RESUMEN
L-type amino acid transporter 1 (LAT1) is upregulated in various cancer types and contributes to disease progression. Previous studies have demonstrated or suggested that hypoxia-inducible factors (HIFs), the key transcription factors in hypoxic responses, control the expression of LAT1 gene in several types of cancer cells. However, this regulatory relationship has not been investigated yet in colorectal cancer (CRC), one of the cancer types in which the increased LAT1 expression holds prognostic significance. In this study, we found that neither LAT1 mRNA nor protein is induced under hypoxic condition (1% O2) in CRC HT-29 cells in vitro, regardless of the prominent HIF-1/2α accumulation and HIFs-dependent upregulation of glucose transporter 1. The hypoxic treatment generally did not increase either the mRNA or protein expression of LAT1 in eight CRC cell lines tested, in contrast to the pronounced upregulation by amino acid restriction. Interestingly, knockdown of von Hippel-Lindau ubiquitin ligase to inhibit the proteasomal degradation of HIFs caused an accumulation of HIF-2α and increased the LAT1 expression in certain CRC cell lines. This study contributes to delineating the molecular mechanisms responsible for the pathological expression of LAT1 in CRC cells, emphasizing the ambiguity of HIFs-dependent transcriptional upregulation of LAT1 across cancer cells.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Neoplasias Colorrectales , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia , Transportador de Aminoácidos Neutros Grandes 1 , Humanos , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células HT29 , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Línea Celular Tumoral , Hipoxia de la Célula , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Transportador de Glucosa de Tipo 1/metabolismo , Transportador de Glucosa de Tipo 1/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia ArribaRESUMEN
Claudin-1 (CLDN1) is highly expressed in human lung adenocarcinoma-derived A549 cells and is involved in the augmentation of chemoresistance. However, the mechanism of chemoresistance is not fully understood. In the tumor microenvironment, cancer cells are exposed to stress conditions such as hypoxia and malnutrition. Here, we investigated the effect of CLDN1 expression on amino acid (AA) flux and chemoresistance using A549 cells. The expression of L-type AA transporters, LAT1 and LAT3, was decreased by CLDN1 silencing in A549 spheroids. A reduction in extracellular AA concentration increased the expression of these AA transporters in two-dimensional (2D) cultured cells. The paracellular AA flux except for Ser, Thr, Tyr, Ala, and Gly was enhanced by CLDN1 silencing. These results suggest that CLDN1 forms a paracellular barrier to some AAs, leading to the elevation of LAT1/3 expression in spheroids. The production of reactive oxygen species in the mitochondria and cytosol was decreased by CLDN1 silencing in spheroids, resulting in downregulation of the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target antioxidant genes. CLDN1 silencing enhanced the cytotoxicity of anticancer drugs including doxorubicin and cisplatin, which was blocked by sulforaphane, an inducer of Nrf2 signaling. Similarly, the anticancer-induced toxicity was enhanced by Nrf2 silencing. In 2D cultured cells, the anticancer-induced toxicity was attenuated by AA deficiency and sulforaphane. We suggest that CLDN1 forms an AA barrier in spheroids, leading to the augmentation of Nrf2-dependent chemoresistance in A549 cells.
