RESUMO
In-vitro to in-vivo correlations (IVIVC), relating in-vitro parameters like IC50 to in-vivo drug exposure in plasma and tumour growth, are widely used in oncology for experimental design and dose decisions. However, they lack a deeper understanding of the underlying mechanisms. Our paper therefore focuses on linking empirical IVIVC relations for small-molecule kinase inhibitors with a semi-mechanistic tumour-growth model. We develop an approach incorporating parameters like the compound's peak-trough ratio (PTR), Hill coefficient of in-vitro dose-response curves, and xenograft-specific properties. This leads to formulas for determining efficacious doses for tumor stasis under linear pharmacokinetics equivalent to traditional empirical IVIVC relations, but enabling more systematic analysis. Our findings reveal that in-vivo xenograft-specific parameters, specifically the growth rate (g) and decay rate (d), along with the average exposure, are generally more significant determinants of tumor stasis and effective dose than the compound's peak-trough ratio. However, as the Hill coefficient increases, the dependency of tumor stasis on the PTR becomes more pronounced, indicating that the compound is more influenced by its maximum or trough values rather than the average exposure. Furthermore, we discuss the translation of our method to predict population dose ranges in clinical studies and propose a resistance mechanism that solely relies on specific in-vivo xenograft parameters instead of IC50 exposure coverage. In summary, our study aims to provide a more mechanistic understanding of IVIVC relations, emphasizing the importance of xenograft-specific parameters and PTR on tumor stasis.
Assuntos
Modelos Teóricos , Neoplasias , Humanos , Neoplasias/tratamento farmacológicoRESUMO
Bleomycin-induced pulmonary fibrosis in mice mimics major hallmarks of idiopathic pulmonary fibrosis. Yet in this model, it spontaneously resolves over time. We studied molecular mechanisms of fibrosis resolution and lung repair, focusing on transcriptional and proteomic signatures and the effect of aging. Old mice showed incomplete and delayed lung function recovery 8 weeks after bleomycin instillation. This shift in structural and functional repair in old bleomycin-treated mice was reflected in a temporal shift in gene and protein expression. We reveal gene signatures and signaling pathways that underpin the lung repair process. Importantly, the downregulation of WNT, BMP, and TGFß antagonists Frzb, Sfrp1, Dkk2, Grem1, Fst, Fstl1, and Inhba correlated with lung function improvement. Those genes constitute a network with functions in stem cell pathways, wound, and pulmonary healing. We suggest that insufficient and delayed downregulation of those antagonists during fibrosis resolution in old mice explains the impaired regenerative outcome. Together, we identified signaling pathway molecules with relevance to lung regeneration that should be tested in-depth experimentally as potential therapeutic targets for pulmonary fibrosis.
Assuntos
Fibrose Pulmonar Idiopática , Transcriptoma , Camundongos , Animais , Transcriptoma/genética , Proteômica , Pulmão , Bleomicina , Camundongos Endogâmicos C57BLRESUMO
Profibrotic and prohomeostatic macrophage phenotypes remain ill-defined, both in vivo and in vitro, impeding the successful development of drugs that reprogram macrophages as an attractive therapeutic approach to manage fibrotic disease. The goal of this study was to reveal profibrotic and prohomeostatic macrophage phenotypes that could guide the design of new therapeutic approaches targeting macrophages to treat fibrotic disease. This study used nintedanib, a broad kinase inhibitor approved for idiopathic pulmonary fibrosis, to dissect lung macrophage phenotypes during fibrosis-linked inflammation by combining in vivo and in vitro bulk and single-cell RNA-sequencing approaches. In the bleomycin model, nintedanib drove the expression of IL-4/IL-13-associated genes important for tissue regeneration and repair at early and late time points in lung macrophages. These findings were replicated in vitro in mouse primary bone marrow-derived macrophages exposed to IL-4/IL-13 and nintedanib. In addition, nintedanib promoted the expression of IL-4/IL-13 pathway genes in human macrophages in vitro. The molecular mechanism was connected to inhibition of the colony stimulating factor 1 (CSF1) receptor in both human and mouse macrophages. Moreover, nintedanib counterbalanced the effects of TNF on IL-4/IL-13 in macrophages to promote expression of IL-4/IL-13-regulated tissue repair genes in fibrotic contexts in vivo and in vitro. This study demonstrates that one of nintedanib's antifibrotic mechanisms is to increase IL-4 signaling in macrophages through inhibition of the CSF1 receptor, resulting in the promotion of tissue repair phenotypes.
