RESUMEN
BACKGROUND: Renal allograft interstitial fibrosis/tubular atrophy (IF/TA) constitutes the principal histopathological characteristic of chronic allograft dysfunction (CAD) in kidney-transplanted patients. While renal vascular endothelial-mesenchymal transition (EndMT) has been verified as an important contributing factor to IF/TA in CAD patients, its underlying mechanisms remain obscure. Through single-cell transcriptomic analysis, we identified Rictor as a potential pivotal mediator for EndMT. This investigation sought to elucidate the role of Rictor/mTORC2 signalling in the pathogenesis of renal allograft interstitial fibrosis and the associated mechanisms. METHODS: The influence of the Rictor/mTOR2 pathway on renal vascular EndMT and renal allograft fibrosis was investigated by cell experiments and Rictor depletion in renal allogeneic transplantation mice models. Subsequently, a series of assays were conducted to explore the underlying mechanisms of the enhanced mitophagy and the ameliorated EndMT resulting from Rictor knockout. RESULTS: Our findings revealed a significant activation of the Rictor/mTORC2 signalling in CAD patients and allogeneic kidney transplanted mice. The suppression of Rictor/mTORC2 signalling alleviated TNFα-induced EndMT in HUVECs. Moreover, Rictor knockout in endothelial cells remarkably ameliorated renal vascular EndMT and allograft interstitial fibrosis in allogeneic kidney transplanted mice. Mechanistically, Rictor knockout resulted in an augmented BNIP3-mediated mitophagy in endothelial cells. Furthermore, Rictor/mTORC2 facilitated the MARCH5-mediated degradation of BNIP3 at the K130 site through K48-linked ubiquitination, thereby regulating mitophagy activity. Subsequent experiments also demonstrated that BNIP3 knockdown nearly reversed the enhanced mitophagy and mitigated EndMT and allograft interstitial fibrosis induced by Rictor knockout. CONCLUSIONS: Consequently, our study underscores Rictor/mTORC2 signalling as a critical mediator of renal vascular EndMT and allograft interstitial fibrosis progression, exerting its impact through regulating BNIP3-mediated mitophagy. This insight unveils a potential therapeutic target for mitigating renal allograft interstitial fibrosis.
Asunto(s)
Fibrosis , Trasplante de Riñón , Diana Mecanicista del Complejo 2 de la Rapamicina , Proteínas de la Membrana , Mitofagia , Proteína Asociada al mTOR Insensible a la Rapamicina , Transducción de Señal , Animales , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Ratones , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Trasplante de Riñón/efectos adversos , Fibrosis/metabolismo , Masculino , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Aloinjertos , Riñón/metabolismo , Riñón/patología , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Proteínas Proto-OncogénicasRESUMEN
RICTOR gene, which encodes the scaffold protein of mTORC2, can be amplified in various tumor types, including squamous cell carcinoma (SCC) of the lung. RICTOR amplification can lead to hyperactivation of mTORC2 and may serve as a targetable genetic alteration, including in lung SCC patients with no PD-L1 expression who are not expected to benefit from immune checkpoint inhibitor therapy. This study aimed to compare RICTOR amplification detected by fluorescence in situ hybridization (FISH) with Rictor and PD-L1 protein expression detected by immunohistochemistry (IHC) in SCC of the lung. The study was complemented by analysis of the publicly available Lung Squamous Cell Carcinoma (TCGA, Firehose legacy) dataset. RICTOR amplification was observed in 20% of our cases and 16% of the lung SCC cases of the TCGA dataset. Rictor and PD-L1 expression was seen in 74% and 44% of the cases, respectively. Rictor IHC showed two staining patterns: membrane staining (16% of the cases) and cytoplasmic staining (58% of the cases). Rictor membrane staining predicted RICTOR amplification as detected by FISH with high specificity (95%) and sensitivity (70%). We did not find any correlation between RICTOR amplification and PD-L1 expression; RICTOR amplification was detected in 18% and 26% of PD-L1 positive and negative cases, respectively. The TCGA dataset analysis showed similar results; RICTOR copy number correlated with Rictor mRNA and protein expression but showed no association with PD-L1 mRNA and protein expression. In conclusion, the correlation between RICTOR amplification and Rictor membrane staining suggests that the latter can potentially be used as a surrogate marker to identify lung SCC cases with RICTOR amplification. Since a significant proportion of PD-L1 negative SCC cases harbor RICTOR amplification, analyzing PD-L1 negative tumors by RICTOR FISH or Rictor IHC can help select patients who may benefit from mTORC2 inhibitor therapy.