Asunto(s)
Adenocarcinoma del Pulmón , Claudina-1 , Resistencia a Antineoplásicos , Neoplasias Pulmonares , Humanos , Células A549 , Claudina-1/metabolismo , Claudina-1/genética , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Aminoácidos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Esferoides Celulares/metabolismo , Esferoides Celulares/efectos de los fármacos , Silenciador del GenRESUMEN
Systemic sclerosis (SSc) is a chronic autoimmune connective tissue disease. Vascular damage is one of the important features of SSc, which affects the progression and prognosis of the disease. MiR-126-3p is an important microRNA (miRNA) that regulates vascular structure and function, which can be transported through exosomes. However, the role of miR-126-3p in vascular damage in SSc is still unclear. Therefore, we focused on the connection between miR-126-3p and vascular damage in SSc, as well as investigated the potential role of miR-126-3p in vascular damage in SSc. First, this study successfully extracted extracellular vesicles from clinical plasma samples and characterized the exosomes within them. Then, we predicted and screened the target pathway mammalian/mechanistic target of rapamycin (mTOR) and the target gene SLC7A5 of miR-126-3p through online databases. Next, we constructed SSc mice for in vivo studies. The results showed that the expression of miR-126-3p was decreased in the plasma exosomes, while the SLC7A5 expression, autophagy, and lipid peroxidation were increased in the aorta. Luciferase reporter gene assays demonstrated that miR-126-3p can bind to SLC7A5, resulting in a decrease in its expression. In vitro experiments have shown that exosomal miR-126-3p can be internalized by human umbilical vein endothelial cells (HUVECs). The miR-126-3p group exhibited enhanced cell viability and tube formation ability, along with increased expression of the vascular formation marker CD31. Additionally, miR-126-3p downregulated the protein expression of SLC7A5 and LC3 in HUVECs, while upregulating the protein expression of mTOR, P62, PPARγ, and CPT-1. However, the effects of miR-126-3p on HUVECs were counteracted by mTOR inhibitors and enhanced by mTOR activators. The results indicated that exosomal miR-126-3p has the potential to protect against vascular injury in SSc by regulating the SLC7A5/mTOR signalling pathway in HUVECs.
Asunto(s)
Exosomas , Células Endoteliales de la Vena Umbilical Humana , MicroARNs , Transducción de Señal , Serina-Treonina Quinasas TOR , MicroARNs/genética , MicroARNs/metabolismo , Humanos , Exosomas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Ratones , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/patología , Masculino , Femenino , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Persona de Mediana Edad , Modelos Animales de Enfermedad , AdultoRESUMEN
Breast cancer is one of the most common malignant tumors worldwide. SLC7A2 is abnormally expressed in multiple cancers. However, its potential in triple negative breast cancer (TNBC) is still unclear. The purpose of this study was to investigate the roles of SLC7A2 and its underlying molecular mechanisms in TNBC. mRNA expression was detected by RT-qPCR. Protein expression was detected by western blot. Co-localization of ACOX1 and TCF1 was determined using FISH assay. Histone crotonylation was performed using in vitro histone crotonylation assay. Functional analysis was performed using CCK-8 and flow cytometry assays. Xenograft assay was conducted to further verify the role of SLC7A2 in TNBC. CD8A expression was detected using immunohistochemistry. We found that SLC7A2 is downregulated in TNBC tumors. Low levels are associated with advanced stages and lymph node metastasis. SLC7A2 expression is positively correlated with CD8A. SLC7A2-mediated lysine catabolism drives the activation of CD8+ T cells. Moreover, SLC7A2 promotes histone crotonylation via upregulating ACOX1. It also promotes interaction between ACOX1 and TCF1, thus promoting antitumor T cell immunity. Additionally, overexpression of SLC7A2 activates CD8+ T cells and enhances the chemosensitivity of anti-PD-1 therapies in vivo. In conclusion, SLC7A2 may function as an antitumor gene in TNBC by activating antitumor immunity, suggesting SLC7A2/ACOX1/TCF1 signaling as a promising therapeutic strategy.
Asunto(s)
Lisina , Neoplasias de la Mama Triple Negativas , Animales , Femenino , Humanos , Ratones , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Lisina/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Upper aerodigestive squamous cell carcinoma (UASCC) is a common and aggressive malignancy with few effective therapeutic options. Here, we investigate amino acid metabolism in this cancer, surprisingly noting that UASCC exhibits the highest methionine level across all human cancers, driven by its transporter LAT1. We show that LAT1 is also expressed at the highest level in UASCC, transcriptionally activated by UASCC-specific promoter and enhancers, which are directly coregulated by SCC master regulators TP63/KLF5/SREBF1. Unexpectedly, unbiased bioinformatic screen identifies EZH2 as the most significant target downstream of the LAT1-methionine pathway, directly linking methionine metabolism to epigenomic reprogramming. Importantly, this cascade is indispensable for the survival and proliferation of UASCC patient-derived tumor organoids. In addition, LAT1 expression is closely associated with cellular sensitivity to inhibition of the LAT1-methionine-EZH2 axis. Notably, this unique LAT1-methionine-EZH2 cascade can be targeted effectively by either pharmacological approaches or dietary intervention in vivo. In summary, this work maps a unique mechanistic cross talk between epigenomic reprogramming with methionine metabolism, establishes its biological significance in the biology of UASCC, and identifies a unique tumor-specific vulnerability which can be exploited both pharmacologically and dietarily.