Assuntos
Fibrose Pulmonar Idiopática , Indóis , Macrófagos , Indóis/farmacologia , Animais , Camundongos , Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Interleucina-4/metabolismo , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismoRESUMO
Interleukin (IL-)6 is the major pro-inflammatory cytokine within the IL-6 family. IL-6 signals via glycoprotein 130 (gp130) and the membrane-bound or soluble IL-6 receptor (IL-6R), referred to as classic or trans-signaling, respectively. Whereas inflammation triggers IL-6 expression, eventually rising to nanogram/ml serum levels, soluble IL-6R (sIL-6R) and soluble gp130 (sgp130) are constitutively present in the upper nanogram/ml range. Calculations based on intermolecular affinities have suggested that systemic IL-6 is immediately trapped in IL-6·sIL-6R and IL-6·sIL-6R·sgp130 complexes, indicating that sIL-6R and sgp130 constitute a buffer system that increases the serum half-life of IL-6 or restricts systemic IL-6 signaling. However, this scenario has not been experimentally validated. Here, we quantified IL-6·sIL-6R and IL-6·sIL-6R·sgp130 complexes over a wide concentration range. The amounts of IL-6 used in this study reflect concentrations found during active inflammatory events. Our results indicated that most IL-6 is free and not complexed with sIL-6R or sgp130, indicating that the level of endogenous sgp130 in the bloodstream is not sufficient to block IL-6 trans-signaling via sIL-6R. Importantly, addition of the single-domain antibody VHH6, which specifically stabilizes IL-6·sIL-6R complexes but did not bind to IL-6 or sIL-6R alone, drove free IL-6 into IL-6·sIL-6R complexes and boosted trans-signaling but not classic signaling, demonstrating that endogenous sIL-6R has at least the potential to form complexes with IL-6. Our findings indicate that even though high concentrations of sIL-6R and sgp130 are present in human serum, the relative ratio of free IL-6 to IL-6·sIL-6R allows for simultaneous classic and trans-signaling.
Assuntos
Receptor gp130 de Citocina/metabolismo , Interleucina-6/metabolismo , Receptores de Interleucina-6/metabolismo , Transdução de Sinais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Humanos , Interleucina-6/sangue , Interleucina-6/imunologia , Receptores de Interleucina-6/sangue , Receptores de Interleucina-6/imunologia , Proteínas Recombinantes/sangue , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Anticorpos de Domínio Único/imunologiaRESUMO
BACKGROUND: Interleukin-6 is a pleiotropic cytokine with high clinical relevance and an important mediator of cellular communication, orchestrating both pro- and anti-inflammatory processes. Interleukin-6-induced signalling is initiated by binding of IL-6 to the IL-6 receptor α and subsequent binding to the signal transducing receptor subunit gp130. This active receptor complex initiates signalling through the Janus kinase/signal transducer and activator of transcription pathway. Of note, IL-6 receptor α exists in a soluble and a transmembrane form. Binding of IL-6 to membrane-bound IL-6 receptor α induces anti-inflammatory classic signalling, whereas binding of IL-6 to soluble IL-6 receptor α induces pro-inflammatory trans-signalling. Trans-signalling has been described to be markedly stronger than classic signalling. Understanding the molecular mechanisms that drive differences between trans- and classic signalling is important for the design of trans-signalling-specific therapies. These differences will be addressed here using a combination of dynamic mathematical modelling and molecular biology. METHODS: We apply an iterative systems biology approach using set-based modelling and validation approaches combined with quantitative biochemical and cell biological analyses. RESULTS: The combination of experimental analyses and dynamic modelling allows to relate the observed differences between IL-6-induced trans- and classic signalling to cell-type specific differences in the expression and ratios of the individual subunits of the IL-6 receptor complex. Canonical intracellular Jak/STAT signalling is indifferent in IL-6-induced trans- and classic signalling. CONCLUSION: This study contributes to the understanding of molecular mechanisms of IL-6 signal transduction and underlines the power of combined dynamical modelling, model-based validation and biological experiments. The opposing pro- and anti-inflammatory responses initiated by IL-6 trans- and classic signalling depend solely on the expression ratios of the subunits of the entire receptor complex. By pointing out the importance of the receptor expression ratio for the strength of IL-6 signalling this study lays a foundation for future precision medicine approaches that aim to selectively block pro-inflammatory trans-signalling. Furthermore, the derived models can be used for future therapy design.
