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Birt-Hogg-Dubé (BHD) syndrome patients are uniquely susceptible to all renal tumour subtypes. The underlying mechanism of carcinogenesis is unclear. To study cancer development in BHD, we used human proximal kidney (HK2) cells and found that long-term folliculin (FLCN) knockdown was required to increase their tumorigenic potential, forming larger spheroids in non-adherent conditions. Transcriptomic and proteomic analysis uncovered links between FLCN, cell cycle control and the DNA damage response (DDR) machinery. HK2 cells lacking FLCN had an altered transcriptome profile with cell cycle control gene enrichment. G1/S cell cycle checkpoint signaling was compromised with heightened protein levels of cyclin D1 (CCND1) and hyperphosphorylation of retinoblastoma 1 (RB1). A FLCN interactome screen uncovered FLCN binding to DNA-dependent protein kinase (DNA-PK). This novel interaction was reversed in an irradiation-responsive manner. Knockdown of FLCN in HK2 cells caused a marked elevation of γH2AX and RB1 phosphorylation. Both CCND1 and RB1 phosphorylation remained raised during DNA damage, showing an association with defective cell cycle control with FLCN knockdown. Furthermore, Flcn-knockdown C. elegans were defective in cell cycle arrest by DNA damage. This work implicates that long-term FLCN loss and associated cell cycle defects in BHD patients could contribute to their increased risk of cancer.
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Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) is a feature of many solid tumours and is a key pathogenic driver in the inherited condition Tuberous Sclerosis Complex (TSC). Modulation of the tumour microenvironment by extracellular vesicles (EVs) is known to facilitate the development of various cancers. The role of EVs in modulating the tumour microenvironment and their impact on the development of TSC tumours, however, remains unclear. This study, therefore, focuses on the poorly defined contribution of EVs to tumour growth in TSC. We characterised EVs secreted from TSC2-deficient and TSC2-expressing cells and identified a distinct protein cargo in TSC2-deficient EVs, containing an enrichment of proteins thought to be involved in tumour-supporting signalling pathways. We show EVs from TSC2-deficient cells promote cell viability, proliferation and growth factor secretion from recipient fibroblasts within the tumour microenvironment. Rapalogs (mTORC1 inhibitors) are the current therapy for TSC tumours. Here, we demonstrate a previously unknown intercellular therapeutic effect of rapamycin in altering EV cargo and reducing capacity to promote cell proliferation in the tumour microenvironment. Furthermore, EV cargo proteins have the potential for clinical applications as TSC biomarkers, and we reveal three EV-associated proteins that are elevated in plasma from TSC patients compared to healthy donor plasma.
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Vesículas Extracelulares , Esclerosis Tuberosa , Humanos , Proteínas Supresoras de Tumor , Esclerosis Tuberosa/metabolismo , Esclerosis Tuberosa/patología , Proteína 2 del Complejo de la Esclerosis Tuberosa , Vesículas Extracelulares/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Microambiente TumoralRESUMEN
This article was originally published under standard licence, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly.
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Delineating the contributions of specific cell signalling cascades to the development and maintenance of tumours has greatly informed our understanding of tumorigenesis and has advanced the modern era of targeted cancer therapy. It has been revealed that one of the key pathways regulating cell growth, the phosphatidylinositol 3-kinase/mechanistic target of rapamycin (PI3K/mTOR) signalling axis, is commonly dysregulated in cancer. With a specific, well-tolerated inhibitor of mTOR available, the impact of inhibiting this pathway at the level of mTOR has been tested clinically. This review highlights some of the promising results seen with mTOR inhibitors in the clinic and assesses some of the challenges that remain in predicting patient outcome following mTOR-targeted therapy.
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To find new anti-cancer drug therapies, we wanted to exploit homeostatic vulnerabilities within Tuberous Sclerosis Complex 2 (TSC2)-deficient cells with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivity. We show that nelfinavir and mefloquine synergize to selectively evoke a cytotoxic response in TSC2-deficient cell lines with mTORC1 hyperactivity. We optimize the concentrations of nelfinavir and mefloquine to a clinically viable range that kill cells that lack TSC2, while wild-type cells tolerate treatment. This new clinically viable drug combination causes a significant level of cell death in TSC2-deficient tumor spheroids. Furthermore, no cell recovery was apparent after drug withdrawal, revealing potent cytotoxicity. Transcriptional profiling by RNA sequencing of drug treated TSC2-deficient cells compared to wild-type cells suggested the cytotoxic mechanism of action, involving initial ER stress and an imbalance in energy homeostatic pathways. Further characterization revealed that supplementation with methyl pyruvate alleviated energy stress and reduced the cytotoxic effect, implicating energy deprivation as the trigger of cell death. This work underpins a critical vulnerability with cancer cells with aberrant signaling through the TSC2-mTORC1 pathway that lack flexibility in homeostatic pathways, which could be exploited with combined nelfinavir and mefloquine treatment.
