RESUMEN
The PTEN tumor suppressor controls cell death and survival by regulating functions of various molecular targets. While the role of PTEN lipid-phosphatase activity on PtdIns(3,4,5)P3 and inhibition of PI3K pathway is well characterized, the biological relevance of PTEN protein-phosphatase activity remains undefined. Here, using knockin (KI) mice harboring cancer-associated and functionally relevant missense mutations, we show that although loss of PTEN lipid-phosphatase function cooperates with oncogenic PI3K to promote rapid mammary tumorigenesis, the additional loss of PTEN protein-phosphatase activity triggered an extensive cell death response evident in early and advanced mammary tumors. Omics and drug-targeting studies revealed that PI3Ks act to reduce glucocorticoid receptor (GR) levels, which are rescued by loss of PTEN protein-phosphatase activity to restrain cell survival. Thus, we find that the dual regulation of GR by PI3K and PTEN functions as a rheostat that can be exploited for the treatment of PTEN loss-driven cancers.
Asunto(s)
Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Fosfohidrolasa PTEN/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , Carcinogénesis , Muerte Celular , Línea Celular Tumoral , Proliferación Celular , Dexametasona/farmacología , Femenino , Humanos , Isoenzimas/metabolismo , Ratones , Modelos Biológicos , Mutación/genética , Organoides/patología , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Estabilidad Proteica , Proteoma/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
The PI3K/MTOR signalling network regulates a broad array of critical cellular processes, including cell growth, metabolism and autophagy. The mechanistic target of rapamycin (MTOR) kinase functions as a core catalytic subunit in two physically and functionally distinct complexes mTORC1 and mTORC2, which also share other common components including MLST8 (also known as GßL) and DEPTOR. Despite intensive research, how mTORC1 and 2 assembly and activity are coordinated, and how they are functionally linked remain to be fully characterized. This is due in part to the complex network wiring, featuring multiple feedback loops and intricate post-translational modifications. Here, we integrate predictive network modelling, in vitro experiments and -omics data analysis to elucidate the emergent dynamic behaviour of the PI3K/MTOR network. We construct new mechanistic models that encapsulate critical mechanistic details, including mTORC1/2 coordination by MLST8 (de)ubiquitination and the Akt-to-mTORC2 positive feedback loop. Model simulations validated by experimental studies revealed a previously unknown biphasic, threshold-gated dependence of mTORC1 activity on the key mTORC2 subunit SIN1, which is robust against cell-to-cell variation in protein expression. In addition, our integrative analysis demonstrates that ubiquitination of MLST8, which is reversed by OTUD7B, is regulated by IRS1/2. Our results further support the essential role of MLST8 in enabling both mTORC1 and 2's activity and suggest MLST8 as a viable therapeutic target in breast cancer. Overall, our study reports a new mechanistic model of PI3K/MTOR signalling incorporating MLST8-mediated mTORC1/2 formation and unveils a novel regulatory linkage between mTORC1 and mTORC2.
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Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular , Péptidos y Proteínas de Señalización Intracelular , Diana Mecanicista del Complejo 2 de la Rapamicina/química , Reproducibilidad de los Resultados , Transducción de Señal , Homóloga LST8 de la Proteína Asociada al mTOR/metabolismoRESUMEN
Amino acid hydroxylation is a common post-translational modification, which generally regulates protein interactions or adds a functional group that can be further modified. Such hydroxylation is currently considered irreversible, necessitating the degradation and re-synthesis of the entire protein to reset the modification. Here we present evidence that the cellular machinery can reverse FIH-mediated asparagine hydroxylation on intact proteins. These data suggest that asparagine hydroxylation is a flexible and dynamic post-translational modification akin to modifications involved in regulating signaling networks, such as phosphorylation, methylation and ubiquitylation.