Assuntos
Receptor gp130 de Citocina/metabolismo , Interleucina-6/metabolismo , Modelos Biológicos , Receptores de Interleucina-6/metabolismo , Transdução de Sinais , Animais , Receptor gp130 de Citocina/genética , Humanos , Interleucina-6/genética , Receptores de Interleucina-6/genéticaRESUMO
Activation of the NLRP3-inflammasome pathway and production of the inflammatory cytokine IL-1B after cellular damage caused by infarct or infection is a key process in several diseases such as acute myocardial infarction and inflammatory bowel disease. However, while the molecular triggers of the NLRP3-pathway after cellular damage are well known, the mechanisms that sustain or confine its activity are currently under investigation. We present here an Ordinary Differential Equation-based model that investigates the mechanisms of inflammasome activation and regulation in monocytes to predict IL-1ß activation kinetics upon a two-step activation by Damage-Associate-Molecular-Particles (DAMP) and extracellular ATP. Assuming both activation signals to be concomitantly present or present with a delay of 12h, the model predicted a transient IL-1ß activation at different concentration levels dependent on signal synchronisation. Introducing a positive feedback loop mediated by active IL-1ß resulted in a sustained IL-1ß activation, hence arguing for a paracrine signalling between inflammatory cells to guarantee a temporally stable inflammatory response. We then investigate mechanisms that control termination of inflammation using two recently identified molecular intervention points in the inflammasome pathway. We found that a more upstream regulation, by attenuating production of the IL-1ß-proform, was more potent in attenuating active IL-1ß production than direct inhibition of the NLRP3-inflammasome. Interestingly, ablating this upstream negative feedback led to a high variability of IL-1ß production in monocytes from different subjects, consistent with a recent pre-clinical study. We finally discuss the relevance and implications of our findings in disease models of acute myocardial infarction and spontaneous colitis.
Assuntos
Inflamassomos/metabolismo , Inflamação/patologia , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Comunicação Parácrina/fisiologia , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Células/patologia , Células Cultivadas , Retroalimentação , Humanos , Interleucina-1beta/biossíntese , Interleucina-1beta/metabolismo , Modelos TeóricosRESUMO
BACKGROUND: Numerous recent studies suggest the potential of circulating MicroRNAs (miRs) in peripheral blood samples as diagnostic or prognostic markers for coronary artery disease (CAD), acute coronary syndrome (ACS) and heart failure (HF). However, literature often remains inconclusive regarding as to which markers are most indicative for which of the above diseases. This shortcoming is mainly due to the lack of a systematic analyses and absence of information on the functional pathophysiological role of these miRs and their target genes. METHODS: We here provide an-easy-to-use scoring approach to investigate the likelihood of regulation of several miRs and their target genes from literature by identifying consensus patterns of regulation. We therefore have screened over 1000 articles that study mRNA markers in cardiovascular and metabolic diseases, and devised a scoring algorithm to identify consensus means for miRs and genes regulation across several studies. We then aimed to identify differential markers between CAD, ACS and HF. RESULTS: We first identified miRs (miR-122, -126, -223, -138 and -370) as commonly regulated within a group of metabolic disease, while investigating cardiac-related pathologies (CAD, ACS, HF) revealed a decisive role of miR-1, -499, -208b, and -133a. Looking at differential markers between cardiovascular disease revealed miR-1, miR-208a and miR-133a to distinguish ACS and CAD to HF. Relating differentially expressed miRs to their putative gene targets using MirTarBase, we further identified HCN2/4 and LASP1 as potential markers of CAD and ACS, but not in HF. Likewise, BLC-2 was found oppositely regulated between CAD and HF. Interestingly, while studying overlap in target genes between CAD, ACS and HF only revealed little similarities, mapping these genes to gene ontology terms revealed a surprising similarity between CAD and ACS compared to HF. CONCLUSION: We conclude that our analysis using gene and miR scores allows the extraction of meaningful markers and the elucidation of differential pathological functions between cardiac diseases and provides a novel approach for literature screening for miR and gene consensus patterns. The analysis is easy to use and extendable upon further emergent literature as we provide an Excel sheet for this analysis to the community.