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Cancer cells lose homeostatic flexibility because of mutations and dysregulated signaling pathways involved in maintaining homeostasis. Tuberous Sclerosis Complex 1 (TSC1) and TSC2 play a fundamental role in cell homeostasis, where signal transduction through TSC1/TSC2 is often compromised in cancer, leading to aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 hyperactivation increases the basal level of endoplasmic reticulum (ER) stress via an accumulation of unfolded protein, due to heightened de novo protein translation and repression of autophagy. We exploit this intrinsic vulnerability of tumor cells lacking TSC2, by treating with nelvinavir to further enhance ER stress while inhibiting the proteasome with bortezomib to prevent effective protein removal. We show that TSC2-deficient cells are highly dependent on the proteosomal degradation pathway for survival. Combined treatment with nelfinavir and bortezomib at clinically relevant drug concentrations show synergy in selectively killing TSC2-deficient cells with limited toxicity in control cells. This drug combination inhibited tumor formation in xenograft mouse models and patient-derived cell models of TSC and caused tumor spheroid death in 3D culture. Importantly, 3D culture assays differentiated between the cytostatic effects of the mTORC1 inhibitor, rapamycin, and the cytotoxic effects of the nelfinavir/bortezomib combination. Through RNA sequencing, we determined that nelfinavir and bortezomib tip the balance of ER protein homeostasis of the already ER-stressed TSC2-deficient cells in favor of cell death. These findings have clinical relevance in stratified medicine to treat tumors that have compromised signaling through TSC and are inflexible in their capacity to restore ER homeostasis.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Neoplasias/patología , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Animales , Bortezomib/farmacología , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/fisiología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Nelfinavir/farmacología , Neoplasias/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Uncontrolled cell growth in Tuberous Sclerosis Complex occurs due to inappropriate activation of mechanistic (mammalian) target of rapamycin complex 1 (mTORC1). The current therapy, rapamycin, produced promising clinical trial results, but patient tumours regrow if treatment is discontinued, revealing rapamycin has cytostatic properties rather than a cytotoxic effect. Taking advantage of the enhanced levels of endoplasmic reticulum (ER) stress present in TSC2-null cells, we investigated drug combinations producing a cytotoxic response. We found a nelfinavir and salinomycin combination specifically killed TSC2-deficient, mTORC1 hyperactive cells. Cytotoxicity was rescued by reducing protein synthesis, either through mTORC1 inhibition or cycloheximide treatment. This indicates that the drug combination targets the cells by tipping the protein homeostasis balance of the already metabolically stressed TSC2-deficient cells in favour of cell death. Furthermore, this drug combination also inhibited tumour formation in TSC2-deficient cell models and caused tumour spheroid death in 3D culture. Importantly, the 3D assay could differentiate the cytostatic agent, rapamycin, from the cytotoxic nelfinavir/salinomycin combination. Sporadic cancer cell lines with hyperactive mTORC1 signalling were also susceptible to this nelfinavir/salinomycin drug combination. This work indicates that the protein homeostasis pathway is an attractive therapeutic target in both Tuberous Sclerosis Complex and mTORC1-driven sporadic cancers.
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Homeostasis/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Nelfinavir/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Piranos/farmacología , Animales , Muerte Celular/efectos de los fármacos , Quimioterapia Combinada , Estrés del Retículo Endoplásmico/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Humanos , Ratones , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Much attention has recently been focussed on the lysosome as a signalling hub. Following the initial discovery that localisation of the nutrient-sensitive kinase, mammalian target of rapamycin complex 1 (mTORC1), to the lysosome was essential for mTORC1 activation, the field has rapidly expanded to reveal the role of the lysosome as a platform permitting the co-ordination of several homeostatic signalling pathways. Much is now understood about how the lysosome contributes to amino acid sensing by mTORC1, the involvement of the energy-sensing kinase, AMP-activated protein kinase (AMPK), at the lysosome and how both AMPK and mTORC1 signalling pathways feedback to lysosomal biogenesis and regeneration following autophagy. This review will cover the classical role of the lysosome in autophagy, the dynamic signalling interactions which take place on the lysosomal surface and the multiple levels of cross-talk which exist between lysosomes, AMPK and mTORC1.
