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1.
J Cancer Res Clin Oncol ; 146(1): 273-285, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31641854

RESUMO

PURPOSE: The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway plays a vital role in cancer development and progression. This study aimed to investigate the relationship between genotype variants in mTORC1 pathway and the risk of brain metastasis (BM) in patients with non-small cell lung cancer (NSCLC). METHODS: We extracted genomic DNA from blood samples of 501 NSCLC patients and genotyped eight single-nucleotide polymorphisms (SNPs) in three core genes [mammalian target of rapamycin (mTOR), mammalian lethal with sec-13 protein 8 (mLST8) and regulatory-associated protein of mTOR (RPTOR)] of the mTORC1 pathway. The associations between these SNPs and the risk of BM development were assessed. RESULTS: The AG/GG genotype of mLST8:rs26865 and TC/CC genotype of mLST8:rs3160 were associated with an increased risk of BM [hazard ratios (HR) 2.938, 95% confidence interval (CI) 1.664-5.189, p < 0.001 and HR = 2.490, 95% CI = 1.543-4.016, p < 0.001, respectively]. These risk polymorphisms had a cumulative effect on BM risk, with two risk genotypes exhibiting the highest increased risk (p < 0.001). Furthermore, these risk SNPs were associated with the lymph node metastasis (N2/3), body mass index (BMI) (≥ 25 kg/m2), high level of squamous cell carcinoma (SCC) antigen and Ki-67 proliferation index. Moreover, patients with AG/GG genotype of mLST8:rs26865 had significantly lower median overall survival than those with AA genotype (12.1 months versus 21.6 months, p = 0.04). CONCLUSIONS: Our results indicate that polymorphisms in mTORC1 pathway were significantly associated with increased risk of BM and may be valuable biomarkers to identify NSCLC patients with a high risk of BM.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundário , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Pessoa de Meia-Idade , Mutação , Polimorfismo de Nucleotídeo Único , Estudos Retrospectivos , Transdução de Sinais
2.
J Int Soc Sports Nutr ; 16(1): 60, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31829236

RESUMO

BACKGROUND: Ursolic acid altered muscle protein metabolism in normal and resting conditions after acute resistance exercise, suggesting that eating fruits rich in ursolic acid could enhance muscle protein synthesis and decrease muscle degradation. Aronia melanocarpa, a member of the family Rosaceae and native to North America and Eastern Canada, is rich in ursolic acid. In this study, we examined the effects of A. melanocarpa extract (AME) supplementation on the mTORC1 signaling pathway and muscle degradation-related factors in rats, both alone and in combination with resistance exercise. METHODS: Male Sprague-Dawley rats were divided into AME and normal chow (NOR) groups. AME group was fed chow providing a dose of 3 g/kg of AME and 115 mg/kg of ursolic acid for 7 days, whereas NOR rats were fed normal powder chow. The right gastrocnemius muscle of each animal was isometrically exercised (5 sets of ten 3-s contractions, with a 7-s interval between contractions and 3-min rest intervals between sets), while the left gastrocnemius muscle served as an internal control. Western blotting and real-time polymerase chain reaction were used to assess expression of factors involved in the mTORC1 signaling pathway and muscle degradation. RESULTS: At 1 h after resistance exercise, phosphorylation of ERK1/2 was significantly increased by AME consumption. At 6 h after resistance exercise, AME consumption significantly increased the phosphorylation of Akt, p70S6K, rpS6, and AMPK. It also increased MAFbx expression. Furthermore, AME significantly increased the phosphorylation of p70S6K and rpS6 in response to resistance exercise. However, AME did not increase muscle protein synthesis (MPS) after resistance exercise. AME did not affect the expression of any of the mediators of protein degradation, with the exception of MAFbx. CONCLUSIONS: Dietary AME enhanced mTORC1 activation in response to resistance exercise without increasing MPS. Moreover, it neither accelerated muscle protein degradation nor otherwise negatively affected protein metabolism. Further study is needed to clarify the effect of the combination of AME and chronic resistance training on muscle hypertrophy.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Músculo Esquelético/efeitos dos fármacos , Condicionamento Físico Animal/fisiologia , Extratos Vegetais/farmacologia , Transdução de Sinais , Triterpenos/farmacologia , Animais , Dieta , Masculino , Músculo Esquelético/fisiologia , Photinia/química , Biossíntese de Proteínas , Ratos Sprague-Dawley
3.
Immunity ; 51(6): 1012-1027.e7, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31668641