Assuntos
Síndrome Coronariana Aguda/sangue , MicroRNA Circulante/sangue , Doença da Artéria Coronariana/sangue , Perfilação da Expressão Gênica/métodos , Insuficiência Cardíaca/sangue , Síndrome Coronariana Aguda/diagnóstico , Síndrome Coronariana Aguda/genética , MicroRNA Circulante/genética , Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/genética , Diagnóstico Diferencial , Regulação da Expressão Gênica , Marcadores Genéticos , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/genética , Humanos , Valor Preditivo dos TestesRESUMO
Computer models allow the mechanistically detailed study of tumour proliferation and its dependency on nutrients. However, the computational study of large vascular tumours requires detailed information on the 3-dimensional vessel network and rather high computation times due to complex geometries. This study puts forward the idea of partitioning vascularised tissue into connected avascular elements that can exchange cells and nutrients between each other. Our method is able to rapidly calculate the evolution of proliferating as well as dead and quiescent cells, and hence a proliferative index, from a given amount and distribution of vascularisation of arbitrary complexity. Applying our model, we found that a heterogeneous vessel distribution provoked a higher proliferative index, suggesting increased malignancy, and increased the amount of dead cells compared to a more static tumour environment when a homogenous vessel distribution was assumed. We subsequently demonstrated that under certain amounts of vascularisation, cell proliferation may even increase when vessel density decreases, followed by a subsequent decrease of proliferation. This effect was due to a trade-off between an increase in compensatory proliferation for replacing dead cells and a decrease of cell population due to lack of oxygen supply in lowly vascularised tumours. Findings were illustrated by an ectopic colorectal cancer mouse xenograft model. Our presented approach can be in the future applied to study the effect of cytostatic, cytotoxic and anti-angiogenic chemotherapy and is ideally suited for translational systems biology, where rapid interaction between theory and experiment is essential.
Assuntos
Modelos Biológicos , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/patologia , Animais , Contagem de Células , Morte Celular , Proliferação de Células , Células HCT116 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microvasos/patologiaRESUMO
PURPOSE OF REVIEW: Microvesicles, in general, and exosomes together with their delivered content in particular, are now being widely recognized as key players in atherosclerosis. We have previously reviewed the role of microvesicles in atherosclerosis pathogenesis, diagnosis and therapy. Here, we focus on the roles of exosomes and discuss their emergent role in mediating activation and response to inflammation, vessel infiltration and induction of coagulation. We will finally give an outlook to discuss novel detection techniques and systems biology based data analyses to investigate exosome-mediated cell-to-cell communication. RECENT FINDINGS: Recent research points to a role of exosomes in delivering apoptotic and inflammatory content between blood cells and vascular cells, with a potential contribution of exosomes secreted by adipose tissue. An atheroprotective role of exosomes in response to coagulation that may contrast with the procoagulatory role of platelet-derived larger microvesicles is envisaged. New detection and separation methods and systems biology techniques are emerging. CONCLUSION: We project that the development of novel detection, separation and analysis mechanism and systems-based analysis methods will further unravel the paracrine and endocrine 'communication protocol' between cellular players in atherosclerosis, mediating inflammation, oxidative stress and apoptosis.