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Asparagina/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Oxigenasas de Función Mixta/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Represoras/metabolismo , Canales Catiónicos TRPV/metabolismo , Tanquirasas/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Humanos , Hidroxilación , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Cinética , Espectrometría de Masas , Metilación , Oxigenasas de Función Mixta/genética , Fosforilación , Unión Proteica , Proteínas Represoras/genética , Transducción de Señal , Canales Catiónicos TRPV/genética , Tanquirasas/genética , UbiquitinaciónRESUMEN
The PI3K/mTOR signalling pathway plays a central role in the governing of cell growth, survival and metabolism. As such, it must integrate and decode information from both external and internal sources to guide efficient decision-making by the cell. To facilitate this, the pathway has evolved an intricate web of complex regulatory mechanisms and elaborate crosstalk with neighbouring signalling pathways, making it a highly non-linear system. Here, we describe the mechanistic biological details that underpin these regulatory mechanisms, covering a multitude of negative and positive feedback loops, feed-forward loops, competing protein interactions, and crosstalk with major signalling pathways. Further, we highlight the non-linear and dynamic network behaviours that arise from these regulations, uncovered through computational and experimental studies. Given the pivotal role of the PI3K/mTOR network in cellular homeostasis and its frequent dysregulation in pathologies including cancer and diabetes, a coherent and systems-level understanding of the complex regulation and consequential dynamic signalling behaviours within this network is imperative for advancing biology and development of new therapeutic approaches.
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Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Homeostasis , Humanos , Neoplasias/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACKGROUND: Triple negative breast cancer (TNBC) accounts for 16% of breast cancers and represents an aggressive subtype that lacks targeted therapeutic options. In this study, mass spectrometry (MS)-based tyrosine phosphorylation profiling identified aberrant FGFR3 activation in a subset of TNBC cell lines. This kinase was therefore evaluated as a potential therapeutic target. METHODS: MS-based tyrosine phosphorylation profiling was undertaken across a panel of 24 TNBC cell lines. Immunoprecipitation and Western blot were used to further characterize FGFR3 phosphorylation. Indirect immunofluorescence and confocal microscopy were used to determine FGFR3 localization. The selective FGFR1-3 inhibitor, PD173074 and siRNA knockdowns were used to characterize the functional role of FGFR3 in vitro. The TCGA and Metabric breast cancer datasets were interrogated to identify FGFR3 alterations and how they relate to breast cancer subtype and overall patient survival. RESULTS: High FGFR3 expression and phosphorylation were detected in SUM185PE cells, which harbor a FGFR3-TACC3 gene fusion. Low FGFR3 phosphorylation was detected in CAL51, MFM-223 and MDA-MB-231 cells. In SUM185PE cells, the FGFR3-TACC3 fusion protein contributed the majority of phosphorylated FGFR3, and largely localized to the cytoplasm and plasma membrane, with staining at the mitotic spindle in a small subset of cells. Knockdown of the FGFR3-TACC3 fusion and wildtype FGFR3 in SUM185PE cells decreased FRS2, AKT and ERK phosphorylation, and induced cell death. Knockdown of wildtype FGFR3 resulted in only a trend for decreased proliferation. PD173074 significantly decreased FRS2, AKT and ERK activation, and reduced SUM185PE cell proliferation. Cyclin A and pRb were also decreased in the presence of PD173074, while cleaved PARP was increased, indicating cell cycle arrest in G1 phase and apoptosis. Knockdown of FGFR3 in CAL51, MFM-223 and MDA-MB-231 cells had no significant effect on cell proliferation. Interrogation of public datasets revealed that increased FGFR3 expression in breast cancer was significantly associated with reduced overall survival, and that potentially oncogenic FGFR3 alterations (eg mutation and amplification) occur in the TNBC/basal, luminal A and luminal B subtypes, but are rare. CONCLUSIONS: These results indicate that targeting FGFR3 may represent a therapeutic option for TNBC, but only for patients with oncogenic FGFR3 alterations, such as the FGFR3-TACC3 fusion. Video abstract.
