and human cancer samples reveal novel mechanisms linking HR defects to increased oxidative metabolism. Triggering oxidative metabolism in HR-defective (HRD) cancer cells emerges as a fundamental characteristic of cancer survival due to its contribution to DNA damage repair, a feature set that highlights additional cancer vulnerabilities, as it emerges as a novel player in determining sensitivity to PARP inhibitors. Results HR deficiency is linked to enhanced OXPHOS gene expression in breast and ovarian cancer To identify metabolic pathways that may be associated with genomic instability due to HR deficiency, we integrated somatic mutational profiles and gene expression data from the breast and ovarian cancer studies of TCGA (Cancer Genome Atlas Research Network, 2011; Cancer Genome Atlas Network, 2012). Tumors were classified according to the presence (S3+) or absence (S3−) of mutational signature 3, originally shown to be characteristic of cases with HR defects and BRCA1/2 mutations (Alexandrov et al, 2013). Next, an association with changes in gene expression of metabolic pathways sets was analyzed using the Gene Set Enrichment Analysis tool (Subramanian et al, 2005) and pathway annotations from the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa et al, 2017). These analyses revealed that OXPHOS and four additional mitochondria-related metabolic sets (Table EV1) are positively associated with the presence of the S3 signature (S3+), that is, with HR defects in breast tumors (gene set expression analysis [GSEA] nominal P = 0.019, Fig 1A, top panel). Moreover, we also complied BRCA1/2 mutation status of TCGA breast cancer datasets from different sources (cBioPortal and TCGA accessible data) (Kraya et al, 2019; Yost et al, 2019) and further analyzed the association with the KEGG oxidative phosphorylation pathway. In multivariate regression analyses including age at diagnosis and tumor stage, overexpression of this pathway was also detected significantly associated with BRCA1/2 mutations (P = 0.013, Fig 1A, middle panel). Consistent with this observation, the single-sample GSEA (ssGSEA) scores of the KEGG OXPHOS gene set were found to be negatively correlated (Pearson's correlation coefficients [PCCs] ≤ −0.15, P < 0.001) with both BRCA1 and BRCA2 expression levels (Fig 1A, bottom panels). Subsequently, a similar metabolic association was observed with high-grade serous ovarian tumors positive for the mutational signature 3: higher OXPHOS gene expression in S3+, HR-defective tumors (false-discovery rate [FDR]-adjusted P = 0.025, Fig 1B, top panel) and with BRCA1/2 mutation status of TCGA ovarian cancer datasets (P < 0.001, Fig 1B, middle panel). The OXPHOS gene set was also found to be negatively correlated (PCCs ≤ −0.18, P < 0.01) with BRCA1 and BRCA2 expression (Fig 1B, bottom panels). Figure 1. HR defects are associated with OXPHOS gene overexpression A, B. GSEA results regarding the association between OXPHOS gene set overexpression and positivity for mutational signature 3 (associated with HR defects) in TCGA breast cancers (A) and TCGA ovarian cancer data (B). Top panel, enrichment score, gene ranking (based on the t-statistic of the expression differences between negative (S3−) and positive (S3+) tumors), and false-discovery rate (FDR)-adjusted P values are shown. Middle panel shows similar GSEA results using as metric the β coefficient of differential expression between BRCA1/2 wild-type and mutant tumors, including the covariates of age at diagnosis and tumor stage. Bottom panels, scatter plots showing the correlations (Pearson's correlation coefficients and P values) between the ssGSEA scores of the OXPHOS gene set and the BRCA1 (top) and BRCA2 (bottom) somatic gene expression values. C. GSEA results of KEGG OXPHOS (top panel) and HRD (bottom panel) signature score comparisons between carboplatin-resistant (left) and carboplatin-sensitive (right) ovarian tumors, using pre-treatment expression data (GSE15622 data). The normalized enrichment scores (NESs) and corresponding P values are indicated. The NES is negative because the comparison is between resistant and sensitive tumors, so negative values mean that expression is higher in the second term (i.e., sensitive tumors). D

Lung cancer is the main cause of cancer death worldwide. Non-Small Cell Lung Carcinoma (NSCLC) is the most common subtype of lung cancer, and the prognosis of NSCLC patients in advanced stages is still very poor. Given the need for new therapies, the metabolism of NSCLC has been widely studied in the past two decades to identify vulnerabilities that could be translated into novel anti-metabolic therapeutic approaches. A number of studies have highlighted the role of glucose and mitochondrial metabolism in the development of NSCLC. The metabolic properties of lung tumors have been characterized in detail in vivo, and they include high glucose and lactate use and high heterogeneity regarding the use of nutrients and mitochondrial pathways. This heterogeneity has also been observed in patients infused with labeled nutrients. We will summarize here the knowledge about the use of amino acids, fatty acids and carbohydrates in NSCLC that could lead to new combination treatments.

Tumors display a high rate of glucose uptake and glycolysis. We investigated how inhibition of glucose metabolism could affect death receptor-mediated apoptosis in human tumor cells of diverse origin. We show that both substitution of glucose for pyruvate and treatment with 2-deoxyglucose enhanced apoptosis induced by tumor necrosis factor (TNF)-α, CD95 agonistic antibody, and TNF-related apoptosis-inducing ligand (TRAIL). Inhibition of glucose metabolism enhanced killing of myeloid leukemia U937, cervical carcinoma HeLa, and breast carcinoma MCF-7 cells upon death receptor ligation. Caspase activation, mitochondrial depolarization, and cytochrome crelease were increased under these conditions. Glucose deprivation-mediated sensitization to apoptosis was prevented in MCF-7 cells overexpressing BCL-2. Interestingly, the human B-lymphoblastoid cell line SKW6.4, a prototype for mitochondria-independent death receptor-induced apoptosis, was also sensitized to anti-CD95 and TRAIL-induced apoptosis under glucose-free conditions. Changes in c-FLIPL and cFLIPs levels were observed in some but not all the cell lines studied following glucose deprivation. Glucose deprivation enhanced death receptor-triggered formation of death-inducing signaling complex and early processing of procaspase-8. Altogether, these results suggest that the glycolytic pathway may be an important target for therapeutic intervention to sensitize tumor cells to selectively toxic soluble death ligands or death ligand-expressing cells of the immune system by facilitating the activation of initiator caspase-8. Tumors display a high rate of glucose uptake and glycolysis. We investigated how inhibition of glucose metabolism could affect death receptor-mediated apoptosis in human tumor cells of diverse origin. We show that both substitution of glucose for pyruvate and treatment with 2-deoxyglucose enhanced apoptosis induced by tumor necrosis factor (TNF)-α, CD95 agonistic antibody, and TNF-related apoptosis-inducing ligand (TRAIL). Inhibition of glucose metabolism enhanced killing of myeloid leukemia U937, cervical carcinoma HeLa, and breast carcinoma MCF-7 cells upon death receptor ligation. Caspase activation, mitochondrial depolarization, and cytochrome crelease were increased under these conditions. Glucose deprivation-mediated sensitization to apoptosis was prevented in MCF-7 cells overexpressing BCL-2. Interestingly, the human B-lymphoblastoid cell line SKW6.4, a prototype for mitochondria-independent death receptor-induced apoptosis, was also sensitized to anti-CD95 and TRAIL-induced apoptosis under glucose-free conditions. Changes in c-FLIPL and cFLIPs levels were observed in some but not all the cell lines studied following glucose deprivation. Glucose deprivation enhanced death receptor-triggered formation of death-inducing signaling complex and early processing of procaspase-8. Altogether, these results suggest that the glycolytic pathway may be an important target for therapeutic intervention to sensitize tumor cells to selectively toxic soluble death ligands or death ligand-expressing cells of the immune system by facilitating the activation of initiator caspase-8. tumor necrosis factor tumor necrosis factor receptor tumor necrosis factor-related apoptosis-inducing ligand tumor necrosis factor-related apoptosis-inducing ligand receptor FLICE-inhibitory protein death-inducing signaling complex 2-deoxy-d-glucose reactive oxygen species phosphatidylinositol 3-kinase fetal bovine serum dichlorodihydrofluorescein diacetate phosphate-buffered saline benzyloxycarbonyl fluoromethyl ketone Apoptotic cell death plays a fundamental role in normal development, tissue homeostasis, and pathological situations (1Evan G. Littlewood T. 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However, sensitivity of tumor cells to death receptor-mediated apoptosis does not always correlate with death receptor expression (11Ashkenazi A. Dixit V.M. Curr. Opin. Cell Biol. 1999; 11: 255-260Crossref PubMed Scopus (1157) Google Scholar,12Dirks W. Schone S. Uphoff C. Quentmeier H. Pradella S. Drexler H.G. Br. J. Haematol. 1997; 96: 584-593Crossref PubMed Scopus (67) Google Scholar). In this respect, understanding the mechanisms that regulate tumor cell sensitivity to death ligand-induced apoptosis should be an important objective in the development of therapies to treat human malignancies. Recent studies (13Thakkar N.S. Potten C.S. Cancer Res. 1993; 53: 2057-2060PubMed Google Scholar, 14Ferrari D. Stepczynska A. Los M. Wesselborg S. Schulze-Osthoff K. J. Exp. Med. 1998; 188: 979-984Crossref PubMed Scopus (194) Google Scholar) have revealed that apoptosis induced by various death receptor-independent treatments is blocked in ATP-depleted cells. Furthermore, although the ATP-dependent steps in apoptosis have not been completely elucidated, it has been suggested that the nuclear transport of pro-apoptotic factors could be one of the processes requiring an active energy metabolism (15Yasuhara N. Eguchi Y. Tachibana T. Imamoto N. Yoneda Y. Tsujimoto Y. Genes Cells. 1997; 2: 55-64Crossref PubMed Scopus (84) Google Scholar). More controversial is the role of ATP in death receptor-mediated apoptosis as completely opposite results have been reported regarding the effect of ATP depletion on CD95-mediated apoptosis (14Ferrari D. Stepczynska A. Los M. Wesselborg S. Schulze-Osthoff K. J. Exp. Med. 1998; 188: 979-984Crossref PubMed Scopus (194) Google Scholar, 16Eguchi Y. Shimizu S. Tsujimoto Y. Cancer Res. 1997; 57: 1835-1840PubMed Google Scholar, 17Leist M. Single B. Castoldi A.F. Kuhnle S. Nicotera P. J. Exp. Med. 1997; 185: 1481-1486Crossref PubMed Scopus (1651) Google Scholar). Thus, some groups have reported (16Eguchi Y. Shimizu S. Tsujimoto Y. Cancer Res. 1997; 57: 1835-1840PubMed Google Scholar, 17Leist M. Single B. Castoldi A.F. Kuhnle S. Nicotera P. J. Exp. Med. 1997; 185: 1481-1486Crossref PubMed Scopus (1651) Google Scholar) that CD95-mediated apoptosis is prevented when cells are depleted of ATP. Under these conditions cell death caused by CD95 activation changes from apoptosis to necrosis (17Leist M. Single B. Castoldi A.F. Kuhnle S. Nicotera P. J. Exp. Med. 1997; 185: 1481-1486Crossref PubMed Scopus (1651) Google Scholar). However, other data have indicated that although prevention of ATP generation completely inhibits caspase activation and apoptosis in response to chemotherapeutic drugs, ATP depletion does not affect CD95-induced apoptosis (14Ferrari D. Stepczynska A. Los M. Wesselborg S. Schulze-Osthoff K. J. Exp. Med. 1998; 188: 979-984Crossref PubMed Scopus (194) Google Scholar). Most of these studies were performed in glucose-free medium, in the presence of the mitochondrial inhibitor oligomycin, to prevent ATP production from both glycolysis and oxidative phosphorylation and thus achieve maximal depletion of cellular ATP (14Ferrari D. Stepczynska A. Los M. Wesselborg S. Schulze-Osthoff K. J. Exp. Med. 1998; 188: 979-984Crossref PubMed Scopus (194) Google Scholar, 16Eguchi Y. Shimizu S. Tsujimoto Y. Cancer Res. 1997; 57: 1835-1840PubMed Google Scholar, 17Leist M. Single B. Castoldi A.F. Kuhnle S. Nicotera P. J. Exp. Med. 1997; 185: 1481-1486Crossref PubMed Scopus (1651) Google Scholar). However, besides causing ATP depletion, inhibition of F0F1-ATPase by oligomycin may interfere in the apoptotic program by preventing cytosol acidification and cytochrome c release from mitochondria (18Matsuyama S. Xu Q. Velours J. Reed J.C. Mol. Cell. 1998; 1: 327-336Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 19Matsuyama S. Llopis J. Deveraux Q.L. Tsien R.Y. Reed J.C. Nat. Cell Biol. 2000; 2: 318-325Crossref PubMed Scopus (636) Google Scholar). Furthermore, oligomycin can cause cell death by apoptosis or necrosis (14Ferrari D. Stepczynska A. Los M. Wesselborg S. Schulze-Osthoff K. J. Exp. Med. 1998; 188: 979-984Crossref PubMed Scopus (194) Google Scholar, 20Wolvetang E.J. Johnson K.L. Krauer K. Ralph S.J. Linnane A.W. FEBS Lett. 1994; 339: 40-44Crossref PubMed Scopus (381) Google Scholar). An elevated number of tumor cells display a high rate of glycolysis under aerobic conditions, and some of them depend on the glycolytic flux to maintain the cellular levels of ATP and metabolism (21Racker E. Am. Sci. 1972; 60: 56-63PubMed Google Scholar, 22Mathupala S.P. Rempel A. Pedersen P.L. J. Bioenerg. Biomembr. 1997; 29: 339-343Crossref PubMed Scopus (227) Google Scholar).In vivo (23Volland S. Amtmann E. Sauer G. Int. J. Cancer. 1992; 52: 384-390Crossref PubMed Scopus (9) Google Scholar) as well as in vitro (23Volland S. Amtmann E. Sauer G. Int. J. Cancer. 1992; 52: 384-390Crossref PubMed Scopus (9) Google Scholar, 24Halicka H.D. Ardelt B. Li X. Melamed M.M. Darzynkiewicz Z. Cancer Res. 1995; 55: 444-449PubMed Google Scholar) studies have revealed that sensitivity of tumor cells to TNF-α is increased under conditions of reduced glucose metabolism. These findings have led some investigators to propose the use of glucose anti-metabolites in combination with TNF-α as a possible anti-tumor treatment (24Halicka H.D. Ardelt B. Li X. Melamed M.M. Darzynkiewicz Z. Cancer Res. 1995; 55: 444-449PubMed Google Scholar). However the mechanism underlying the facilitation of TNF-α action by 2-deoxy-d-glucose (2-DG) has not been characterized. More recently, it was reported that glucose deprivation enhances TRAIL-induced apoptosis by down-regulating the expression of cFLIP through the ceramide-AKT-FLIP pathway (25Nam S. Amoscato A. Lee Y. Oncogene. 2002; 21: 337-346Crossref PubMed Google Scholar). In this report, we have examined the effect of glucose deprivation on apoptosis induced upon activation of different death receptors of the TNF/nerve growth factor receptor family in human tumor cells whose ATP levels are largely dependent on the activity of the glycolytic pathway. The results obtained indicated that under conditions of glucose metabolism inhibition, death receptor-induced apoptosis was considerably enhanced in U937 myeloid leukemic, SKW6.4 B-lymphoblastoid, MCF-7 breast carcinoma and HeLa cervical carcinoma cells. Inhibition of glucose metabolism promoted the activation of both mitochondria-dependent and -independent pathways of death receptor-induced apoptosis. Finally, our data indicate that the sensitization mechanism probably involves the increased processing of apical procaspase-8 at the DISC upon death receptor activation. RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Invitrogen. Etoposide, 4′,6′-diamidino-2-phenylindole, RPMI 1640 glucose-free medium, 2-deoxy-d-glucose (2-DG), streptavidin-agarose beads, mouse anti-α-tubulin antibody, insulin, and LY294002 were obtained from Sigma. CH-11 monoclonal antibody (IgM) reacting with CD95 was from Upstate Biotechnology, Inc. (Lake Placid, NY). Human TRAIL and TNF-α were obtained from PeproTech (London, UK). Mouse anti-human caspase-8 monoclonal antibody was purchased from Cell Diagnostica (Münster, Germany). Rabbit anti-cleaved caspase-9 and anti-cleaved caspase-3 polyclonal antibodies were obtained from New England Biolabs (Beverly, MA). AKT and phospho-AKT (Ser-473) antibodies were from Cell Signaling(Beverly, MA). Anti-X chromosome-linked inhibitor of apoptosis protein and cellular inhibitor of apoptosis protein antibodies were donated by Dr. Douglas R. Green (La Jolla Institute for Allergy and Immunology, San Diego, CA). Anti-cFLIP antibody NF6 was provided by Dr. M. Peter (University of Chicago). Mouse monoclonal antibodies to BAX and cytochrome c were obtained from Pharmingen. Anti-AIF was a gift of Dr. Santos Susin (CNRS, Villejuif, France). Caspases inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (Z-VAD-FMK) was from Enzyme System Inc. (Dublin, CA). 3,3′-Dihexyloxacarbocyanine iodide(3) and dichlorodihydrofluorescein diacetate (H2DCFDA) were purchased from Molecular Probes (Eugene, OR).l-Buthionine-(SR)-sulfoximine was a generous gift from Dr. Isabel Fabregat, Universidad Complutense (Madrid, Spain). The human SKW6.4 B-lymphoblastoid cell line was provided by Dr. Katja Zimmermann (La Jolla Institute for Allergy and Immunology, San Diego, CA). The human breast tumor cell line MCF-7 was kindly donated by Dr. M. Ruiz de Almodovar (Department of Radiology, University of Granada). Human U937 myeloid leukemic, SKW6.4 B-lymphoblastoid, HeLa cervical carcinoma, and MCF-7 breast carcinoma cells were maintained in RPMI medium containing 10% fetal bovine serum, 1 mm glutamine, and gentamycin at 37 °C in a humidified 5% CO2, 95% air incubator. MCF-7 cells stably overexpressing human BCL-2 (MCF-7BCL-2) and mock-transfected cells (MCF-7neo) were generated as described (26Ruiz-Ruiz C. Munoz-Pinedo C. Lopez-Rivas A. Cancer Res. 2000; 60: 5673-5680PubMed Google Scholar). Incubation under glucose-free conditions was performed by washing cells twice in glucose-free RPMI 1640 medium and incubating them in the same medium with 1 mm glutamine, 2 mm pyruvate, and 5% dialyzed FBS. Control cultures were incubated with 2 g/liter glucose instead of pyruvate. Treatment with 2-DG was performed in normal RPMI 1640 medium with 1 mm glutamine and 10% FBS. Hypodiploid apoptotic cells were detected by flow cytometry according to published procedures (27Gong J. Traganos F. Darzynkiewicz Z. Anal. Biochem. 1994; 218: 314-319Crossref PubMed Scopus (649) Google Scholar). Basically, cells were washed with phosphate-buffered saline (PBS), fixed in cold 70% ethanol, and then stained with propidium iodide while treating with RNase. Quantitative analysis of sub-G1 cells was carried out in a FACScan cytometer using the Cell Quest software (BD Biosciences). Phosphatidylserine exposure on the surface of apoptotic cells was examined by flow cytometry after staining with annexin-V-FLUOS (Roche Molecular Biochemicals), following instructions provided by the manufacturer. Viable MCF-7 and HeLa cells were determined by the crystal violet method as described (26Ruiz-Ruiz C. Munoz-Pinedo C. Lopez-Rivas A. Cancer Res. 2000; 60: 5673-5680PubMed Google Scholar). Chromatin condensation was assessed after staining of cellular DNA with 4′,6′-diamidino-2-phenylindole (alone or in combination with 1 μm propidium iodide) and viewing the cell preparations under a Zeiss Axiophot fluorescent microscope. ATP was determined luminometrically as described (14Ferrari D. Stepczynska A. Los M. Wesselborg S. Schulze-Osthoff K. J. Exp. Med. 1998; 188: 979-984Crossref PubMed Scopus (194) Google Scholar). For measurements of mitochondrial depolarization, cells were collected by centrifugation and resuspended in PBS with 40 nm 3,3′-dihexyloxacarbocyanine iodide(3) and 5 μm propidium iodide and incubated for 15 min at room temperature. Generation of ROS was quantified by adding H2DCFDA to the culture medium at a concentration of 5 μm, for the last 30 min of treatment. Similar results were obtained when staining with H2DCFDA was performed at room temperature in PBS. Quantitative analyses of ΔΨm(mitochondrial membrane potential) and ROS production were carried out in a FACScan cytometer using the Cell Quest software. MCF-7 and HeLa cells were detached with RPMI/EDTA, washed with PBS, and collected by centrifugation. U937 and SKW6.4 cells were washed with PBS. Protein content was measured before lysing the cells in Laemmli sample buffer under reducing conditions. Cell lysates were sonicated, and proteins were resolved on SDS-polyacrylamide minigels and detected as described (26Ruiz-Ruiz C. Munoz-Pinedo C. Lopez-Rivas A. Cancer Res. 2000; 60: 5673-5680PubMed Google Scholar). For measurements of cytochrome c release from mitochondria and BAX redistribution, cells were lysed and cytosolic fractions were separated from mitochondria and nuclei as described previously (28Chandra J. Niemer I. Gilbreath J. Kliche K.O. Andreeff M. Freireich E.J. Keating M. McConkey D.J. Blood. 1998; 92: 4220-4229Crossref PubMed Google Scholar). Equal protein loading in each lane was verified by incubating membranes with anti-α-tubulin antibody. DISC precipitation was performed using biotin-tagged recombinant TRAIL (Bio-TRAIL) (29MacFarlane M. Harper N. Snowden R. Dyer M. Barnett G. Pringle J. Cohen G. Oncogene. 2002; 21: 6809-6810Crossref PubMed Scopus (169) Google Scholar, 30Harper N. Farrow N. Kapstein A. Cohen G. MacFarlane M. J. Biol. Chem. 2001; 276: 34743-34752Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), which was a gift from Nick Harper (MRC Toxicology Unit, University of Leicester, UK). U937 cells (75 × 106 cells/treatment) were washed twice in sterile PBS and incubated for 12 h in glucose-free RPMI 1640 medium with 1 mm glutamine, 2 mm pyruvate, and 5% dialyzed FBS. Control U937 cells were incubated in the same medium with 2 g/liter glucose instead of pyruvate. After this incubation, the same number of control and glucose-deprived cells were treated with Bio-TRAIL for the times indicated in the figure legends. DISC formation was then stopped, and unbound TRAIL was removed by washing the cells three times in ice-cold PBS. Cells were lysed in 3 ml of lysis buffer (30 mmTris/HCl, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100, containing CompleteTM protease inhibitors (Roche Molecular Biochemicals)) for 30 min on ice followed by centrifugation at 15,000 × g for 10 min at 4 °C. To provide an unstimulated receptor control, Bio-TRAIL was added to lysates from untreated cells. The TRAIL DISC was then precipitated using 30 μl of streptavidin-agarose beads at 4 °C overnight. Precipitates were washed six times with lysis buffer, and receptor complexes were eluted with 30 μl of sample buffer. Western blotting was performed as described above. To address the question of the effect of ATP depletion on death receptor-mediated apoptosis, we first tested whether glucose removal was sufficient to deplete intracellular ATP in U937 cells. Incubation of U937 cells in a glucose-free RPMI 1640 medium supplemented with pyruvate, glutamine, and 5% dialyzed FBS, as described under “Experimental Procedures,” led to a marked drop in ATP levels (Fig.1). The ATP decrease occurred more rapidly when cells were incubated under the same conditions with 1% dialyzed serum (Fig. 1a). Because viability of cultures upon glucose deprivation was better maintained in medium containing 5% dialyzed FBS, this serum concentration was used in all experiments shown in the present work. When U937 cells were incubated in the absence of glucose and pyruvate, cells died rapidly, probably due to the lack of a carbon source rather than lack of ATP (not shown). We next examined the effect of glucose deprivation on apoptosis induced either by the DNA-damaging drug etoposide or by activators of different death receptors. We observed that in cultures of U937 cells treated with the DNA-damaging drug etoposide under glucose-deprived conditions apoptosis was inhibited (Fig. 1b), in agreement with reported data (14Ferrari D. Stepczynska A. Los M. Wesselborg S. Schulze-Osthoff K. J. Exp. Med. 1998; 188: 979-984Crossref PubMed Scopus (194) Google Scholar). In contrast, when apoptosis was induced by ligation of CD95, TNFR, or TRAILR, we observed a marked increase in the percentage of apoptotic cells in glucose-free medium, as measured by sub-G1 DNA content (Fig. 1b). Inhibition of etoposide-induced apoptosis and enhancement of death receptor-induced apoptosis were also observed in cells incubated under glucose-free conditions with 1% FBS (not shown), in which the ATP content was very low (10% of normal level). It was previously reported that 2-DG, a non-metabolizable glucose analogue that inhibits glucose metabolism, enhanced TNF-α-induced apoptosis in U937 cells (24Halicka H.D. Ardelt B. Li X. Melamed M.M. Darzynkiewicz Z. Cancer Res. 1995; 55: 444-449PubMed Google Scholar). We have confirmed these data and demonstrated that anti-CD95- and TRAIL-induced apoptosis was also clearly enhanced when U937 cells were incubated in the presence of 2-DG (Fig. 1c). At the concentration used (5 mm), 2-DG alone did not have a considerable effect on cell death. ATP loss upon 2-DG treatment occurred with similar kinetics to that observed in glucose removal experiments, although the decrease in ATP levels after 20 h of 2-DG treatment was 50% (not shown). To establish further that the death process promoted by glucose metabolism inhibition was apoptosis, U937 cells were treated for 20 h with 2-DG in the presence of death receptor activators, and several features of apoptosis were examined. Results shown in Fig.1c indicate that this death was dependent on caspase activation, as it was completely inhibited by the caspase inhibitor Z-VAD-FMK. Furthermore, results not shown indicated that there was an increase in the number of cells showing nuclear condensation and fragmentation in cultures incubated in the presence of both 2-DG and death receptor activator. In these experiments, 2-DG alone did not have a significant effect on cell death (not shown). Under these conditions, a low percentage of cells (<10% in all cases) was stained with propidium iodide, indicating loss of membrane integrity. However, these cells also displayed marked nuclear apoptotic changes suggesting that the observed changes in membrane permeability were probably due to secondary necrosis. Finally, we determined death receptor-induced translocation of phosphatidylserine to the external side of the plasma membrane when glucose uptake and utilization were inhibited by 2-DG. Results not shown indicated that annexin-V binding to cells upon death receptor triggering was also enhanced in the presence of 2-DG. We next tested whether the regulation by glucose metabolism of death receptor-induced apoptosis was cell type-specific. Accordingly, we carried out some experiments on tumor cells of non-hematopoietic origin such as human breast tumor MCF-7 cells and human cervical carcinoma HeLa cells. MCF-7 cells are very resistant to CD95-mediated apoptosis mainly because they express very low levels of CD95 at the cell surface (26Ruiz-Ruiz C. Munoz-Pinedo C. Lopez-Rivas A. Cancer Res. 2000; 60: 5673-5680PubMed Google Scholar). Therefore, to analyze the role of glucose metabolism in death receptor-induced apoptosis in MCF-7 cells, we used TRAIL as an apoptosis trigger. Data shown in Fig. 2a demonstrate that both glucose withdrawal and 2-DG facilitated the activation by TRAIL of caspase-dependent loss of viability in cultures of breast tumor MCF-7 cells. Likewise, glucose removal from the medium strongly enhanced CD95-mediated cell death in cervical carcinoma HeLa cells (Fig. 2b). Death receptor-induced apoptosis can occur by at least two different pathways (31Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar). One of them involving the mitochondria is facilitated when protein synthesis is inhibited and is inhibited in cells overexpressing anti-apoptotic BCL-2 family members (32Scaffidi C. Schmitz I. Zha J. Korsmeyer S.J. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 22532-22538Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). In this respect, death receptor-induced apoptosis in U937 cells is markedly enhanced in the presence of protein synthesis inhibitors (33Noguchi K. Naito M. Kataoka S. Yonehara S. Tsuruo T. Cell Growth Differ. 1995; 6: 1271-1277PubMed Google Scholar) and inhibited in clones overexpressing BCL-2 (34Monney L. Otter I. Olivier R. Ravn U. Mirzasaleh H. Fellay I. Poirier G.G. Borer C. Biochem. Biophys. Res. Commun. 1996; 221: 340-345Crossref PubMed Scopus (63) Google Scholar). These results suggest that the mitochondrial pathway of apoptosis is required for death receptor-induced apoptosis in U937 cells. In order to understand the step in death receptor-triggered cell death that is modulated by glucose levels, we first examined whether or not inhibition of glucose metabolism may regulate the mitochondria-regulated apoptotic pathway. To this end we analyzed in U937 cells the depolarization of mitochondrial membrane and the release of cytochrome c into the cytosol, two mitochondrial parameters that have been largely implicated in death receptor-mediated apoptosis (35Brahma C.A. Ian T. Streets K. Trautwein C. Brenner D.A. Lemasters J.J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (368) Google Scholar). Fig. 3a shows that although death receptor activation induced mitochondrial depolarization in U937 cells in the absence of 2-DG, this event was further stimulated upon ligation of death receptors in cells incubated in the presence of 2-DG. Likewise, release of cytochrome cinto the cytosol of U937 cells was facilitated in the presence of glucose anti-metabolite (Fig. 3b). In breast tumor MCF-7 cells the mitochondrial pathway is also important in death receptor-triggered apoptosis because these cells are deficient in caspase-3 expression, which is required to activate the non-mitochondrial pathway after death receptor activation (31Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar). Furthermore, in MCF-7 cells overexpressing BCL-2 or BCL-xL, both CD95 (26Ruiz-Ruiz C. Munoz-Pinedo C. Lopez-Rivas A. Cancer Res. 2000; 60: 5673-5680PubMed Google Scholar) and TRAILR-induced apoptosis (36Srinivasula S.M. Datta P. Fan X.J. Fernandes-Alnemri T. Huang Z. Alnemri E.S. J. Biol. Chem. 2000; 275: 36152-36157Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar) are considerably inhibited, indicating a role of mitochondria in death receptor-mediated apoptosis. In MCF-7BCL-2 cells, we examined the effect of glucose deprivation in TRAIL-induced apoptosis. As shown in Fig.3c, TRAIL induced significant death when added to glucose-free mock-transfected cultures (MCF-7neo). Results presented in Fig. 3c demonstrate that overexpression of BCL-2 completely abrogated apoptosis induced by the death ligand in cells deprived of glucose. These results also suggest that in those cell lines in which mitochondria are involved in death receptor-induced apoptosis, glucose deprivation does not overcome BCL-2 protection. The human SKW6.4 B-lymphoblastoid cell line has been extensively used in studies of apoptosis activation by death receptors, particularly CD95. These cells do not require mitochondria-regulated events to undergo apoptosis upon death receptor activation (31Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar). In order to get further insight into the mechanism of glucose depletion-promoted enhancement of death receptor-induced apoptosis, we examined the sensitivity of SKW6.4 cells to CD95 antibody and recombinant TRAIL. As shown in Fig. 3d, incubation of these cells in glucose-free medium increased their sensitivity to both CD95 antibody and TRAIL. Altogether, these results suggested that glucose deprivation must regula