Asunto(s)
Carcinoma de Células Escamosas , Regulación Neoplásica de la Expresión Génica , Transportador de Aminoácidos Neutros Grandes 1 , Metionina , Metionina/metabolismo , Humanos , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Línea Celular Tumoral , Epigénesis Genética , Epigenómica/métodos , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Ratones , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Animales , Proliferación Celular , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Reprogramación Celular/genéticaRESUMEN
Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Supervivencia Celular , Ganglios Espinales , Compuestos de Metilmercurio , Células de Schwann , Animales , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Compuestos de Metilmercurio/toxicidad , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Nervios Periféricos/metabolismo , Nervios Periféricos/efectos de los fármacos , Masculino , Ratas , Proteína 2 Asociada a Resistencia a Múltiples MedicamentosRESUMEN
The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino acids, hormones and drugs. Its dysfunction is associated with many cancers and immune/neurological disorders. Here, we apply native mass spectrometry (MS)-based approaches to provide evidence of super-dimer formation (LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we discover four endogenous phosphatidylethanolamine (PE) molecules at the interface and C-terminus of both LAT1 subunits. We find that interfacial PE binding is regulated by 4F2hc-R183 and is critical for regulation of palmitoylation on neighbouring LAT1-C187. Combining native MS with mass photometry (MP), we reveal that super-dimerization is sensitive to pH, and modulated by complex N-glycans on the 4F2hc subunit. We further validate the dynamic assemblies of LAT1-4F2hc on plasma membrane and in the lysosome. Together our results link PTM and lipid binding with regulation and localisation of the LAT1-4F2hc super-dimer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Cadena Pesada de la Proteína-1 Reguladora de Fusión , Transportador de Aminoácidos Neutros Grandes 1 , Lipoilación , Proteínas de la Membrana , Fosfatidiletanolaminas , Humanos , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Fosfatidiletanolaminas/metabolismo , Lisosomas/metabolismo , Membrana Celular/metabolismo , Sistema de Transporte de Aminoácidos y+/metabolismo , Sistema de Transporte de Aminoácidos y+/genética , Células HEK293 , Multimerización de Proteína , Unión Proteica , Espectrometría de Masas , Mutagénesis Sitio-Dirigida , Concentración de Iones de HidrógenoRESUMEN
Our previous studies have shown that upregulation of SLC7A1 in epithelial ovarian cancer (EOC) tumor cells significantly increases cancer cell proliferation, migration, and cisplatin resistance; however, the molecular mechanism by which SLC7A1 functions in EOC remains unknown. In later studies, we found that SLC7A1 is also highly expressed in the interstitial portion of high-grade serous ovarian cancer (HGSOC), but the significance of this high expression in the interstitial remains unclear. Here, we showed the Interstitial high expression of SLC7A1 in HGSOC by immunohistochemistry. SLC7A1 enriched in cancer-associated fibroblasts (CAFs) was upregulated by TGF-ß1. Transwell assay, scratch assay, cck8 assay and cell adhesion assay showed that SLC7A1 highly expressed in CAFs promoted tumor cells invasion, migration and metastasis in vitro. The effect of SLC7A1 on MAPK and EMT pathway proteins in ovarian cancer (OC) was verified by RNA sequencing and western blotting. Overexpression of SLC7A1 in OC is involved in MAPK/ ERK pathway and EMT. In general, in HGSOC, CAFs overexpressing SLC7A1 supported the migration and invasion of tumor cells; SLC7A1 is highly expressed in ovarian cancer and is involved in ERK phosphorylation and EMT signaling in MAPK signaling pathway. This suggests that SLC7A1 may be a potential therapeutic target for OC metastasis.