Asunto(s)
Antígeno B7-H1 , Biomarcadores de Tumor , Carcinoma de Células Escamosas , Amplificación de Genes , Neoplasias Pulmonares , Proteína Asociada al mTOR Insensible a la Rapamicina , Humanos , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Femenino , Masculino , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Persona de Mediana Edad , Anciano , Hibridación Fluorescente in Situ/métodos , Pronóstico , Anciano de 80 o más AñosRESUMEN
Mutations in the phosphatase and tensin homolog (PTEN) gene are associated with severe neurodevelopmental disorders. Loss of PTEN leads to hyperactivation of the mechanistic target of rapamycin (mTOR), which functions in two distinct protein complexes, mTORC1 and mTORC2. The downstream signaling mechanisms that contribute to PTEN mutant phenotypes are not well delineated. Here, we show that pluripotent stem cell-derived PTEN mutant human neurons, neural precursors, and cortical organoids recapitulate disease-relevant phenotypes, including hypertrophy, electrical hyperactivity, enhanced proliferation, and structural overgrowth. PTEN loss leads to simultaneous hyperactivation of mTORC1 and mTORC2. We dissect the contribution of mTORC1 and mTORC2 by generating double mutants of PTEN and RPTOR or RICTOR, respectively. Our results reveal that the synergistic hyperactivation of both mTORC1 and mTORC2 is essential for the PTEN mutant human neural phenotypes. Together, our findings provide insights into the molecular mechanisms that underlie PTEN-related neural disorders and highlight novel therapeutic targets.
Asunto(s)
Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Neuronas , Organoides , Fosfohidrolasa PTEN , Humanos , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Organoides/metabolismo , Neuronas/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Mutación/genética , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Transducción de Señal , Proliferación Celular , Proteína Reguladora Asociada a mTOR/metabolismo , Proteína Reguladora Asociada a mTOR/genética , FenotipoRESUMEN
BACKGROUND: The main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF-mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM. METHODS: After analyzing The Cancer Genome Atlas MM patients' database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAFV600E MM cell lines on their response to BRAF/MEKi. We performed proteomic screening to identify proteins modulated by changes in RICTOR expression, and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. RESULTS: Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAFV600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi. CONCLUSIONS: Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase of BRAFV600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low RICTOR tumors. Importantly, our findings indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.
Asunto(s)
Resistencia a Antineoplásicos , Diana Mecanicista del Complejo 2 de la Rapamicina , Melanoma , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas B-raf , Proteína Asociada al mTOR Insensible a la Rapamicina , Animales , Humanos , Ratones , Línea Celular Tumoral , Regulación hacia Abajo , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Melanoma/genética , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteómica/métodos , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidoresRESUMEN
BACKGROUND: The mTOR (mechanistic target of rapamycin) is an essential regulator of fundamental biological processes. mTOR forms 2 distinct complexes, mTORC1 (mTOR complex 1) when it binds with RAPTOR (Regulatory-associated Protein of mTOR) and mTORC2 (mTOR complex 2) when it associates with RICTOR (Rapamycin-insesitive companion of mTOR). Due to the previous link between the mTOR pathway, aldosterone, and blood pressure (BP), we anticipated that variants in the mTOR complex might be associated with salt-sensitive BP. METHODS: BP and other parameters were assessed after a one-week liberal Na+ (200 mmol/d) and a one-week restricted Na+ (10 mmol/d) diet in 608 White subjects from the Hypertensive Pathotype cohort, single-nucleotide variants in MTOR, RPTOR, and RICTOR genes were obtained for candidate genes analyses. RESULTS: The analysis revealed a significant association between a single nucleotide variants within the RPTOR gene and BP. Individuals carrying the RPTOR rs9901846 homozygous risk allele (AA) and heterozygous risk allele (GA) exhibited a 5 mm Hg increase in systolic BP on a liberal diet compared with nonrisk allele individuals (GG), but only in women. This single nucleotide variants effect was more pronounced on the restricted diet and present in both sexes, with AA carriers having a 9 mm Hg increase and GA carriers having a 5 mm Hg increase in systolic BP compared with GG. Interestingly, there were no significant associations between MTOR or RICTOR gene variants and BP. CONCLUSIONS: The RPTOR gene variation is associated with elevated BP in White participants, regardless of salt intake, specifically in females.
