RESUMEN
For a short period during early development of mammalian embryos, both X chromosomes in females are active, before dosage compensation is ensured through X-chromosome inactivation. In female mouse embryonic stem cells (mESCs), which carry two active X chromosomes, increased X-dosage affects cell signaling and impairs differentiation. The underlying mechanisms, however, remain poorly understood. To dissect X-dosage effects on the signaling network in mESCs, we combine systematic perturbation experiments with mathematical modeling. We quantify the response to a variety of inhibitors and growth factors for cells with one (XO) or two X chromosomes (XX). We then build models of the signaling networks in XX and XO cells through a semi-quantitative modeling approach based on modular response analysis. We identify a novel negative feedback in the PI3K/AKT pathway through GSK3. Moreover, the presence of a single active X makes mESCs more sensitive to the differentiation-promoting Activin A signal and leads to a stronger RAF1-mediated negative feedback in the FGF-triggered MAPK pathway. The differential response to these differentiation-promoting pathways can explain the impaired differentiation propensity of female mESCs.
Asunto(s)
Células Madre Embrionarias , Células Madre Embrionarias de Ratones , Femenino , Animales , Masculino , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias/metabolismo , Caracteres Sexuales , Glucógeno Sintasa Quinasa 3 , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Diferenciación Celular/genética , MamíferosRESUMEN
Very high risk neuroblastoma is characterised by increased MAPK signalling, and targeting MAPK signalling is a promising therapeutic strategy. We used a deeply characterised panel of neuroblastoma cell lines and found that the sensitivity to MEK inhibitors varied drastically between these cell lines. By generating quantitative perturbation data and mathematical modelling, we determined potential resistance mechanisms. We found that negative feedbacks within MAPK signalling and via the IGF receptor mediate re-activation of MAPK signalling upon treatment in resistant cell lines. By using cell-line specific models, we predict that combinations of MEK inhibitors with RAF or IGFR inhibitors can overcome resistance, and tested these predictions experimentally. In addition, phospho-proteomic profiling confirmed the cell-specific feedback effects and synergy of MEK and IGFR targeted treatment. Our study shows that a quantitative understanding of signalling and feedback mechanisms facilitated by models can help to develop and optimise therapeutic strategies. Our findings should be considered for the planning of future clinical trials introducing MEKi in the treatment of neuroblastoma.
Asunto(s)
Retroalimentación , Modelos Biológicos , Neuroblastoma/metabolismo , Transducción de Señal , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Sistema de Señalización de MAP Quinasas , Neuroblastoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/metabolismoRESUMEN
Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80-540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with four clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system.
Asunto(s)
Modelos Animales de Enfermedad , Neuroblastoma/genética , Neuroblastoma/patología , Neoplasias Abdominales/genética , Neoplasias Abdominales/patología , Animales , Femenino , Heterogeneidad Genética , Xenoinjertos , Humanos , Masculino , Ratones , Ratones SCID , Neoplasias Torácicas/genética , Neoplasias Torácicas/patología , Células Tumorales CultivadasRESUMEN
Motivation: Intracellular signalling is realized by complex signalling networks, which are almost impossible to understand without network models, especially if feedbacks are involved. Modular Response Analysis (MRA) is a convenient modelling method to study signalling networks in various contexts. Results: We developed the software package STASNet (STeady-STate Analysis of Signalling Networks) that provides an augmented and extended version of MRA suited to model signalling networks from incomplete perturbation schemes and multi-perturbation data. Using data from the Dialogue on Reverse Engineering Assessment and Methods challenge, we show that predictions from STASNet models are among the top-performing methods. We applied the method to study the effect of SHP2, a protein that has been implicated in resistance to targeted therapy in colon cancer, using a novel dataset from the colon cancer cell line Widr and a SHP2-depleted derivative. We find that SHP2 is required for mitogen-activated protein kinase signalling, whereas AKT signalling only partially depends on SHP2. Availability and implementation: An R-package is available at https://github.com/molsysbio/STASNet. Supplementary information: Supplementary data are available at Bioinformatics online.