Assuntos
Aterosclerose/metabolismo , Exossomos/fisiologia , Microvasos/metabolismo , Animais , Apoptose , Células Endoteliais/fisiologia , Transferência Genética Horizontal , Humanos , Microvasos/patologia , Estresse Oxidativo , Comunicação Parácrina , Transdução de SinaisRESUMO
Excitotoxicity is a condition occurring during cerebral ischemia, seizures, and chronic neurodegeneration. It is characterized by overactivation of glutamate receptors, leading to excessive Ca(2+)/Na(+) influx into neurons, energetic stress, and subsequent neuronal injury. We and others have previously investigated neuronal populations to study how bioenergetic parameters determine neuronal injury; however, such experiments are often confounded by population-based heterogeneity and the contribution of effects of non-neuronal cells. Hence, we here characterized bioenergetics during transient excitotoxicity in rat and mouse primary neurons at the single-cell level using fluorescent sensors for intracellular glucose, ATP, and activation of the energy sensor AMP-activated protein kinase (AMPK). We identified ATP depletion and recovery to energetic homeostasis, along with AMPK activation, as surprisingly rapid and plastic responses in two excitotoxic injury paradigms. We observed rapid recovery of neuronal ATP levels also in the absence of extracellular glucose, or when glycolytic ATP production was inhibited, but found mitochondria to be critical for fast and complete energetic recovery. Using an injury model of oxygen and glucose deprivation, we identified a similarly rapid bioenergetics response, yet with incomplete ATP recovery and decreased AMPK activity. Interestingly, excitotoxicity also induced an accumulation of intracellular glucose, providing an additional source of energy during and after excitotoxicity-induced energy depletion. We identified this to originate from extracellular, AMPK-dependent glucose uptake and from intracellular glucose mobilization. Surprisingly, cells recovering their elevated glucose levels faster to baseline survived longer, indicating that the plasticity of neurons to adapt to bioenergetic challenges is a key indicator of neuronal viability.
Assuntos
Metabolismo Energético/fisiologia , Glucose/deficiência , Hipóxia , Neurônios/fisiologia , Imagem Óptica , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Células Cultivadas , Cerebelo/citologia , Córtex Cerebral/citologia , Desoxiglucose/farmacocinética , Glucose/metabolismo , Ácido Glutâmico/farmacologia , Glicina/toxicidade , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Neurônios/efeitos dos fármacos , Neurotoxinas/farmacologia , Ratos , Análise de Célula ÚnicaRESUMO
Positive feedback loops are common regulatory elements in metabolic and protein signalling pathways. The length of such feedback loops determines stability and sensitivity to network perturbations. Here we provide a mathematical analysis of arbitrary length positive feedback loops with protein production and degradation. These loops serve as an abstraction of typical regulation patterns in protein signalling pathways. We first perform a steady state analysis and, independently of the chain length, identify exactly two steady states that represent either biological activity or inactivity. We thereby provide two formulas for the steady state protein concentrations as a function of feedback length, strength of feedback, as well as protein production and degradation rates. Using a control theory approach, analysing the frequency response of the linearisation of the system and exploiting the Small Gain Theorem, we provide conditions for local stability for both steady states. Our results demonstrate that, under some parameter relationships, once a biological meaningful on steady state arises, it is stable, while the off steady state, where all proteins are inactive, becomes unstable. We apply our results to a three-tier feedback of caspase activation in apoptosis and demonstrate how an intermediary protein in such a loop may be used as a signal amplifier within the cascade. Our results provide a rigorous mathematical analysis of positive feedback chains of arbitrary length, thereby relating pathway structure and stability.
Assuntos
Apoptose/fisiologia , Caspases/fisiologia , Retroalimentação Fisiológica/fisiologia , Modelos Biológicos , Transdução de Sinais/fisiologia , CinéticaRESUMO
Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can induce extrinsic apoptosis, resulting in caspase-8 activation, but may also initiate transcription-dependent prosurvival signaling. Proteasome inhibitors were suggested to promote TRAIL signal transduction through the death-inducing signaling complex (DISC) by modulating the relative abundance of core DISC components, thereby enhancing caspase-8 activation and apoptosis. To test this hypothesis, we quantified the changes in DISC protein levels as an early consequence of proteasome inhibition in HeLa cervical cancer cells and, based on these data, mathematically modeled the proapoptotic TRAIL signaling toward caspase-8 activation. Modeling results surprisingly suggested that caspase-8 activation might be delayed in presence of proteasome inhibitors, in particular at submaximal TRAIL doses. Subsequent FRET-based single cell time-lapse imaging at conditions where transcription dependent prosurvival signaling was blocked confirmed this hypothesis: caspase-8 activity was delayed by hours in the presence of proteasome inhibitors epoxomicin or bortezomib. Corresponding delays were detected for effector caspase processing and cell death. Contrary to current models, we therefore provide evidence that synergies between TRAIL and proteasome inhibitors do not result from changes in the levels of core DISC signaling proteins.