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Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/fisiopatologíaRESUMEN
Prediction of drug combinations that effectively target cancer cells is a critical challenge for cancer therapy, in particular for triple-negative breast cancer (TNBC), a highly aggressive breast cancer subtype with no effective targeted treatment. As signalling pathway networks critically control cancer cell behaviour, analysis of signalling network activity and crosstalk can help predict potent drug combinations and rational stratification of patients, thus bringing therapeutic and prognostic values. We have previously showed that the non-receptor tyrosine kinase PYK2 is a downstream effector of EGFR and c-Met and demonstrated their crosstalk signalling in basal-like TNBC. Here we applied a systems modelling approach and developed a mechanistic model of the integrated EGFR-PYK2-c-Met signalling network to identify and prioritize potent drug combinations for TNBC. Model predictions validated by experimental data revealed that among six potential combinations of drug pairs targeting the central nodes of the network, including EGFR, c-Met, PYK2 and STAT3, co-targeting of EGFR and PYK2 and to a lesser extent of EGFR and c-Met yielded strongest synergistic effect. Importantly, the synergy in co-targeting EGFR and PYK2 was linked to switch-like cell proliferation-associated responses. Moreover, simulations of patient-specific models using public gene expression data of TNBC patients led to predictive stratification of patients into subgroups displaying distinct susceptibility to specific drug combinations. These results suggest that mechanistic systems modelling is a powerful approach for the rational design, prediction and prioritization of potent combination therapies for individual patients, thus providing a concrete step towards personalized treatment for TNBC and other tumour types.
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Antineoplásicos , Receptores ErbB/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal , Neoplasias de la Mama Triple Negativas , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biología Computacional , Bases de Datos Genéticas , Sinergismo Farmacológico , Femenino , Perfilación de la Expresión Génica , Humanos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
The notion of feedback is fundamental for understanding signal transduction networks. Feedback loops attenuate or amplify signals, change the network dynamics and modify the input-output relationships between the signal and the target. Negative feedback provides robustness to noise and adaptation to perturbations, but as a double-edged sword can prevent effective pathway inhibition by a drug. Positive feedback brings about switch-like network responses and can convert analog input signals into digital outputs, triggering cell fate decisions and phenotypic changes. We show how a multitude of protein-protein interactions creates hidden feedback loops in signal transduction cascades. Drug treatments that interfere with feedback regulation can cause unexpected adverse effects. Combinatorial molecular interactions generated by pathway crosstalk and feedback loops often bypass the block caused by targeted therapies against oncogenic mutated kinases. We discuss mechanisms of drug resistance caused by network adaptations and suggest that development of effective drug combinations requires understanding of how feedback loops modulate drug responses.
Asunto(s)
Antineoplásicos/uso terapéutico , Retroalimentación Fisiológica , Neoplasias/tratamiento farmacológico , Transducción de Señal , Animales , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Retroalimentación Fisiológica/efectos de los fármacos , Homeostasis/efectos de los fármacos , Humanos , Transducción de Señal/efectos de los fármacosRESUMEN
Post-translational modification of cellular proteins by ubiquitin is a pivotal regulatory event that controls not only protein degradation, but also a variety of non-proteolytic functions. Ubiquitination is involved in a broad array of physiological processes, and its dysregulation has been associated with many human diseases, including neuronal disorders and cancers. Ubiquitin-mediated signalling has thus come to the forefront of biomedical research. It is increasingly apparent that ubiquitination is a highly complex and dynamic process, evidenced by a myriad of ways of ubiquitin chain formation, tightly regulatory mechanisms involving E3 ligases and deubiquitinating enzymes and extensive crosstalk with other post-translational modifications. To unravel the complexity of ubiquitination and understand the dynamic properties of ubiquitin-mediated signalling are challenging, but critical topics in ubiquitin research, which will undoubtedly benefit our effort in developing strategies that could target ubiquitin signalling for therapeutics. Computational modelling and model-based approaches are emerging as promising tools that help tackle the complexity and provide useful frameworks for quantitative and dynamical analysis of ubiquitin signalling. In this article, I will discuss recent advances in our understanding of the dynamic behaviour of ubiquitination from both theoretical and experimental studies, and aspects of ubiquitin signalling that may have major dynamical consequences. It is expected the discussed issues will be of relevant interest to both the ubiquitin and systems biology fields.