The use of live cell microscopy has made a number of contributions to the study of apoptosis. Many of the tools and techniques are available that allow us to image the key events that occur during cell death including mitochondrial outer membrane permeabilization, mitochondrial transmembrane potential changes, translocation of Bcl-2 family members, caspase activation, phosphatidylserine flip and plasma membrane rupture. We discuss these techniques here and highlight the advantages and drawbacks of using such approaches to study apoptosis.

Apoptosis induced by most stimuli proceeds through the mitochondrial pathway. One such stimulus is nutrient deprivation. In this study we studied death induced by glucose deprivation in cells deficient in Bax and Bak. These cells cannot undergo mitochondrial outer membrane permeabilization (MOMP) during apoptosis, but they undergo necrosis when treated with MOMP-dependent apoptotic stimuli. We find in these cells that glucose deprivation, rather than inducing necrosis, triggered apoptosis. Cell death required caspase activation as inhibition of caspases with peptidic inhibitors prevented death. Glucose deprivation-induced death displayed many hallmarks of apoptosis, such as caspase cleavage and activity, phosphatidyl-serine exposure and cleavage of caspase substrates. Neither overexpression of Bcl-xL nor knockdown of caspase-9 prevented death. However, transient or stable knockdown of caspase-8 or overexpression of CrmA inhibited apoptosis. Cell death was not inhibited by preventing death receptor-ligand interactions, by overexpression of c-FLIP or by knockdown of RIPK1. Glucose deprivation induced apoptosis in the human tumor cell line HeLa, which was prevented by knockdown of caspase-8. Thus, we have found that glucose deprivation can induce a death receptor-independent, caspase-8-driven apoptosis, which is engaged to kill cells that cannot undergo MOMP.

In response to nutrient shortage or organelle damage, cells undergo macroautophagy. Starvation of glucose, an essential nutrient, is thought to promote autophagy in mammalian cells. We thus aimed to determine the role of autophagy in cell death induced by glucose deprivation. Glucose withdrawal induces cell death that can occur by apoptosis (in Bax, Bak-deficient mouse embryonic fibroblasts or HeLa cells) or by necrosis (in Rh4 rhabdomyosarcoma cells). Inhibition of autophagy by chemical or genetic means by using 3-methyladenine, chloroquine, a dominant negative form of ATG4B or silencing Beclin-1, Atg7, or p62 indicated that macroautophagy does not protect cells undergoing necrosis or apoptosis upon glucose deprivation. Moreover, glucose deprivation did not induce autophagic flux in any of the four cell lines analyzed, even though mTOR was inhibited. Indeed, glucose deprivation inhibited basal autophagic flux. In contrast, the glycolytic inhibitor 2-deoxyglucose induced prosurvival autophagy. Further analyses indicated that in the absence of glucose, autophagic flux induced by other stimuli is inhibited. These data suggest that the role of autophagy in response to nutrient starvation should be reconsidered. Background: Autophagy is a response to nutrient deprivation. Results: Inhibition of autophagy does not sensitize cells to apoptotic or necrotic cell death induced by glucose starvation. Moreover, glucose deprivation inhibits autophagy. Conclusion: 2-Deoxyglucose, but not glucose deprivation, induces autophagy. Significance: Not all forms of starvation induce cytoprotective autophagy in mammalian cells.

Autophagy is a cellular recycling and stress response that degrades organelles and long-lived proteins and serves to protect cells from the potential damage induced by dysfunctional organelles and protein aggregates.1 Autophagy can also be used as a recycling or salvage process to provide amino acids, nucleotides and other building blocks to protect cells from some, but not all, forms of starvation.2, 3