Asunto(s)
Carcinoma Epitelial de Ovario , Cistadenocarcinoma Seroso , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Transportador de Aminoácidos Neutros Grandes 1 , Neoplasias Ováricas , Femenino , Humanos , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinoma Epitelial de Ovario/patología , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patología , Cistadenocarcinoma Seroso/genética , Progresión de la Enfermedad , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Sistema de Señalización de MAP Quinasas , Clasificación del Tumor , Invasividad Neoplásica , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Proteins overexpressed in early-stage cancers may serve as early diagnosis and prognosis markers as well as targets for cancer therapies. In this study, we examined the expression of an essential amino acid carrier SLC7A5 (LAT1, CD98, or 4F2 light chain) in cancer tissue from two well-annotated cohorts of 575 cases of early-stage and 106 cases of late-stage colorectal cancer patients. Immunohistochemistry showed SLC7A5 overexpression in 72.0% of early-stage and 56.6% of late-stage cases. SLC7A5 expression was not influenced by patient gender, age, location, or mismatch repair status, although it appeared to be slightly less prevalent in tumors of mucinous differentiation or with lymphovascular invasion. Statistical analyses revealed a positive correlation between SLC7A5 overexpression and both overall survival and disease-free survival in early-stage but not late-stage cancers. Co-expression analyses of the TCGA and CPTAC colorectal cancer cohorts identified a network of gene transcripts positively related to SLC7A5, with its heterodimer partner SLC3A2 having the highest co-expression score. Network analysis uncovered the SLC7A network to be significantly associated with ncRNA such as tRNA processing and the mitotic cell cycle. Since SLC7A5 is also a marker of activated lymphocytes such as NK, T, and B lymphocytes, SLC7A5 overexpression in early colorectal cancers might trigger a strong anti-tumor immune response which could results in better clinical outcome. Overall, our study provides clear evidence of differential SLC7A5 expression and its prognostic value for early-stage colorectal cancer, although the understanding of its functions in colorectal tumorigenesis and cancer immunity is currently rather limited and awaits further characterization.
Asunto(s)
Biomarcadores de Tumor , Neoplasias Colorrectales , Transportador de Aminoácidos Neutros Grandes 1 , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Supervivencia sin Enfermedad , Cadena Pesada de la Proteína-1 Reguladora de Fusión , Regulación Neoplásica de la Expresión Génica , Inmunohistoquímica , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Estadificación de Neoplasias , PronósticoRESUMEN
Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.
Asunto(s)
Barrera Hematorretinal , Células Endoteliales , Factores de Transcripción Forkhead , Neovascularización Retiniana , Animales , Masculino , Ratones , Angiogénesis , Barrera Hematorretinal/metabolismo , Células Endoteliales/metabolismo , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Pericitos/metabolismo , Retina/metabolismo , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/genética , Neovascularización Retiniana/patología , Vasos Retinianos/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The intestine is critical for not only processing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell (IEC)-specific knockout (ΔIEC) of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt and Slc7a5ΔIEC reduces mTORC1 signaling. Surprisingly, adult Slc7a5ΔIEC intestinal crypts have increased cell proliferation but reduced mature Paneth cells. Goblet cells, the other major secretory cell type in the small intestine, are increased in the crypts but reduced in the villi. Analyses with scRNA-seq and electron microscopy have revealed dedifferentiation of Paneth cells in Slc7a5ΔIEC mice, leading to markedly reduced secretory granules with little effect on Paneth cell number. Thus, SLC7A5 likely regulates secretory cell differentiation to affect stem cell niche and indirectly regulate cell proliferation.
Asunto(s)
Sistemas de Transporte de Aminoácidos , Transportador de Aminoácidos Neutros Grandes 1 , Animales , Ratones , Diferenciación Celular/genética , Proliferación Celular/genética , Transportador de Aminoácidos Neutros Grandes 1/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genéticaRESUMEN
Cancer-associated fibroblasts (CAFs) can promote the crosstalk between cancer cells and tumor microenvironment by exosomes. METTL3-mediated N6-methyladenine (m6A) modification has been proved to promote the progression of non-small cell lung cancer (NSCLC). Here, we focused on the impacts of CAFs-derived exosomes and METTL3-mediated m6A modification on NSCLC progression. Functional analyses were conducted using Cell Counting Kit-8, EdU, colony formation, sphere formation and transwell assays, respectively. Glutamine metabolism was evaluated by detecting glutamate consumption, and the production of intercellular glutamate and α-ketoglutarate (α-KG). qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. Exosomes were isolated by kits. The methylated RNA immunoprecipitation assay detected the m6A modification profile of Amino acid transporter LAT1 (SLC7A5) mRNA. The NSCLC mouse model was established to conduct in vivo experiments. We found that CAFs promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells. METTL3 was enriched in CAFs and was packaged into exosomes. After knockdown of METTL3 in CAF exosomes, it was found the oncogenic effects of CAFs on NSCLC cells were suppressed. CAFs elevated m6A levels in NSCLC cells. Mechanistically, exosomal METTL3-induced m6A modification in SLC7A5 mRNA and stabilized its expression in NSCLC cells. Moreover, SLC7A5 overexpression abolished the inhibitory effects of exosomal METTL3-decreased CAFs on NSCLC cells. In addition, METTL3 inhibition in CAF exosomes impeded NSCLC growth in vivo. In all, CAFs-derived exosomal METTL3 promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells by inducing SLC7A5 m6A modification.