Asunto(s)
Ubiquitina/metabolismo , Humanos , Procesamiento Proteico-Postraduccional , Proteínas , Biología de Sistemas , UbiquitinaciónRESUMEN
The detailed, atomistic-level understanding of molecular signaling along the tumor-suppressive Hippo signaling pathway that controls tissue homeostasis by balancing cell proliferation and death through apoptosis is a promising avenue for the discovery of novel anticancer drug targets. The activation of kinases such as Mammalian STE20-Like Protein Kinases 1 and 2 (MST1 and MST2)-modulated through both homo- and heterodimerization (e.g. interactions with Ras association domain family, RASSF, enzymes)-is a key upstream event in this pathway and remains poorly understood. On the other hand, RASSFs (such as RASSF1A or RASSF5) act as important apoptosis activators and tumor suppressors, although their exact regulatory roles are also unclear. We present recent molecular studies of signaling along the Ras-RASSF-MST pathway, which controls growth and apoptosis in eukaryotic cells, including a variety of modern molecular modeling and simulation techniques. Using recently available structural information, we discuss the complex regulatory scenario according to which RASSFs perform dual signaling functions, either preventing or promoting MST2 activation, and thus control cell apoptosis. Here, we focus on recent studies highlighting the special role being played by the specific interactions between the helical Salvador/RASSF/Hippo (SARAH) domains of MST2 and RASSF1a or RASSF5 enzymes. These studies are crucial for integrating atomistic-level mechanistic information about the structures and conformational dynamics of interacting proteins, with information available on their system-level functions in cellular signaling.
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Unión Proteica , Animales , Apoptosis , Modelos Moleculares , Proteínas Serina-Treonina Quinasas , Transducción de SeñalRESUMEN
RASSF enzymes act as key apoptosis activators and tumor suppressors, being downregulated in many human cancers, although their exact regulatory roles remain unknown. A key downstream event in the RASSF pathway is the regulation of MST kinases, which are main effectors of RASSF-induced apoptosis. The regulation of MST1/2 includes both homo- and heterodimerization, mediated by helical SARAH domains, though the underlying molecular interaction mechanism is unclear. Here, we study the interactions between RASSF1A, RASSF5, and MST2 SARAH domains by using both atomistic molecular simulation techniques and experiments. We construct and study models of MST2 homodimers and MST2-RASSF SARAH heterodimers, and we identify the factors that control their high molecular stability. In addition, we also analyze both computationally and experimentally the interactions of MST2 SARAH domains with a series of synthetic peptides particularly designed to bind to it, and hope that our approach can be used to address some of the challenging problems in designing new anti-cancer drugs.
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Proteínas Portadoras/química , Proteínas Portadoras/ultraestructura , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/química , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/ultraestructura , Proteínas de Drosophila/química , Proteínas de Drosophila/ultraestructura , Simulación del Acoplamiento Molecular , Sitios de Unión , Dimerización , Activación Enzimática , Unión Proteica , Conformación Proteica , Dominios ProteicosRESUMEN
Cyclooxygenase 2 (COX2), a key regulatory enzyme of the prostaglandin/eicosanoid pathway, is an important target for anti-inflammatory therapy. It is highly induced by pro-inflammatory cytokines in a Nuclear factor kappa B (NFκB)-dependent manner. However, the mechanisms determining the amplitude and dynamics of this important pro-inflammatory event are poorly understood. Furthermore, there is significant difference between human and mouse COX2 expression in response to the inflammatory stimulus tumor necrosis factor alpha (TNFα). Here, we report the presence of a molecular logic AND gate composed of two NFκB response elements (NREs) which controls the expression of human COX2 in a switch-like manner. Combining quantitative kinetic modeling and thermostatistical analysis followed by experimental validation in iterative cycles, we show that the human COX2 expression machinery regulated by NFκB displays features of a logic AND gate. We propose that this provides a digital, noise-filtering mechanism for a tighter control of expression in response to TNFα, such that a threshold level of NFκB activation is required before the promoter becomes active and initiates transcription. This NFκB-regulated AND gate is absent in the mouse COX2 promoter, most likely contributing to its differential graded response in promoter activity and protein expression to TNFα. Our data suggest that the NFκB-regulated AND gate acts as a novel mechanism for controlling the expression of human COX2 to TNFα, and its absence in the mouse COX2 provides the foundation for further studies on understanding species-specific differential gene regulation.