Asunto(s)
Presión Sanguínea , Hipertensión , Proteína Reguladora Asociada a mTOR , Cloruro de Sodio Dietético , Femenino , Humanos , Masculino , Proteínas Portadoras/genética , Hipertensión/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Nucleótidos/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Proteína Reguladora Asociada a mTOR/genética , Proteína Reguladora Asociada a mTOR/metabolismo , Sirolimus , Cloruro de Sodio Dietético/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Población BlancaRESUMEN
The increasing knowledge of molecular alterations in malignancies, including mutations and regulatory failures in the mTOR (mechanistic target of rapamycin) signaling pathway, highlights the importance of mTOR hyperactivity as a validated target in common and rare malignancies. This review summarises recent findings on the characterization and prognostic role of mTOR kinase complexes (mTORC1 and mTORC2) activity regarding differences in their function, structure, regulatory mechanisms, and inhibitor sensitivity. We have recently identified new tumor types with RICTOR (rapamycin-insensitive companion of mTOR) amplification and associated mTORC2 hyperactivity as useful potential targets for developing targeted therapies in lung cancer and other newly described malignancies. The activity of mTOR complexes is recommended to be assessed and considered in cancers before mTOR inhibitor therapy, as current first-generation mTOR inhibitors (rapamycin and analogs) can be ineffective in the presence of mTORC2 hyperactivity. We have introduced and proposed a marker panel to determine tissue characteristics of mTOR activity in biopsy specimens, patient materials, and cell lines. Ongoing phase trials of new inhibitors and combination therapies are promising in advanced-stage patients selected by genetic alterations, molecular markers, and/or protein expression changes in the mTOR signaling pathway. Hopefully, the summarized results, our findings, and the suggested characterization of mTOR activity will support therapeutic decisions.
Asunto(s)
Neoplasias Pulmonares , Serina-Treonina Quinasas TOR , Humanos , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Sirolimus/farmacología , Factores de Transcripción/metabolismoRESUMEN
Dysfunction of the mechanistic target of rapamycin (mTOR) signaling pathway is implicated in neuropsychiatric disorders including depression and anxiety. Most studies have been focusing on neurons, and the function of mTOR signaling pathway in astrocytes is less investigated. mTOR forms two distinct complexes, mTORC1 and mTORC2, with key scaffolding protein Raptor and Rictor, respectively. The ventral tegmental area (VTA), a vital component of the brain reward system, is enrolled in regulating both depression and anxiety. In the present study, we aimed to examine the regulation effect of VTA astrocytic mTOR signaling pathway on depression and anxiety. We specifically deleted Raptor or Rictor in VTA astrocytes in mice and performed a series of behavioral tests for depression and anxiety. Deletion of Raptor and Rictor both decreased the immobility time in the tail suspension test and the latency to eat in the novelty suppressed feeding test, and increased the horizontal activity and the movement time in locomotor activity. Deletion of Rictor decreased the number of total arm entries in the elevated plus-maze test and the vertical activity in locomotor activity. These data suggest that VTA astrocytic mTORC1 plays a role in regulating depression-related behaviors and mTORC2 is involved in both depression and anxiety-related behaviors. Our results indicate that VTA astrocytic mTOR signaling pathway might be new targets for the treatment of psychiatric disorders.