RESUMO

Regulatory T (Treg) cells are critical mediators of immune tolerance whose activity depends upon T cell receptor (TCR) and mTORC1 kinase signaling, but the mechanisms that dictate functional activation of these pathways are incompletely understood. Here, we showed that amino acids license Treg cell function by priming and sustaining TCR-induced mTORC1 activity. mTORC1 activation was induced by amino acids, especially arginine and leucine, accompanied by the dynamic lysosomal localization of the mTOR and Tsc complexes. Rag and Rheb GTPases were central regulators of amino acid-dependent mTORC1 activation in effector Treg (eTreg) cells. Mice bearing RagA-RagB- or Rheb1-Rheb2-deficient Treg cells developed a fatal autoimmune disease and had reduced eTreg cell accumulation and function. RagA-RagB regulated mitochondrial and lysosomal fitness, while Rheb1-Rheb2 enforced eTreg cell suppressive gene signature. Together, these findings reveal a crucial requirement of amino acid signaling for licensing and sustaining mTORC1 activation and functional programming of Treg cells.


Assuntos
Arginina/metabolismo , Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Ciclo Celular , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Tolerância Imunológica/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/citologia
4.
Nat Med ; 25(11): 1684-1690, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31636454

RESUMO

Dysregulation of the mammalian target of rapamycin (mTOR) signaling, which is mediated by two structurally and functionally distinct complexes, mTORC1 and mTORC2, has been implicated in several neurological disorders1-3. Individuals carrying loss-of-function mutations in the phosphatase and tensin homolog (PTEN) gene, a negative regulator of mTOR signaling, are prone to developing macrocephaly, autism spectrum disorder (ASD), seizures and intellectual disability2,4,5. It is generally believed that the neurological symptoms associated with loss of PTEN and other mTORopathies (for example, mutations in the tuberous sclerosis genes TSC1 or TSC2) are due to hyperactivation of mTORC1-mediated protein synthesis1,2,4,6,7. Using molecular genetics, we unexpectedly found that genetic deletion of mTORC2 (but not mTORC1) activity prolonged lifespan, suppressed seizures, rescued ASD-like behaviors and long-term memory, and normalized metabolic changes in the brain of mice lacking Pten. In a more therapeutically oriented approach, we found that administration of an antisense oligonucleotide (ASO) targeting mTORC2's defining component Rictor specifically inhibits mTORC2 activity and reverses the behavioral and neurophysiological abnormalities in adolescent Pten-deficient mice. Collectively, our findings indicate that mTORC2 is the major driver underlying the neuropathophysiology associated with Pten-deficiency, and its therapeutic reduction could represent a promising and broadly effective translational therapy for neurological disorders where mTOR signaling is dysregulated.


Assuntos
Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Doenças do Sistema Nervoso/genética , PTEN Fosfo-Hidrolase/genética , Serina-Treonina Quinases TOR/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Humanos , Mutação com Perda de Função/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Knockout , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , PTEN Fosfo-Hidrolase/deficiência , Proteína Companheira de mTOR Insensível à Rapamicina/antagonistas & inibidores , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína 1 do Complexo Esclerose Tuberosa/genética
5.
Endocrinology ; 160(12): 3001-3017, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31599935

RESUMO

A growing body of evidence implies a pivotal role of sirtuin-1 (Sirt1) in chondrocyte function and homeostasis; however, its underlying mechanisms mediating chondrogenesis, which is an essential process for physiological skeletal growth, are still poorly understood. In the current study, we generated TamCartSirt1-/- [Sirt1 conditional knockout (cKO)] mice to explore the role of Sirt1 during postnatal endochondral ossification. Compared with control mice, cKO mice exhibited growth retardation associated with inhibited chondrocyte proliferation and hypertrophy, as well as activated apoptosis. These effects were regulated by hyperactivation of mammalian target of rapamycin complex 1 (mTORC1) signaling, and thereby inhibition of autophagy and induction of endoplasmic reticulum stress in growth plate chondrocytes. IP injection of the mTORC1 inhibitor rapamycin to mice with Sirt1 deletion partially neutralized such inhibitory effects of Sirt1 ablation on longitudinal bone growth, indicating the causative link between SIRT1 and mTORC1 signaling in the growth plate. Mechanistically, SIRT1 interacted with tuberous sclerosis complex 2 (TSC2), a key upstream negative regulator of mTORC1 signaling, and loss of Sirt1 inhibited TSC2 expression, resulting in hyperactivated mTORC1 signaling in chondrocytes. In conclusion, our findings suggest that loss of Sirt1 may trigger mTORC1 signaling in growth plate chondrocytes and contributes to growth retardation, thus indicating that SIRT1 is an important regulator during chondrogenesis and providing new insights into the clinical potential of SIRT1 in bone development.