The role of interferon (IFN)-γ as a sensitizing agent in apoptosis induced by ligation of death receptors has been evaluated in human myeloid leukemia cells. Incubation of U937 cells with IFN-γ sensitized these cells to apoptosis induced by tumor necrosis factor-α, agonistic CD95 antibody, and tumor necrosis factor-related apoptosis-inducing ligand. Other human myeloid leukemic cells were also sensitized by IFN-γ to death receptor-mediated apoptosis. Treatment of U937 cells with IFN-γ up-regulated the expression of caspase-8 and potently synergized with death receptor ligation in the processing of caspase-8 and BID cleavage. Concomitantly, a marked down-regulation of BCL-2 protein was also observed in cells incubated with IFN-γ. Furthermore, the caspase-dependent generation of a 23-kDa fragment of BCL-2 protein, the release of cytochrome c from mitochondria and the activation of caspase-9 were also enhanced upon death receptor ligation in IFN-γ-treated cells. Ectopically expressed Bcl-2 protein inhibited IFN-γ-induced sensitization to apoptosis. In summary, these results indicate that IFN-γ sensitizes human myeloid leukemic cells to a death receptor-induced, mitochondria-mediated pathway of apoptosis. The role of interferon (IFN)-γ as a sensitizing agent in apoptosis induced by ligation of death receptors has been evaluated in human myeloid leukemia cells. Incubation of U937 cells with IFN-γ sensitized these cells to apoptosis induced by tumor necrosis factor-α, agonistic CD95 antibody, and tumor necrosis factor-related apoptosis-inducing ligand. Other human myeloid leukemic cells were also sensitized by IFN-γ to death receptor-mediated apoptosis. Treatment of U937 cells with IFN-γ up-regulated the expression of caspase-8 and potently synergized with death receptor ligation in the processing of caspase-8 and BID cleavage. Concomitantly, a marked down-regulation of BCL-2 protein was also observed in cells incubated with IFN-γ. Furthermore, the caspase-dependent generation of a 23-kDa fragment of BCL-2 protein, the release of cytochrome c from mitochondria and the activation of caspase-9 were also enhanced upon death receptor ligation in IFN-γ-treated cells. Ectopically expressed Bcl-2 protein inhibited IFN-γ-induced sensitization to apoptosis. In summary, these results indicate that IFN-γ sensitizes human myeloid leukemic cells to a death receptor-induced, mitochondria-mediated pathway of apoptosis. interferon tumor necrosis factor tumor necrosis factor-related apoptosis-inducing ligand interleukin-1-β-converting enzyme death-inducing signaling complex poly(ADP-ribose) polymerase benzyloxycarbonyl-Val-Ala-Asp-(OMe)fluoromethyl ketone FLICE-inhibitory protein interferon regulatory factor-1 monoclonal antibody polymerase chain reaction reverse transcription phosphate-buffered saline fas-associated death domain Interferons (IFNs)1 are a family of natural glycoproteins that share antiviral, immunomodulatory, and anti-proliferative effects (1Stark G.R. Kerr I.M. Williams B.R.G. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3380) Google Scholar). In mice, a defect in the transcriptional regulation of IFN-dependent genes causes a marked increase in the number of myeloid cells and hematologic alterations similar to chronic human myelogenous leukemia (2Gabriele L. Phung J. Fukumoto J. Segal D. Wang I.M. Giannakakou P. Giese N.A. Ozato K. Morse III, H.C. J. Exp. Med. 1999; 190: 411-422Crossref PubMed Scopus (101) Google Scholar). In both chronic and acute human myeloid leukemias, a decrease of IFN-modulated transcriptional activity has been reported (3Schmidt M. Nagel S. Proba J. Thiede C. Ritter M. Waring J.F. Rosenbauer F. Huhn D. Wittig B. Horak I. Neubauer A. Blood. 1998; 91: 22-29Crossref PubMed Google Scholar, 4Green W.B. Slovak M.L. Chen I.M. Pallavicini M. Hecht J.L. Willman C.L. Leukemia. 1999; 13: 1960-1971Crossref PubMed Scopus (54) Google Scholar). 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In tumor cell lines, IFN-γ can induce or modulate cell death either as a single agent or in combination with chemotherapeutic drugs (9Ossina N.K. Cannas A. Powers V.C. Fitzpatrick P.A. Knight J.D. Gilbert J.R. Shekhtman E.M. Tomei L.D. Umansky S.R. Kiefer M.C. J. Biol. Chem. 1997; 272: 16351-16357Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Apoptosis is an active form of cell death that plays a fundamental role in normal development, tissue homeostasis, and pathological situations (10Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1362) Google Scholar, 11Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6191) Google Scholar, 12Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar). CD95 (Fas/Apo-1) receptor, a member of TNF/nerve growth factor receptor family (13Itoh N. Yonehara S. Ishii A. Yonehara M. Mizushima S.I. Sameshima M. Hase A. Seto Y. 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Chem. 1996; 271: 12687-12690Abstract Full Text Full Text PDF PubMed Scopus (1649) Google Scholar) upon binding to death-domain containing receptors, TRAIL-R1 and TRAIL-R2 (also known as DR4 and DR5, respectively) (18Pan G. Ni J. Wei Y.-F., Yu, G.-L. Gentz R. Dixit V.M. Science. 1997; 277: 815-818Crossref PubMed Scopus (1381) Google Scholar, 19Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit V.M. Science. 1997; 276: 111-113Crossref PubMed Scopus (1561) Google Scholar, 20Sheridan J.P. Marsters S.A. Pitti R.M. Gurney A. Skubatch M. Baldwin D. Ramakrishnan L. Gray C.L. Baker K. Wood W.I. Goddard A.D. Godowski P. Ashkenazi A. Science. 1997; 277: 818-821Crossref PubMed Scopus (1529) Google Scholar, 21Walczak H. Degli-Esposti M.A. Johnson R.S. Smolak P.J. Waugh J.Y. Boiani N. Timour M.S. Gerhart M.J. Schooley K.A. Smith C.A. Goodwin R.G. Rauch C.T. EMBO J. 1997; 16: 5386-5397Crossref PubMed Scopus (1018) Google Scholar). Although the expression of CD95L seems to be more restricted to lymphoid cells (15Suda T. Takahashi T. Golstein P. Nagata S. Cell. 1993; 75: 1169-1178Abstract Full Text PDF PubMed Scopus (2448) Google Scholar), TRAIL transcripts are detectable in many normal organs and tissues (16Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.P. Nicholl J.K. Sutherland G.R. Smith T.D. Rauch C. Smith C.A. Goodwin R.G. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2656) Google Scholar), suggesting that this ligand may be non-toxic to normal cells. Death receptors are expressed in many tumor cells that can therefore be killed by the appropriate ligands (22Ashkenazi A. Dixit V.M. Curr. Opin. Cell Biol. 1999; 11: 255-260Crossref PubMed Scopus (1155) Google Scholar). However, expression of death receptors is not always sufficient to allow an apoptotic response since there are examples of tumor cells, including myeloid leukemic cells, that express significant levels of death receptors in the plasma membrane but are resistant to death ligands (22Ashkenazi A. Dixit V.M. Curr. Opin. Cell Biol. 1999; 11: 255-260Crossref PubMed Scopus (1155) Google Scholar, 23Dirks W. Schone S. Uphoff C. Quentmeier H. Pradella S. Drexler H.G. Br. J. Haematol. 1997; 96: 584-593Crossref PubMed Scopus (67) Google Scholar). Understanding the mechanisms that sensitize tumor cells to death ligand-induced apoptosis could therefore be an important objective in the development of therapies to treat malignancies like human myelogenous leukemias. In this respect, IFN-γ and IFN-α can up-regulate the expression of a number of apoptosis-related proteins in different types of cells (9Ossina N.K. Cannas A. Powers V.C. Fitzpatrick P.A. Knight J.D. Gilbert J.R. Shekhtman E.M. Tomei L.D. Umansky S.R. Kiefer M.C. J. Biol. Chem. 1997; 272: 16351-16357Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar,24Spanaus K.S. Schlapbach R. Fontana A. Eur. J. Immunol. 1998; 28: 4398-4408Crossref PubMed Google Scholar, 25Kayagaki N. Yamaguchi N. Nakayama M. Eto H. Okumura K. Yagita H. J. Exp. Med. 1999; 189: 1451-1460Crossref PubMed Scopus (435) Google Scholar). In certain cancer cells, including U937 myeloid leukemic cells, it has been reported that IFN-γ induces sensitization to CD95-mediated apoptosis by up-regulating the expression of ICE/caspase-1 (26Keane M.M. Ettenberg S.A. Lowrey G.A. Russell E.K. Lipkowitz S. Cancer Res. 1996; 56: 4791-4798PubMed Google Scholar, 27Tamura T. Ueda S. Yoshida M. Matsuzaki M. Mohri H. Okubo T. Biochem. Biophys. Res. Commun. 1996; 229: 21-26Crossref PubMed Scopus (107) Google Scholar). However, more recent data have demonstrated that caspase-1/ICE is not involved in the proteolytic cascade activated upon CD95 cross-linking at the cell surface by CD95L or CD95 antibody (28Smith D.J. McGuire M.J. Tocci M.J. Thiele D.L. J. Immunol. 1997; 158: 163-170PubMed Google Scholar, 29Chow S.C. Slee E.A. MacFarlane M. Cohen G.M. Exp. Cell Res. 1999; 246: 491-500Crossref PubMed Scopus (15) Google Scholar, 30Los M. Wesselborg S. Schulze-Osthoff K. Immunity. 1999; 10: 629-639Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). The above data prompted us to investigate the effects of IFN-γ on death receptor-induced apoptosis in the human U937 myeloid leukemic cell line. We were particularly interested to ascertain whether IFN-γ could enhance the sensitivity of these cells to TRAIL-induced apoptosis, in view of the importance of TRAIL as a rather selective anti-tumor protein (31Walczak H. Miller R.E. Ariail K. Gliniak B. Griffith T.S. Kubin M. Chin W. Jones J. Woodward A. Le T. Smith C. Smolak P. Goodwin R.G. Rauch C.T. Schuh J.C. Lynch D.H. Nat. Med. 1999; 5: 157-163Crossref PubMed Scopus (2233) Google Scholar). In this report we show that IFN-γ sensitizes U937 cells and other human myeloid leukemic cell lines to CD95-, TNF-R-, and TRAIL-R-mediated apoptosis. Following treatment of U937 cells with IFN-γ, we have observed a significant up-regulation of caspase-8 and a marked down-regulation of BCL-2 protein. Furthermore, we demonstrate that in U937 cells, IFN-γ facilitates several biochemical events involved in death receptor-induced mitochondria-mediated apoptosis. RPMI 1640 medium and fetal bovine serum were obtained from Life Technologies, Inc. CH-11 monoclonal antibody (mAb) reacting with CD95 was from Upstate Biotechnology Inc. (Lake Placid, NY). Human IFN-γ, human TNF-α and recombinant human TRAIL were obtained from PreproTech EC Ltd (London, United Kingdom). Mouse anti-BAX mAb, mouse anti-BAD mAb, and mouse anti-cytochromec mAb were obtained from PharMingen (San Diego, CA). Mouse anti-BCL-2 mAb was from Dako (Glostrup, Denmark). Mouse anti-human caspase-8 mAb and rabbit anti-caspase-9 polyclonal antibodies were purchased from Cell Diagnostica (Münster, Germany) and StressGen Biotechnologies Corp. (Victoria, Canada), respectively. Goat polyclonal anti-caspase-3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antiserum against PARP was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Mouse anti-FADD mAb was from Transduction Laboratories (Lexington, KY). Rabbit anti-BID polyclonal antibody was generously provided by Dr. X. Wang (Howard Hughes Medical Institute, Dallas, TX). Rabbit polyclonal antibodies to caspase-9 p37 fragment and caspase-3 p17 subunit were obtained from New England Biolabs (Beverly, MA). Monoclonal antibody to alpha-tubulin was purchased from Sigma (Poole, United Kingdom). Benzyloxycarbonyl-Val-Ala-Asp- (OMe)fluoromethyl ketone (Z-VAD-fmk) was from Enzyme System Inc. (Dublin, CA). The various human myeloid leukemic cell lines used in this study were maintained in culture in RPMI 1640 medium containing 10% fetal calf serum and 1 mml-glutamine, at 37 °C in a humidified 5% CO2, 95% air incubator. Cell viability was determined by the trypan blue dye exclusion method. Analysis by flow cytometry of hypodiploid apoptotic cells was performed on a FACScan cytometer using the Cell Quest software (Becton Dickinson, Mountain View, CA), after extraction of the degraded DNA from apoptotic cells following a recently described method (32Gong J. Traganos F. Darzynkiewicz Z. Anal. Biochem. 1994; 218: 314-319Crossref PubMed Scopus (649) Google Scholar). Phosphatidylserine exposure on the surface of apoptotic cells was detected by flow cytometry after staining with Annexin-V-FLUOS (Roche Molecular Biochemicals). Analysis of DNA cleavage into oligonucleosome-length fragments was performed following a method described previously (33Ruiz-Ruiz M.C. Oliver F.J. Izquierdo M. Lopez-Rivas A. Mol. Immunol. 1995; 32: 947-955Crossref PubMed Scopus (14) Google Scholar). For measurements of cytochrome c release from mitochondria, cells were lysed and cytosolic fractions were separated from mitochondria as described (34Chandra J. Niemer I. Gilbreath J. Kliche K.O. Andreeff M. Freireich E.J. Keating M. McConkey D.J. Blood. 1998; 92: 4220-4229Crossref PubMed Google Scholar). Cytosolic proteins (40 μg of protein) were mixed with Laemmli buffer and resolved on 12% SDS-polyacrylamide minigels. Cytochromec was determined by Western blot analysis as described below. Cells were pelleted and lysed in Laemmli buffer. After sonication, proteins were resolved on 7.5% SDS-polyacrylamide minigels for determination of PARP cleavage or 12% SDS for analysis of other proteins, and electrophoretically transferred onto Immobilon (Millipore). Membranes were blocked with 5% milk powder in PBS plus 0.1% Tween 20 (PBS/Tween) for 1 h and washed with PBS/Tween. For protein detection, immunoblots were probed with α-tubulin mAb (1:40000), BAX mAb (1 μg/ml), BAD mAb (1:500), cytochrome c mAb (0.5 μg/ml), polyclonal antiserum against PARP (1:2000), caspase-3 antibody (1:1000), caspase-8 mAb (1:200), caspase-9 antibody (1:1000), caspase-9 (37-kDa fragment, 1:500), caspase-3 (17-kDa subunit, 1:500), BID antibody (1:2000), or BCL-2 mAb (1:1000). After washing, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:2000; Dako) or horseradish peroxidase-conjugated anti-mouse Ig (1:2000, Dako). Bound antibody was visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech), according to manufacturer's instructions. Total RNA was isolated from cells with Trizol reagent (Life Technologies, Inc.) as recommended by the supplier. cDNAs were synthesized from 2 μg of total RNA using a RT-PCR kit (PerkinElmer Life Sciences) with the supplied oligo(dT) primer under conditions described by the manufacturer. PCR reactions were performed using the following primers: TRAIL-R1 sense, 5′-CTGAGCAACGCAGACTCGCTGTCCAC-3′; TRAIL-R1 antisense, 5′-TCCAAGGACACGGCAGAGCCTGTGCCAT-3′; TRAIL-R2 sense, 5′-GCCTCATGGACAATGAGATAAAGGTGGCT-3′; TRAIL-R2 antisense, 5′-CCAAATCTCAAAGTACGCACAAACGG-3′; BAK sense, 5′-CCTGTTTGAGAGTGGCATC-3′; BAK antisense, 5′-TCGTACCACAAACTGGCCCA-3′; IRF-1 sense, 5′-CTTAAGAACCAGGCAACCTCTGCCTTC-3′; IRF-1 antisense, 5′-GATATCTGGCAGGGAGTTCATG-3′; BCL-2 sense, 5′-AGATGTCCAGCCAGCTGC ACCTGAC-3′; BCL-2 antisense, 5′AGATAGGCACCAGGGTGAGCAAGCT-3′; β-actin sense, 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′; and β-actin antisense, 5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′, giving products of 506, 502, 266, 406, 367, and 661 base pairs, respectively. Expression of β-actin was used as a control of RNA integrity and equal gel loading. Cycle conditions for all PCR reactions were 1 min at 95 °C, 1 min at 55 °C, and 1 min at 72 °C for 30 cycles. FLIP was analyzed by RT-PCR as described recently (35Yeh J.H. Hsu S.C. Han S.H. Lai M.Z. J. Exp. Med. 1998; 188: 1795-1802Crossref PubMed Scopus (119) Google Scholar). Activation of death receptors in tumor cells by appropriate ligands or agonistic antibodies results in the death of target cells by apoptosis (22Ashkenazi A. Dixit V.M. Curr. Opin. Cell Biol. 1999; 11: 255-260Crossref PubMed Scopus (1155) Google Scholar). However, some tumor cells are not very sensitive to death receptor-mediated apoptosis unless protein synthesis is inhibited (36Owen-Schaub L.B. Radinsky R. Kruzel E. Berry K. Yonehara S. Cancer Res. 1994; 54: 1580-1586PubMed Google Scholar). This is also the case for the human promyelocytic cell line U937 (Fig.1). As shown in Fig. 1, U937 cells were markedly sensitized to CD95-mediated death by co-treatment with the protein synthesis inhibitor cycloheximide. In order to find a physiologically relevant sensitizing agent, in this work we have examined the ability of IFN-γ to modulate the apoptotic response of human U937 myeloid leukemic cells upon death receptor ligation in the cell surface. IFN-γ can induce an anti-proliferative response in a variety of tumor cells (1Stark G.R. Kerr I.M. Williams B.R.G. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3380) Google Scholar). It could also activate an apoptotic program or sensitize cells to apoptosis induced by other stimuli (27Tamura T. Ueda S. Yoshida M. Matsuzaki M. Mohri H. Okubo T. Biochem. Biophys. Res. Commun. 1996; 229: 21-26Crossref PubMed Scopus (107) Google Scholar, 37Deiss L.P. Galinka H. Berissi H. Cohen O. Kimchi A. EMBO J. 1996; 15: 3861-3870Crossref PubMed Scopus (419) Google Scholar, 38Sato T. Selleri C. Anderson S. Young N.S. Maciejewski J.P. Br. J. Haematol. 1997; 97: 356-365Crossref PubMed Scopus (62) Google Scholar), although the mechanism underlying the sensitization process remains unclear. Results shown in Fig. 2 indicate that pre-incubation of U937 cells with IFN-γ (10 units/ml) for 24 h markedly sensitized these leukemic cells to apoptosis upon death receptor activation. In these experiments we observed that IFN-γ facilitated the generation of hypodiploid apoptotic cells by a subsequent treatment with TNF-α, CD95 agonistic antibody, or TRAIL (Fig. 2 a). This effect was paralleled by the externalization of phosphatidylserine in the plasma membrane of U937 cells (data not shown). Other apoptotic features like activation of caspase-3 (Fig.2 b), DNA fragmentation in a ladder pattern (Fig.2 c), and PARP cleavage (Fig. 2 d) were also markedly enhanced by IFN-γ. In the experiments involving CD95 IgM, an irrelevant IgM antibody did not cooperate with IFN-γ in the induction of apoptosis (results not shown).Figure 2IFN -γ sensitizes U937 cells to death receptor-mediated apoptosis. Cells were pre-incubated for 24 h with 10 units/ml IFN-γ and treated for an additional 24-h period with CD95 mAb (20 ng/ml), TNF-α (1 ng/ml), or TRAIL (10 ng/ml). Apoptotic features were determined as described under “Experimental Procedures.” a, percentage of apoptotic cells as determined by cytofluorimetric analysis of DNA content.b, caspase-3 activation, measured by the generation of 17-kDa subunit. c, DNA fragmentation into oligonucleosome-length fragments. d, proteolytic cleavage of poly(ADP-ribose) polymerase. In a, error bars represent S.D. from three independent experiments. Inb–d, the results illustrate a representative experiment from at least three different experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Sensitization by IFN-γ to death receptor-induced apoptosis was also observed in two other human myeloid leukemic cell lines examined. Results shown in Fig. 3 demonstrate that incubation of either HL-60 (Fig. 3 a) or THP-1 (Fig.3 b) cells with IFN-γ sensitized them to a subsequent treatment with TNF-α or CD95 antibody. It has been reported that IFN-γ can elevate the expression of CD95 in several acute myelogenous leukemic cell lines, including U937 cells (39Sumimoto S. Ishigami T. Horiguchi Y. Yonehara S. Kanazashi S. Heike T. Katamura K. Mayumi M. Cell Immunol. 1994; 153: 184-193Crossref PubMed Scopus (29) Google Scholar, 40Munker R. Andreeff M. Cytokines Mol. Ther. 1996; 2: 147-159PubMed Google Scholar) and that this effect could explain the enhanced sensitivity of IFN-γ-treated cells to apoptosis mediated by CD95 receptors. However, U937 cells express a high number of CD95 molecules in their surface, and in our experiments we have observed only a slight increase in CD95 expression in IFN-γ-treated cells (data not shown). Furthermore, we have determined by RT-PCR the expression of pro-apoptotic TRAIL receptors (DR4 and DR5) in U937 cells treated with IFN-γ (Fig.4). As a control of IFN-γ action, we determined the expression of the transcription factor IRF-1, an IFN-γ-regulated gene (Fig. 4). Expression of TRAIL receptors was not significantly elevated by IFN-γ in U937 cells after 24 h (Fig.4) or 48 h (data not shown). We also examined by RT-PCR the levels of TRAIL decoy receptors DcR1 and DcR2 in cells incubated in the presence of IFN-γ for 24 h. Results not shown indicated that the cellular levels of these anti-apoptotic receptors did not change upon IFN-γ treatment. Although we have not investigated the expression of TNF-R in U937 cells, the above results suggested that the sensitization to apoptosis observed in IFN-γ-treated cells should be probably related to changes in the intracellular levels of apoptosis regulators rather than to an increase in the expression of death receptors. Regulation of the expression and/or activity of the death receptor-inducing signaling complex (DISC) components could be a strategy used by tumor cells to escape from the host immune system (41Thome M. Schneider P. Hofmann K. Fickenscher H. Meinl E. Neipel F. Mattmann C. Burns K. Bodmer J.L. Schroter M. Scaffidi C. Krammer P.H. Peter M.E. Tschopp J. Nature. 1997; 386: 517-521Crossref PubMed Scopus (1144) Google Scholar, 42Griffith T.S. Chin W.A. Jackson G.C. Lynch D.H. Kubin M.Z. J. Immunol. 1998; 161: 2833-2840PubMed Google Scholar, 43Teitz T. Wei T. Valentine M.B. Vanin E.F. Grenet J. Valentine V.A. Behm F.G. Look A.T. Lahti J.M. Kidd V.J. Nat. Med. 2000; 6: 529-535Crossref PubMed Scopus (699) Google Scholar). Caspase-8, the most apical caspase required in death receptor-mediated apoptosis (44Varfolomeev E.E. Schuchmann M. Luria V. Chiannilkulchai N. Beckmann J.S. Mett I.L. Rebrikov D. Brodianski V.M. Kemper O.C. Kollet O. Lapidot T. Soffer D. Sobe T. Avraham K.B. Goncharov T. Holtmann H. Lonai P. Wallach D. Immunity. 1998; 9: 267-276Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar), is also a cellular target of oncogenic viruses, to protect transformed cells from death receptor-induced apoptosis (45Chen P. Tian J. Kovesdi I. Bruder J.T. J. Biol. Chem. 1998; 273: 5815-5820Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The intracellular signaling mechanism involved in the activation of apoptosis by death receptors comprises different activities (46Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5154) Google Scholar). The adapter protein FADD is responsible for coupling death receptors to the initiator caspase-8 (47Baker S.J. Reddy E.P. Oncogene. 1996; 12: 1-9PubMed Google Scholar). We have examined the levels of both FADD and caspase-8 in U937 cells following treatment with IFN-γ. Results in Fig. 5indicate that procaspase-8, but not FADD, was up-regulated in these leukemic cells after 24 h of incubation in the presence of IFN-γ. This could be relevant in the mechanism of IFN-γ-induced sensitization of U937 cells to death receptor-mediated apoptosis as overexpression of procaspase-8 by transfection has been reported to facilitate apoptosis (48Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar). On the contrary, the cellular levels of procaspase-9, which plays an important role in the mitochondria-mediated pathway of apoptosis, and procaspase-3, responsible for many of the nuclear changes during apoptosis, did not change in U937 cells treated with IFN-γ for up to 48 h (Fig.5). To further characterize the cellular modulators of apoptosis that may be responsible for the sensitizing action of IFN-γ in U937 cells, we analyzed the expression of the apoptosis inhibitor FLIP and several pro-apoptotic members of the BCL-2 family. As shown in Fig.6 (a and b), IFN-γ treatment did not modify the cellular levels of FLIP, BAK, BAX, BAD, or BID, determined either by RT-PCR or Western blot analysis. In these experiments, the cellular expression of IRF-1 mRNA was up-regulated by IFN-γ, serving as an internal control of IFN-γ activity. Interestingly, when we determined the expression of anti-apoptotic BCL-2 protein, a marked decline in the cellular levels of this anti-apoptotic protein was observed following IFN-γ treatment (Fig.7). A decreased level of BCL-2 was clearly observed after 24 h of IFN-γ treatment and remained low for at least 48 h. This effect was also observed at the mRNA level (Fig. 7). Although we cannot exclude an IFN-γ-induced decrease of BCL-2 mRNA stability, these results may suggest a negative regulation of BCL-2 gene transcription by IFN-γ as described recently (49Stephanou A. Brar B.K. Knight R.A. Latchman D.S. Cell Death Differ. 2000; 7: 329-330Crossref PubMed Scopus (151) Google Scholar). Reduction in the levels of BCL-2 could be an important event in the regulation of cellular sensitivity to stress treatments, which operate through a mitochondrial pathway of apoptosis (50Green D. Kroemer G. Trends Cell Biol. 1998; 8: 267-271Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar). A decrease in cellular BCL-2 protein levels could also sensitize type II cells to CD95-mediated apoptosis (51Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2631) Google Scholar). CD95 type II cells are also characterized by a markedly increased sensitivity to death receptor-induced apoptosis upon inhibition of protein synthesis (52Scaffidi C. Schmitz I. Zha J. Korsmeyer S.J. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 22532-22538Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). Sensitivity of U937 cells to death receptor-induced apoptosis is considerably enhanced by cycloheximide treatment (Fig. 1) (53Noguchi K. Naito M. Kataoka S. Yonehara S. Tsuruo T. Cell Growth Differ. 1995; 6: 1271-1277PubMed Google Scholar), which suggests that U937 cells are probably type II cells (52Scaffidi C. Schmitz I. Zha J. Korsmeyer S.J. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 22532-22538Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). In order to ascertain the steps in death receptor-mediated apoptosis that are affected by IFN-γ treatment, we first examined the activation of caspase-8 by analyzing the processing of this caspase into various specific proteolytic fragments. As shown in Fig.8 a, activation of either death receptor clearly synergized with IFN-γ in the stimulation of procaspase-8 processing. By immunoblot analysis of caspase-8 in treated cells, we detected both the 55- and 53-kDa inactive proforms corresponding to caspase-8a and -8b as well as the 43- and 41-kDa intermediate products corresponding to cleavage of both caspase-8a and -8b between the large and small subunits. We also detected the presence of the large 18-kDa subunit, which would lead upon combination with the small 10-kDa subunit to the assembly of the active caspase (54Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1041) Google Scholar). Caspase-8 is the most upstream caspase required in death receptor-mediated apoptosis (44Varfolomeev E.E. Schuchmann M. Luria V. Chiannilkulchai N. Beckmann J.S. Mett I.L. Rebrikov D. Brodianski V.M. Kemper O.C. Kollet O. Lapidot T. Soffer D. Sobe T. Avraham K.B. Goncharov T. Holtmann H. Lonai P. Wallach D. Immunity. 1998; 9: 267-276Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar), although it could also be activated downstream of mitochondria t