Asunto(s)
Fibroblastos Asociados al Cáncer , Carcinoma de Pulmón de Células no Pequeñas , Proliferación Celular , Exosomas , Glutamina , Neoplasias Pulmonares , Metiltransferasas , Invasividad Neoplásica , Animales , Humanos , Ratones , Adenosina/análogos & derivados , Adenosina/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Modelos Animales de Enfermedad , Exosomas/metabolismo , Exosomas/genética , Expresión Génica/genética , Glutamina/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Transportador de Aminoácidos Neutros Grandes 1/fisiología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Metiltransferasas/metabolismo , Metiltransferasas/genética , Metiltransferasas/fisiología , Invasividad Neoplásica/genética , Microambiente Tumoral/genéticaRESUMEN
L-type amino acid transporter 1 (LAT1) is recognized as a promising target for cancer therapy; however, the cellular adaptive response to its pharmacological inhibition remains largely unexplored. This study examined the adaptive response to LAT1 inhibition using nanvuranlat, a high-affinity LAT1 inhibitor. Proteomic analysis revealed the activation of a stress-induced transcription factor ATF4 following LAT1 inhibition, aligning with the known cellular responses to amino acid deprivation. This activation was linked to the GCN2-eIF2α pathway which regulates translation initiation. Our results show that ATF4 upregulation counteracts the suppressive effect of nanvuranlat on cell proliferation in pancreatic ductal adenocarcinoma cell lines, suggesting a role for ATF4 in cellular adaptation to LAT1 inhibition. Importantly, dual targeting of LAT1 and ATF4 exhibited more substantial anti-proliferative effects in vitro than individual treatments. This study underscores the potential of combining LAT1 and ATF4 inhibition as a therapeutic strategy in cancer treatment.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Regulación hacia Arriba , Proteómica , Aminoácidos/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Transportador de Aminoácidos Neutros Grandes 1/genética , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Línea Celular Tumoral , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismoRESUMEN
BACKGROUND: Increased adhesivity of red blood cells (RBCs) to endothelial cells (ECs) may contribute to organ dysfunction in malaria, sickle cell disease, and diabetes. RBCs normally export nitric oxide (NO)-derived vascular signals, facilitating blood flow. S-nitrosothiols (SNOs) are thiol adducts formed in RBCs from precursor NO upon the oxygenation-linked allosteric transition in hemoglobin. RBCs export these vasoregulatory SNOs on demand, thereby regulating regional blood flow and preventing RBC-EC adhesion, and the large (system L) neutral amino acid transporter 1 (LAT1; SLC7A5) appears to mediate SNO export by RBCs. METHODS: To determine the role of LAT1-mediated SNO import by ECs generally and of LAT1-mediated SNO import by ECs in RBC SNO-dependent modulation of RBC sequestration and blood oxygenation in vivo, we engineered LAT1fl/fl; Cdh5-Cre+ mice, in which the putative SNO transporter LAT1 can be inducibly depleted (knocked down, KD) specifically in ECs ("LAT1ECKD"). RESULTS: We show that LAT1 in mouse lung ECs mediates cellular SNO uptake. ECs from LAT1ECKD mice (tamoxifen-induced LAT1fl/fl; Cdh5-Cre+) import SNOs poorly ex vivo compared with ECs from wild-type (tamoxifen-treated LAT1fl/fl; Cdh5-Cre-) mice. In vivo, endothelial depletion of LAT1 increased RBC sequestration in the lung and decreased blood oxygenation after RBC transfusion. CONCLUSION: This is the first study showing a role for SNO transport by LAT1 in ECs in a genetic mouse model. We provide the first direct evidence for the coordination of RBC SNO export with EC SNO import via LAT1. SNO flux via LAT1 modulates RBC-EC sequestration in lungs after transfusion, and its disruption impairs blood oxygenation by the lung.