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Proteínas Quinasas Activadas por AMP/metabolismo , Lisosomas/metabolismo , Complejos Multiproteicos/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Autofagia/fisiología , Aumento de la Célula , Homeostasis/fisiología , Humanos , Diana Mecanicista del Complejo 1 de la RapamicinaRESUMEN
The mammalian target of rapamycin complex 1 (mTORC1) is the key signaling hub that regulates cellular protein homeostasis, growth, and proliferation in health and disease. As a prerequisite for activation of mTORC1 by hormones and mitogens, there first has to be an available pool of intracellular amino acids. Arginine, an amino acid essential during mammalian embryogenesis and early development is one of the key activators of mTORC1. Herein, we demonstrate that arginine acts independently of its metabolism to allow maximal activation of mTORC1 by growth factors via a mechanism that does not involve regulation of mTORC1 localization to lysosomes. Instead, arginine specifically suppresses lysosomal localization of the TSC complex and interaction with its target small GTPase protein, Rheb. By interfering with TSC-Rheb complex, arginine relieves allosteric inhibition of Rheb by TSC. Arginine cooperates with growth factor signaling which further promotes dissociation of TSC2 from lysosomes and activation of mTORC1. Arginine is the main amino acid sensed by the mTORC1 pathway in several cell types including human embryonic stem cells (hESCs). Dependence on arginine is maintained once hESCs are differentiated to fibroblasts, neurons, and hepatocytes, highlighting the fundamental importance of arginine-sensing to mTORC1 signaling. Together, our data provide evidence that different growth promoting cues cooperate to a greater extent than previously recognized to achieve tight spatial and temporal regulation of mTORC1 signaling.
Cells need to be able to sense and respond to signals from their environment. A group (or complex) of conserved proteins called mTORC1 acts a key signaling hub that regulates cell growth and many other processes. This complex can be activated by many different signals from outside the cell. However, mTORC1 can only be activated by these signals if there is also a good supply of amino acids which are needed to make new proteins within the cell. The amino acids are thought to be presented to mTORC1 on the outer surface of cellular compartments known as lysosomes. A protein called Rheb on the surface of the lysosomes activates mTORC1, while a protein complex called TSC inhibits the activity of Rheb to regulate mTORC1 activity. Previous studies have shown that some amino acids influence whether mTORC1 can be activated by affecting whether it is localized to the lysosomes or not. Here, Carroll et al. explored how an amino acid called arginine regulates mTORC1. The experiments show that arginine is the major amino acid that influences whether mTORC1 can be activated in several different types of human cell. When cells were deprived of arginine, the activity of the complex was strongly suppressed. However, microscopy showed that arginine had no effect on whether mTORC1 was found at the lysosomes or not, which suggests that arginine might be acting in a different way to other amino acids. Further experiments found that a lack of arginine led to an increase in the number of TSC complexes at the lysosomes. This led to the inhibition of Rheb and therefore prevented mTORC1 from being activated. Together, Carroll et al.'s findings provide evidence that the different signals that regulate mTORC1 signaling cooperate to a greater extent than previously thought. A future challenge will be to understand the molecular details of how the arginine is detected.
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Arginina/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Complejos Multiproteicos/metabolismo , Neuropéptidos/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Diferenciación Celular , Células Madre Embrionarias/fisiología , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteína Homóloga de Ras Enriquecida en el Cerebro , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
Tuberous sclerosis complex (TSC) is a rare genetic disorder where patients develop benign tumours in several organ systems. Central to TSC pathology is hyper-activation of the mammalian target of rapamycin complex 1 (mTORC1) signalling pathway, which is a key controller of cell growth. As a result, TSC model systems are a valuable tool for examining mTORC1-driven cellular processes. The immunosuppressant, rapamycin, is a specific inhibitor of mTORC1 and has shown promise as a therapeutic agent in TSC as well as in malignancy. This review will focus on the cellular processes controlled by mTORC1 and how TSC-deficient cell lines and mouse models have broadened our understanding of the mTORC1 signalling network. It will also discuss how our knowledge of TSC signalling can help us understand sporadic conditions where mTORC1 activity is implicated in disease onset or progression, and the possibility of using rapamycin to treat sporadic disease.
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Esclerosis Tuberosa/patología , Animales , Autofagia , Proliferación Celular/fisiología , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/metabolismo , Neoplasias/genética , Neoplasias/patología , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/metabolismoRESUMEN
Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Homólogo de la Proteína 1 Relacionada con la Autofagia , Síndrome de Birt-Hogg-Dubé/metabolismo , Síndrome de Birt-Hogg-Dubé/patología , Proteínas Portadoras/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/deficiencia , Proteína Sequestosoma-1 , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/deficienciaRESUMEN
The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPK-dependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α-mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation.