Altered metabolism is a hallmark of cancer that opens new therapeutic possibilities. 2‐deoxyglucose (2‐ DG ) is a non‐metabolizable glucose analog tested in clinical trials and is frequently used in experimental settings to mimic glucose starvation. However, in the present study, conducted in a rhabdomyosarcoma cell line, we find that 2‐ DG induces classical nuclear apoptotic morphology and caspase‐dependent cell death, whereas glucose deprivation drives cells toward necrotic cell death. Necrosis induced by glucose deprivation did not resemble necroptosis or ferroptosis and was not prevented by antioxidants. Both stimuli promote endoplasmic reticulum stress. Moreover, the transcription factor ATF 4 is found to mediate both the apoptosis induced by 2‐ DG and the glycosylation inhibitor tunicamycin, as well as the necrosis provoked by glucose withdrawal. Several hexoses partially prevented glucose deprivation‐induced necrosis in rhabdomyosarcoma, although only mannose prevented apoptosis induced by 2‐ DG . In both cases, a reduction of cell death was associated with decreased levels of ATF 4. Our results confirm previous data indicating the differential effects of these two forms with respect to inhibiting glucose metabolism, and they place endoplasmic reticulum stress as the critical mediator of glucose starvation‐induced cell death.

In this report, we have assessed the role of IFN-gamma as a sensitizing agent in apoptosis mediated by activation of death receptor CD95 in breast tumor cells. Treatment of the tumor cell lines MCF-7 and MDA-MB231 with IFN-gamma significantly facilitated apoptosis induced by CD95 receptor ligation at the plasma membrane, independently of p53 status. In contrast, IFN-gamma treatment did not enhance the apoptotic effect of the DNA-damaging drug, doxorubicin. Analysis of apoptosis regulators indicated that caspase-8 mRNA and protein levels were up-regulated in both of the cell lines after treatment with IFN-gamma. Furthermore, IFN-gamma sensitized MCF-7 and MDA-MB231 cells to CD95-mediated activation of caspase-8, induction of cytochrome c release from mitochondria, and processing of caspase-9. Release of cytochrome c, caspases activation, and apoptosis were prevented in MCF-7 cells overexpressing Bcl-2. Altogether these results indicate that IFN-gamma, maybe through the elevation of caspase-8 levels, sensitizes human breast tumor cells to a death receptor-mediated, mitochondria-operated pathway of apoptosis.

DeathBase: a database on structure, evolution and function of proteins involved in apoptosis and other forms of cell death

Abstract During the last decades, the knowledge of cell death mechanisms involved in anticancer therapy has grown exponentially. However, in many studies, cell death is still described in an incomplete manner. The frequent use of indirect proliferation assays, unspecific probes, or bulk analyses leads too often to misunderstandings regarding cell death events. There is a trend to focus on molecular or genetic regulations of cell demise without a proper characterization of the phenotype that is the object of this study. Sometimes, cancer researchers can feel overwhelmed or confused when faced with such a corpus of detailed insights, nomenclature rules, and debates about the accuracy of a particular probe or assay. On the basis of the information available, we propose a simple guide to distinguish forms of cell death in experimental settings using cancer cell lines. Cancer Res; 75(6); 913–7. ©2015 AACR.

Cancer cells escape, suppress and exploit the host immune system to sustain themselves, and the tumor microenvironment (TME) actively dampens T cell function by various mechanisms. Over the last years, new immunotherapeutic approaches, such as adoptive chimeric antigen receptor (CAR) T cell therapy and immune checkpoint inhibitors, have been successfully applied for refractory malignancies that could only be treated in a palliative manner previously. Engaging the anti-tumor activity of the immune system, including CAR T cell therapy to target the CD19 B cell antigen, proved to be effective in acute lymphocytic leukemia. In low-grade hematopoietic B cell malignancies, such as chronic lymphocytic leukemia, clinical outcomes have been tempered by cancer-induced T cell dysfunction characterized in part by a state of metabolic lethargy. In multiple myeloma, novel antigens such as BCMA and CD38 are being explored for CAR T cells. In solid cancers, T cell-based immunotherapies have been applied successfully to melanoma and lung cancers, whereas application in e.g., breast cancer lags behind and is modestly effective as yet. The main hurdles for CAR T cell immunotherapy in solid tumors are the lack of suitable antigens, anatomical inaccessibility, and T cell anergy due to immunosuppressive TME. Given the wide range of success and failure of immunotherapies in various cancer types, it is crucial to comprehend the underlying similarities and distinctions in T cell dysfunction. Hence, this review aims at comparing selected, distinct B cell-derived versus solid cancer types and at describing means by which malignant cells and TME might dampen T cell anti-tumor activity, with special focus on immunometabolism. Drawing a meaningful parallel between the efficacy of immunotherapy and the extent of T cell dysfunction will shed light on areas where we can improve immune function to battle cancer.

Recent technological advances and the application of high-throughput mutation and transcriptome analyses have improved our understanding of cancer diseases, including non-small cell lung cancer. For instance, genomic profiling has allowed the identification of mutational events which can be treated with specific agents. However, detection of DNA alterations does not fully recapitulate the complexity of the disease and it does not allow selection of patients that benefit from chemo- or immunotherapy. In this context, transcriptional profiling has emerged as a promising tool for patient stratification and treatment guidance. For instance, transcriptional profiling has proven to be especially useful in the context of acquired resistance to targeted therapies and patients lacking targetable genomic alterations. Moreover, the comprehensive characterization of the expression level of the different pathways and genes involved in tumor progression is likely to better predict clinical benefit from different treatments than single biomarkers such as PD-L1 or tumor mutational burden in the case of immunotherapy. However, intrinsic technical and analytical limitations have hindered the use of these expression signatures in the clinical setting. In this review, we will focus on the data reported on molecular classification of non-small cell lung cancer and discuss the potential of transcriptional profiling as a predictor of survival and as a patient stratification tool to further personalize treatments.

Interleukin-8 (IL-8/CXCL8) is a pro-angiogenic and pro-inflammatory chemokine that plays a role in cancer development. Non-small cell lung carcinoma (NSCLC) produces high amounts of IL-8, which is associated with poor prognosis and resistance to chemo-radio and immunotherapy. However, the signaling pathways that lead to IL-8 production in NSCLC are unresolved. Here, we show that expression and release of IL-8 are regulated autonomously by TRAIL death receptors in several squamous and adenocarcinoma NSCLC cell lines. NSCLC constitutively secrete IL-8, which could be further enhanced by glucose withdrawal or by treatment with TRAIL or TNFα. In A549 cells, constitutive and inducible IL-8 production was dependent on NF-κB and MEK/ERK MAP Kinases. DR4 and DR5, known regulators of these signaling pathways, participated in constitutive and glucose deprivation-induced IL-8 secretion. These receptors were mainly located intracellularly. While DR4 signaled through the NF-κB pathway, DR4 and DR5 both regulated the ERK-MAPK and Akt pathways. FADD, caspase-8, RIPK1, and TRADD also regulated IL-8. Analysis of mRNA expression data from patients indicated that IL-8 transcripts correlated with TRAIL, DR4, and DR5 expression levels. Furthermore, TRAIL receptor expression levels also correlated with markers of angiogenesis and neutrophil infiltration in lung squamous carcinoma and adenocarcinoma. Collectively, these data suggest that TRAIL receptor signaling contributes to a pro-tumorigenic inflammatory signature associated with NSCLC.

Bc1-2 protein is a potent anti-apoptotic protein that inhibits a mitochondria-operated pathway of apoptosis in many cells. DNA damaging agents and death receptor ligands can activate this mitochondrial apoptotic mechanism. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been suggested to escape from the inhibitory action of Bc1-2 protein. We show that in human breast tumor MCF-7 cells, TRAIL induced a mitochondrial pathway of apoptosis that involved cytochrome c release from mitochondria and activation of caspase 9. The DNA damaging drug doxorubicin also activated this mitochondria-regulated mechanism of apoptosis, which was inhibited in Bc1-2-overexpressing cells. We also demonstrate that in MCF-7 cells Bc1-2 might confer resistance to TRAIL-induced apoptosis, depending on the expression levels of the anti-apoptotic protein. These results indicate that enhanced expression of Bc1-2 in tumor cells can render these cells less sensitive not only to chemotherapeutic drugs but also to TRAIL.

Apolipoproteins of the L family are lipid-binding proteins whose function is largely unknown. Apolipoprotein L1 and apolipoprotein L6 have been recently described as novel pro-death BH3-only proteins that are also capable of regulating autophagy. In an in-silico screening to discover novel putative BH3-only proteins, we identified yet another member of the apolipoprotein L family, apolipoprotein L2 (ApoL2), as a BH3 motif-containing protein. ApoL2 has been suggested to behave as a BH3-only protein and mediate cell death induced by interferon-gamma or viral infection. As previously described, we observed that ApoL2 protein was induced by interferon-gamma. However, knocking down its expression in HeLa cells did not regulate cell death induced by interferon-gamma. Overexpression of ApoL2 did not induce cell death on its own. ApoL2 did not sensitize or protect cells from overexpression of the BH3-only proteins Bmf or Noxa. Furthermore, siRNA against ApoL2 did not alter sensitivity to a variety of death stimuli. We could, however, detect a weak interaction between ApoL2 and Bcl-2 by immunoprecipitation of the former, suggesting a role of ApoL2 in a Bcl-2-regulated process like autophagy. However, in contrast to what has been described about its homologs ApoL1 and ApoL6, ApoL2 did not regulate autophagy. Thus, the role, if any, of ApoL2 in cell death remains to be clarified.

Unregulated growth and replication as well as an abnormal microenvironment, leads to elevated levels of stress which is a common trait of cancer.By inducing both energy and endoplasmic reticulum (ER) stress, 2-Deoxy-glucose (2-DG) is particularly well-suited to take advantage of the therapeutic window that heightened stress in tumors provides.Under hypoxia, blocking glycolysis with 2-DG leads to significant lowering of ATP resulting in energy stress and cell death in numerous carcinoma cell types.In contrast, under normoxia, 2-DG at a low-concentration is not toxic in most carcinomas tested, but induces growth inhibition, which is primarily due to ER stress.Here we find a synergistic toxic effect in several tumor cell lines in vitro combining 2-DG with fenofibrate (FF), a drug that has been safely used for over 40 years to lower cholesterol in patients.This combination induces much greater energy stress than either agent alone, as measured by ATP reduction, increased p-AMPK and downregulation of mTOR.Inhibition of mTOR results in blockage of GRP78 a critical component of the unfolded protein response which we speculate leads to greater ER stress as observed by increased p-eIF2α.Moreover, to avoid an insulin response and adsorption by the liver, 2-DG is delivered by slow-release pump yielding significant anti-tumor control when combined with FF.Our results provide promise for developing this combination clinically and others that combine 2-DG with agents that act synergistically to selectively increase energy and ER stress to a level that is toxic to numerous tumor cell types.