Assuntos
Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Caspase 8/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Pirazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Antineoplásicos/farmacologia , Bortezomib , Caspase 8/genética , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Humanos , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
Deregulation of apoptosis is a hallmark of carcinogenesis. We here combine live cell imaging and systems modeling to investigate caspase-dependent apoptosis execution subsequent to mitochondrial outer membrane permeabilization (MOMP) in several cancer cell lines. We demonstrate that, although most cell lines that underwent MOMP also showed robust and fast activation of executioner caspases and apoptosis, the colorectal cancer cell lines LoVo and HCT-116 Smac(-/-), similar to X-linked inhibitor of apoptosis protein (XIAP)-overexpressing HeLa (HeLa XIAP(Adv)) cells, only showed delayed and often no caspase activation, suggesting apoptosis impairment subsequent to MOMP. Employing APOPTO-CELL, a recently established model of apoptosis subsequent to MOMP, this impairment could be understood by studying the systemic interaction of five proteins that are present in the apoptosis pathway subsequent to MOMP. Using APOPTO-CELL as a tool to study detailed molecular mechanisms during apoptosis execution in individual cell lines, we demonstrate that caspase-9 was the most important regulator in DLD-1, HCT-116, and HeLa cells and identified additional cell line-specific co-regulators. Developing and applying a computational workflow for parameter screening, systems modeling identified that apoptosis execution kinetics are more robust against changes in reaction kinetics in HCT-116 and HeLa than in DLD-1 cells. Our systems modeling study is the first to draw attention to the variability in cell specific protein levels and reaction rates and to the emergent effects of such variability on the efficiency of apoptosis execution and on apoptosis impairment subsequent to MOMP.
Assuntos
Apoptose , Caspases/metabolismo , Membranas Mitocondriais/enzimologia , Neoplasias/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Transferência Ressonante de Energia de Fluorescência/métodos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Cinética , Microscopia/métodos , Modelos Estatísticos , Método de Monte Carlo , Prognóstico , Especificidade por Substrato , Análise de Sistemas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
The pro-apoptotic proteins Bax and Bak are essential for executing programmed cell death (apoptosis), yet the mechanism of their activation is not properly understood at the structural level. For the first time in cell death research, we calculated intra-protein charge transfer in order to study the structural alterations and their functional consequences during Bax activation. Using an electronegativity equalization model, we investigated the changes in the Bax charge profile upon activation by a functional peptide of its natural activator protein, Bim. We found that charge reorganizations upon activator binding mediate the exposure of the functional sites of Bax, rendering Bax active. The affinity of the Bax C-domain for its binding groove is decreased due to the Arg94-mediated abrogation of the Ser184-Asp98 interaction. We further identified a network of charge reorganizations that confirms previous speculations of allosteric sensing, whereby the activation information is conveyed from the activation site, through the hydrophobic core of Bax, to the well-distanced functional sites of Bax. The network was mediated by a hub of three residues on helix 5 of the hydrophobic core of Bax. Sequence and structural alignment revealed that this hub was conserved in the Bak amino acid sequence, and in the 3D structure of folded Bak. Our results suggest that allostery mediated by charge transfer is responsible for the activation of both Bax and Bak, and that this might be a prototypical mechanism for a fast activation of proteins during signal transduction. Our method can be applied to any protein or protein complex in order to map the progress of allosteric changes through the proteins' structure.