Assuntos
Condrócitos/fisiologia , Lâmina de Crescimento/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Osteogênese , Sirtuína 1/fisiologia , Animais , Desenvolvimento Ósseo , Condrogênese , Feminino , Masculino , Camundongos , Camundongos Knockout , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
6.
Anticancer Res ; 39(9): 4829-4835, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519585

RESUMO

BACKGROUND/AIM: Chronic lymphocytic leukemia (CLL) still remains an incurable disease as the cells evade apoptosis, which is an obstacle for current therapeutic approaches. Therefore, our aim was to identify an ideal target of leukemic cell growth for developing inhibitors. MATERIALS AND METHODS: Mouse lymphocytic leukemia cell line L1210, human Toledo cells and a DBA/2 mouse graft model were used to analyze the activity of dual mTORC1/2 inhibitor AZD2014s. Western blotting and flow cytometry were performed to determine the mechanism. RESULTS: AZD2014 inhibited L1210 and human Toledo cell proliferation. Treatment with AZD2014 reduced the phosphorylation levels of S6K1 and 4EBP1 and the protein levels of Rictor, a component of the mTORC2 pathway. AZD2014 induced cell cycle arrest at the G0-G1 phase by reducing the expression of cyclin D1 and CDK4. Oral administration of AZD2014 significantly inhibited the growth of L1210 cell grafts in DBA/2 mice. CONCLUSION: The mTORC1/2 inhibitor may be a better therapeutic agent compared to PI3K/mTORC1 inhibitors for treating patients with CLL.


Assuntos
Antineoplásicos/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Benzamidas , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Leucemia Linfoide/tratamento farmacológico , Leucemia Linfoide/metabolismo , Leucemia Linfoide/patologia , Masculino , Camundongos , Morfolinas/farmacologia , Pirimidinas
7.
Nat Cell Biol ; 21(10): 1206-1218, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548609

RESUMO

Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes by the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signalling are unknown. Here, we show that oxysterol binding protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA and VAPB, deliver cholesterol across ER-lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-interacting cholesterol carriers, the recruitment of mTORC1 by the Rag GTPases is inhibited owing to impaired transport of cholesterol to lysosomes. By contrast, OSBP-mediated cholesterol trafficking drives constitutive mTORC1 activation in a disease model caused by the loss of the lysosomal cholesterol transporter, Niemann-Pick C1 (NPC1). Chemical and genetic inactivation of OSBP suppresses aberrant mTORC1 signalling and restores autophagic function in cellular models of Niemann-Pick type C (NPC). Thus, ER-lysosome contacts are signalling hubs that enable cholesterol sensing by mTORC1, and targeting the sterol-transfer activity of these signalling hubs could be beneficial in patients with NPC.


Assuntos
Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Doenças de Niemann-Pick/metabolismo , Receptores de Esteroides/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Receptores de Esteroides/genética , Transdução de Sinais , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
8.
Biochem Soc Trans ; 47(4): 1173-1185, 2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31383818

RESUMO

Lysosomes are the main degradative compartments of mammalian cells and serve as platforms for cellular nutrient signaling and sterol transport. The diverse functions of lysosomes and their adaptation to extracellular and intracellular cues are tightly linked to the spatiotemporally controlled synthesis, turnover and interconversion of lysosomal phosphoinositides, minor phospholipids that define membrane identity and couple membrane dynamics to cell signaling. How precisely lysosomal phosphoinositides act and which effector proteins within the lysosome membrane or at the lysosomal surface recognize them is only now beginning to emerge. Importantly, mutations in phosphoinositide metabolizing enzyme cause lysosomal dysfunction and are associated with numerous diseases ranging from neurodegeneration to cancer. Here, we discuss the phosphoinositides and phosphoinositide metabolizing enzymes implicated in lysosome function and homeostasis and outline perspectives for future research.