The oxidative stress response pathway in mammals is mediated by the transcription factor nuclear factor, erythroid 2 like 2 (NFE2L2/NRF2). NRF2 is a leucine zipper transcription factor expressed in all cell types at low basal levels under unstressed conditions.1Rojo de la Vega M. Chapman E. Zhang D.D. NRF2 and the Hallmarks of Cancer.Cancer Cell. 2018; 34: 21-43Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar Under homeostatic conditions, low cellular levels of NRF2 are maintained via proteasomal degradation through binding to kelch-like ECH associated protein 1 (KEAP1). KEAP1 binds NRF2 as a dimer through its C-terminal kelch domain. Through its N-terminal, KEAP1 interacts with Cullen 3 (CUL3), which serves as a scaffold for the E3 ubiquitin ligase ring box 1 (RBX1).2Shibata T. Ohta T. Tong K.I. et al.Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy.Proc Natl Acad Sci U S A. 2008; 105: 13568-13573Crossref PubMed Scopus (568) Google Scholar Oxidative stress induces the oxidation of KEAP1 and modifies its conformation thereby releasing NRF2. Upon release, NRF2 translocates and accumulates in the nucleus, promoting the expression of genes that are involved in the cellular antioxidant, detoxification, and metabolic pathways, including nucleotide synthesis.3DeNicola G.M. Chen P.H. Mullarky E. et al.NRF2 regulates serine biosynthesis in non–small cell lung cancer.Nat Genet. 2015; 47: 1475-1481Crossref PubMed Scopus (452) Google Scholar In cancer, genomic alterations of NFE2L2, KEAP1, and CUL3 lead to constitutive activation of NRF2-dependent gene transcription that promotes cellular resistance to oxidative stress, xenobiotic efflux, proliferation, and metabolic reprogramming.4Menegon S. Columbano A. Giordano S. The dual roles of NRF2 in cancer.Trends Mol Med. 2016; 22: 578-593Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 5Cloer E.W. Goldfarb D. Schrank T.P. et al.NRF2 activation in cancer: from DNA to protein.Cancer Res. 2019; 79: 889-898Crossref PubMed Scopus (98) Google Scholar Somatic mutations in NFE2L2 and KEAP1 are found in 3.5% to 15% and 12% to 17%, respectively, of patients with NSCLC.6Cancer Genome Atlas Research NetworkComprehensive genomic characterization of squamous cell lung cancers.Nature. 2012; 489: 519-525Crossref PubMed Scopus (2916) Google Scholar, 7Cancer Genome Atlas Research NetworkComprehensive molecular profiling of lung adenocarcinoma.Nature. 2014; 511: 543-550Crossref PubMed Scopus (3530) Google Scholar Most patients with NSCLC with mutant KEAP1/NFE2L2 are smokers and have metastatic disease. Mutations in KEAP1 and NFE2L2 are generally mutually exclusive. KEAP1 mutations have been detected throughout the whole gene sequence and are more likely to occur in lung adenocarcinoma. NFE2L2 mutations generally cluster in specific regions (DLG or ETGE motifs), lead to aberrant cellular accumulation of NRF2, and are more common in squamous cell carcinoma.8Frank R. Scheffler M. Merkelbach-Bruse S. et al.Clinical and pathological characteristics of KEAP1- and NFE2L2-mutated non–small cell lung carcinoma (NSCLC).Clin Cancer Res. 2018; 24: 3087-3096Crossref PubMed Scopus (89) Google Scholar KEAP1 mutations are enriched in driver-negative lung adenocarcinoma but co-occur with clinically relevant molecular alterations such as KRAS and EGFR mutations or serine/threonine kinase 11 (STK11) loss.7Cancer Genome Atlas Research NetworkComprehensive molecular profiling of lung adenocarcinoma.Nature. 2014; 511: 543-550Crossref PubMed Scopus (3530) Google Scholar, 9Romero R. Sayin V.I. Davidson S.M. et al.Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis.Nat Med. 2017; 23: 1362-1368Crossref PubMed Scopus (338) Google Scholar, 10Galan-Cobo A. Sitthideatphaiboon P. Qu X. et al.LKB1 and KEAP1/NRF2 pathways cooperatively promote metabolic reprogramming with enhanced glutamine dependence in KRAS-mutant lung adenocarcinoma.Cancer Res. 2019; 79: 3251-3267Crossref PubMed Scopus (137) Google Scholar The role of the KEAP1/NRF2 pathway in tumor cell protection has been well established in vitro, and deregulation of this pathway promotes metastases in animal models. In this issue of the Journal, Goeman et al.11Goeman F. De Nicola F. Scalera S. et al.Mutations in the KEAP1-NFE2L2 pathway define a molecular subset of rapidly progressing lung adenocarcinoma.J Throac Oncol. 2019; 14: 1924-1934Abstract Full Text Full Text PDF Scopus (44) Google Scholar studied the clinical implications of KEAP1 and NFE2L2 mutations in a cohort of patients with advanced NSCLC treated with first-line chemotherapy. They found that the frequency of KEAP1 and NFE2L2 mutations was 18% and 7%, respectively, and those mutations were generally mutually exclusive to actionable alterations in EGFR or ALK genes. As the authors did not assess additional genomic or epigenetic mechanisms leading to NRF2 activation, the frequency of KEAP1/NRF2 deregulation may have been underestimated. Indeed, loss of heterozygosity of the KEAP1 locus at 19p13 and epigenetic silencing of the KEAP1 promoter are relatively frequent events in NSCLC.12Singh A. Misra V. Thimmulappa R.K. et al.Dysfunctional KEAP1-NRF2 interaction in non–small-cell lung cancer.PLoS Med. 2006; 3: e420Crossref PubMed Scopus (820) Google Scholar, 13Muscarella L.A. Barbano R. D'Angelo V. et al.Regulation of KEAP1 expression by promoter methylation in malignant gliomas and association with patient's outcome.Epigenetics. 2011; 6: 317-325Crossref PubMed Scopus (80) Google Scholar Nevertheless, Goeman et al.11Goeman F. De Nicola F. Scalera S. et al.Mutations in the KEAP1-NFE2L2 pathway define a molecular subset of rapidly progressing lung adenocarcinoma.J Throac Oncol. 2019; 14: 1924-1934Abstract Full Text Full Text PDF Scopus (44) Google Scholar found that patients harboring mutations in the stress response pathway had significantly worse clinical outcomes in terms of progression-free survival and overall survival than those with wild-type disease, even after adjusting for clinical covariates. These results are consistent with previous studies in early stage NSCLC, where NFE2L2 mutations had been associated with poor overall survival in patients with lung squamous cell carcinoma.2Shibata T. Ohta T. Tong K.I. et al.Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy.Proc Natl Acad Sci U S A. 2008; 105: 13568-13573Crossref PubMed Scopus (568) Google Scholar Additionally, transcriptional signatures derived from KEAP1-mutant tumors have been correlated with overall survival in lung adenocarcinoma.3DeNicola G.M. Chen P.H. Mullarky E. et al.NRF2 regulates serine biosynthesis in non–small cell lung cancer.Nat Genet. 2015; 47: 1475-1481Crossref PubMed Scopus (452) Google Scholar, 9Romero R. Sayin V.I. Davidson S.M. et al.Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis.Nat Med. 2017; 23: 1362-1368Crossref PubMed Scopus (338) Google Scholar Goeman et al.11Goeman F. De Nicola F. Scalera S. et al.Mutations in the KEAP1-NFE2L2 pathway define a molecular subset of rapidly progressing lung adenocarcinoma.J Throac Oncol. 2019; 14: 1924-1934Abstract Full Text Full Text PDF Scopus (44) Google Scholar also observed that patients with lung adenocarcinoma harboring KEAP1/NFE2L2 mutations were more likely to experience earlier disease progression to chemotherapy. The authors validated these findings in independent cohorts of NSCLC patients. Previous research has shown that genomic alterations in KEAP1 confer resistance to conventional treatment, such as chemotherapy, radiotherapy, or tyrosine kinase inhibitors. Congruently, abrogation of NRF2 expression by small-interference RNA knock-down sensitized cancer cells to oxidative stress and chemotherapy.2Shibata T. Ohta T. Tong K.I. et al.Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy.Proc Natl Acad Sci U S A. 2008; 105: 13568-13573Crossref PubMed Scopus (568) Google Scholar, 14Wang X.J. Sun Z. Villeneuve N.F. et al.Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2.Carcinogenesis. 2008; 29: 1235-1243Crossref PubMed Scopus (609) Google Scholar KEAP1 loss in lung cancer models conferred resistance to cisplatin and radiotherapy with higher resistance to oxidative stress.15Ohta T. Iijima K. Miyamoto M. et al.Loss of Keap1 function activates Nrf2 and provides advantages for lung cancer cell growth.Cancer Res. 2008; 68: 1303-1309Crossref PubMed Scopus (503) Google Scholar, 16Jeong Y. Hoang N.T. Lovejoy A. et al.Role of KEAP1/NRF2 and TP53 mutations in lung squamous cell carcinoma development and radiation resistance.Cancer Discov. 2017; 7: 86-101Crossref PubMed Scopus (190) Google Scholar KEAP1 loss also modulates the response to BRAF, MEK, EGFR, and ALK inhibition in lung cancer cells harboring BRAF, KRAS, EGFR and ALK receptor tyrosine kinase (ALK) alterations.17Krall E.B. Wang B. Munoz D.M. et al.KEAP1 loss modulates sensitivity to kinase targeted therapy in lung cancer.Elife. 2017; 6 (pii:e18970)Google Scholar In addition, KEAP1 mutations have been associated with resistance to chemotherapy and to immune checkpoint inhibitors in patients harboring KRAS mutations.18Arbour K.C. Jordan E. Kim H.R. et al.Effects of co-occurring genomic alterations on outcomes in patients with KRAS-mutant non–small cell lung cancer.Clin Cancer Res. 2018; 24: 334-340Crossref PubMed Scopus (207) Google Scholar A recent study found that KEAP1 mutations were enriched in patients with high tumor mutational burden (TMB) lacking T-cell infiltration, suggesting that KEAP1 mutations can promote immune evasion. Moreover, tumors with this genomic alteration are generally cold immune tumors.19Cristescu R. Mogg R. Ayers M. et al.Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy.Science. 2018; 362 (pii:eaar3593)Crossref PubMed Scopus (1086) Google Scholar In this sense, KEAP1 mutations have been associated with worse clinical outcomes in patients with lung adenocarcinoma treated with pembrolizumab plus carboplatin and pemetrexed; current standard of care for the first-line treatment of driver negative nonsquamous NSCLC.20Skoulidis F. Arbour K.C. Hellmann M.D. et al.Association of STK11/LKB1 genomic alterations with lack of benefit from the addition of pembrolizumab to platinum doublet chemotherapy in non-squamous non–small cell lung cancer.J Clin Oncol. 2019; 37 (abstr 102)Google Scholar All these studies therefore support that high NRF2 activity and KEAP1 loss define a molecular subtype of treatment-resistant and rapidly progressing NSCLC. In addition to the higher resistance to oxidative stress associated with NRF2 activation, the aggressive behavior and treatment resistance of tumors harboring KEAP1/NFE2L2 mutations might be explained by features such as proficient DNA repair or metabolic rewiring. In this sense, Goeman et al.11Goeman F. De Nicola F. Scalera S. et al.Mutations in the KEAP1-NFE2L2 pathway define a molecular subset of rapidly progressing lung adenocarcinoma.J Throac Oncol. 2019; 14: 1924-1934Abstract Full Text Full Text PDF Scopus (44) Google Scholar examined the relationship between KEAP1/NFE2L2 status and the DNA-damage response pathway (tumor protein 53 [TP53], ATM serine/threonine kinase [ATM], and ATR serine/threonine kinase [ATR]), since they hypothesized that levels of oxidative stress should be related to DNA repair capabilities. They found that tumors with KEAP1/NFE2L2 mutations had higher expression of phospho-ATM and phospho-ATR, suggesting that they were proficient in repairing DNA damage, and they also found that TP53 alterations were mutually exclusive with KEAP1/NFE2L2 mutations. A recent study showed that KEAP1 loss and NRF2 activation promotes metastasis in lung adenocarcinoma by accumulating BTB domain and CNC homolog 1 (BACH1), a transcription regulator that not only promotes a pro-metastatic transcriptional program, but also stimulates glycolysis-dependent lung cancer metastasis.21Lignitto L. LeBoeuf S.E. Homer H. et al.Nrf2 activation promotes lung cancer metastasis by inhibiting the degradation of Bach1.Cell. 2019; 178: 316-329 e318Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 22Wiel C. Le Gal K. Ibrahim M.X. et al.BACH1 stabilization by antioxidants stimulates lung cancer metastasis.Cell. 2019; 178: 330-345 e322Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar Novel therapeutic approaches are needed to target KEAP1/NFE2L2 mutations that promote an aggressive phenotype characterized by enhanced antioxidant ability and resistance to chemotherapy and immunotherapy. In this regard, exploiting metabolic vulnerabilities and metabolic rewiring towards synthesis of the antioxidant glutathione may open novel therapeutic avenues in those tumors. Interestingly, KEAP1-mutant cancer cells show decreased intracellular glutamate and increased glutamine consumption.9Romero R. Sayin V.I. Davidson S.M. et al.Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis.Nat Med. 2017; 23: 1362-1368Crossref PubMed Scopus (338) Google Scholar, 23Sayin V.I. LeBoeuf S.E. Singh S.X. et al.Activation of the NRF2 antioxidant program generates an imbalance in central carbon metabolism in cancer.Elife. 2017; 6 (pii:e28083)Crossref PubMed Google Scholar This makes these lung cancer cells highly dependent on the glutamine transporter solute carrier family 1 member 5 (SLC1A5), which leads toward higher sensitivity to glutaminase inhibition. A glutaminase inhibitor (CB-839) is currently being studied in a phase I/II clinical trial for metastatic NSCLC and could potentially become an option for these patients. NRF2 also promotes the intracellular accumulation of cysteine, which is maintained at optimal levels by degradation through cysteine dioxygenase type 1 (CDO1) that produces toxic by-products and reduces nicotinamide adenine dinucleotide phosphate hydrogen (NADPH). KEAP1-mutant cells inactivate CDO1 epigenetically to avoid toxicity, and this suggests that cysteine metabolism could be manipulated to selectively promote toxicity of KEAP1-mutant cells.24Kang Y.P. Torrente L. Falzone A. et al.Cysteine dioxygenase 1 is a metabolic liability for non–small cell lung cancer.Elife. 2019; 8 (pii:e45572)Google Scholar In conclusion, the work of Goeman et al.11Goeman F. De Nicola F. Scalera S. et al.Mutations in the KEAP1-NFE2L2 pathway define a molecular subset of rapidly progressing lung adenocarcinoma.J Throac Oncol. 2019; 14: 1924-1934Abstract Full Text Full Text PDF Scopus (44) Google Scholar is relevant because it adds to the understanding of the stress response signaling pathway, showing that the presence of genomic alterations in this pathway is associated with poor prognosis and resistance to conventional antitumor treatments in NSCLC; however, unfortunately, this has not yet translated into effective treatment for patients with KEAP1/NFE2L2-altered tumors. We believe that the incorporation of next-generation sequencing into the clinic will increase the number of patients identified as having a NRF2 hyperactive tumor, and this in turn will lead to the investigation of novel therapeutic strategies. Dr. Nadal received support from the SLT006/17/00127 grant, funded by the Department of Health of the Generalitat de Catalunya by the call “Acció instrumental d’intensificació de professionals de la salut” and the PROYBAR17005NADA project funded by the AECC Barcelona (Spanish Association Against Cancer Barcelona). The authors thank CERCA Program/Generalitat de Catalunya for their institutional support and grant 2017SGR448. Copyediting editorial support was provided by Aurora O’Brate. Mutations in the KEAP1-NFE2L2 Pathway Define a Molecular Subset of Rapidly Progressing Lung AdenocarcinomaJournal of Thoracic OncologyVol. 14Issue 11PreviewMolecular characterization studies revealed recurrent kelch like ECH associated protein 1 gene (KEAP1)/nuclear factor, erythroid 2 like 2 gene (NFE2L2) alterations in NSCLC. These genes encode two interacting proteins (a stress response pathway [SRP]) that mediate a cytoprotective response to oxidative stress and xenobiotics. Nevertheless, whether KEAP1/NFE2L2 mutations have an impact on clinical outcomes is unclear. Full-Text PDF Open Archive

We have monitored and imaged cell death induced in human leukemic U937 cells over time using three-color confocal imaging. Three different apoptotic inducers, anti-Fas, TNF-α and Etoposide were used. Individual cascaded events such as loss of mitochondrial transmembrane potential, exposure of phosphatidyl-serine, membrane blebbing and permeabilization of the cell membrane have been observed in real time with different individual cells. From the results, an interesting heterogeneicity in the apoptotic phenotype has been observed. The CRISC method is easy to use and provides biologist with a powerful additional tool to study in real-time processes of several hours of duration such as apoptosis. We predict that the period of cell viability obtained after protein coating of the PDMS devices (>80 h) will also allow monitoring of other biological processes of longer duration or long onset time, such as mitosis, phagocytosis and differentiation.

Abstract Chemokine (C–C motif) ligand 2 (CCL2) has been associated with chronic metabolic diseases. We aimed to investigate whether Ccl2 gene overexpression is involved in the regulation of signaling pathways in metabolic organs. Biochemical and histological analyses were used to explore tissue damage in cisgenic mice that overexpressed the Ccl2 gene. Metabolites from energy and one-carbon metabolism in liver and muscle extracts were measured by targeted metabolomics. Western blot analysis was used to explore the AMP-activated protein kinase (AMPK) and mammalian target of rapamycin pathways. Ccl2 overexpression resulted in steatosis, decreased AMPK activity and altered mitochondrial dynamics in the liver. These changes were associated with decreased oxidative phosphorylation and alterations in the citric acid cycle and transmethylation. In contrast, AMPK activity and its downstream mediators were increased in muscle, where we observed an increase in oxidative phosphorylation and increased concentrations of different metabolites associated with ATP synthesis. In conclusion, Ccl2 overexpression induces distinct metabolic alterations in the liver and muscle that affect mitochondrial dynamics and the regulation of energy sensors involved in cell homeostasis. These data suggest that CCL2 may be a therapeutic target in metabolic diseases.

Natural killer cells (NK) are important effectors of anti-tumor immunity, activated either by the downregulation of HLA-I molecules on tumor cells and/or the interaction of NK-activating receptors with ligands that are overexpressed on target cells upon tumor transformation (including NKG2D and NKP30). NK kill target cells by the vesicular delivery of cytolytic molecules such as Granzyme-B and Granulysin activating different cell death pathways, which can be Caspase-3 dependent or Caspase-3 independent. Multiple myeloma (MM) remains an incurable neoplastic plasma-cell disorder. However, we previously reported the encouraging observation that cord blood-derived NK (CB-NK), a new source of NK, showed anti-tumor activity in an in vivo murine model of MM and confirmed a correlation between high levels of NKG2D expression by MM cells and increased efficacy of CB-NK in reducing tumor burden. We aimed to characterize the mechanism of CB-NK-mediated cytotoxicity against MM cells. We show a Caspase-3- and Granzyme-B-independent cell death, and we reveal a mechanism of transmissible cell death between cells, which involves lipid–protein vesicle transfer from CB-NK to MM cells. These vesicles are secondarily transferred from recipient MM cells to neighboring MM cells amplifying the initial CB-NK cytotoxicity achieved. This indirect cytotoxicity involves the transfer of NKG2D and NKP30 and leads to lysosomal cell death and decreased levels of reactive oxygen species in MM cells. These findings suggest a novel and unique mechanism of CB-NK cytotoxicity against MM cells and highlight the importance of lipids and lipid transfer in this process. Further, these data provide a rationale for the development of CB-NK-based cellular therapies in the treatment of MM.