Asunto(s)
Transducción de Señal , Programas Informáticos , Línea Celular Tumoral , Neoplasias del Colon , Biología Computacional , Humanos , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genéticaRESUMEN
Motivation: Signal-transduction networks are often aberrated in cancer cells, and new anti-cancer drugs that specifically target oncogenes involved in signaling show great clinical promise. However, the effectiveness of such targeted treatments is often hampered by innate or acquired resistance due to feedbacks, crosstalks or network adaptations in response to drug treatment. A quantitative understanding of these signaling networks and how they differ between cells with different oncogenic mutations or between sensitive and resistant cells can help in addressing this problem. Results: Here, we present Comparative Network Reconstruction (CNR), a computational method to reconstruct signaling networks based on possibly incomplete perturbation data, and to identify which edges differ quantitatively between two or more signaling networks. Prior knowledge about network topology is not required but can straightforwardly be incorporated. We extensively tested our approach using simulated data and applied it to perturbation data from a BRAF mutant, PTPN11 KO cell line that developed resistance to BRAF inhibition. Comparing the reconstructed networks of sensitive and resistant cells suggests that the resistance mechanism involves re-establishing wild-type MAPK signaling, possibly through an alternative RAF-isoform. Availability and implementation: CNR is available as a python module at https://github.com/NKI-CCB/cnr. Additionally, code to reproduce all figures is available at https://github.com/NKI-CCB/CNR-analyses. Supplementary information: Supplementary data are available at Bioinformatics online.
Asunto(s)
Programas Informáticos , Redes Neurales de la Computación , Transducción de SeñalRESUMEN
The RAF-MEK-ERK signalling pathway controls fundamental, often opposing cellular processes such as proliferation and apoptosis. Signal duration has been identified to play a decisive role in these cell fate decisions. However, it remains unclear how the different early and late responding gene expression modules can discriminate short and long signals. We obtained both protein phosphorylation and gene expression time course data from HEK293 cells carrying an inducible construct of the proto-oncogene RAF By mathematical modelling, we identified a new gene expression module of immediate-late genes (ILGs) distinct in gene expression dynamics and function. We find that mRNA longevity enables these ILGs to respond late and thus translate ERK signal duration into response amplitude. Despite their late response, their GC-rich promoter structure suggested and metabolic labelling with 4SU confirmed that transcription of ILGs is induced immediately. A comparative analysis shows that the principle of duration decoding is conserved in PC12 cells and MCF7 cells, two paradigm cell systems for ERK signal duration. Altogether, our findings suggest that ILGs function as a gene expression module to decode ERK signal duration.
Asunto(s)
Regulación de la Expresión Génica , Sistema de Señalización de MAP Quinasas/genética , ARN Mensajero/metabolismo , Animales , Simulación por Computador , Secuencia Rica en GC , Células HEK293 , Semivida , Humanos , Células MCF-7 , Modelos Teóricos , Familia de Multigenes , Células PC12 , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , Ratas , Transducción de Señal/genética , Quinasas raf/genéticaRESUMEN
MOTIVATION: Impedance-based technologies are advancing methods for measuring proliferation of adherent cell cultures non-invasively and in real time. The analysis of the resulting data has so far been hampered by inappropriate computational methods and the lack of systematic data to evaluate the characteristics of the assay. RESULTS: We used a commercially available system for impedance-based growth measurement (xCELLigence) and compared the reported cell index with data from microscopy. We found that the measured signal correlates linearly with the cell number throughout the time of an experiment with sufficient accuracy in subconfluent cell cultures. The resulting growth curves for various colon cancer cells could be well described with the empirical Richards growth model, which allows for extracting quantitative parameters (such as characteristic cycle times). We found that frequently used readouts like the cell index at a specific time or the area under the growth curve cannot be used to faithfully characterize growth inhibition. We propose to calculate the average growth rate of selected time intervals to accurately estimate time-dependent IC50 values of drugs from growth curves. CONTACT: nils.bluethgen@charite.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Bioensayo/métodos , Células/citología , Adhesión Celular , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Impedancia Eléctrica , Humanos , Concentración 50 Inhibidora , Modelos Biológicos , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de TiempoRESUMEN
The epidermal growth factor receptor (EGFR) signaling network is activated in most solid tumors, and small-molecule drugs targeting this network are increasingly available. However, often only specific combinations of inhibitors are effective. Therefore, the prediction of potent combinatorial treatments is a major challenge in targeted cancer therapy. In this study, we demonstrate how a model-based evaluation of signaling data can assist in finding the most suitable treatment combination. We generated a perturbation data set by monitoring the response of RAS/PI3K signaling to combined stimulations and inhibitions in a panel of colorectal cancer cell lines, which we analyzed using mathematical models. We detected that a negative feedback involving EGFR mediates strong cross talk from ERK to AKT. Consequently, when inhibiting MAPK, AKT activity is increased in an EGFR-dependent manner. Using the model, we predict that in contrast to single inhibition, combined inactivation of MEK and EGFR could inactivate both endpoints of RAS, ERK and AKT. We further could demonstrate that this combination blocked cell growth in BRAF- as well as KRAS-mutated tumor cells, which we confirmed using a xenograft model.