Assuntos
Homeostase , Lisossomos/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Autofagossomos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Transporte Biológico , Humanos , Membranas Intracelulares/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Esteróis/metabolismo
9.
Hematol Oncol ; 37(4): 352-359, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31385336

RESUMO

Patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) who are unfit for or relapsed postautologous stem-cell transplantation have poor outcomes. Historically, mTORC1 inhibitors have produced responses in approximately 30% of patients in this setting. mTORC1 inhibitor efficacy may be limited by resistance mechanisms including AKT activation by mTORC2. To date, dual mTORC1/2 inhibitors targeting both the TORC1 and TORC2 complexes have not been investigated in DLBCL. This phase II trial investigated the oral dual mTORC1/2 inhibitor vistusertib in an intermittent dosing schedule of 125 mg b.d. for 2 days per week. Thirty patients received vistusertib and six received vistusertib-rituximab for up to six cycles (28-day cycles). Two partial responses were achieved on monotherapy. Durations of response were 57 and 62 days, respectively, for these patients. 19% had stable disease within six cycles. In the monotherapy arm, the median progression-free survival was1.69 (95% confidence interval [CI] 1.61-2.14) months and median overall survival was 6.58 (95% CI 3.81-not reached) months, respectively. The median duration of response or stable disease across the trial duration was 153 days (95% CI 112-not reached). Tumour responses according to positron emission tomography/computed tomography versus computed tomography were concordant. There were no differences noted in tumour volume response according to cell of origin by either gene expression profiling or immunohistochemistry. Vistusertib ± rituximab was well tolerated; across 36 patients 86% of adverse events were grade (G) 1-2. Common vistusertib-related adverse events were similar to those described with mTORC1 inhibitors: nausea (47% G1-2), diarrhoea (27% G1-2, 6% G3), fatigue (30% G1-2, 3% G3), mucositis (25% G1-2, 6% G3), vomiting (17% G1-2), and dyspepsia (14% G1-2). Dual mTORC1/2 inhibitors do not clearly confer an advantage over mTORC1 inhibitors in relapsed or refractory DLBCL. Potential resistance mechanisms are discussed within.


Assuntos
Antineoplásicos/efeitos adversos , Benzamidas/efeitos adversos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Terapia de Alvo Molecular , Morfolinas/efeitos adversos , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases/efeitos adversos , Pirimidinas/efeitos adversos , Terapia de Salvação , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Subpopulações de Linfócitos B/patologia , Benzamidas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Feminino , Gastroenteropatias/induzido quimicamente , Humanos , Estimativa de Kaplan-Meier , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Morfolinas/uso terapêutico , Células-Tronco Neoplásicas/patologia , Intervalo Livre de Progressão , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Rituximab/administração & dosagem , Rituximab/efeitos adversos
10.
Biochim Biophys Acta Gene Regul Mech ; 1862(9): 194414, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31394264

RESUMO

Iron is an essential micronutrient that participates as a cofactor in a broad range of metabolic processes including mitochondrial respiration, DNA replication, protein translation and lipid biosynthesis. Adaptation to iron deficiency requires the global reorganization of cellular metabolism directed to optimize iron utilization. The budding yeast Saccharomyces cerevisiae has been widely used to characterize the responses of eukaryotic microorganisms to iron depletion. In this report, we used a genomic approach to investigate the contribution of transcription rates to the modulation of mRNA levels during adaptation of yeast cells to iron starvation. We reveal that a decrease in the activity of all RNA polymerases contributes to the down-regulation of many mRNAs, tRNAs and rRNAs. Opposite to the general expression pattern, many genes including components of the iron deficiency response, the mitochondrial retrograde pathway and the general stress response display a remarkable increase in both transcription rates and mRNA levels upon iron limitation, whereas genes encoding ribosomal proteins or implicated in ribosome biogenesis exhibit a pronounced fall. This expression profile is consistent with an activation of the environmental stress response. The phosphorylation stage of multiple regulatory factors strongly suggests that the conserved nutrient signaling pathway TORC1 is inhibited during the progress of iron deficiency. These results suggest an intricate crosstalk between iron metabolism and the TORC1 pathway that should be considered in many disorders.