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Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Síndrome de Birt-Hogg-Dubé/etiología , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/metabolismo , Línea Celular , Transformación Celular Neoplásica , Metabolismo Energético , Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signalling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by peroxisomal biogenesis factors 19 and 5 (PEX19 and PEX5), respectively, and peroxisome-localized TSC functioned as a Rheb GTPase-activating protein (GAP) to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, and abrogated peroxisome localization, Rheb GAP activity and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS, and peroxisome-localization-deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for the TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signalling organelle involved in regulation of mTORC1.
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Autofagia , Regulación Enzimológica de la Expresión Génica , Complejos Multiproteicos/genética , Peroxisomas/metabolismo , Especies Reactivas de Oxígeno , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Animales , Línea Celular , Células HEK293 , Humanos , Células MCF-7 , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas de la Membrana/metabolismo , Ratones , Complejos Multiproteicos/metabolismo , Unión Proteica , Ratas , Serina-Treonina Quinasas TOR/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
In order for cells to divide in a proficient manner, they must first double their biomass, which is considered to be the main rate-limiting phase of cell proliferation. Cell growth requires an abundance of energy and biosynthetic precursors such as lipids and amino acids. Consequently, the energy and nutrient status of the cell is acutely monitored and carefully maintained. mTORC1 [mammalian (or mechanistic) target of rapamycin complex 1] is often considered to be the master regulator of cell growth that enhances cellular biomass through up-regulation of protein translation. In order for cells to control cellular homoeostasis during growth, there is close signalling interplay between mTORC1 and two other protein kinases, AMPK (AMP-activated protein kinase) and ULK1 (Unc-51-like kinase 1). This kinase triad collectively senses the energy and nutrient status of the cell and appropriately dictates whether the cell will actively favour energy- and amino-acid-consuming anabolic processes such as cellular growth, or energy- and amino-acid-generating catabolic processes such as autophagy. The present review discusses important feedback mechanisms between these three homoeostatic protein kinases that orchestrate cell growth and autophagy, with a particular focus on the mTORC1 component raptor (regulatory associated protein of mammalian target of rapamycin), as well as the autophagy-initiating kinase ULK1.
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Adenilato Quinasa/metabolismo , Metabolismo Energético , Homeostasis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Autofagia , Homólogo de la Proteína 1 Relacionada con la Autofagia , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Transducción de SeñalRESUMEN
Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , Transducción de Señal , Homólogo de la Proteína 1 Relacionada con la Autofagia , Proteínas de Ciclo Celular , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos , Fosfoproteínas/metabolismo , Fosforilación , Proteína Reguladora Asociada a mTOR , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Especificidad por Sustrato , Serina-Treonina Quinasas TORRESUMEN
Tuberous sclerosis complex (TSC) is a genetic condition characterized by the growth of benign tumours in multiple organs, including the brain and kidneys, alongside intellectual disability and seizures. Identification of a causative mutation in TSC1 or TSC2 is important for accurate genetic counselling in affected families, but it is not always clear from genetic data whether a sequence variant is pathogenic or not. In vitro functional analysis could provide support for determining whether an unclassified TSC1 or TSC2 variant is disease-causing. We have performed a detailed functional analysis of four patient-derived TSC2 mutations, E92V, R505Q, H597R and L1624P. One mutant, E92V, functioned similarly to wild-type TSC2, whereas H597R and L1624P had abnormal function in all assays, consistent with available clinical and segregation information. One TSC2 mutation, R505Q, was identified in a patient with intellectual disability, seizures and autistic spectrum disorder but who did not fulfil the diagnostic criteria for TSC. The R505Q mutation was also found in two relatives, one with mild learning difficulties and one without apparent phenotypic abnormality. R505Q TSC2 exhibited partially disrupted function in our assays. These data highlight the difficulties of assessing pathogenicity of a mutation and suggest that multiple lines of evidence, both genetic and functional, are required to assess the pathogenicity of some mutations.