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Neoplasias Colorrectales/metabolismo , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Modelos Genéticos , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Ensayos de Selección de Medicamentos Antitumorales , Quimioterapia Combinada , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Ratones , Ratones Desnudos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Trasplante Heterólogo , Carga Tumoral/efectos de los fármacos , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Protein levels within signal transduction pathways vary strongly from cell to cell. Here, we analysed how signalling pathways can still process information quantitatively despite strong heterogeneity in protein levels. We systematically perturbed the protein levels of Erk, the terminal kinase in the MAPK signalling pathway in a panel of human cell lines. We found that the steady-state phosphorylation of Erk is very robust against perturbations of Erk protein level. Although a multitude of mechanisms exist that may provide robustness against fluctuating protein levels, we found that one single feedback from Erk to Raf-1 accounts for the observed robustness. Surprisingly, robustness is provided through a fast post-translational mechanism although variation of Erk levels occurs on a timescale of days.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Retroalimentación Fisiológica , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Proto-Oncogénicas c-raf , Secuencia de Bases , Línea Celular , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/genética , Silenciador del Gen , Humanos , Cómputos Matemáticos , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Fosforilación/efectos de los fármacos , Fosforilación/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , TransfecciónRESUMEN
Targeted inhibition of aberrant signaling is an important treatment strategy in cancer, but responses are often short-lived. Multi-drug combinations have the potential to mitigate this, but to avoid toxicity such combinations must be selective and given at low dosages. Here, we present a pipeline to identify promising multi-drug combinations. We perturbed an isogenic PI3K mutant and wild-type cell line pair with a limited set of drugs and recorded their signaling state and cell viability. We then reconstructed their signaling networks and mapped the signaling response to changes in cell viability. The resulting models, which allowed us to predict the effect of unseen combinations, indicated that no combination selectively reduces the viability of the PI3K mutant cells. However, we were able to validate 25 of the 30 combinations that we predicted to be anti-selective. Our pipeline enables efficient prioritization of multi-drug combinations from the enormous search space of possible combinations.
RESUMEN
In colorectal cancer (CRC), the prevalence of NRAS mutations (5-9%) is inferior to that of KRAS mutations (40-50%). NRAS mutations feature lately during tumour progression and drive resistance to anti-EGFR therapy in KRAS wild-type tumours. To elucidate specific functions of NRAS mutations in CRC, we expressed doxycycline-inducible G12D and Q61K mutations in the CRC cell line Caco-2. A focused phospho-proteome analysis based on the Bio-Plex platform, which interrogated the activity of MAPK, PI3K, mTOR, STAT, p38, JNK and ATF2, did not reveal significant differences between Caco-2 cells expressing NRASG12D, NRASQ61K and KRASG12V. However, phenotypic read-outs were different. The NRAS Q61K mutation promoted anchorage-independent proliferation and tumorigenicity, similar to features driven by canonical KRAS mutations. In contrast, expression of NRASG12D resulted in reduced proliferation and apoptosis. At the transcriptome level, we saw upregulation of cytokines and chemokines. IL1A, IL11, CXCL8 (IL-8) and CCL20 exhibited enhanced secretion into the culture medium. In addition, RNA sequencing results indicated activation of the IL1-, JAK/STAT-, NFκB- and TNFα signalling pathways. These results form the basis for an NRASG12D-driven inflammatory phenotype in CRC.