Asunto(s)
Células Endoteliales , Transfusión de Eritrocitos , Eritrocitos , Transportador de Aminoácidos Neutros Grandes 1 , S-Nitrosotioles , Animales , Eritrocitos/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Células Endoteliales/metabolismo , S-Nitrosotioles/metabolismo , Ratones , Humanos , Pulmón/metabolismo , Óxido Nítrico/metabolismo , Ratones Endogámicos C57BL , Adhesión CelularRESUMEN
The urea cycle is frequently rewired in cancer cells to meet the metabolic demands of cancer. Elucidation of the underlying mechanism by which oncogenic signaling mediates urea cycle reprogramming could help identify targetable metabolic vulnerabilities. In this study, we discovered that oncogenic activation of KRAS in non-small cell lung cancer (NSCLC) silenced the expression of argininosuccinate synthase 1 (ASS1), a urea cycle enzyme that catalyzes the production of arginine from aspartate and citrulline, and thereby diverted the utilization of aspartate to pyrimidine synthesis to meet the high demand for DNA replication. Specifically, KRAS signaling facilitated a hypoacetylated state in the promoter region of the ASS1 gene in a histone deacetylase 3-dependent manner, which in turn impeded the recruitment of c-MYC for ASS1 transcription. ASS1 suppression in KRAS-mutant NSCLC cells impaired the biosynthesis of arginine and rendered a dependency on the arginine transmembrane transporter SLC7A1 to import extracellular arginine. Depletion of SLC7A1 in both patient-derived organoid and xenograft models inhibited KRAS-driven NSCLC growth. Together, these findings uncover the role of oncogenic KRAS in rewiring urea cycle metabolism and identify SLC7A1-mediated arginine uptake as a therapeutic vulnerability for treating KRAS-mutant NSCLC. SIGNIFICANCE: ASS1 deficiency is induced by mutant KRAS in NSCLC to facilitate DNA synthesis and creates a dependency on SLC7A1, revealing dietary arginine restriction and SLC7A1 inhibition as potential therapeutic strategies.
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Arginina , Argininosuccinato Sintasa , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Arginina/metabolismo , Ratones , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Argininosuccinato Sintasa/metabolismo , Argininosuccinato Sintasa/genética , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Proliferación CelularRESUMEN
BACKGROUND: The development of radioresistance seriously hinders the efficacy of radiotherapy in lung cancer. However, the underlying mechanisms by which radioresistance occurs are still incompletely understood. The N6-Methyladenosine (m6A) modification of RNA is involved in cancer progression, but its role in lung cancer radioresistance remains elusive. This study aimed to identify m6A regulators involved in lung cancer radiosensitivity and further explore the underlying mechanisms to identify therapeutic targets to overcome lung cancer radioresistance. METHODS: Bioinformatic mining was used to identify the m6A regulator IGF2BP2 involved in lung cancer radiosensitivity. Transcriptome sequencing was used to explore the downstream factors. Clonogenic survival assays, neutral comet assays, Rad51 foci formation assays, and Annexin V/propidium iodide assays were used to determine the significance of FBW7/IGF2BP2/SLC7A5 axis in lung cancer radioresistance. Chromatin immunoprecipitation (ChIP)-qPCR analyses, RNA immunoprecipitation (RIP) and methylated RNA immunoprecipitation (MeRIP)-qPCR analyses, RNA pull-down analyses, co-immunoprecipitation analyses, and ubiquitination assays were used to determine the feedback loop between IGF2BP2 and SLC7A5 and the regulatory effect of FBW7/GSK3ß on IGF2BP2. Mice models and tissue microarrays were used to verify the effects in vivo. RESULTS: We identified IGF2BP2, an m6A "reader", that is overexpressed in lung cancer and facilitates radioresistance. We showed that inhibition of IGF2BP2 impairs radioresistance in lung cancer both in vitro and in vivo. Furthermore, we found that IGF2BP2 enhances the stability and translation of SLC7A5 mRNA through m6A modification, resulting in enhanced SLC7A5-mediated transport of methionine to produce S-adenosylmethionine. This feeds back upon the IGF2BP2 promoter region by further increasing the trimethyl modification at lysine 4 of histone H3 (H3K4me3) level to upregulate IGF2BP2 expression. We demonstrated that this positive feedback loop between IGF2BP2 and SLC7A5 promotes lung cancer radioresistance through the AKT/mTOR pathway. Moreover, we found that the ubiquitin ligase FBW7 functions with GSK3ß kinase to recognize and degrade IGF2BP2. CONCLUSIONS: Collectively, our study revealed that the m6A "reader" IGF2BP2 promotes lung cancer radioresistance by forming a positive feedback loop with SLC7A5, suggesting that IGF2BP2 may be a potential therapeutic target to control radioresistance in lung cancer.