Asunto(s)
Astrocitos , Área Tegmental Ventral , Humanos , Ratones , Animales , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Área Tegmental Ventral/metabolismo , Astrocitos/metabolismo , Depresión , Complejos Multiproteicos/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Proteínas Portadoras/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína Reguladora Asociada a mTOR/metabolismo , Factores de Transcripción/metabolismo , AnsiedadRESUMEN
The mineralocorticoid receptor (MR) is a transcription factor for genes mediating diverse, cell-specific functions, including trophic effects as well as promoting fluid/electrolyte homeostasis. It was reported that in intercalated cells, phosphorylation of the MR at serine 843 (S843) by Unc-51-like kinase (ULK1) inhibits MR activation and that phosphorylation of ULK1 by mechanistic target of rapamycin (mTOR) inactivates ULK1, and thereby prevents MR inactivation. We extended these findings with studies in M1 mouse cortical collecting duct cells stably expressing the rat MR and a reporter gene. Pharmacological inhibition of ULK1 dose-dependently increased ligand-induced MR transactivation, while ULK1 activation had no effect. Pharmacological inhibition of mTOR and CRISPR/gRNA gene knockdown of rapamycin-sensitive adapter protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) decreased phosphorylated ULK1 and ligand-induced activation of the MR reporter gene, as well as transcription of endogenous MR-target genes. As predicted, ULK1 inhibition had no effect on aldosterone-mediated transcription in M1 cells with the mutated MR-S843A (alanine cannot be phosphorylated). In contrast, mTOR inhibition dose-dependently decreased transcription in the MR-S843A cells, though not as completely as in cells with the wild-type MR-S843. mTOR, Raptor, and Rictor coprecipitated with the MR and addition of aldosterone increased their phosphorylated, active state. These results suggest that mTOR significantly regulates MR activity in at least 2 ways: by suppressing MR inactivation by ULK1, and by a yet ill-defined mechanism that involves direct association with MR. They also provide new insights into the diverse functions of ULK1 and mTOR, 2 key enzymes that monitor the cell's energy status.
Asunto(s)
Aldosterona , Receptores de Mineralocorticoides , Animales , Ratones , Ratas , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Ligandos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Complejos Multiproteicos/metabolismo , Fosforilación , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Proteína Reguladora Asociada a mTOR , ARN Guía de Sistemas CRISPR-Cas , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Cardiomyocyte apoptosis and cardiac fibrosis are the leading causes of mortality in patients with ischemic heart disease. As such, these processes represent potential therapeutic targets to treat heart failure resulting from ischemic insult. We previously demonstrated that the mitochondrial acetyltransferase protein GCN5L1 regulates cardiomyocyte cytoprotective signaling in ischemia-reperfusion injury in vivo and hypoxia-reoxygenation injury in vitro. The current study investigated the mechanism underlying GCN5L1-mediated regulation of the Akt/mTORC2 cardioprotective signaling pathway. Rictor protein levels in cardiac tissues from human ischemic heart disease patients were significantly decreased relative to non-ischemic controls. Rictor protein levels were similarly decreased in cardiac AC16 cells following hypoxic stress, while mRNA levels remained unchanged. The reduction in Rictor protein levels after hypoxia was enhanced by the knockdown of GCN5L1, and was blocked by GCN5L1 overexpression. These findings correlated with changes in Rictor lysine acetylation, which were mediated by GCN5L1 acetyltransferase activity. Rictor degradation was regulated by proteasomal activity, which was antagonized by increased Rictor acetylation. Finally, we found that GCN5L1 knockdown restricted cytoprotective Akt signaling, in conjunction with decreased mTOR abundance and activity. In summary, these studies suggest that GCN5L1 promotes cardioprotective Akt/mTORC2 signaling by maintaining Rictor protein levels through enhanced lysine acetylation.