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Neoplasias Colorrectales/genética , GTP Fosfohidrolasas/genética , Proteínas de la Membrana/genética , Apoptosis , Células CACO-2 , Proliferación Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/fisiopatología , Citocinas/genética , Citocinas/metabolismo , GTP Fosfohidrolasas/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Mutación , Oncogenes , Transducción de SeñalRESUMEN
Resistance against radio(chemo)therapy-induced cell death is a major determinant of oncological treatment failure and remains a perpetual clinical challenge. The underlying mechanisms are manifold and demand for comprehensive, cancer entity- and subtype-specific examination. In the present study, resistance against radiotherapy was systematically assessed in a panel of human head-and-neck squamous cell carcinoma (HNSCC) cell lines and xenotransplants derived thereof with the overarching aim to extract master regulators and potential candidates for mechanism-based pharmacological targeting. Clonogenic survival data were integrated with molecular and functional data on DNA damage repair and different cell fate decisions. A positive correlation between radioresistance and early induction of HNSCC cell senescence accompanied by NF-κB-dependent production of distinct senescence-associated cytokines, particularly ligands of the CXCR2 chemokine receptor, was identified. Time-lapse microscopy and medium transfer experiments disclosed the non-cell autonomous, paracrine nature of these mechanisms, and pharmacological interference with senescence-associated cytokine production by the NF-κB inhibitor metformin significantly improved radiotherapeutic performance in vitro and in vivo. With regard to clinical relevance, retrospective analyses of TCGA HNSCC data and an in-house HNSCC cohort revealed that elevated expression of CXCR2 and/or its ligands are associated with impaired treatment outcome. Collectively, our study identifies radiation-induced tumor cell senescence and the NF-κB-dependent production of distinct senescence-associated cytokines as critical drivers of radioresistance in HNSCC whose therapeutic targeting in the context of multi-modality treatment approaches should be further examined and may be of particular interest for the subgroup of patients with elevated expression of the CXCR2/ligand axis.
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Senescencia Celular , Neoplasias de Cabeza y Cuello , Tolerancia a Radiación , Receptores de Interleucina-8B , Carcinoma de Células Escamosas de Cabeza y Cuello , Línea Celular Tumoral , Citocinas , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Ligandos , FN-kappa B , Receptores de Interleucina-8B/metabolismo , Estudios Retrospectivos , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapiaRESUMEN
In colorectal cancer, oncogenic mutations transform a hierarchically organized and homeostatic epithelium into invasive cancer tissue lacking visible organization. We sought to define transcriptional states of colorectal cancer cells and signals controlling their development by performing single-cell transcriptome analysis of tumors and matched non-cancerous tissues of twelve colorectal cancer patients. We defined patient-overarching colorectal cancer cell clusters characterized by differential activities of oncogenic signaling pathways such as mitogen-activated protein kinase and oncogenic traits such as replication stress. RNA metabolic labeling and assessment of RNA velocity in patient-derived organoids revealed developmental trajectories of colorectal cancer cells organized along a mitogen-activated protein kinase activity gradient. This was in contrast to normal colon organoid cells developing along graded Wnt activity. Experimental targeting of EGFR-BRAF-MEK in cancer organoids affected signaling and gene expression contingent on predictive KRAS/BRAF mutations and induced cell plasticity overriding default developmental trajectories. Our results highlight directional cancer cell development as a driver of non-genetic cancer cell heterogeneity and re-routing of trajectories as a response to targeted therapy.