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Ciclooxigenasa 2/genética , Regulación de la Expresión Génica , Modelos Teóricos , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , Elementos de Respuesta/genética , Animales , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HEK293 , Células HT29 , Humanos , Ratones , FN-kappa B/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
RAF kinases are key players in the MAPK signaling pathway and are important targets for personalized cancer therapy. RAF dimerization is part of the physiological activation mechanism, together with phosphorylation, and is known to convey resistance to RAF inhibitors. Herein, molecular dynamics simulations are used to show that phosphorylation of a key N-terminal acidic (NtA) motif facilitates RAF dimerization by introducing several interprotomer salt bridges between the αC-helix and charged residues upstream of the NtA motif. Additionally, we show that the R-spine of RAF interacts with a conserved Trp residue in the vicinity of the NtA motif, connecting the active sites of two protomers and thereby modulating the cooperative interactions in the RAF dimer. Our findings provide a first structure-based mechanism for the auto-transactivation of RAF and could be generally applicable to other kinases, opening new pathways for overcoming dimerization-related drug resistance.
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Activación Transcripcional , Quinasas raf/metabolismo , Dimerización , Fosforilación , Conformación Proteica , Quinasas raf/químicaRESUMEN
BACKGROUND: Serum response factor (SRF) is an important transcription factor in castrate-resistant prostate cancer (CRPC). Since CRPC is associated with androgen receptor (AR) hypersensitivity, we investigated the relationship between SRF and AR. MATERIALS AND METHODS: Transcriptional activity was assessed by luciferase assay. Cell proliferation was measured by MTT and flow cytometry. Protein expression in patients was assessed by immunohistochemistry. RESULTS: To investigate AR involvement in SRF response to androgen, AR expression was down-regulated using siRNA. This resulted in the abrogation of SRF induction post-DHT. Moreover, DHT stimulation failed to induce SRF transcriptional activity in AR-negative PC346 DCC cells, which was only restored following AR over-expression. Next, SRF expression was down-regulated by siRNA, resulting in AR increased transcriptional activity in castrate-resistant LNCaP Abl cells but not in the parental LNCaP. This negative feedback loop in the resistant cells was confirmed by immunohistochemistry which showed a negative correlation between AR and SRF expression in CRPC bone metastases and a positive correlation in androgen-naïve prostatectomies. Cell proliferation was next assessed following SRF inhibition, demonstrating that SRF inhibition is more effective than AR inhibition in castrate-resistant cells. CONCLUSION: Our data support SRF as a promising therapeutic target in combination with current treatments.
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Adenocarcinoma/metabolismo , Proliferación Celular/fisiología , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Factor de Respuesta Sérica/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dihidrotestosterona/farmacología , Regulación hacia Abajo , Humanos , Masculino , Próstata/efectos de los fármacos , Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Interferente Pequeño , Receptores Androgénicos/genética , Factor de Respuesta Sérica/genéticaRESUMEN
Activation of the hypoxia-inducible factor (HIF) pathway is a critical step in the transcriptional response to hypoxia. Although many of the key proteins involved have been characterised, the dynamics of their interactions in generating this response remain unclear. In the present study, we have generated a comprehensive mathematical model of the HIF-1α pathway based on core validated components and dynamic experimental data, and confirm the previously described connections within the predicted network topology. Our model confirms previous work demonstrating that the steps leading to optimal HIF-1α transcriptional activity require sequential inhibition of both prolyl- and asparaginyl-hydroxylases. We predict from our model (and confirm experimentally) that there is residual activity of the asparaginyl-hydroxylase FIH (factor inhibiting HIF) at low oxygen tension. Furthermore, silencing FIH under conditions where prolyl-hydroxylases are inhibited results in increased HIF-1α transcriptional activity, but paradoxically decreases HIF-1α stability. Using a core module of the HIF network and mathematical proof supported by experimental data, we propose that asparaginyl hydroxylation confers a degree of resistance upon HIF-1α to proteosomal degradation. Thus, through in vitro experimental data and in silico predictions, we provide a comprehensive model of the dynamic regulation of HIF-1α transcriptional activity by hydroxylases and use its predictive and adaptive properties to explain counter-intuitive biological observations.