On glucose restriction, epithelial cells can undergo entosis, a cell-in-cell cannibalistic process, to allow considerable withstanding to this metabolic stress. Thus, we hypothesized that reduced protein glycosylation might participate in the activation of this cell survival pathway. Glucose deprivation promoted entosis in an MCF7 breast carcinoma model, as evaluated by direct inspection under the microscope, or revealed by a shift to apoptosis + necrosis in cells undergoing entosis treated with a Rho-GTPase kinase inhibitor (ROCKi). In this context, curbing protein glycosylation defects with N-acetyl-glucosamine partially rescued entosis, whereas limiting glycosylation in the presence of glucose with tunicamycin or NGI-1, but not with other unrelated ER-stress inducers such as thapsigargin or amino-acid limitation, stimulated entosis. Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is upregulated by glucose deprivation, thereby enhancing cell survival. Therefore, we presumed that PEPCK-M could play a role in this process by offsetting key metabolites into glycosyl moieties using alternative substrates. PEPCK-M inhibition using iPEPCK-2 promoted entosis in the absence of glucose, whereas its overexpression inhibited entosis. PEPCK-M inhibition had a direct role on total protein glycosylation as determined by Concanavalin A binding, and the specific ratio of fully glycosylated LAMP1 or E-cadherin. The content of metabolites, and the fluxes from 13C-glutamine label into glycolytic intermediates up to glucose-6-phosphate, and ribose- and ribulose-5-phosphate, was dependent on PEPCK-M content as measured by GC/MS. All in all, we demonstrate for the first time that protein glycosylation defects precede and initiate the entosis process and implicates PEPCK-M in this survival program to dampen the consequences of glucose deprivation. These results have broad implications to our understanding of tumor metabolism and treatment strategies.

There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy.We aimed to investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM.Based on publicly available transcriptomic data of MPM, patients with CDK4 or CDK6 overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell models evaluated. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest, thereby increasing cell senescence and increased the expression of interferon signalling pathway and tumour antigen presentation process in culture models of MPM. In vivo preclinical studies showed that palbociclib significantly reduced tumour growth and prolonged overall survival using distinct xenograft models of MPM implanted in athymic mice.Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of MPM.

Stress signaling in response to oxygen/glucose deprivation (OGD) and ischemic injury activates a group of pro-apoptotic genes, the Bcl-2 homology domain 3 (BH3)-only proteins, which are capable of activating the mitochondrial apoptosis pathway. Targeted studies previously identified the BH3-only proteins Puma, Bim and Bid to have a role in ischemic/hypoxic neuronal injury. We here investigated the transcriptional activation of pro-apoptotic BH3-only proteins after OGD-induced injury in murine neocortical neurons. We observed a potent and early upregulation of noxa at mRNA and protein level, and a significant increase in Bmf protein levels during OGD in neocortical neurons and in the ipsilateral cortex of mice subjected to transient middle cerebral artery occlusion (tMCAO). Surprisingly, gene deficiency in noxa reduced neither OGD- nor glutamate-induced neuronal injury in cortical neurons and failed to influence infarct size or neurological deficits after tMCAO. In contrast, bmf deficiency induced significant protection against OGD- or glutamate-induced injury in cultured neurons, and bmf-deficient mice showed reduced neurological deficits after tMCAO in vivo. Collectively, our data not only point to a role of Bmf as a BH3-only protein contributing to excitotoxic and ischemic neuronal injury but also demonstrate that the early and potent induction of noxa does not influence ischemic neuronal injury.

Correction to: Cell Death and Disease (2012) 3, e303; doi:10.1038/cddis.2012.41; published online 3 May 2012

Cell death can occur through different mechanisms, defined by their nature and physiological implications. Correct assessment of cell death is crucial for cancer therapy success. Sarcomas are a large and diverse group of neoplasias from mesenchymal origin. Among cell death types, apoptosis is by far the most studied in sarcomas. Albeit very promising in other fields, regulated necrosis and other cell death circumstances (as so-called ‘autophagic cell death’ or ‘mitotic catastrophe’) have not been yet properly addressed in sarcomas. Cell death is usually quantified in sarcomas by unspecific assays and in most cases the precise sequence of events remains poorly characterized. In this review our main objective is to put into context the most recent sarcoma cell death findings in the more general landscape of different cell death modalities.

Background: Natural killer (NK) cells are important anti-tumor cells of our innate immune system.Their anti-cancer activity is mediated through interaction of a wide array of activating and inhibitory receptors with their ligands on tumor cells.After activation, NK cells also secrete a variety of pro-inflammatory molecules that contribute to the final immune response by modulating other innate and adaptive immune cells.In this regard, external proteins from NK cell secretome and the mechanisms by which they mediate these responses are poorly defined.Methods: TRANS-stable-isotope labeling of amino acids in cell culture (TRANS-SILAC) combined with proteomic was undertaken to identify early materials transferred between cord blood-derived NK cells (CB-NK) and multiple myeloma (MM) cells.Further in vitro and in vivo studies with knock-down of histones and CD138, overexpression of histones and addition of exogenous histones were undertaken to confirm TRANS-SILAC results and to determine functional roles of this material transferred. Results:We describe a novel mechanism by which histones are actively released by NK cells early after contact with MM cells.We show that extracellular histones bind to the heparan sulfate proteoglycan CD138 on the surface of MM cells to promote the creation of immune-tumor cell clusters bringing immune and MM cells into close proximity, and thus facilitating not only NK but also T lymphocyte anti-MM activity.Conclusion: This study demonstrates a novel immunoregulatory role of NK cells against MM cells mediated by histones, and an additional role of NK cells modulating T lymphocytes activity that will open up new avenues to design future immunotherapy clinical strategies.

Impairments in protein folding in the endoplasmic reticulum (ER) lead to a condition called ER stress, which can trigger apoptosis via the mitochondrial or the death receptor (extrinsic) pathway. There is controversy concerning involvement of the death receptor (DR)4 and DR5-Caspase-8 -Bid pathway in ER stress-mediated cell death, and this axis has not been fully studied in B-cell malignancies. Using three B-cell lines from Mantle Cell Lymphoma, Waldenström's macroglobulinemia and Multiple Myeloma origins, we engineered a set of CRISPR KOs of key components of these cell death pathways to address this controversy. We demonstrate that DR4 and/or DR5 are essential for killing via TRAIL, however, they were dispensable for ER-stress induced-cell death, by Thapsigargin, Brefeldin A or Bortezomib, as were Caspase-8 and Bid. In contrast, the deficiency of Bax and Bak fully protected from ER stressors. Caspase-8 and Bid were cleaved upon ER-stress stimulation, but this was DR4/5 independent and rather a result of mitochondrial-induced feedback loop subsequent to Bax/Bak activation. Finally, combined activation of the ER-stress and TRAIL cell-death pathways was synergistic with putative clinical relevance for B-cell malignancies.

The KEAP1-NFE2L2 pathway modulates cell homeostasis under stress conditions. NFE2L2/NRF2 is a transcription factor that acts as a master regulator of the cellular antioxidant response. Under homeostatic conditions, NRF2 binds to KEAP1, a tumor suppressor and ROS-sensing protein that eliminates NRF2 by means of proteasomal degradation.1 In normal cells, NRF2 may play a protective role and prevent carcinogenesis by eliminating ROS or repairing oxidative damage. Nevertheless, cancer cells take advantage of these physiological mechanisms to counteract stress and rely on NRF2 to promote cancer progression and metastasis.

Abstract Patient stratification according to drug responses, together with the discovery of novel antitumor targets, is leading to a new era of personalized cancer treatments. With the aim of identifying emerging pathways and the challenges faced by clinicians during clinical trials, the IDIBELL Cancer Conference on Personalized Cancer Medicine took place in Barcelona on December 3–4, 2012. This conference brought together speakers working in different areas of cancer research (epigenetics, metabolism and the mTOR pathway, cell death and the immune system, clinical oncology) to discuss the latest developments in personalized cancer medicine. Cancer Res; 73(14); 4185–9. ©2013 AACR.

Chemotherapeutic drugs that inhibit the synthesis of DNA precursor thymidine triphosphate cause apoptosis, although the mechanism underlying this process remains rather unknown. Here, we describe thymineless death of human myeloid leukemia U937 cells treated with the thymidylate‐synthase inhibitor 5 ′ ‐fluoro‐2 ′ ‐deoxyuridine (FUdR). This apoptotic process was shown to be independent of p53, reactive oxygen species generation and CD95 activation. Caspases were activated downstream of cytochrome c but upstream of mitochondrial depolarization. Furthermore, FUdR‐induced apoptosis required the presence of glucose in the culture medium at a step upstream of the release of cytochrome c from mitochondria.

Treatment of haematopoietic BA/F3 cells with the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (FUdR) activated apoptosis through a mechanism that required continuous protein synthesis and was inhibited by Bcl-2 over-expression. Analysis of p53 levels in cells treated with FUdR indicated a marked accumulation of this protein. Accumulation of p53 was also observed in cells over-expressing Bcl-2. In BA/F3 cells transfected with a cDNA coding for the human papilloma virus protein E6, p53 accumulation after FUdR treatment was inhibited markedly. However, apoptosis was induced in both control and E6 cells to a similar extent. The role of the CD95/CD95 ligand (CD95L) system in FUdR-induced apoptosis was also assessed. As determined by reverse transcriptase PCR, BA/F3 expressed a low constitutive level of CD95L mRNA, which decreased following FUdR treatment. Moreover, blocking CD95-CD95L interactions with antagonistic CD95 monoclonal antibody did not prevent drug-induced apoptosis. Furthermore, analysis of caspase involvement showed important differences in apoptosis induced by CD95-triggering or FUdR treatment. In summary, these results suggest that apoptosis induced by thymineless stress in haematopoietic BA/F3 cells occurs by a mechanism that does not require accumulation of p53 and which is independent of CD95-CD95L interactions.

Nutrient starvation or inhibition of cellular metabolism can induce cancer cell death. This can be measured by a variety of methods. We describe here four simple methods to measure cell death in culture by using microscopy, western blot, and flow cytometry. We also provide tools to differentiate between different forms of cell death like apoptosis and necrosis by using chemical inhibitors.

Novel therapeutic approaches are needed to improve the clinical outcome of patients with malignant pleural mesothelioma (MPM). In the current study, we investigate the antitumor activity of CDK4/6 inhibitors in preclinical models of MPM. MPM cell lines (H28, H226, H2052, H2452, MSTO-211H) and primary cultures (ICO_MPM1, ICO_MPM2, ICO_MPM3) were treated with abemaciclib or palbociclib for 24 and 72 hours. Cell viability was evaluated by cell counting and crystal violet assays. Cell death and cell cycle distribution were analyzed by flow cytometry and senescence was quantified by β-galactosidase expression. For transcriptomic studies, mRNA expression was assessed through RNA sequencing analysis. Gene set enrichment analysis (GSEA) was used to identify signaling pathways deregulated in MSTO-211H cells treated with CDK4/6 inhibitors. MSTO-211H cells were implanted subcutaneously in athymic mice that were randomly assigned to the following cohorts (n=7): i) vehicle; ii) cisplatin + pemetrexed; iii) palbociclib alone and iv) palbociclib + gemcitabine. Tumors’ size and mice weight was monitored during 4 weeks to evaluate efficacy. Treatment with abemaciclib or palbociclib at 100nM induced a significant decrease in cell proliferation (mean 50.9% ± 7.6; mean 47.3% ± 9.9, respectively) in distinct MPM cell models, including cells derived from patients who progressed to prior cisplatin and pemetrexed. Both CDK4/6 inhibitors induced G1-phase cell cycle arrest, while cell death was slightly affected (up to 1-5%). At concentrations ranging from 250 to 500nM, the percentage of senescent cells was increased after abemaciclib (15-26%) and palbociclib (18-25%) treatment in all the analyzed cell models. GSEA revealed that CDK4/6 inhibitors promote interferon signaling pathway and MHC presentation. In the in vivo experiment, a significant reduction in tumor growth was observed in response to palbociclib alone or combined with gemcitabine for 4 weeks (vehicle = 1335.8±586.4 mm3; cisplatin + pemetrexed= 726±573.5 mm3; palbociclib = 479±235.7 mm3; palbociclib + gemcitabine = 517±487.4 mm3; p< 0.05). CDK4/6 inhibitors reduce cell proliferation in culture models of MPM mainly by blocking cell proliferation at G1 and by inducing senescence. Palbociclib alone or combined with gemcitabine reduces in vivo tumor growth of subcutaneously implanted MSTO-211H cells compared to chemotherapy.

Correction to: Cell Death and Disease (2012) 3, e303; doi:10.1038/cddis.2012.41; published online 3 May 2012 Since the publication of this article the authors have noticed MG Vander-Heiden name was incorrect. The error has now been rectified. The article with the corrected authors list appears online, together with this corrigendum.

Entosis is an atypical form of cell death that occurs when a cell engulfs and kills another cell. A recent article by Overholtzer and colleagues indicates that glucose deprivation promotes entosis. AMP-activated protein kinase (AMPK) activation in the loser cells triggers their engulfment and elimination by winner cells, which endure starvation. Entosis is an atypical form of cell death that occurs when a cell engulfs and kills another cell. A recent article by Overholtzer and colleagues indicates that glucose deprivation promotes entosis. AMP-activated protein kinase (AMPK) activation in the loser cells triggers their engulfment and elimination by winner cells, which endure starvation. Entosis Is Induced by Glucose StarvationHamann et al.Cell ReportsJuly 05, 2017In BriefEntosis has been shown to occur in human cancers and promotes cell competition. Hamann et al. now show that nutrient deprivation, in the form of glucose withdrawal, induces entosis to support the outgrowth of winner cells that feed off of losers. Full-Text PDF Open Access

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Brain metastases (BM) are a frequent complication in patients with non-small lung cancer (NSCLC). Treating BM is challenging as the blood-brain barrier can limit intracranial drug delivery. Data from different trials suggest that antibody-drug conjugates (ADC) can have clinically relevant intracranial activity. This study aims to characterize the gene expression of ADC targets in NSCLC-BM to guide treatment decisions. Gene expression data from 125 BM resected from patients with NSCLC were obtained from 3 public datasets. Gene expression of 14 ADC targets (ERBB2, ERBB3, TACSTD2, MET, GPNMB, PTK7, MSLN, CD276, TF, AXL, EGFR, SLC34A2, CEACAM5 and ROR2) was evaluated to identify those consistently expressed across the cohorts. Additionally, hierarchical clustering was used to stratify NSCLC-BM, in each dataset separately, based on the combined relative gene expression of the common 25% most variable genes: MSLN, CEACAM5 and SLC34A2. Furthermore, association between clusters and clinical covariates was assessed for one of the datasets with available data (N=63). Gene expression evaluation showed that TACSTD2 (Trop-2) was ubiquitously highly expressed across datasets, suggesting that TACSTD2 could be a promising ADC target for NSCLC-BM. Contrarily, ROR2 showed consistently lower expression and would probably not be a good candidate in NSCLC-BM. Transcriptional patterns from the 3 most variable ADC targets (MSLN, CEACAM5, and SLC34A2) yielded common subgroups in all datasets defined by the following expression levels (respectively): High/High/High, Low/High/High, High/Low/Low. These groups did not correlate with clinical covariates. Gene expression evaluation of ADC targets in NSCLC-BM resulted in the identification of TACSTD2 expression across all datasets. TACSTD2 might be a promising ADC target for patients with NSCLC with BM. Further validation is warranted in terms of protein expression. Our results underscore the potential value of gene expression profiling to identify new targets and improve patient selection in the context of NSCLC-BM ADC therapy.

&lt;div&gt;Abstract&lt;p&gt;Alveolar and embryonal rhabdomyosarcomas are childhood tumors that do not respond well to current chemotherapies. Here, we report that the glycolytic inhibitor 2-deoxyglucose (2-DG) can efficiently promote cell death in alveolar, but not embryonal, rhabdomyosarcoma cell lines. Notably, 2-DG also induced cell differentiation accompanied by downregulation of PAX3/FOXO1a, the chromosome translocation–encoded fusion protein that is a central oncogenic driver in this disease. Cell death triggered by 2-DG was associated with its ability to activate Bax and Bak. Overexpression of the antiapoptotic Bcl-2 homologues Bcl-x&lt;sub&gt;L&lt;/sub&gt; and Mcl-1 prevented apoptosis, indicating that cell death proceeds through the mitochondrial pathway. Mechanistic investigations indicated that Mcl-1 downregulation and Noxa upregulation were critical for 2-DG–induced apoptosis. In addition, 2-DG promoted eIF2α phosphorylation and inactivation of the mTOR pathway. Mcl-1 loss and cell death were prevented by downregulation of the endoplasmic reticulum (ER) stress–induced protein ATF4 and by incubating cells in the presence of mannose, which reverted 2-DG–induced ER stress but not ATP depletion. Thus, energetic stresses created by 2-DG were not the primary cause of cell death. Together, our findings suggest that glycolysis inhibitors such as 2-DG may be highly effective in treating alveolar rhabdomyosarcoma and that Noxa could offer a prognostic marker to monitor the efficacy of such agents. &lt;i&gt;Cancer Res; 71(21); 6796–806. ©2011 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Cancer cells are markedly different from normal cells with regards to how their metabolic pathways are used to fuel cellular growth and survival. Two basic metabolites that exemplify these differences through increased uptake and altered metabolic usage are glucose and glutamine. These molecules can be catabolized to manufacture many of the building blocks required for active cell growth and proliferation. The alterations in the metabolic pathways necessary to sustain this growth have been linked to therapeutic resistance, a trait that is correlated with poor patient outcomes. By targeting the metabolic pathways that import, catabolize, and synthesize essential cellular components, drug-resistant cancer cells can often be resensitized to anticancer treatments. The specificity and efficacy of agents directed at the unique aspects of cancer metabolism are expected to be high; and may, when in used in combination with more traditional therapeutics, present a pathway to surmount resistance within tumors that no longer respond to current forms of treatment. &lt;i&gt;Cancer Res; 73(9); 2709–17. ©2013 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Cancer cells are markedly different from normal cells with regards to how their metabolic pathways are used to fuel cellular growth and survival. Two basic metabolites that exemplify these differences through increased uptake and altered metabolic usage are glucose and glutamine. These molecules can be catabolized to manufacture many of the building blocks required for active cell growth and proliferation. The alterations in the metabolic pathways necessary to sustain this growth have been linked to therapeutic resistance, a trait that is correlated with poor patient outcomes. By targeting the metabolic pathways that import, catabolize, and synthesize essential cellular components, drug-resistant cancer cells can often be resensitized to anticancer treatments. The specificity and efficacy of agents directed at the unique aspects of cancer metabolism are expected to be high; and may, when in used in combination with more traditional therapeutics, present a pathway to surmount resistance within tumors that no longer respond to current forms of treatment. &lt;i&gt;Cancer Res; 73(9); 2709–17. ©2013 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Alveolar and embryonal rhabdomyosarcomas are childhood tumors that do not respond well to current chemotherapies. Here, we report that the glycolytic inhibitor 2-deoxyglucose (2-DG) can efficiently promote cell death in alveolar, but not embryonal, rhabdomyosarcoma cell lines. Notably, 2-DG also induced cell differentiation accompanied by downregulation of PAX3/FOXO1a, the chromosome translocation–encoded fusion protein that is a central oncogenic driver in this disease. Cell death triggered by 2-DG was associated with its ability to activate Bax and Bak. Overexpression of the antiapoptotic Bcl-2 homologues Bcl-x&lt;sub&gt;L&lt;/sub&gt; and Mcl-1 prevented apoptosis, indicating that cell death proceeds through the mitochondrial pathway. Mechanistic investigations indicated that Mcl-1 downregulation and Noxa upregulation were critical for 2-DG–induced apoptosis. In addition, 2-DG promoted eIF2α phosphorylation and inactivation of the mTOR pathway. Mcl-1 loss and cell death were prevented by downregulation of the endoplasmic reticulum (ER) stress–induced protein ATF4 and by incubating cells in the presence of mannose, which reverted 2-DG–induced ER stress but not ATP depletion. Thus, energetic stresses created by 2-DG were not the primary cause of cell death. Together, our findings suggest that glycolysis inhibitors such as 2-DG may be highly effective in treating alveolar rhabdomyosarcoma and that Noxa could offer a prognostic marker to monitor the efficacy of such agents. &lt;i&gt;Cancer Res; 71(21); 6796–806. ©2011 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

In Xavier Bosch's interview “Spain's science minister sees future in telecom” (News Focus, 31 Jan., p. 653), Josep Pique, Spanish Minister of Science and Technology, asserts that “[n]ow there are many more scientists from abroad working in Spain than there are Spanish scientists abroad.”