Asunto(s)
Neoplasias Colorrectales , Neoplasias Colorrectales/genética , Humanos , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos , Mutación , OncogenesRESUMEN
HRAS, NRAS, and KRAS isoforms are almost identical proteins that are ubiquitously expressed and activate a common set of effectors. In vivo studies have revealed that they are not biologically redundant; however, the isoform specificity of Ras signaling remains poorly understood. Using a novel panel of isogenic SW48 cell lines endogenously expressing wild-type or G12V-mutated activated Ras isoforms, we have performed a detailed characterization of endogenous isoform-specific mutant Ras signaling. We find that despite displaying significant Ras activation, the downstream outputs of oncogenic Ras mutants are minimal in the absence of growth factor inputs. The lack of mutant KRAS-induced effector activation observed in SW48 cells appears to be representative of a broad panel of colon cancer cell lines harboring mutant KRAS. For MAP kinase pathway activation in KRAS-mutant cells, the requirement for coincident growth factor stimulation occurs at an early point in the Raf activation cycle. Finally, we find that Ras isoform-specific signaling was highly context dependent and did not conform to the dogma derived from ectopic expression studies.
Asunto(s)
Proteínas ras/genética , Proteínas ras/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Genes ras , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mutación , Isoformas de Proteínas , Transducción de Señal/fisiologíaRESUMEN
Aberrant cell signaling can cause cancer and other diseases and is a focal point of drug research. A common approach is to infer signaling activity of pathways from gene expression. However, mapping gene expression to pathway components disregards the effect of post-translational modifications, and downstream signatures represent very specific experimental conditions. Here we present PROGENy, a method that overcomes both limitations by leveraging a large compendium of publicly available perturbation experiments to yield a common core of Pathway RespOnsive GENes. Unlike pathway mapping methods, PROGENy can (i) recover the effect of known driver mutations, (ii) provide or improve strong markers for drug indications, and (iii) distinguish between oncogenic and tumor suppressor pathways for patient survival. Collectively, these results show that PROGENy accurately infers pathway activity from gene expression in a wide range of conditions.
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Expresión Génica , Genes Relacionados con las Neoplasias , Genómica/métodos , Neoplasias/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos , Células HEK293 , Humanos , Mutación , Neoplasias/mortalidadRESUMEN
Genomic features are used as biomarkers of sensitivity to kinase inhibitors used widely to treat human cancer, but effective patient stratification based on these principles remains limited in impact. Insofar as kinase inhibitors interfere with signaling dynamics, and, in turn, signaling dynamics affects inhibitor responses, we investigated associations in this study between cell-specific dynamic signaling pathways and drug sensitivity. Specifically, we measured 14 phosphoproteins under 43 different perturbed conditions (combinations of 5 stimuli and 7 inhibitors) in 14 colorectal cancer cell lines, building cell line-specific dynamic logic models of underlying signaling networks. Model parameters representing pathway dynamics were used as features to predict sensitivity to a panel of 27 drugs. Specific parameters of signaling dynamics correlated strongly with drug sensitivity for 14 of the drugs, 9 of which had no genomic biomarker. Following one of these associations, we validated a drug combination predicted to overcome resistance to MEK inhibitors by coblockade of GSK3, which was not found based on associations with genomic data. These results suggest that to better understand the cancer resistance and move toward personalized medicine, it is essential to consider signaling network dynamics that cannot be inferred from static genotypes. Cancer Res; 77(12); 3364-75. ©2017 AACR.
Asunto(s)
Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Humanos , Modelos Estadísticos , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
During early development of female mouse embryos, both X chromosomes are transiently active. X gene dosage is then equalized between the sexes through the process of X chromosome inactivation (XCI). Whether the double dose of X-linked genes in females compared with males leads to sex-specific developmental differences has remained unclear. Using embryonic stem cells with distinct sex chromosome compositions as a model system, we show that two X chromosomes stabilize the naive pluripotent state by inhibiting MAPK and Gsk3 signaling and stimulating the Akt pathway. Since MAPK signaling is required to exit the pluripotent state, differentiation is paused in female cells as long as both X chromosomes are active. By preventing XCI or triggering it precociously, we demonstrate that this differentiation block is released once XX cells have undergone X inactivation. We propose that double X dosage interferes with differentiation, thus ensuring a tight coupling between X chromosome dosage compensation and development.