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Proteína 7 que Contiene Repeticiones F-Box-WD , Transportador de Aminoácidos Neutros Grandes 1 , Neoplasias Pulmonares , Proteínas de Unión al ARN , Animales , Ratones , Línea Celular Tumoral , Glucógeno Sintasa Quinasa 3 beta/genética , Transportador de Aminoácidos Neutros Grandes 1/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , ARN , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Proteínas de Unión al ARN/genética , Tolerancia a RadiaciónRESUMEN
The Developmental Origins of Health and Disease hypothesis sustains that exposure to different stressors during prenatal development prepares the offspring for the challenges to be encountered after birth. We studied the gestational period as a particularly vulnerable window where different stressors can have strong implications for fetal programming of the offspring's life-long metabolic status via alterations of specific placentally expressed nutrient transporters. To study this mechanism, we used a murine prenatal stress model, human preeclampsia, early miscarriage, and healthy placental tissue samples, in addition to in vitro models of placental cells. In stressed mice, placental overexpression of L-type amino acid transporter 1 (Lat1) and subsequent global placental DNA hypermethylation was accompanied by fetal and adult hypothalamic dysregulation in global DNA methylation and gene expression as well as long-term metabolic abnormalities exclusively in female offspring. In human preeclampsia, early miscarriage, and under hypoxic conditions, placental LAT1 was significantly upregulated, leading to increased methionine uptake and global DNA hypermethylation. Remarkably, subgroups of healthy term placentas with high expression of stress-related genes presented increased levels of placental LAT1 mRNA and protein, DNA and RNA hypermethylation, increased methionine uptake capacity, one-carbon metabolic pathway disruption, higher methionine concentration in the placenta and transport to the fetus specifically in females. Since LAT1 mediates the intracellular accumulation of methionine, global DNA methylation, and one-carbon metabolism in the placenta, our findings hint at a major sex-specific global response to a variety of prenatal stressors affecting placental function, epigenetic programming, and life-long metabolic disease and provide a much-needed insight into early-life factors predisposing females/women to metabolic disorders.
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Epigénesis Genética , Desarrollo Fetal , Predisposición Genética a la Enfermedad , Transportador de Aminoácidos Neutros Grandes 1 , Enfermedades Metabólicas , Metionina , Placenta , Adulto , Animales , Femenino , Humanos , Masculino , Ratones , Embarazo , Aborto Espontáneo , Proteínas Adaptadoras Transductoras de Señales , Enfermedades Metabólicas/genética , Metionina/metabolismo , Placenta/metabolismo , Preeclampsia , Racemetionina , Metilación de ADN , Transportador de Aminoácidos Neutros Grandes 1/genética , Transportador de Aminoácidos Neutros Grandes 1/metabolismoRESUMEN
Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer-related death worldwide. The purpose of this study was to discover novel molecular pathways and potential prognosis biomarkers. To achieve this, we acquired five microarray datasets from the Gene Expression Omnibus (GEO) database. We identified differentially expressed genes between CRC and adjacent normal tissue samples and further validated them using The Cancer Genome Atlas (TCGA) database. Using various analytical approaches, including the construction of a competing endogenous RNA (ceRNA) network, Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway analyses, as well as survival analysis, we identified key genes and pathways associated with the diagnosis and prognosis of CRC. We obtained a total of 185 differentially expressed genes, comprising 17 lncRNAs, 30 miRNAs, and 138 mRNAs. The ceRNA network consisted of 17 lncRNAs, 25 miRNAs, and 7 mRNAs. Among the 7 mRNAs involved in the ceRNA network, SLC7A5 and KRT80 were found to be upregulated, while ADIPOQ, CCBE1, KCNB1, CADM2, and CHRDL1 were downregulated in CRC. Further analysis revealed that ADIPOQ and SLC7A5 are involved in the AMPK and mTOR signaling pathway, respectively. In addition, survival analysis demonstrated a statistically significant relationship between ADIPOQ, SLC7A5, and overall survival rates in CRC patients. In conclusion, our findings suggest that downregulation of ADIPOQ and upregulation of SLC7A5 in tumor cells lead to increased mTORC1 activity, reduced autophagy, enhanced angiogenesis, and ultimately contribute to cancer progression and decreased survival in CRC patients.