Assuntos
Anemia Ferropriva/genética , Proteínas de Ligação a DNA/genética , Ferro/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Adaptação Fisiológica/genética , Anemia Ferropriva/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/genética , Humanos , Fosforilação , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética
11.
Adv Exp Med Biol ; 1152: 283-292, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31456190

RESUMO

Based on the insights gleaned from decades of research, it seems clear that mechanistic target of rapamycin (mTOR) is an essential signaling node that integrates environmental clues for regulation of cell survival, metabolism and proliferation of the cells. However, overwhelmingly increasing scientific evidence has added a new layer of intricacy to already complicated and versatile signaling pathway of mTOR. Deregulation of spatio-temporally controlled mTOR-driven pathway played contributory role in breast cancer development and progression. Pharmacologists and molecular biologists have specifically emphasized on the identification and development of mTOR-pathway inhibitors. In this chapter we have attempted to provide an overview of the most recent findings related to therapeutic targeting of mTOR-associated mTORC1 and mTORC2 in breast cancer. We have also comprehensively summarized regulation of mTOR and its partners by microRNAs in breast cancer.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Feminino , Humanos , MicroRNAs/genética , Terapia de Alvo Molecular , Metástase Neoplásica
12.
Nat Cell Biol ; 21(9): 1093-1101, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31451768

RESUMO

Mechanistic target of rapamycin (mTOR) kinase functions in two multiprotein complexes: lysosomal mTOR complex 1 (mTORC1) and mTORC2 at the plasma membrane. mTORC1 modulates the cell response to growth factors and nutrients by increasing protein synthesis and cell growth, and repressing the autophagy-lysosomal pathway1-4; however, dysfunction in mTORC1 is implicated in various diseases3,5,6. mTORC1 activity is regulated by phosphoinositide lipids7-10. Class I phosphatidylinositol-3-kinase (PI3K)-mediated production of phosphatidylinositol-3,4,5-trisphosphate6,11 at the plasma membrane stimulates mTORC1 signalling, while local synthesis of phosphatidylinositol-3,4-bisphosphate by starvation-induced recruitment of class II PI3K-ß (PI3KC2-ß) to lysosomes represses mTORC1 activity12. How the localization and activity of PI3KC2-ß are regulated by mitogens is unknown. We demonstrate that protein kinase N (PKN) facilitates mTORC1 signalling by repressing PI3KC2-ß-mediated phosphatidylinositol-3,4-bisphosphate synthesis downstream of mTORC2. Active PKN2 phosphorylates PI3KC2-ß to trigger PI3KC2-ß complex formation with inhibitory 14-3-3 proteins. Conversely, loss of PKN2 or inactivation of its target phosphorylation site in PI3KC2-ß represses nutrient signalling via mTORC1. These results uncover a mechanism that couples mTORC2-dependent activation of PKN2 to the regulation of mTORC1-mediated nutrient signalling by local lipid signals.


Assuntos
Lipídeos , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais/fisiologia , Proliferação de Células/fisiologia , Fibroblastos/metabolismo , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
13.
J Agric Food Chem ; 67(34): 9510-9521, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31382738

RESUMO

Glutamate (Glu) is a critical nutritional regulator of intestinal epithelial homeostasis. In addition, intestinal stem cells (ISCs) at crypt bases are known to play important roles in maintaining the renewal and homeostasis of the intestinal epithelium, and the aspects of communication between Glu and ISCs are still unknown. Here, we identify Glu and mammalian target of rapamycin complex 1 (mTORC1) as essential regulators of ISC expansion. The results showed that extracellular Glu promoted ISC expansion, indicated by increased intestinal organoid forming efficiency and budding efficiency as well as cell proliferation marker Ki67 immunofluorescence and differentiation marker Keratin 20 (KRT20) expression. Moreover, the insulin receptor (IR) mediating phosphorylation of the insulin receptor substrate (IRS) and downstream signaling phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway was involved in this response in ISCs. As expected, Glu-induced mTORC1 signaling activation was observed in the intestinal porcine enterocyte cell line (IPEC-J2), and Glu activated the PI3K/Akt/mTORC1 pathway. Accordingly, PI3K inhibition partially suppressed Glu-induced mTORC1 activation. In addition, Glu increased the phosphorylation levels of IR and IRS, and inhibiting IR downregulated the IRS/PI3K/Akt pathway. Collectively, our findings first indicate that extracellular Glu activates mTORC1 via the IR/IRS/PI3K/Akt pathway and stimulates ISC expansion, providing a new perspective for regulating the growth and health of the intestinal epithelium.