Nutrient unavailability is associated with pathologies such as cancer, miocardial infarction and stroke. When cells are subjected to acute starvation they may die by apoptosis or necrosis. We are studying the mechanism by which cells die when glucose metabolism is inhibited by employing 2-deoxyglucose (a non-metabolizable glucose analog) or by depriving cells of glucose. In rhabdomyosarcoma cell lines, glucose deprivation induces necrosis, while 2-deoxyglucose induces Noxa-mediated mitochondrial apoptosis. Addition of different carbon/energy sources to 2-deoxyglucose-treated cells indicated that apoptosis is not associated with loss of ATP but rather with endoplasmic reticulum stress and the eIF2-alpha-ATF4 pathway. 2-DG promoted Mcl-1 loss, probably due to general inhibition of translation, since both eIF2-alpha phosphorylation and inactivation of the mTOR pathway were observed.

Treatment of haematopoietic BA/F3 cells with the thymidylate synthase inhibitor 5-fluoro-2′-deoxyuridine (FUdR) activated apoptosis through a mechanism that required continuous protein synthesis and was inhibited by Bcl-2 over-expression. Analysis of p53 levels in cells treated with FUdR indicated a marked accumulation of this protein. Accumulation of p53 was also observed in cells over-expressing Bcl-2. In BA/F3 cells transfected with a cDNA coding for the human papilloma virus protein E6, p53 accumulation after FUdR treatment was inhibited markedly. However, apoptosis was induced in both control and E6 cells to a similar extent. The role of the CD95/CD95 ligand (CD95L) system in FUdR-induced apoptosis was also assessed. As determined by reverse transcriptase PCR, BA/F3 expressed a low constitutive level of CD95L mRNA, which decreased following FUdR treatment. Moreover, blocking CD95–CD95L interactions with antagonistic CD95 monoclonal antibody did not prevent drug-induced apoptosis. Furthermore, analysis of caspase involvement showed important differences in apoptosis induced by CD95-triggering or FUdR treatment. In summary, these results suggest that apoptosis induced by thymineless stress in haematopoietic BA/F3 cells occurs by a mechanism that does not require accumulation of p53 and which is independent of CD95–CD95L interactions.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Disruption of protein folding in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR)—a signaling network that ultimately determines cell fate. Initially, UPR signaling aims at cytoprotection and restoration of ER homeostasis; that failing, it drives apoptotic cell death. ER stress initiates apoptosis through intracellular activation of death receptor 5 (DR5) independent of its canonical extracellular ligand Apo2L/TRAIL; however, the mechanism underlying DR5 activation is unknown. In cultured human cells, we find that misfolded proteins can directly engage with DR5 in the ER-Golgi intermediate compartment, where DR5 assembles pro-apoptotic caspase 8-activating complexes. Moreover, peptides used as a proxy for exposed misfolded protein chains selectively bind to the purified DR5 ectodomain and induce its oligomerization. These findings indicate that misfolded proteins can act as ligands to activate DR5 intracellularly and promote apoptosis. We propose that cells can use DR5 as a late protein-folding checkpoint before committing to a terminal apoptotic fate. eLife digest Proteins are chains of building blocks called amino acids, folded into a flexible 3D shape that is critical for its biological activity. This shape depends on many factors, but one is the chemistry of the amino acids. Because the internal and external environments of cells are mostly water-filled, correctly folded proteins often display so-called hydrophilic (or ‘water-loving’) amino acids on their surface, while tucking hydrophobic (or ‘water-hating’) amino acids on the inside. A compartment within the cell called the endoplasmic reticulum folds the proteins that are destined for the outside of the cell. It can handle a steady stream of protein chains, but a sudden increase in demand for production, or issues with the underlying machinery, can stress the endoplasmic reticulum and hinder protein folding. This is problematic because incorrectly folded proteins cannot work as they should and can be toxic to the cell that made them or even to other cells. Many cells handle this kind of stress by activating a failsafe alarm system called the unfolded protein response. It detects the presence of incorrectly shaped proteins and sends signals that try to protect the cell and restore protein folding to normal. If that fails within a certain period of time, it switches to signals that tell the cell to safely self-destruct. That switch, from protection to self-destruction, involves a protein called death receptor 5, or DR5 for short. DR5 typically triggers the cell’s self-destruct program by forming molecular clusters at the cell’s surface, in response to a signal it receives from the exterior. During a failed unfolded protein response, DR5 seems instead to act in response to signals from inside the cell, but it was not clear how this works. To find out, Lam et al. stressed the endoplasmic reticulum in human cells by forcing it to fold a lot of proteins. This revealed that DR5 sticks to misfolded proteins when they leave the endoplasmic reticulum. In response, DR5 molecules form clusters that trigger the cell's self-destruct program. DR5 directly recognized hydrophobic amino acids on the misfolded protein’s surface that would normally be hidden inside. When Lam et al. edited these hydrophobic regions to become hydrophilic, the DR5 molecules could no longer detect them as well. This stopped the cells from dying so easily when they were under stress. It seems that DR5 decides the fate of the cell by detecting proteins that were incorrectly folded in the endoplasmic reticulum. Problems with protein folding occur in many human diseases, including metabolic conditions, cancer and degenerative brain disorders. Future work could reveal whether controlling the activation of DR5 could help to influence if and when cells die. The next step is to understand how DR5 interacts with incorrectly folded proteins at the atomic level. This could aid the design of drugs that specifically target such receptors. Introduction Proper folding of transmembrane and secreted proteins is critical to cell function and intercellular communication. Quality control of protein folding begins in the endoplasmic reticulum (ER) and responds to increased protein-folding demand during physiological or pathophysiological stresses. Accumulation of unfolded or misfolded proteins in the ER, known as ER stress, activates the unfolded protein response (UPR) – a network of intracellular signaling pathways that initially mount cytoprotective response to restore ER homeostasis but can ultimately switch to a pro-apoptotic program under irresolvable stress (Walter and Ron, 2011; Tabas and Ron, 2011). Two key UPR sensors, IRE1 and PERK coordinate the decision between cell survival and death through the delayed upregulation of the apoptosis-initiating protein death receptor 5 (DR5) (Lu et al., 2014; Chang et al., 2018). During ER stress, IRE1 and PERK oligomerize upon directly binding to misfolded proteins, leading to their activation (Karagöz et al., 2017; Wang et al., 2018). PERK activation causes the selective translation of ATF4 and CHOP, which, in addition to upregulating genes that enhance the folding capacity of the ER, promotes the transcription of pro-apoptotic DR5 (Harding et al., 2003; Yamaguchi and Wang, 2004). The pro-apoptotic signal is initially counteracted by regulated IRE1-dependent mRNA decay (RIDD) that degrades DR5 mRNA (Lu et al., 2014). Upon prolonged ER stress, PERK exerts negative feedback on IRE1 activity attenuating RIDD, thus de-repressing DR5 synthesis to drive cell commitment to apoptosis (Chang et al., 2018). DR5 is a pro-apoptotic member of the tumor necrosis factor receptor (TNFR) superfamily that signals from the plasma membrane into the cell in response to extracellular cues (Sheridan et al., 1997; Walczak et al., 1997; Ashkenazi, 1998). It is constitutively expressed in various tissue types and forms auto-inhibited dimers in its resting state, analogous to other members of the TNFR family (Spierings et al., 2004; Pan et al., 2019; Vanamee and Faustman, 2018). In its canonical mode of activation, binding of the homotrimeric extracellular ligand TRAIL (also known as Apo2L) (Wiley et al., 1995; Pitti et al., 1996) assembles DR5 into higher-order oligomers (Hymowitz et al., 1999; Mongkolsapaya et al., 1999; Valley et al., 2012). Consequently, DR5 forms intracellular scaffolds in which its cytosolic death domains recruit the adaptor protein FADD and pro-caspase 8 into the ‘death-inducing signaling complex’ (DISC) (Kischkel et al., 2000; Sprick et al., 2000; Jin et al., 2009; Dickens et al., 2012). Upon DISC-mediated dimerization, pro-caspase 8 molecules undergo regulated auto-proteolysis to form active initiator caspase 8 (Muzio et al., 1998). Activated caspase 8 frequently induces the intrinsic mitochondrial apoptotic pathway by truncating Bid, a pro-apoptotic Bcl2 protein, to cause Bax-mediated permeabilization of the mitochondrial outer membrane (Wei et al., 2001; LeBlanc et al., 2002). While DR5 and caspase 8 are both required for apoptosis during ER stress, we (Lu et al., 2014; Lam et al., 2018), along with other independent groups (Cazanave et al., 2011; Iurlaro et al., 2017; Dufour et al., 2017), found unexpectedly that TRAIL is dispensable for this DR5 activation. Indeed, upon ER stress, most newly synthesized DR5 molecules never make it to the plasma membrane but remain intracellular and thus inaccessible to extracellular ligands (Lu et al., 2014; Iurlaro et al., 2017). Given that at physiological levels DR5 is auto-inhibited until activated by a ligand, it remained a mystery how DR5 is activated in response to ER stress, prompting us to interrogate its intracellular mechanism of activation. Results Misfolded proteins induce DR5-dependent apoptosis and can assemble DR5-caspase 8 signaling complexes To examine the mechanism of cell death driven by an unmitigated protein folding burden, we induced the exogenous expression of a GFP-tagged form of the glycoprotein myelin protein zero (MPZ) in epithelial cells (Figure 1A). MPZ initially folds in the ER and then travels to the plasma membrane to mediate membrane adhesion in myelin-forming Schwann cells, where it is normally expressed. Mutations of MPZ that impair folding and cause its intracellular retention activate the UPR, leading to apoptosis in a manner dependent on CHOP (Pennuto et al., 2008). We found that in epithelial cells, titration of even non-mutant, GFP-tagged MPZ to high expression levels resulted in its intracellular accumulation, indicating a compromised MPZ folding state (Figure 1A). Folding-compromised MPZ induced a dose-dependent upregulation of the UPR transcriptional target genes CHOP, BiP, and DR5 (Figure 1—figure supplement 1A). Upregulated DR5 was retained intracellularly (Figure 1A, Figure 1—figure supplement 1B) and occurred concomitantly with cleavage of caspase 8and its downstream target caspase 3 (Figure 1B). By contrast, low levels of MPZ-GFP expression that exhibited proper plasma membrane localization did not induce caspase 8 or 3 activity (Figure 1A, B). To determine when caspase 8 became active relative to cytoprotective UPR signaling, we assessed IRE1 activity during high MPZ-GFP expression through analysis of XBP1 mRNA splicing. As expected, IRE1-mediated XBP1 mRNA splicing initiated a few hours post-transfection with MPZ-GFP and later attenuated (Figure 1—figure supplement 1C). The upregulation of DR5, caspase activity, and PARP cleavage (another indicator of apoptotic progression) occurred after the attenuation of IRE1 activity, consistent with the hallmarks of terminal pro-apoptotic UPR signaling (Figure 1—figure supplement 1D–1E). Figure 1 with 5 supplements see all Download asset Open asset Misfolded proteins induce DR5-dependent apoptosis and assemble DR5-caspase 8 signaling complexes. (A) Confocal images of epithelial cells HCT116 fixed 24 hr post-transfection with 0.25–1.0 μg of a plasmid containing myelin protein zero (MPZ) tagged with a C-terminal monomeric EGFP or 1.0 μg of the empty vector showing MPZ-GFP fluorescence (green) and immunofluorescence with an antibody against DR5 (red) (scale bar = 5 μm). (B) Western blot of HCT116 cell lysates harvested 24 hr post-transfection with a titration of MPZ-GFP plasmid or the empty vector (C8 = caspase 8, cC3 = cleaved caspase 3). p55 represents full-length, inactive C8; p43 indicates a C8 intermediate after release of the active p10 subunit, and p29 corresponds to the released p18 and p10 subunits. (C) Western blot of HCT116 cells transfected with siRNA against a non-targeting (Nt) control or DR5 (48 hr) followed by the empty vector -/+ 100 nM thapsigargin (Tg), 1.0 μg MPZ-GFP, or cytosolic GFP (24 hr; * denotes degradation products; L and S denote the long and short isoforms of DR5, respectively; FL and C denote full-length and cleaved PARP, respectively). (D) Average percent of annexin V staining for HCT116 cells transfected as described in C) from n = 3 biological replicates (error bars = SEM; * indicates p<0.05; ns indicates p=0.46 as analyzed by unpaired t-test with equal SD). See Figure 1—figure supplement 4D for gating. (E) Top: Caspase 8 activity in size exclusion chromatography fractions from lysates of HCT116 cells transfected with MPZ-GFP or cytosolic GFP (24 hr). Bottom: Size exclusion fractions were pooled according to dotted grid lines and immunoblotted for DR5 and GFP (* denotes degradation products). (F) Immunoprecipitation of GFP-tagged proteins from lysates of HCT116 transfected with MPZ-GFP, cytosolic GFP, or the empty vector (L and S denote the long and short isoforms of DR5, respectively). The percent of total DR5 recovered has been quantified in Figure 1—figure supplement 5C. (G) Fold change in caspase 8 activity relative to the empty vector control for beads with immunoprecipitated contents shown in Figure 1F (error bars = SEM for n = 3 biological replicates; * indicates p=0.023 and ns indicates p=0.83 as calculated by unpaired t-tests with equal SD). Figure 1—source data 1 Caspase glo 8 measurements for IP of MPZ-GFP vs GFP. This zip archive contains the measured luminescent units for caspase glo 8 activity shown in Figure 1G (IP beads) and Figure 1—figure supplement 3C (input lysates). Coomassie gels used to normalize lysate concentration are included as. tif files. https://cdn.elifesciences.org/articles/52291/elife-52291-fig1-data1-v2.zip Download elife-52291-fig1-data1-v2.zip Figure 1—source data 2 Westerns and quantification of DR5 recovered on IPs. This zip archive contains images of the Western blots and measurements used to quantify the amount of DR5 in the IP samples relative to the input lysate. https://cdn.elifesciences.org/articles/52291/elife-52291-fig1-data2-v2.zip Download elife-52291-fig1-data2-v2.zip Figure 1—source data 3 FCS files and quantification of annexin V staining for MPZ-GFP. This zip archive contains FCS files from n = 3 biological replicates of HCT116 transfected with the conditions outlined in Figure 1D. The excel file contains the quantification of annexin V staining exported frow FlowJo. https://cdn.elifesciences.org/articles/52291/elife-52291-fig1-data3-v2.zip Download elife-52291-fig1-data3-v2.zip Figure 1—source data 4 qPCR analysis of MPZ-GFP titration. This zip archive contains the compiled excel file for qPCR data shown in Figure 1—figure supplement 1A along with the Prism 6 file used to perform multiple t-tests with Holm-Sidak correction for multiple comparisons. https://cdn.elifesciences.org/articles/52291/elife-52291-fig1-data4-v2.zip Download elife-52291-fig1-data4-v2.zip Figure 1—source data 5 Caspase glo 8 measurements for time course of MPZ-GFP transfection. This zip archive contains the measured luminescent units for caspase glo 8 activity shown in Figure 1—figure supplement 1E and the tif file of the Coomassie blue-stained gel used to normalize lysate concentrations. https://cdn.elifesciences.org/articles/52291/elife-52291-fig1-data5-v2.zip Download elife-52291-fig1-data5-v2.zip Figure 1—source data 6 qPCR and cell death measurement for CHOP expression. This zip archive contains the qPCR analysis from CHOP expression in Figure 1—figure supplement 2B, and brightfield images of Trypan Blue staining measured on the Countess II for n = 3 biological replicates, summarized in Figure 1—figure supplement 2D. https://cdn.elifesciences.org/articles/52291/elife-52291-fig1-data6-v2.zip Download elife-52291-fig1-data6-v2.zip Figure 1—source data 7 qPCR analysis of INS and RHO-GFP expression. This zip archive contains the compiled excel file for qPCR data shown in Figure 1—figure supplement 4A along with the Prism 6 file used to perform multiple t-tests with Holm-Sidak correction for multiple comparisons. https://cdn.elifesciences.org/articles/52291/elife-52291-fig1-data7-v2.zip Download elife-52291-fig1-data7-v2.zip Figure 1—source data 8 FCS files and quantification of annexin V staining for INS and RHO. This zip archive contains FCS files from n = 3 biological replicates of HCT116 transfected with the conditions outlined in Figure 1—figure supplement 4E. The excel file contains the quantification of annexin V staining exported frow FlowJo. https://cdn.elifesciences.org/articles/52291/elife-52291-fig1-data8-v2.zip Download elife-52291-fig1-data8-v2.zip Figure 1—source data 9 Caspase glo 8 measurements for IP of INS and RHO-GFP. This zip archive contains the measured luminescent units for caspase glo 8 activity shown in Figures 1S5B (input lysates and IP beads). Coomassie gels used to normalize lysate concentration are included as. tif files. https://cdn.elifesciences.org/articles/52291/elife-52291-fig1-data9-v2.zip Download elife-52291-fig1-data9-v2.zip To determine if DR5 was required for apoptosis during this sustained protein misfolding stress, we acutely depleted DR5 mRNA by siRNA prior to overexpressing MPZ-GFP. Knockdown of DR5 significantly reduced PARP cleavage and annexin V staining following overexpression of MPZ-GFP (Figure 1C, D), which was not observed in control experiments expressing cytosolic GFP. To determine if upregulation of DR5 was sufficient to induce apoptosis, we increased DR5 levels in the absence of ER stress through ectopic expression of CHOP. Comparable levels of CHOP-induced DR5 protein in the absence of ER stress drove drastically lower levels of PARP cleavage and trypan blue staining (demarking apoptotic cells) compared to the presence of misfolded-protein stress (Figure 1—figure supplement 2A and C–D). These results show that DR5 activation does not occur spontaneously after its upregulation but requires additional input signals conveyed by ER stress. To assess the molecular composition of activated DR5 assemblies formed in response to ER stress, we measured caspase 8 activity in cell extracts fractionated through size exclusion chromatography. We detected increased caspase 8 activity in high-molecular w8 (MW) fractions of cells transfected with MPZ-GFP relative to GFP (Figure 1E). The fractions contained DR5 complexes and co-eluted with full-length MPZ-GFP but not GFP-degradation products (Figure 1E, lanes 2 and 4). Pull-down of DR5 from cell lysates enriched for FADD and MPZ-GFP (Figure 1—figure supplement 3A), suggesting that the co-elution of DR5 and MPZ-GFP in the high MW fractions resulted from their physical association. To test if MPZ physically interacted with activated DR5 complexes, we immunoprecipitated MPZ-GFP and detected DR5, FADD, and caspase 8 (both full-length p55 and its cleaved form p43) (Figure 1F, Figure 1—figure supplement 3B). Furthermore, MPZ-GFP immunoprecipitates contained 2–3-fold more caspase 8 activity compared to empty beads (Figure 1G, Figure 1—figure supplement 3C), indicating that they contained assembled DISC in a similar degree as seen after affinity purification of TRAIL-ligated DR5 (Hughes et al., 2013). In contrast, pull-down of cytosolic GFP did not enrich for DR5, FADD, or caspase activity (Figure 1F, G), confirming the selectivity for ER-folded MPZ-GFP. To determine if misfolded proteins generally induced caspase activity through association with DR5, we overexpressed GFP-tagged forms of two other ER-trafficked proteins, rhodopsin (RHO) and proinsulin (INS), which are also associated with CHOP-dependent cell death pathologies (Chiang et al., 2016; Oyadomari et al., 2002). Sustained overexpression of both RHO-GFP and INS-GFP upregulated BiP and CHOP mRNAs (Figure 1—figure supplement 4A) and induced XBP1 mRNA splicing (Figure 1—figure supplement 4B). Both proteins formed SDS-insoluble aggregates and induced PARP cleavage and annexin V staining in a DR5-dependent manner (Figure 1—figure supplement 4C–4E). By contrast, immunoprecipitation of RHO-GFP enriched for DR5 protein and caspase 8 activity more robustly than INS-GFP (Figure 1—figure supplement 5), despite inducing DR5-dependent apoptosis to a similar extent. This indicates that misfolded proteins differ in their propensity to directly engage the DR5-assembled DISC, and that other misfolded substrates—caused by the ectopically overexpressed ER-trafficked protein—may mediate direct DR5 activation. Thus, as exemplified by MPZ and RHO, a selective subset of misfolded proteins in the secretory pathway can engage DR5 to form oligomeric complexes that induce caspase 8 activation. Misfolded protein engages DR5 at the ER-Golgi intermediate compartment, inducing active DR5 signaling clusters To explore where within in the cell DR5 associated with misfolded protein, we used confocal imaging of fixed cells for immunofluorescence. These analyses revealed that intracellular MPZ-GFP and DR5 appeared in discrete puncta that often overlapped (Figure 2A). DR5 siRNA knockdown eliminated the DR5 signal, confirming the specificity of the DR5 antibody (Figure 2—figure supplement 1A, right panel). Similarly, overexpression of RHO also resulted in intracellular puncta that frequently co-localized with DR5 clusters (Figure 2—figure supplement 1B). Quantification of the mean Pearson’s correlation per cell demonstrated statistically significant overlap with DR5 signal for both GFP-tagged MPZ and RHO (Figure 2—figure supplement 1C), indicating that these misfolded proteins accumulate in the same compartment as DR5. Figure 2 with 2 supplements see all Download asset Open asset Misfolded protein engages DR5 at the ER-Golgi intermediate compartment, inducing active DR5 signaling clusters. (A) Top: Immunofluorescence of HCT116 cells transfected with MPZ-GFP (green) for 24 hr and stained with anti-DR5 (red, scale bar 5 μm). Bottom: Enlargements of the inset stepping through the z-plane in 0.5 μm increments (scale bar 2 μm). (B) Subcellular fractionation of lysate expressing MPZ-GFP, where IRE1 marks the ER, Giantin marks the Golgi, Sec31A and Sec23A mark COPII vesicles, and ERGIC53 and RCAS1 correspond to ER-Golgi intermediate compartment. Bands of the expected size are indicated by “– “and bands that may represent a modified or degraded protein are indicated by *. (C) Average caspase activity of each fraction from subcellular gradient centrifugation in (B) normalized to total lysate (input) measured by caspase 8 substrate luminescence (n = 3 biological replicates, error bars = SEM; ns1 indicates p=0.079, * denotes p=0.015, and ns indicates p=0.31 from unpaired t-tests with equal SD). (D) Top: Immunostaining of DR5 and ERGIC53 in fixed HCT116 cells transfected with MPZ-GFP for 24 hr as in (A). Bottom: Merged images with ERGIC53 in magenta or cyan to depict overlapping signal as white (scale bar = 5 μm, insets scale bar = 2 μm). (E) Immunostaining of DR5 and giantin in fixed HCT116 cells expressing MPZ-GFP. Giantin is magenta in the overlay with MPZ (green) or cyan in the overlay with DR5 (red). Bottom row enlarges the inset marked in the merges images to show little overlapping signal with giantin (scale bar = 5 μm, inset scale bar = 1 μm). (F) Box-whisker plots quantifying the Pearson’s correlation per cell between DR5 and ERGIC53 (mean = 0.61 ± 0.03) or giantin (mean = 0.14 ± 0.02) within MPZ-positive cells (N > 55), where whiskers correspond to minimum and maximum values of the data (**** indicates p<0.001). Figure 2—source data 1 Caspase activity for fractions of iodixanol gradient. This excel file contains the caspase glo 8 luminescent units of the fractionation samples (n = 3 biological replicates) shown in Figure 2C. https://cdn.elifesciences.org/articles/52291/elife-52291-fig2-data1-v2.xlsx Download elife-52291-fig2-data1-v2.xlsx Previous findings suggested that DR5 is retained near the Golgi apparatus during ER stress (Lu et al., 2014). We confirmed co-localization with the purported Golgi marker RCAS1, as previously reported (Figure 2—figure supplement 1D). However, we observed little overlap in DR5 staining with another cis-Golgi marker, giantin (Figure 2E). To resolve this discrepancy, we employed subcellular fractionation as an orthogonal biochemical approach. Separating organelle membranes revealed that RCAS1, DR5, and MPZ-GFP co-sedimented in fractions containing ERGIC53, a marker of the ER-Golgi intermediate compartment (ERGIC), but not with those containing giantin (Figure 2B). Notably, a portion of FADD, a cytosolic protein expected to exclusively remain in the topmost, cytosolic fraction, migrated into the second fraction of the gradient, indicating its association with the ERGIC membranes. Consistent with the presence of FADD, the first and second ERGIC-associated fractions harbored the majority of the caspase 8 activity in the cell lysate (Figure 2C), indicating the presence of active DR5 DISCs. Moreover, immunofluorescence with quantification of the mean correlation per cell demonstrated the co-localization of DR5 with the ERGIC rather than with the Golgi (Figure 2D and F). To determine when DR5 accumulates at the ERGIC relative to misfolded proteins, we compared the immunofluorescence of cells fixed at 20 hr (before the onset of caspase activity) and at 24 hr post-transfection (after the onset of caspase activity, Figure 1—figure supplement 1E). Intracellular puncta of MPZ appeared at 20 hr, preceding the appearance of DR5 clusters at 24 hr (Figure 2—figure supplement 2A). Between 20 and 24 hr, the correlation of DR5 and ERGIC53 increased, whereas the correlation of MPZ with ERGIC53 remained steady, indicating that DR5 accumulated after saturation of MPZ levels at the ERGIC (Figure 2—figure supplement 2B–2C). By contrast, the mean Pearson’s correlation with giantin approached zero for both MPZ and DR5 at 24 hr post-transfection (Figure 2—figure supplement 2B, Figure 2F). These results confirm the localization of DR5 and misfolded protein at the ERGIC under conditions of unmitigated ER stress. Polypeptide sequences of mammalian ER-trafficked protein directly bind to the DR5 ectodomain and induce its oligomerization With evidence of a physical association between misfolded protein and active DR5 oligomers at the ERGIC, we asked how misfolded proteins and DR5 interact. Considering the precedence that (i) DR5 binds unstructured peptides mimicking TRAIL (Kajiwara, 2004; Pavet et al., 2010) and (ii) that UPR sensors can directly bind misfolded protein to sense ER stress (Karagöz et al., 2017; Wang et al., 2018; Gardner and Walter, 2011), we hypothesized that DR5 may directly recognize unstructured regions of misfolded proteins through its ectodomain (ECD) that would project into the ERGIC lumen. Probing a peptide array with purified recombinant Fc-tagged DR5 ECD revealed promiscuous recognition of amino acid sequences throughout the ectodomain of MPZ and within extracellular loops of RHO (Figure 3A, Figure 3—figure supplement 1A–1B). Quantification of the relative signal intensity revealed that DR5-binding sequences were enriched for aliphatic and aromatic residues whereas polar and acidic residues were excluded (Figure 3—figure supplement 1C), reminiscent of qualities that become surface-exposed in misfolded or unfolded proteins. Figure 3 with 2 supplements see all Download asset Open asset Direct binding of exposed ER-trafficked protein sequences to the DR5 ECD is sufficient to induce oligomerization. (A) A peptide array tiled with sequences from the ectodomain of myelin protein zero (MPZ) and extracellular loops from rhodopsin (RHO) was incubated with Fc-tagged DR5 ectodomain domain (long isoform, 500 nM). Signal was obtained by probing with anti-Fc. (B) Coomassie stained SDS-PAGE gel of pulldown on Fc-tagged DR5L ECD (55 kDa) or TNFR1 ECD (65 kDa) incubated with increasing concentrations of the MPZ-ectoVD peptide (apparent MW of 10 kDa, see 'Amino acid sequences of MPZ-derived peptides' for sequence). (C) Fluorescence quenching of AlexaFluor647-DR5L (green) or TNFR1 ECD (blue) was measured with increasing MPZ-ecto peptide to quantify the binding affinity, whereas quenching was not observed with the mutated MPZ-ectoTyr→Glu peptide (magenta) (N = 3, error bars are SD). DR5L ECD binds to the MPZ-ecto peptide with a K1/2 of 109 ± 11 μM with a hill coefficient of 2.6 ± 0.5. (D) SDS-PAGE of recombinant FLAG-tagged DR5L ECD (25 kDa, 10 μM) incubated with MPZ-ecto peptide at the noted concentrations and treated with the amine crosslinker BS3 (100 μM), probed with anti-FLAG. (E) Size exclusion chromatographs of absorbance at 280 nm for 25 μM recombinant DR5L ECD alone (black), pre-incubated with 100 μM fluorescein-conjugated MPZ-ecto peptide (green) or 100 μM fluorescein-conjugated MPZ-ectoTyr→Glu peptide (magenta). (F) SDS-PAGE gels scanned for fluorescence and then stained with Coomassie for eluted size exclusion fractions in (E). Green outlines (top pair) correspond to fractions from DR5L pre-incubated with MPZ-ecto peptide, and magenta outlines (bottom pair) correspond to DR5L with MPZ-ectoTyr→Glu peptide. Lane marked by “-“ denotes a blank lane between the input and 7 ml fraction to minimize spillover of signal from input sample. Arrowheads mark detectable peptide fluorescence in the indicated fractions. Figure 3—source data 1 Sequences and quantification of peptides probed with Fc-DR5 ECD on the peptide array. This excel file contains the peptide sequences of the peptide array shown in Figure 3A, the quantification of DR5 ECD detected for each spot, and the analysis for enriched amino acids in Figure 3—figure supplement 1. https://cdn.elifesciences.org/articles/52291/elife-52291-fig3-data1-v2.xlsx Download elife-52291-fig3-data1-v2.xlsx To validate the specificity of DR5 interactions on the array, we performed pull-down assays on the MPZ-derived peptide exhibiting the strongest signal (spots C18-C19 in Figure 3A, hereon referred to as MPZ-ecto) with recombinant Fc-tagged DR5 ECD versus TNFR1 ECD as a selectivity control. The MPZ-ecto peptide bound specifically to the DR5 ECD but not the TNFR1 ECD (Figure 3B). Under equilibrium conditions, interaction with MPZ-ecto peptide quenched fluorescently labeled DR5 ECD but not fluorescently labeled TNFR1 ECD, yielding an apparent binding affinity of K1/2 = 109 μM±11 μM with a Hill coefficient of 2.6 (Figure 3C, Figure 3—figure supplement 2A). Adding excess unlabeled DR5 ECD restored fluorescence (Figure 3—figure supplement 2B), indicating that the quenching reflected a specific and reversible interaction between the DR5 ECD and the MPZ-ecto peptide. Moreover, mutation of two aromatic amino acids (both Tyr) to disfavored acidic amino acids (Glu) abrogated binding (Figure 3C), demonstrati

