Starvation and antimetabolic therapy promote cytokine release and recruitment of immune cells.

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.

Exploiting metabolic vulnerabilities of Non small cell lung carcinoma.

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.

In the Hunger Games, the Winner Takes Everything.

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.

Efficacy of CDK4/6 inhibitors in preclinical models of malignant pleural mesothelioma.

BACKGROUND: There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy. METHODS: We aimed to investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM. RESULTS: 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. CONCLUSIONS: 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.

Glucose-starved cells do not engage in prosurvival autophagy.

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.

Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis.

In addition to their role in cellular bioenergetics, mitochondria also initiate common forms of programmed cell death (apoptosis) through the release of proteins such as cytochrome c from the intermembrane and intracristal spaces. The release of these proteins is studied in populations of cells by western blotting mitochondrial and cytoplasmic fractions of cellular extracts, and in single cells by fluorescence microscopy using fluorescent indicators and fusion proteins. However, studying the changes in ultrastructure associated with release of proteins requires the higher resolution provided by transmission electron microscopy. Here, we have used fluorescence microscopy to characterize the state of apoptosis in HeLa cells treated with etoposide followed by electron microscopy and three-dimensional electron microscope tomography of the identical cells to study the sequence of structural changes. We have identified a remodelling of the inner mitochondrial membrane into many separate vesicular matrix compartments that accompanies release of proteins; however, this remodelling is not required for efficient release of cytochrome c. Swelling occurs only late in apoptosis after release of cytochrome c and loss of the mitochondrial membrane potential.

NF-κB and TET2 promote macrophage reprogramming in hypoxia that overrides the immunosuppressive effects of the tumor microenvironment.

Macrophages orchestrate tissue homeostasis and immunity. In the tumor microenvironment (TME), macrophage presence is largely associated with poor prognosis because of their reprogramming into immunosuppressive cells. We investigated the effects of hypoxia, a TME-associated feature, on the functional, epigenetic, and transcriptional reprogramming of macrophages and found that hypoxia boosts their immunogenicity. Hypoxic inflammatory macrophages are characterized by a cluster of proinflammatory genes undergoing ten-eleven translocation-mediated DNA demethylation and overexpression. These genes are regulated by NF-κB, while HIF1α dominates the transcriptional reprogramming, demonstrated through ChIP-seq and pharmacological inhibition. In bladder and ovarian carcinomas, hypoxic inflammatory macrophages are enriched in immune-infiltrated tumors, correlating with better patient prognoses. Coculture assays and cell-cell communication analyses support that hypoxic-activated macrophages enhance T cell-mediated responses. The NF-κB-associated hypomethylation signature is displayed by a subset of hypoxic inflammatory macrophages, isolated from ovarian tumors. Our results challenge paradigms regarding the effects of hypoxia on macrophages and highlight actionable target cells to modulate anticancer immune responses.

Oxidative stress modulates mitochondrial failure and cyclophilin D function in X-linked adrenoleukodystrophy.

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.

Negligible role of TRAIL death receptors in cell death upon endoplasmic reticulum stress in B-cell malignancies.

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.

Signaling pathways that regulate life and cell death: evolution of apoptosis in the context of self-defense.

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.

Disruption of the M80-Fe ligation stimulates the translocation of cytochrome c to the cytoplasm and nucleus in nonapoptotic cells.

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.

Glycosylation defects, offset by PEPCK-M, drive entosis in breast carcinoma cells.

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.

TRAIL receptors promote constitutive and inducible IL-8 secretion in non-small cell lung carcinoma.

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.

Hematopoietic versus Solid Cancers and T Cell Dysfunction: Looking for Similarities and Distinctions.

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.

Gene Expression Profiling as a Potential Tool for Precision Oncology in Non-Small Cell Lung 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.

The anti-cancer drug ABTL0812 induces ER stress-mediated cytotoxic autophagy by increasing dihydroceramide levels in cancer cells.

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.

Chemokine C-C motif ligand 2 overexpression drives tissue-specific metabolic responses in the liver and muscle of mice.

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.

Tumors defective in homologous recombination rely on oxidative metabolism: relevance to treatments with PARP inhibitors.

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.

Measuring the Activation of Cell Death Pathways upon Inhibition of Metabolism.

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.