Assuntos
Modelos Biológicos , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Apoptose/fisiologia , Biologia Computacional , Simulação por Computador , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
OBJECTIVE: Key to the clinical management of colorectal cancer is identifying tools which aid in assessing patient prognosis and determining more effective and personalised treatment strategies. We evaluated whether an experimental systems biology strategy which analyses the susceptibility of cancer cells to undergo caspase activation can be exploited to predict patient responses to 5-fluorouracil-based chemotherapy and to case-specifically identify potential alternative targeted treatments to reactivate apoptosis. DESIGN: We quantified five essential apoptosis-regulating proteins (Pro-Caspases 3 and 9, APAF-1, SMAC and XIAP) in samples of Stage II (n = 13) and III (n=17) tumour and normal colonic (n = 8) tissue using absolute quantitative immunoblotting and employed systems simulations of apoptosis signalling to predict the susceptibility of tumour cells to execute apoptosis. Additional systems analyses assessed the efficacy of novel apoptosis-inducing therapeutics such as XIAP antagonists, proteasome inhibitors and Pro-Caspase-3-activating compounds in restoring apoptosis execution in apoptosis-incompetent tumours. RESULTS: Comparisons of caspase activity profiles demonstrated that the likelihood of colorectal tumours to undergo apoptosis decreases with advancing disease stage. Systems-level analysis correctly predicted positive or negative outcome in 85% (p=0.004) of colorectal cancer patients receiving 5-fluorouracil based chemotherapy and significantly outperformed common uni- and multi-variate statistical approaches. Modelling of individual patient responses to novel apoptosis-inducing therapeutics revealed markedly different inter-individual responses. CONCLUSIONS: Our study represents the first proof-of-concept example demonstrating the significant clinical potential of systems biology-based approaches for predicting patient outcome and responsiveness to novel targeted treatment paradigms.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Neoplasias Colorretais/tratamento farmacológico , Técnicas de Apoio para a Decisão , Fluoruracila/uso terapêutico , Modelos Biológicos , Biologia de Sistemas , Idoso , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Caspases/metabolismo , Quimioterapia Adjuvante , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/fisiopatologia , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Estadiamento de Neoplasias , Análise de Componente Principal , Resultado do TratamentoRESUMO
The effect of combination therapies in many cancers has often been shown to be superior to that of monotherapies. This success is commonly attributed to drug synergies. Combinations of two (or more) drugs in xenograft tumor growth inhibition (TGI) studies are typically designed at fixed doses for each compound. The available methods for assessing synergy in such study designs are based on combination indices (CI) and model-based analyses. The former methods are suitable for screening exercises but are difficult to verify in in vivo studies, while the latter incorporate drug synergy in semi-mechanistic frameworks describing disease progression and drug action but are unsuitable for screening. In the current study, we proposed the empirical radius additivity (Rad-add) score, a novel CI for synergy detection in fixed-dose xenograft TGI combination studies. The Rad-add score approximates model-based analysis performed using the semi-mechanistic constant-radius growth TGI model. The Rad-add score was compared with response additivity, defined as the addition of the two response values, and the bliss independence model in combination studies derived from the Novartis PDX dataset. The results showed that the bliss independence and response additivity models predicted synergistic interactions with high and low probabilities, respectively. The Rad-add score predicted synergistic probabilities that appeared to be between those predicted with response additivity and the Bliss model. We believe that the Rad-add score is particularly suitable for assessing synergy in the context of xenograft combination TGI studies, as it combines the advantages of CI approaches suitable for screening exercises with those of semi-mechanistic TGI models based on a mechanistic understanding of tumor growth.
RESUMO
Cytotoxic CD8 +T lymphocytes (CTLs) are key players of adaptive anti-tumor immunity based on their ability to specifically recognize and destroy tumor cells. Many cancer immunotherapies rely on unleashing CTL function. However, tumors can evade killing through strategies which are not yet fully elucidated. To provide deeper insight into tumor evasion mechanisms in an antigen-dependent manner, we established a human co-culture system composed of tumor and primary immune cells. Using this system, we systematically investigated intrinsic regulators of tumor resistance by conducting a complementary CRISPR screen approach. By harnessing CRISPR activation (CRISPRa) and CRISPR knockout (KO) technology in parallel, we investigated gene gain-of-function as well as loss-of-function across genes with annotated function in a colon carcinoma cell line. CRISPRa and CRISPR KO screens uncovered 187 and 704 hits, respectively, with 60 gene hits overlapping between both. These data confirmed the role of interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α) and autophagy pathways and uncovered novel genes implicated in tumor resistance to killing. Notably, we discovered that ILKAP encoding the integrin-linked kinase-associated serine/threonine phosphatase 2 C, a gene previously unknown to play a role in antigen specific CTL-mediated killing, mediate tumor resistance independently from regulating antigen presentation, IFN-γ or TNF-α responsiveness. Moreover, our work describes the contrasting role of soluble and membrane-bound ICAM-1 in regulating tumor cell killing. The deficiency of membrane-bound ICAM-1 (mICAM-1) or the overexpression of soluble ICAM-1 (sICAM-1) induced resistance to CTL killing, whereas PD-L1 overexpression had no impact. These results highlight the essential role of ICAM-1 at the immunological synapse between tumor and CTL and the antagonist function of sICAM-1.