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Neoplasias Colorrectales , MicroARNs , ARN Largo no Codificante , Humanos , Transportador de Aminoácidos Neutros Grandes 1/genética , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Redes Reguladoras de Genes , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Angiogénesis , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , Neoplasias Colorrectales/genética , Biología Computacional , Regulación Neoplásica de la Expresión GénicaRESUMEN
PURPOSE: Thyroid cancer metabolic characteristics vary depending on the molecular subtype determined by mutational status. We aimed to investigate the molecular subtype-specific metabolic characteristics of thyroid cancers. EXPERIMENTAL DESIGN: An integrative multi-omics analysis was conducted, incorporating transcriptomics, metabolomics, and proteomics data obtained from human tissues representing distinct molecular characteristics of thyroid cancers: BRAF-like (papillary thyroid cancer with BRAFV600E mutation; PTC-B), RAS-like (follicular thyroid cancer with RAS mutation; FTC-R), and ATC-like (anaplastic thyroid cancer with BRAFV600E or RAS mutation; ATC-B or ATC-R). To validate our findings, we employed tissue microarray of human thyroid cancer tissues and performed in vitro analyses of cancer cell phenotypes and metabolomic assays after inducing genetic knockdown. RESULTS: Metabolic properties differed between differentiated thyroid cancers of PTC-B and FTC-R, but were similar in dedifferentiated thyroid cancers of ATC-B/R, regardless of their mutational status. Tricarboxylic acid (TCA) intermediates and branched-chain amino acids (BCAA) were enriched with the activation of TCA cycle only in FTC-R, whereas one-carbon metabolism and pyrimidine metabolism increased in both PTC-B and FTC-R and to a great extent in ATC-B/R. However, the protein expression levels of the BCAA transporter (SLC7A5) and a key enzyme in one-carbon metabolism (SHMT2) increased in all thyroid cancers and were particularly high in ATC-B/R. Knockdown of SLC7A5 or SHMT2 inhibited the migration and proliferation of thyroid cancer cell lines differently, depending on the mutational status. CONCLUSIONS: These findings define the metabolic properties of each molecular subtype of thyroid cancers and identify metabolic vulnerabilities, providing a rationale for therapies targeting its altered metabolic pathways in advanced thyroid cancer.
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Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Humanos , Transportador de Aminoácidos Neutros Grandes 1/genética , Multiómica , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Mutación , Fenotipo , Carbono/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer cell survival under nutrient limitation. Here we discovered that missense mutant p53 oncoproteins stimulate de novo serine/glycine synthesis and essential amino acids intake, promoting breast cancer growth. Mechanistically, mutant p53, unlike the wild-type counterpart, induces the expression of serine-synthesis-pathway enzymes and L-type amino acid transporter 1 (LAT1)/CD98 heavy chain heterodimer. This effect is exacerbated by amino acid shortage, representing a mutant p53-dependent metabolic adaptive response. When cells suffer amino acids scarcity, mutant p53 protein is stabilized and induces metabolic alterations and an amino acid transcriptional program that sustain cancer cell proliferation. In patient-derived tumor organoids, pharmacological targeting of either serine-synthesis-pathway and LAT1-mediated transport synergizes with amino acid shortage in blunting mutant p53-dependent growth. These findings reveal vulnerabilities potentially exploitable for tackling breast tumors bearing missense TP53 mutations.