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Mutación/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Adolescente , Animales , Células Cultivadas , Niño , Preescolar , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/fisiopatología , Genotipo , Células HEK293 , Humanos , Lactante , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Fenotipo , Fosforilación/genética , Multimerización de Proteína/genética , Estabilidad Proteica , Proteínas/metabolismo , Serina-Treonina Quinasas TOR , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Eritropoyetina/metabolismo , Transducción de Señal/fisiología , Activación Transcripcional/fisiología , Animales , Neoplasias de la Mama/genética , Carcinoma/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Inhibidores Enzimáticos/farmacología , Eritropoyetina/farmacología , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Janus Quinasa 2/efectos de los fármacos , Janus Quinasa 2/metabolismo , Invasividad Neoplásica/genética , Invasividad Neoplásica/fisiopatología , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Ratas , Receptores de Eritropoyetina/genética , Factor de Transcripción STAT5/efectos de los fármacos , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Proteínas ras/efectos de los fármacos , Proteínas ras/metabolismoRESUMEN
The mammalian target of rapamycin (mTOR) Ser/Thr kinase signals in at least two multiprotein complexes distinguished by their different partners and sensitivities to rapamycin. Acute rapamycin inhibits signaling by mTOR complex 1 (mTORC1) but not mTOR complex 2 (mTORC2), which both promote cell growth, proliferation, and survival. Although mTORC2 regulation remains poorly defined, diverse cellular mitogens activate mTORC1 signaling in a manner that requires sufficient levels of amino acids and cellular energy. Before the identification of distinct mTOR complexes, mTOR was reported to autophosphorylate on Ser-2481 in vivo in a rapamycin- and amino acid-insensitive manner. These results suggested that modulation of mTOR intrinsic catalytic activity does not universally underlie mTOR regulation. Here we re-examine the regulation of mTOR Ser-2481 autophosphorylation (Ser(P)-2481) in vivo by studying mTORC-specific Ser(P)-2481 in mTORC1 and mTORC2, with a primary focus on mTORC1. In contrast to previous work, we find that acute rapamycin and amino acid withdrawal markedly attenuate mTORC1-associated mTOR Ser(P)-2481 in cycling cells. Although insulin stimulates both mTORC1- and mTORC2-associated mTOR Ser(P)-2481 in a phosphatidylinositol 3-kinase-dependent manner, rapamycin acutely inhibits insulin-stimulated mTOR Ser(P)-2481 in mTORC1 but not mTORC2. By interrogating diverse mTORC1 regulatory input, we find that without exception mTORC1-activating signals promote, whereas mTORC1-inhibitory signals decrease mTORC1-associated mTOR Ser(P)-2481. These data suggest that mTORC1- and likely mTORC2-associated mTOR Ser-2481 autophosphorylation directly monitors intrinsic mTORC-specific catalytic activity and reveal that rapamycin inhibits mTORC1 signaling in vivo by reducing mTORC1 catalytic activity.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Sirolimus/farmacología , Células 3T3-L1 , Animales , Anticuerpos/farmacología , Catálisis , Línea Celular Transformada , Fibroblastos/citología , Humanos , Péptidos y Proteínas de Señalización Intracelular/inmunología , Riñón/citología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Fosforilación , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas , Conejos , Serina/metabolismo , Serina-Treonina Quinasas TOR , Factores de Transcripción/metabolismoRESUMEN
The mammalian target of rapamycin (mTOR) pathway is implicated in a number of human diseases, but the pathway details are not fully understood. Here we elucidate the interactions between various proteins involved in mTOR complex 1 (mTORC1). An in vitro mTORC1 kinase assay approach was used to probe the role of the mTORC1 component Raptor and revealed that certain Raptor mutations disrupt binding to eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and prevent its subsequent phosphorylation by mTOR. Interestingly, we show that a point mutation in the highly conserved Raptor RNC domain still allows binding to mTOR but prevents Raptor association and mTOR-dependent phosphorylation of 4E-BP1, indicating that this Raptor domain facilitates substrate recognition by mTORC1. This Raptor RNC domain mutant also dominantly inhibits mTORC1 signalling to 4E-BP1, S6K1 and HIF1alpha in vivo. We further characterise the functions of the mTORC1 signalling (TOS) and RAIP motifs of 4E-BP1, which are involved in substrate recognition by Raptor and phosphorylation by mTORC1. We show that an mTOR mutant, L1460P, responds to insulin even in nutrient-deprived conditions and is resistant to inhibition by inactive RagB-RagC heterodimers that mimic nutrient withdrawal suggesting that this region of mTOR is involved in sensing the permissive amino acid input. We found that FKBP38 inhibits mTOR(L1460P), while the mTOR(E2419K) kinase domain mutant was resistant to FKBP38 inhibition. Finally, we show that activation of mTORC1 by both Rheb and RhebL1 is impaired by FKBP38. Our work demonstrates the value of an in vitro mTORC1 kinase assay to characterise cell signalling components of mTORC1 involved in recognition and phosphotransfer to mTORC1 substrates.