Assuntos
Ácido Glutâmico/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Mucosa Intestinal/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Células-Tronco/metabolismo , Animais , Proteínas Substratos do Receptor de Insulina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Receptor de Insulina/genética , Transdução de Sinais , Suínos
14.
Hypertension ; 74(4): 936-946, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31378107

RESUMO

Endothelial cells regulate vascular tone by producing both relaxing and contracting factors to control the local blood flow. Hypertension is a common side effect of mTORC1 (mammalian target of rapamycin complex 1) inhibitors. However, the role of endothelial mTORC1 in hypertension remains elusive. The present study aimed to determine the role of endothelial mTORC1 in Ang II (angiotensin II)-induced hypertension and the underlying mechanism. Endothelial mTORC1 activity was increased by Ang II both in vitro and in vivo. Blood pressure was higher in Tie-2-Cre-mediated regulatory associated protein of mTOR (mammalian target of rapamycin; Raptor) heterozygous-deficient (Tie2Cre-RaptorKD) mice than control mice both before and after Ang II infusion. Acetylcholine-evoked endothelium-dependent relaxation of mesenteric arteries was impaired in Tie2Cre-RaptorKD mice. Treatment with indomethacin or a specific COX (cyclooxygenase)-2 inhibitor, NS-398, but not L-NG-nitroarginine methyl ester reduced endothelium-dependent relaxation in Raptorflox/- mice to a similar extent as in Tie2Cre-RaptorKD mice. Metabolomic profiling revealed that the plasma content of prostaglandin E2 was reduced in Tie2Cre-RaptorKD mice with or without Ang II infusion. In endothelial cells, reduction of the protein level of YAP (yes-associated protein) with siRNA-mediated RPTOR deficiency was autophagy dependent and transcriptionally regulated the expression of COX-2 and mPGES-1 (microsomal prostaglandin E synthase-1). Hence, overexpression of YAP in endothelial cells enhanced the mRNA and protein levels of COX-2 and mPGES-1 and reversed the endothelial dysfunction and hypertension in Tie2Cre-RaptorKD mice. The present results demonstrate that suppression of mTORC1 activity in endothelial cells reduces prostaglandin E2 production and causes hypertension by reducing YAP-mediated COX-2/mPGES-1 expression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Pressão Sanguínea/fisiologia , Endotélio Vascular/metabolismo , Hipertensão/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais/fisiologia , Angiotensina II , Animais , Pressão Sanguínea/efeitos dos fármacos , Proteínas de Ciclo Celular , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Hipertensão/induzido quimicamente , Indometacina/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Camundongos , Prostaglandina-E Sintases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia
15.
Int J Mol Sci ; 20(15)2019 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-31382550

RESUMO

Reactive oxygen species (ROS) are highly reactive signaling molecules that maintain redox homeostasis in mammalian cells. Dysregulation of redox homeostasis under pathological conditions results in excessive generation of ROS, culminating in oxidative stress and the associated oxidative damage of cellular components. ROS and oxidative stress play a vital role in the pathogenesis of acute kidney injury and chronic kidney disease, and it is well documented that increased oxidative stress in patients enhances the progression of renal diseases. Oxidative stress activates autophagy, which facilitates cellular adaptation and diminishes oxidative damage by degrading and recycling intracellular oxidized and damaged macromolecules and dysfunctional organelles. In this review, we report the current understanding of the molecular regulation of autophagy in response to oxidative stress in general and in the pathogenesis of kidney diseases. We summarize how the molecular interactions between ROS and autophagy involve ROS-mediated activation of autophagy and autophagy-mediated reduction of oxidative stress. In particular, we describe how ROS impact various signaling pathways of autophagy, including mTORC1-ULK1, AMPK-mTORC1-ULK1, and Keap1-Nrf2-p62, as well as selective autophagy including mitophagy and pexophagy. Precise elucidation of the molecular mechanisms of interactions between ROS and autophagy in the pathogenesis of renal diseases may identify novel targets for development of drugs for preventing renal injury.


Assuntos
Lesão Renal Aguda/genética , Autofagia/genética , Estresse Oxidativo/genética , Insuficiência Renal Crônica/genética , Lesão Renal Aguda/patologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Fator 2 Relacionado a NF-E2/genética , Proteínas Quinases/genética , Proteínas de Ligação a RNA/genética , Espécies Reativas de Oxigênio/metabolismo , Insuficiência Renal Crônica/patologia
16.
Kobe J Med Sci ; 64(6): E200-E209, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-31327863

RESUMO

Mammalian target of rapamycin complex 1 (mTORC1), a protein complex containing the serine/threonine kinase mTOR, integrates various growth stimulating signals. mTORC1 is expressed in intestinal epithelial cells (IECs), whereas the physiological roles of this protein complex in homeostasis of IECs remain virtually unknown. We here generated mice, in which tuberous sclerosis complex 2 (Tsc2), a negative regulator of mTORC1, was specifically ablated in IECs (Tsc2 CKO mice). Ablation of Tsc2 enhanced the phosphorylation of mTORC1 downstream molecules such as ribosomal S6 protein and 4E-BP1 in IECs. Tsc2 CKO mice manifested the enhanced proliferative activity of IECs in intestinal crypts as well as the promoted migration of these cells along the crypt-villus axis. The mutant mice also manifested the increased apoptotic rate of IECs as well as the increased ectopic Paneth cells, which are one of the major differentiated IECs. In addition, in vitro study showed that ablation of Tsc2 promoted the development of intestinal organoids without epidermal growth factor, while mTORC1 inhibitor, rapamycin, diminished this phenotype. Our results thus suggest that Tsc2-mTORC1 signaling regulates the proliferation, migration, and positioning of IECs, and thereby contributes to the proper regulation of intestinal homeostasis.


Assuntos
Homeostase , Mucosa Intestinal/citologia , Animais , Proliferação de Células , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Camundongos , Transdução de Sinais/fisiologia , Proteína 2 do Complexo Esclerose Tuberosa/fisiologia
17.
Life Sci ; 232: 116665, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31323273

RESUMO

AIMS: Overexpression of the mechanistic target of rapamycin (mTOR), a member of the PIKK (phosphoinositide kinase-related kinase) family, protects cardiomyocytes from cell death induced by pathological stimuli such as ischemia. We previously reported that posttranslational modification of mTOR plays an important role in regulating cardiac mTOR expression. The aim of this study was to see if Tel2 (telomere maintenance 2), a protein that regulates the abundance of PIKKs, confers similar cardioprotective effects as mTOR. Tel2 is not well-characterized in cardiomyocytes, therefore we examined the effects of Tel2 on cardiomyocyte viability under ischemic stress conditions. MATERIALS AND METHODS: We overexpressed Tel2 or silenced Tel2 with siRNA in the HL-1 cardiomyocyte cell line to survey the effects of Tel2 overexpression and downregulation on cell survival during hypoxia. Adult mouse cardiomyocytes transfected with Tel2 adenoviruses were used to test whether Tel2 sufficiently prevented cardiomyocyte cell death against hydrogen peroxide (H2O2). KEY FINDINGS: Overexpressing Tel2 increased mTOR expression with a concomitant increase in mTOR Complex 1 (mTORC1) and mTORC2 activity in HL-1 cells. Tel2 deletion decreased mTOR expression, and mTORC1 and mTORC2 activity accordingly. In both HL-1 cells and adult mouse cardiomyocytes, Tel2 overexpression protected cardiomyocytes under ischemic stress. These effects were mTOR-dependent, as mTOR inhibitors blunted the effects of Tel2. While gene silencing of Tel2 did not affect cell survival under normoxia, Tel2 silencing made cardiomyocytes more vulnerable to cell death under hypoxia. SIGNIFICANCE: Upregulating Tel2 expression increases mTOR-mediated cardiomyocyte survival and targeting Tel2 could be another therapeutic strategy against ischemic heart disease.


Assuntos
Sobrevivência Celular/fisiologia , Miócitos Cardíacos/citologia , Proteínas de Ligação a Telômeros/fisiologia , Adenoviridae/genética , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Inativação Gênica , Peróxido de Hidrogênio/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Transdução de Sinais , Proteínas de Ligação a Telômeros/genética , Transfecção
18.
Nat Commun ; 10(1): 3194, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324799

RESUMO

Rapamycin, an inhibitor of mechanistic Target Of Rapamycin Complex 1 (mTORC1), extends lifespan and shows strong potential for the treatment of age-related diseases. However, rapamycin exerts metabolic and immunological side effects mediated by off-target inhibition of a second mTOR-containing complex, mTOR complex 2. Here, we report the identification of DL001, a FKBP12-dependent rapamycin analog 40x more selective for mTORC1 than rapamycin. DL001 inhibits mTORC1 in cell culture lines and in vivo in C57BL/6J mice, in which DL001 inhibits mTORC1 signaling without impairing glucose homeostasis and with substantially reduced or no side effects on lipid metabolism and the immune system. In cells, DL001 efficiently represses elevated mTORC1 activity and restores normal gene expression to cells lacking a functional tuberous sclerosis complex. Our results demonstrate that highly selective pharmacological inhibition of mTORC1 can be achieved in vivo, and that selective inhibition of mTORC1 significantly reduces the side effects associated with conventional rapalogs.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Animais , Linhagem Celular , Descoberta de Drogas , Expressão Gênica/efeitos dos fármacos , Humanos , Sistema Imunitário/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteômica , Transdução de Sinais/efeitos dos fármacos , Sirolimo/química , Serina-Treonina Quinases TOR , Esclerose Tuberosa
19.
Cell Mol Neurobiol ; 39(8): 1207-1215, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31342232

RESUMO

The KDEL (Lys-Asp-Glu-Leu) receptors (KDELRs), proteins with seven transmembrane domains, are primarily responsible for endoplasmic reticulum (ER) homeostasis. Recent studies have found additional function of KDELRs in growth, cellular secretory traffic, immune response, and autophagy; however, its role in tumorigenesis is still poorly understood. Here, we showed that KDELR2 is highly expressed in glioblastoma (GBM) tissues. Reviewing the expression of KDELR2 in TCGA and REMBRANDT database, we found that higher expression of KDELR2 is associated with shorter survival of GBM patients. We explored the effect of KDELR2 on tumorigenesis in GBM cells and animal model (nude mice), and identified KDELR2 as oncogene promoting cell proliferation. Additionally, KDELR2 expression in GBM cells correlated positively with HIF1alpha (HIF1α) expression, and we demonstrated by ChIP-qPCR and luciferase reporter assay that the upstream region of the KDELR2 gene is directly targeted by HIF1alpha. Taken together, our data suggest that KDELR2 is a target gene downstream of HIF1-alpha driving the malignancy of GBM and could eventually serve as a therapeutic target for the treatment of GBM patients.


Assuntos
Neoplasias Encefálicas/patologia , Carcinogênese/patologia , Glioblastoma/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Nus , Análise de Sobrevida , Transcrição Genética , Regulação para Cima/genética , Proteínas de Transporte Vesicular/genética
20.
Oncogene ; 38(35): 6270-6282, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31316145

RESUMO

The involvement of mammalian target of rapamycin (mTOR) in the positive regulation of oncogenesis has been well documented and thus mTOR has emerged as an attractive cancer therapeutic target. Although rapamycin and its analogues (rapalogs) are FDA-approved for the treatment of certain cancers, major success in targeting mTOR, particularly with new generation mTOR kinase inhibitors, for the effective treatment of cancers has not been achieved. Hence, a thorough understanding of the biology of the mTOR axis in cancer is still needed. It is now recognized that programmed death-ligand 1 (PD-L1) expression on cancer cells is a critical mechanism contributing to immunosuppression and immune escape via interacting with program death-1 (PD-1) on immune cells. This study has revealed a previously undiscovered role of the mTOR complex 1 (mTORC1)/p70 S6 kinase (p70S6K) in the negative regulation of PD-L1 on cancer cells and tissues. We demonstrate that disruption of this signaling pathway with mTOR inhibitors, raptor knockdown or p70S6K inhibitors elevated PD-L1 levels in some lung and other cancer cell lines. Elevation of PD-L1 by inhibition of mTORC1/p70S6K signaling is likely due to suppression of ß-TrCP-mediated proteasomal degradation of PD-L1, because inhibition of either mTORC1 or p70S6K facilitated ß-TrCP degradation accompanied with enhanced PD-L1 protein stabilization. Our current findings indicate the complexity of the mTOR axis in cancer, which should be considered when targeting this axis for effective cancer treatment. Our findings also suggest a strong scientific rationale for enhancing PD-1/PD-L1-targeted cancer immunotherapy through co-targeting mTORC1/p70S6K signaling.


Assuntos
Antígeno B7-H1/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Neoplasias/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Sirolimo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Células A549 , Antígeno B7-H1/genética , Benzoxazóis/farmacologia , Cicloeximida/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HEK293 , Humanos , Leupeptinas/farmacologia , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Morfolinas/farmacologia , Naftiridinas/farmacologia , Neoplasias/genética , Neoplasias/patologia , Estabilidade Proteica/efeitos dos fármacos , Pirimidinas/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Células Tumorais Cultivadas
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