Asunto(s)
Isquemia Miocárdica , Proteínas Proto-Oncogénicas c-akt , Humanos , Acetilación , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Hipoxia/metabolismo , Lisina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteínas Mitocondriales/metabolismo , Isquemia Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Mutations in TP53 gene is considered a main driver of hepatocellular carcinoma (HCC). While TP53 mutations are the leading cause of p53 dysfunction, their occurrence rates may drop to approximately 10% in cohorts without hepatitis B virus and aflatoxin exposure. This observation suggests that the deactivation of wild-type p53 (p53wt) may be a critical factor in the majority of HCC cases. However, the mechanism undermining p53wt activity in the liver remains unclear. METHODS: Microarray analysis and luciferase assay were utilized to confirm target associations. Gain- and/or loss-of-function methods were employed to assess alterations in signaling pathways. Protein interactions were analyzed by molecular immunological methods and further visualized by confocal microscopy. Bioinformatic analysis was performed to analyze clinical significance. Tumor xenograft nude mice were used to validate the findings in vivo. RESULTS: Our study highlights the oncogenic role of Rictor, a key component of the mammalian target of rapamycin complex 2 (mTORC2), in hepatocytes. Rictor exerts its oncogenic function by binding to p53wt and subsequently blocking p53wt activity based on p53 status, requiring the involvement of mTOR. Moreover, we observed a dynamic nucleocytoplasmic distribution pattern of Rictor, characterized by its translocation from the nucleus (in precancerous lesions) to the cytoplasm (in HCCs) during malignant transformation. Notably, Rictor is directly targeted by the liver-enriched microRNA miR-192, and the disruption of the miR-192-Rictor-p53-miR-192 signaling axis was consistently observed in both human and rat HCC models. Clinical analysis associated lower miR-192/higher Rictor with shorter overall survival and more advanced clinical stages (P < 0.05). In mice, xenograft tumors overexpressing miR-192 exhibited lower Rictor expression levels, leading to higher p53 activity, and these tumors displayed slower growth compared to untreated HCC cells. CONCLUSIONS: Rictor dynamically shuttles between the nucleus and cytoplasm during HCC development. Its pivotal oncogenic role involves binding and inhibiting p53wt activity within the nucleus in early hepatocarcinogenesis. Targeting Rictor presents a promising strategy for HCC based on p53 status.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroARNs , Proteína Asociada al mTOR Insensible a la Rapamicina , Animales , Humanos , Ratones , Ratas , Carcinogénesis/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Genes p53 , Hepatocitos/patología , Neoplasias Hepáticas/patología , Ratones Desnudos , MicroARNs/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismoRESUMEN
Alterations in mTOR signalling molecules, including RICTOR amplification, have been previously described in many cancers, particularly associated with poor prognosis. In this study, RICTOR copy number variation (CNV) results of diagnostic next-generation sequencing (NGS) were analysed in 420 various human malignant tissues. RICTOR amplification was tested by Droplet Digital PCR (ddPCR) and validated using the "gold standard" fluorescence in situ hybridisation (FISH). Additionally, the consequences of Rictor protein expression were also studied by immunohistochemistry. RICTOR amplification was presumed in 37 cases with CNV ≥ 3 by NGS, among these, 16 cases (16/420; 3.8%) could be validated by FISH, however, ddPCR confirmed only 11 RICTOR-amplified cases with lower sensitivity. Based on these, neither NGS nor ddPCR could replace traditional FISH in proof of RICTOR amplification. However, NGS could be beneficial to highlight potential RICTOR-amplified cases. The obtained results of the 14 different tumour types with FISH-validated RICTOR amplification demonstrate the importance of RICTOR amplification in a broad spectrum of tumours. The newly described RICTOR-amplified entities could initiate further collaborative studies with larger cohorts to analyse the prevalence of RICTOR amplification in rare diseases. Finally, our and further work could help to improve and expand future therapeutic opportunities for mTOR-targeted therapies.
Asunto(s)
Variaciones en el Número de Copia de ADN , Neoplasias , Humanos , Neoplasias/genética , Serina-Treonina Quinasas TOR/genética , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Amplificación de GenesRESUMEN
The mechanistic target of rapamycin (mTOR) kinase is a central regulator of cell growth and metabolism. It is the catalytic subunit of two distinct large protein complexes, mTOR complex 1 (mTORC1) and mTORC2. mTOR activity is subjected to tight regulation in response to external nutrition and growth factor stimulation. As an important mechanism of signaling transduction, the 'second messenger' cyclic nucleotides including cAMP and cGMP and their associated cyclic nucleotide-dependent kinases, including protein kinase A (PKA) and protein kinase G (PKG), play essential roles in mediating the intracellular action of a variety of hormones and neurotransmitters. They have also emerged as important regulators of mTOR signaling in various physiological and disease conditions. However, the mechanism by which cAMP and cGMP regulate mTOR activity is not completely understood. In this review, we will summarize the earlier work establishing the ability of cAMP to dampen mTORC1 activation in response to insulin and growth factors and then discuss our recent findings demonstrating the regulation of mTOR signaling by the PKA- and PKG-dependent signaling pathways. This signaling framework represents a new non-canonical regulation of mTOR activity that is independent of AKT and could be a novel mechanism underpinning the action of a variety of G protein-coupled receptors that are linked to the mTOR signaling network. We will further review the implications of these signaling events in the context of cardiometabolic disease, such as obesity, non-alcoholic fatty liver disease, and cardiac remodeling. The metabolic and cardiac phenotypes of mouse models with targeted deletion of Raptor and Rictor, the two essential components for mTORC1 and mTORC2, will be summarized and discussed.