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Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Oxigenasas de Función Mixta/metabolismo , Modelos Biológicos , Proteínas Represoras/metabolismo , Biología Computacional , Células HEK293 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/farmacología , Oxígeno/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteolisis , ARN Interferente Pequeño/genética , Proteínas Represoras/genética , Proteínas Represoras/farmacología , Transducción de Señal , Activación Transcripcional/genéticaRESUMEN
Signal processing by intracellular kinases controls near all biological processes but how signal pathway functions evolve with changed cellular context is poorly understood. Functional specificity of c-Jun N-terminal Kinases (JNK) are partly encoded by signal strength. Here we reveal that intracellular pH (pHi) is a significant component of the JNK network and defines signal response to specific stimuli. We show pHi regulates JNK activity in response to cell stress, with the relationship between pHi and JNK activity dependent on specific stimuli and upstream kinases activated. Using the optogenetic clustering tag CRY2, we show that an increase in pHi promotes the light-induced phase transition of ASK1 to augment JNK activation. While increased pHi similarly promoted CRY2-tagged JNK2 to form light-induced condensates, this attenuated JNK activity. Mathematical modelling of feedback signalling incorporating pHi and differential contributions by ASK1 and JNK2 condensates was sufficient to delineate signal responses to specific stimuli. Taking pHi and ASK1/JNK2 signal contributions into consideration may delineate oncogenic versus tumour suppressive JNK functions and cancer cell drug responses.
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MAP Quinasa Quinasa Quinasa 5 , Concentración de Iones de Hidrógeno , MAP Quinasa Quinasa Quinasa 5/metabolismo , MAP Quinasa Quinasa Quinasa 5/genética , Humanos , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/genética , Estrés Fisiológico , Transducción de Señal , Animales , Optogenética , Sistema de Señalización de MAP QuinasasRESUMEN
Oncogenic FGFR4 signaling represents a potential therapeutic target in various cancer types, including triple negative breast cancer (TNBC) and hepatocellular carcinoma (HCC). However, resistance to FGFR4 single-agent therapy remains a major challenge, emphasizing the need for effective combinatorial treatments. Our study sought to develop a comprehensive computational model of FGFR4 signaling and provide network-level insights into resistance mechanisms driven by signaling dynamics. An integrated approach, combining computational network modeling with experimental validation, uncovered potent AKT reactivation following FGFR4 targeting in TNBC cells. Analyzing the effects of co-targeting specific network nodes by systematically simulating the model predicted synergy of co-targeting FGFR4 and AKT or specific ErbB kinases, which was subsequently confirmed through experimental validation; however, co-targeting FGFR4 and PI3K was not synergistic. Protein expression data from hundreds of cancer cell lines was incorporated to adapt the model to diverse cellular contexts. This revealed that while AKT rebound was common, it was not a general phenomenon. For example, ERK reactivation occurred in certain cell types, including an FGFR4-driven HCC cell line, where there is a synergistic effect of co-targeting FGFR4 and MEK but not AKT. In summary, this study offers key insights into drug-induced network remodeling and the role of protein expression heterogeneity in targeted therapy responses. These findings underscore the utility of computational network modeling for designing cell type-selective combination therapies and enhancing precision cancer treatment.
RESUMEN
The transcriptional co-activator YAP forms complexes with distinct transcription factors, controlling cell fate decisions, such as proliferation and apoptosis. However, the mechanisms underlying its context-dependent function are poorly defined. This study explores the interplay between the TGF-ß and Hippo pathways and their influence on YAP's association with specific transcription factors. By integrating iterative mathematical modeling with experimental validation, we uncover molecular switches, predominantly controlled by RASSF1A and ITCH, which dictate the formation of YAP-SMAD (proliferative) and YAP-p73 (apoptotic) complexes. Our results show that RASSF1A enhances the formation of apoptotic complexes, whereas ITCH promotes the formation of proliferative complexes. Notably, higher levels of ITCH transform YAP-SMAD activity from a transient to a sustained state, impacting cellular behaviors. Extending these findings to various breast cancer cell lines highlights the role of cellular context in YAP regulation. Our study provides new insights into the mechanisms of YAP transcriptional activities and their therapeutic implications.