Abstract ABTL0812 is a first-in-class small molecule with anticancer activity currently in Phase 2a clinical evaluation in patients with advanced endometrial and squamous NSCLC. We have previously described that ABTL0812 induces TRIB3 pseudokinase expression, resulting in inhibition of the Akt-mTORC1 axis and autophagy-mediated cancer cell death. However, classical PI3K/Akt/mTOR inhibitors do not induce an autophagy as strong as ABTL0812 does, therefore we aimed to further elucidate the molecular mechanism responsible for the cytotoxic autophagy which causes ABTL0812 anticancer activity. ABTL0812 induced UPR hallmarks ATF4, CHOP and TRIB3 in vitro in lung, endometrial and pancreatic cancer cell lines, as well as in non-tumor cells. Nevertheless, therapeutic concentrations of ABTL0812 did not induce cytotoxic autophagy in non-tumor cells. Furthermore, genetic or pharmacological inhibition of the UPR resulted in impaired ABTL0812 cytotoxicity in cancer cells. Expression of UPR markers (ATF4, CHOP and TRIB3) in response to ABTL0812 treatment were also validated in xenograft models. In order to uncover the precise molecular mechanism involved in ABTL0812-induced UPR and cytotoxic autophagy we undertook a comprehensive sphingolipidomic analysis, since changes in sphingolipids have been reported to contribute to activation of both UPR and autophagy. ABTL0812 treatment resulted in increased levels of long chain dihydroceramides in cultured cancer cells as well as in vivo. Mechanistically, ABTL0812 impaired desaturase-1 activity (Des-1), the enzyme that introduces a 4,5-trans-double bond in the sphingolipid backbone of dihydroceramides to generate ceramides. Accordingly, in vitro incubation of cancer cells with dihydroceramides resulted in activation of UPR, autophagy and cytotoxicity. Of interest, we observed that tumor cells showed higher Des-1 expression levels than non-tumor cells. Finally, we showed that Des-1 inhibition (GT11) and mTORC1 inhibition (everolimus) collaborate to promote autophagy and cancer cell death, simulating ABTL0812 activity. Furthermore, we have validated the increased expression of CHOP and TRIB3 mRNA levels in blood samples from ABTL0812 treated patients. These biomarkers are currently used as pharmacodynamic biomarkers in the ongoing phase 2 clinical trial in patients with squamous NSCLC and endometrial cancer. To our knowledge, this is the first time that UPR markers are reported to change in human blood in response to any drug treatment. In conclusion, we have shown that ABTL0812 triggers a sustained ER stress and UPR activation mediated by the impairment of Des-1 activity which collaborates with mTORC1 inhibition to induce cytotoxic autophagy in cancer cells, which offers improved anticancer activity over just inhibiting mTORC1. Citation Format: Pau Muñoz-Guardiola, Josefina Casas, Elisabet Megías-Roda, Hector Perez-Montoyo, Sonia Solé-Sánchez, Marc Yeste-Velasco, Tatiana Erazo, Nora Diéguez-Martínez, Sergio Espinosa-Gil, Guillermo Yoldi, Cristina Muñoz-Pinedo, Miguel F. Segura, Jose Alfon, Gemma Fabriàs, Guillermo Velasco, Carles Domenech, Jose M. Lizcano. The anticancer drug ABTL0812 induces cancer cell death by impairing Akt/mTORC1 axis and inducing ER stress-mediated cytotoxic autophagy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1234.

ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased <i>DDIT3</i> and <i>TRIB3</i> mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer.

ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased <i>DDIT3</i> and <i>TRIB3</i> mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer. 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ⁹-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response.

Abstract There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy. Here, we investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM. Based on publicly available transcriptomic data of MPM, patients with CDK4 or CDK6 overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell models. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest thereby increasing cell senescence and increased the expression of interferon signaling pathway and tumor antigen presentation process in culture models of MPM. In vivo preclinical studies showed that palbociclib significantly reduced tumor growth and prolonged overall survival in a platinum-naïve and platinum resistant MPM mouse model. Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of MPM.

ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased <i>DDIT3</i> and <i>TRIB3</i> mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer. 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ⁹-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response.

El desbalance en los niveles de nucleotidos trifosfato induce apoptosis en celulas hematopoyeticas. En este trabajo se ha estudiado la implicacion de la proteina supresora de tumores p53 y la relacion con la via de apoptosis inducida por ligandos de muerte. La timidina exogena, pero no la uridina, inhibio la muerte inducida por el inhibidor de la timidilato-sintasa 5-fluoro-2'deoxiuridina (FUdR). La acumulacion de p53 no fue necesaria para este proceso de muerte. Se observo liberacion de citocromo c durante este proceso, y esto se produjo en ausencia de activacion de caspasas. La caspasa/8 se activo, pero esta activacion no resulto esencial para el desarrollo de la apoptosis. La despolarizacion mitocondrial observada ocurrio como consecuencia, no como causa, de la activacion de caspasas. La inhibicion de la glicolisis produjo la inhibicion de la muerte inducida por FUdR, inhibiendose este proceso en un punto anterior a la liberacion de citocromo c. Sin embargo, en las mismas condiciones, se potencio la apoptosis inducida por activacion de receptores de muerte de la familia del receptor del Factor de Necrosis Tumoral alga (TNF-alfa). Se demostro que las celulas, en condiciones de bajo ATP, seguian muriendo por apoptosis, no por necrosis. En medio sin glucoso, o en presencia del inhibidor de la glicolisis 2-deoxiglucosa, se observo sensibilizacion de varias lineas tumorales a la apoptosis inducida por TNF-alfa, TRAIL y un anticuerpo activador de Fas. Tanto la activacion de caspasas como la via mitocondrial estaban potenciadas en estas condiciones, y se determino que incluso la activacion de la caspasa-8 apical ocurria antes en ausencia de glucosa que en medio de cultivo con glucosa. Este fenomeno se observo en varias lineas tumorales de distinto origen, lo que sugiere nuevas posibildiades terapeuticas del uso combiando de inhibidores de glicolisis y ligandos de muerte que matan selectivamente ce