Asunto(s)
Enfermedades Cardiovasculares , Complejos Multiproteicos , Sirolimus , Serina-Treonina Quinasas TOR , Animales , Ratones , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Complejos Multiproteicos/metabolismo , Nucleótidos Cíclicos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Group 3 innate lymphoid cells (ILC3s) are mediators of intestinal immunity and barrier function. Recent studies have investigated the role of the mammalian target of rapamycin complex (mTOR) in ILC3s, whereas the mTORC1-related mechanisms and crosstalk between mTORC1 and mTORC2 involved in regulating ILC3 homeostasis remain unknown. In this study, we found that mTORC1 but not mTORC2 was critical in ILC3 development, IL-22 production, and ILC3-mediated intestinal homeostasis. Single-cell RNA sequencing revealed that mTORC1 deficiency led to disruption of ILC3 heterogeneity, showing an increase in differentiation into ILC1-like phenotypes. Mechanistically, mTORC1 deficiency decreased the expression of NFIL3, which is a critical transcription factor responsible for ILC3 development. The activities of both mTORC1 and mTORC2 were increased in wild-type ILC3s after activation by IL-23, whereas inhibition of mTORC1 by Raptor deletion or rapamycin treatment resulted in increased mTORC2 activity. Previous studies have demonstrated that S6K, the main downstream target of mTORC1, can directly phosphorylate Rictor to dampen mTORC2 activity. Our data found that inhibition of mTORC1 activity by rapamycin reduced Rictor phosphorylation in ILC3s. Reversing the increased mTORC2 activity via heterozygous or homozygous knockout of Rictor in Raptor-deleted ILC3s resulted in severe ILC3 loss and complete susceptibility to intestinal infection in mice with mTORC1 deficiency (100% mortality). Thus, mTORC1 acts as a rheostat of ILC3 heterogeneity, and mTORC2 protects ILC3s from severe loss of cells and immune activity against intestinal infection when mTORC1 activity is diminished.
Asunto(s)
Inmunidad Innata , Linfocitos , Ratones , Animales , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Proteína Reguladora Asociada a mTOR/genética , Factores de Transcripción/metabolismo , Sirolimus/farmacología , Mamíferos/metabolismoRESUMEN
The importance of the network defined by phosphatidylinositol-3-kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) downstream of Receptor Tyrosine Kinase (RTK) has been recognized for many years. However, the central role of RICTOR (rapamycin-insensitive companion of mTOR) in this pathway has only recently come to light. The function of RICTOR in pan-cancer still needs to be systematically elucidated. In this study, we examined RICTOR's molecular characteristics and clinical prognostic value by pan-cancer analysis. Our findings indicate that RICTOR was overexpressed in twelve cancer types, and a high RICTOR expression was linked to poor overall survival. Moreover, the CRISPR Achilles' knockout analysis revealed that RICTOR was a critical gene for the survival of many tumor cells. Function analysis revealed that RICTOR-related genes were mainly involved in TOR signaling and cell growth. We further demonstrated that the RICTOR expression was significantly influenced by genetic alteration and DNA-methylation in multiple cancer types. Additionally, we found a positive relationship between RICTOR expression and the immune infiltration of macrophages and cancer-associated fibroblasts in Colon adenocarcinoma and Head and Neck squamous cell carcinoma. Finally, we validated the ability of RICTOR in sustaining tumor growth and invasion in the Hela cell line using cell-cycle analysis, the cell proliferation assay, and wound-healing assay. Our pan-cancer analysis highlights the critical role of RICTOR in tumor progression and its potential as a prognostic marker for various cancer types.