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Utility of PI3Kα inhibitors like BYL719 is limited by the acquisition of genetic and non-genetic mechanisms of resistance which cause disease recurrence. Several combination therapies based on PI3K inhibition have been proposed, but a way to systematically prioritize them for breast cancer treatment is still missing. By integrating published and in-house studies, we have developed in silico models that quantitatively capture dynamics of PI3K signaling at the network-level under a BYL719-sensitive versus BYL719 resistant-cell state. Computational predictions show that signal rewiring to alternative components of the PI3K pathway promote resistance to BYL719 and identify PDK1 as the most effective co-target with PI3Kα rescuing sensitivity of resistant cells to BYL719. To explore whether PI3K pathway-independent mechanisms further contribute to BYL719 resistance, we performed phosphoproteomics and found that selection of high levels of the cell cycle regulator p21 unexpectedly promoted drug resistance in T47D cells. Functionally, high p21 levels favored repair of BYL719-induced DNA damage and bypass of the associated cellular senescence. Importantly, targeted inhibition of the check-point inhibitor CHK1 with MK-8776 effectively caused death of p21-high T47D cells, thus establishing a new vulnerability of BYL719-resistant breast cancer cells. Together, our integrated studies uncover hidden molecular mediators causing resistance to PI3Kα inhibition and provide a framework to prioritize combination therapies for PI3K-mutant breast cancer.
RESUMEN
The PEAK family of pseudokinases, comprising PEAK1-3, are signalling scaffolds that play oncogenic roles in several poor prognosis human cancers, including triple negative breast cancer (TNBC). However, therapeutic targeting of pseudokinases is challenging due to their lack of catalytic activity. To address this, we screened for PEAK1 effectors by affinity purification and mass spectrometry, identifying calcium/calmodulin-dependent protein kinase 2 (CAMK2)D and CAMK2G. PEAK1 promoted CAMK2D/G activation in TNBC cells via a novel feed-forward mechanism involving PEAK1/PLCγ1/Ca 2+ signalling and direct binding via a consensus CAMK2 interaction motif in the PEAK1 N-terminus. In turn, CAMK2 phosphorylated PEAK1 to enhance association with PEAK2, which is critical for PEAK1 oncogenic signalling. To achieve pharmacologic targeting of PEAK1/CAMK2, we repurposed RA306, a second generation CAMK2 inhibitor under pre-clinical development for treatment of cardiovascular disease. RA306 demonstrated on-target activity against CAMK2 in TNBC cells and inhibited PEAK1-enhanced migration and invasion in vitro . Moreover, RA306 significantly attenuated TNBC xenograft growth and blocked metastasis in a manner mirrored by CRISPR-mediated PEAK1 ablation. Overall, these studies establish PEAK1 as a critical cell signalling nexus, identify a novel mechanism for regulation of Ca 2+ signalling and its integration with tyrosine kinase signals, and identify CAMK2 as a therapeutically 'actionable' target downstream of PEAK1.
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Oxygen is a crucial molecule for cellular function. When oxygen demand exceeds supply, the oxygen sensing pathway centred on the hypoxia inducible factor (HIF) is switched on and promotes adaptation to hypoxia by up-regulating genes involved in angiogenesis, erythropoiesis and glycolysis. The regulation of HIF is tightly modulated through intricate regulatory mechanisms. Notably, its protein stability is controlled by the oxygen sensing prolyl hydroxylase domain (PHD) enzymes and its transcriptional activity is controlled by the asparaginyl hydroxylase FIH (factor inhibiting HIF-1).To probe the complexity of hypoxia-induced HIF signalling, efforts in mathematical modelling of the pathway have been underway for around a decade. In this paper, we review the existing mathematical models developed to describe and explain specific behaviours of the HIF pathway and how they have contributed new insights into our understanding of the network. Topics for modelling included the switch-like response to decreased oxygen gradient, the role of micro environmental factors, the regulation by FIH and the temporal dynamics of the HIF response. We will also discuss the technical aspects, extent and limitations of these models. Recently, HIF pathway has been implicated in other disease contexts such as hypoxic inflammation and cancer through crosstalking with pathways like NFκB and mTOR. We will examine how future mathematical modelling and simulation of interlinked networks can aid in understanding HIF behaviour in complex pathophysiological situations. Ultimately this would allow the identification of new pharmacological targets in different disease settings.