Assuntos
Neoplasias do Colo , Linfócitos T Citotóxicos , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Fator de Necrose Tumoral alfa , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Interferon gama/farmacologia , Morte CelularRESUMO
Mitochondria are pivotal for cellular bioenergetics, but are also a core component of the cell death machinery. Hypothesis-driven research approaches have greatly advanced our understanding of the role of mitochondria in cell death and cell survival, but traditionally focus on a single gene or specific signalling pathway at a time. Predictions originating from these approaches become limited when signalling pathways show increased complexity and invariably include redundancies, feedback loops, anisotropies or compartmentalisation. By introducing methods from theoretical chemistry, control theory, and biophysics, computational models have provided new quantitative insights into cell decision processes and have led to an increased understanding of the key regulatory principles of apoptosis. In this review, we describe the currently applied modelling approaches, discuss the suitability of different modelling techniques, and evaluate their contribution to the understanding of the mitochondrial apoptosis pathway. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.
Assuntos
Apoptose , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Modelos Teóricos , Transdução de Sinais , Animais , HumanosRESUMO
Many anticancer drugs activate caspases via the mitochondrial apoptosis pathway. Activation of this pathway triggers a concomitant bioenergetic crisis caused by the release of cytochrome-c (cyt-c). Cancer cells are able to evade these processes by altering metabolic and caspase activation pathways. In this study, we provide the first integrated system study of mitochondrial bioenergetics and apoptosis signalling and examine the role of mitochondrial cyt-c release in these events. In accordance with single-cell experiments, our model showed that loss of cyt-c decreased mitochondrial respiration by 95% and depolarised mitochondrial membrane potential ΔΨ(m) from -142 to -88 mV, with active caspase-3 potentiating this decrease. ATP synthase was reversed under such conditions, consuming ATP and stabilising ΔΨ(m). However, the direction and level of ATP synthase activity showed significant heterogeneity in individual cancer cells, which the model explained by variations in (i) accessible cyt-c after release and (ii) the cell's glycolytic capacity. Our results provide a quantitative and mechanistic explanation for the protective role of enhanced glucose utilisation for cancer cells to avert the otherwise lethal bioenergetic crisis associated with apoptosis initiation.
Assuntos
Citocromos c/metabolismo , Metabolismo Energético , Glucose/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Apoptose , Caspase 3/metabolismo , Linhagem Celular , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Potenciais da Membrana , Mitocôndrias/enzimologia , Modelos TeóricosRESUMO
The IGF-1 mediated Akt/mTOR pathway has been recently proposed as mediator of skeletal muscle growth and a positive feedback between Akt and mTOR was suggested to induce homogeneous growth signals along the whole spatial extension of such long cells. Here we develop two biologically justified approximations which we study under the presence of four different initial conditions that describe different paradigms of IGF-1 receptor-induced Akt/mTOR activation. In first scenario the activation of the feedback cascade was assumed to be mild or protein turnover considered to be high. In turn, in the second scenario the transcriptional regulation was assumed to maintain defined levels of inactive pro-enzymes. For both scenarios, we were able to obtain closed-form formulas for growth signal progression in time and space and found that a localised initial signal maintains its Gaussian shape, but gets delocalised and exponentially degraded. Importantly, mathematical treatment of the reaction diffusion system revealed that diffusion filtered out high frequencies of spatially periodic initiator signals suggesting that the muscle cell is robust against fluctuations in spatial receptor expression or activation. However, neither scenario was consistent with the presence of stably travelling signal waves. Our study highlights the role of feedback loops in spatiotemporal signal progression and results can be applied to studies in cell proliferation, cell differentiation and cell death in other spatially extended cells.