Asunto(s)
Adenocarcinoma , Neoplasias del Colon , Humanos , Células HeLa , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/metabolismo , Sirolimus , PronósticoRESUMEN
Ingestion of arsenic interferes with spermatogenesis and increases the risk of male infertility, but the underlying mechanism remines unclear. In this study, we investigated spermatogenic injury with a focus on blood-testis barrier (BTB) disruption by administrating 5 mg/L and 15 mg/L arsenic orally to adult male mice for 60 d. Our results showed that arsenic exposure reduced sperm quality, altered testicular architecture, and impaired Sertoli cell junctions at the BTB. Analysis of BTB junctional proteins revealed that arsenic intake downregulated Claudin-11 expression and increased protein levels of ß-catenin, N-cadherin, and Connexin-43. Aberrant localization of these membrane proteins was also observed in arsenic-treated mice. Meanwhile, arsenic exposure altered the components of Rictor/mTORC2 pathway in mouse testis, including inhibition of Rictor expression, reduced phosphorylation of protein kinase Cα (PKCα) and protein kinase B (PKB), and elevated matrix metalloproteinase-9 (MMP-9) levels. Furthermore, arsenic also induced testicular lipid peroxidative damage, inhibited antioxidant enzyme (T-SOD) activity, and caused glutathione (GSH) depletion. Our findings suggest that disruption of BTB integrity is one of the main factors responsible for the decline in sperm quality caused by arsenic. PKCα-mediated rearrangement of actin filaments and PKB/MMP-9-increased barrier permeability jointly contribute to arsenic-induced BTB disruption.
Asunto(s)
Arsénico , Ratones , Masculino , Animales , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Arsénico/toxicidad , Arsénico/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteína Quinasa C-alfa/metabolismo , Barrera Hematotesticular/metabolismo , Semen , Testículo/metabolismo , Espermatogénesis , Factores de Transcripción/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismoRESUMEN
The mTORC2 pathway plays a critical role in promoting tumor progression in human colorectal cancer (CRC). The regulatory mechanisms for this signaling pathway are only partially understood. We previously identified UBXN2A as a novel tumor suppressor protein in CRCs and hypothesized that UBXN2A suppresses the mTORC2 pathway, thereby inhibiting CRC growth and metastasis. We first used murine models to show that haploinsufficiency of UBXN2A significantly increases colon tumorigenesis. Induction of UBXN2A reduces AKT phosphorylation downstream of the mTORC2 pathway, which is essential for a plethora of cellular processes, including cell migration. Meanwhile, mTORC1 activities remain unchanged in the presence of UBXN2A. Mechanistic studies revealed that UBXN2A targets Rictor protein, a key component of the mTORC2 complex, for 26S proteasomal degradation. A set of genetic, pharmacological, and rescue experiments showed that UBXN2A regulates cell proliferation, apoptosis, migration, and colon cancer stem cells (CSCs) in CRC. CRC patients with a high level of UBXN2A have significantly better survival, and high-grade CRC tissues exhibit decreased UBXN2A protein expression. A high level of UBXN2A in patient-derived xenografts and tumor organoids decreases Rictor protein and suppresses the mTORC2 pathway. These findings provide new insights into the functions of an ubiquitin-like protein by inhibiting a dominant oncogenic pathway in CRC.
Asunto(s)
Neoplasias del Colon , Humanos , Ratones , Animales , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Neoplasias del Colon/patología , Línea Celular Tumoral , Células Madre Neoplásicas/patología , Transducción de Señal , Factores de Transcripción/genética , Carcinogénesis/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ubiquitinas/metabolismoRESUMEN
Pharmacological vitamin C (VC) is a potential natural compound for cancer treatment. However, the mechanism underlying its antitumor effects remains unclear. In this study, we found that pharmacological VC significantly inhibits the mTOR (including mTORC1 and mTORC2) pathway activation and promotes GSK3-FBXW7-mediated Rictor ubiquitination and degradation by increasing the cellular ROS. Moreover, we identified that HMOX1 is a checkpoint for pharmacological-VC-mediated mTOR inactivation, and the deletion of FBXW7 or HMOX1 suppresses the regulation of pharmacological VC on mTOR activation, cell size, cell viability, and autophagy. More importantly, it was observed that the inhibition of mTOR by pharmacological VC supplementation in vivo produces positive therapeutic responses in tumor growth, while HMOX1 deficiency rescues the inhibitory effect of pharmacological VC on tumor growth. These results demonstrate that VC influences cellular activities and tumor growth by inhibiting the mTOR pathway through Rictor and HMOX1, which may have therapeutic potential for cancer treatment.
Asunto(s)
Ácido Ascórbico , Neoplasias , Humanos , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Ácido Ascórbico/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Factores de Transcripción/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismoRESUMEN
The existing studies by our team demonstrated the pro-recovery effect of 3-Acetyl-11-keto-beta-boswellic acid (AKBA) on a sciatic nerve injury. To further investigate the role of AKBA in peripheral nerve injury repair, The TMT quantitative proteomics technique was used to obtain differentially significant proteins in a Sham group, Model group, and AKBA group. After that, three time points (5, 14, and 28 d) and four groups (Sham + AKBA, Sham, Model, and AKBA) were set up, and immunoblotting, immunofluorescence, and cellular assays were applied to investigate the expression of CDC42, Rac1, RhoA, and Rictor in the sciatic nerve at different time points for each group in more depth. The results showed that AKBA enriched the cellular components of the myelin sheath and axon regeneration after a sciatic nerve injury and that AKBA upregulated CDC42 and Rac1 and downregulated RhoA expression 5 d after a sciatic nerve injury, promoting axon regeneration and improving the repair of a sciatic nerve injury in rats. Rictor is regulated by AKBA and upregulated in PC12 cells after AKBA action. Our findings provide a new basis for AKBA treatment of a peripheral nerve injury.
Asunto(s)
Traumatismos de los Nervios Periféricos , Neuropatía Ciática , Animales , Ratas , Axones , Regeneración Nerviosa/fisiología , Proteína Asociada al mTOR Insensible a la Rapamicina , Nervio Ciático/lesiones , Neuropatía Ciática/tratamiento farmacológicoRESUMEN
Non-small cell lung cancer (NSCLC) is one of the deadliest cancers in the world. Metastasis is considered one of the leading causes of treatment failure and death in NSCLC patients. A crucial factor of promoting metastasis in epithelium-derived carcinoma has been considered as epithelial-mesenchymal transition (EMT). Rictor, one of the components of mTORC2, has been reportedly involved in EMT and metastasis of human malignancies. However, the regulatory mechanisms of Rictor, Rictor-mediated EMT and metastasis in cancers remain unknown. Our present study indicates that Rictor is highly expressed in human NSCLC cell lines and tissues and is regulated, at least partially, at the transcriptional level. Knockdown of Rictor expression causes phenotype alterations through EMT, which is accompanied by the impairment of migration and invasion ability in NSCLC cells. Additionally, we have cloned and identified the human Rictor core promoter for the first time and confirmed that transcription factor KLF4 directly binds to the Rictor promoter and transcriptionally upregulated Rictor expression. Knockdown of KLF4 results in Rictor's downregulation accompanied by a series of characteristic changes of mesenchymal-epithelial transition (MET) and significantly decreases migration, invasion as well as metastasis of NSCLC cells. Re-introducing Rictor in KLF4-knockdown NSCLC cells partially reverses the epithelial phenotype to the mesenchymal phenotype and attenuates the inhibition of cell migration and invasion caused by KLF4 knocking down. Knockdown of KLF4 prevents mTOR/Rictor interaction and metastasis of NSCLC in vivo. The understanding of the regulator upstream of Rictor may provide an opportunity for the development of new inhibitors and the rational design of combination regimens based on different metastasis-related molecular targets and finally prevents cancer metastasis.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Proteína Asociada al mTOR Insensible a la Rapamicina , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Humanos , Factor 4 Similar a Kruppel/genética , Neoplasias Pulmonares/patología , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2-interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain-of-function and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase.