RESUMO
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.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia , Transportador 1 de Aminoácidos Neutros Grandes , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células HT29 , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Linhagem Celular Tumoral , Hipóxia Celular , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 1/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
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.
Assuntos
Exossomos , Células Endoteliais da Veia Umbilical Humana , MicroRNAs , Transdução de Sinais , Serina-Treonina Quinases TOR , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , Exossomos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Camundongos , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Masculino , Feminino , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Pessoa de Meia-Idade , Modelos Animais de Doenças , AdultoRESUMO
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.
Assuntos
Adenocarcinoma de Pulmão , Claudina-1 , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares , Humanos , Células A549 , Claudina-1/metabolismo , Claudina-1/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Aminoácidos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Esferoides Celulares/metabolismo , Esferoides Celulares/efeitos dos fármacos , Inativação GênicaRESUMO
Aggressive natural killer cell leukemia (ANKL) is a rare hematological malignancy with a fulminant clinical course. Our previous study revealed that ANKL cells proliferate predominantly in the liver sinusoids and strongly depend on transferrin supplementation. In addition, we demonstrated that liver-resident ANKL cells are sensitive to PPMX-T003, an anti-human transferrin receptor 1 inhibitory antibody, whereas spleen-resident ANKL cells are resistant to transferrin receptor 1 inhibition. However, the microenvironmental factors that regulate the iron dependency of ANKL cells remain unclear. In this study, we first revealed that the anti-neoplastic effect of PPMX-T003 was characterized by DNA double-strand breaks in a DNA replication-dependent manner, similar to conventional cytotoxic agents. We also found that the influx of extracellular amino acids via LAT1 stimulated sensitivity to PPMX-T003. Taken together, we discovered that the amount of extracellular amino acid influx through LAT1 was the key environmental factor determining the iron dependency of ANKL cells via adjustment of their mTOR/Myc activity, which provides a good explanation for the different sensitivity to PPMX-T003 between liver- and spleen-resident ANKL cells, as the liver sinusoid contains abundant amino acids absorbed from the gut.
Assuntos
Aminoácidos , Ferro , Células Matadoras Naturais , Transportador 1 de Aminoácidos Neutros Grandes , Humanos , Ferro/metabolismo , Células Matadoras Naturais/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Aminoácidos/metabolismo , Receptores da Transferrina/metabolismo , Camundongos , Animais , Fígado/metabolismo , Fígado/patologiaRESUMO
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.
Assuntos
Carcinoma de Células Escamosas , Regulação Neoplásica da Expressão Gênica , Transportador 1 de Aminoácidos Neutros Grandes , Metionina , Metionina/metabolismo , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Linhagem Celular Tumoral , Epigênese Genética , Epigenômica/métodos , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Camundongos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Animais , Proliferação de Células , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Reprogramação Celular/genéticaRESUMO
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.
Assuntos
Lisina , Neoplasias de Mama Triplo Negativas , Animais , Feminino , Humanos , Camundongos , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Lisina/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
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.
Assuntos
Carcinoma Epitelial do Ovário , Cistadenocarcinoma Seroso , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Transportador 1 de Aminoácidos Neutros Grandes , Neoplasias Ovarianas , Feminino , Humanos , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Cistadenocarcinoma Seroso/genética , Progressão da Doença , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Sistema de Sinalização das MAP Quinases , Gradação de Tumores , Invasividade Neoplásica , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
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.
Assuntos
Biomarcadores Tumorais , Neoplasias Colorretais , Transportador 1 de Aminoácidos Neutros Grandes , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Intervalo Livre de Doença , Cadeia Pesada da Proteína-1 Reguladora de Fusão , Regulação Neoplásica da Expressão Gênica , Imuno-Histoquímica , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Estadiamento de Neoplasias , PrognósticoRESUMO
O-([18F]Fluoroethyl)-l-tyrosine ([18F]FET) is actively transported into the brain and cancer cells by LAT1 and possibly other amino acid transporters, which enables brain tumor imaging by positron emission tomography (PET). However, tumor delivery of this probe in the presence of competing amino acids may be limited by a relatively low affinity for LAT1. The aim of the present work was to evaluate the meta-substituted [18F]FET analog m-[18F]FET and the methyl ester [18F]FET-OMe, which were designed to improve tumor delivery by altering the physicochemical, pharmacokinetic, and/or transport properties. Both tracers could be prepared with good radiochemical yields of 41-56% within 66-90 min. Preclinical evaluation with [18F]FET as a reference tracer demonstrated reduced in vitro uptake of [18F]FET-OMe by U87 glioblastoma cells and no advantage for in vivo tumor imaging. In contrast, m-[18F]FET showed significantly improved in vitro uptake and accelerated in vivo tumor accumulation in an orthotopic glioblastoma model. As such, our work identifies m-[18F]FET as a promising alternative to [18F]FET for brain tumor imaging that deserves further evaluation with regard to its transport properties and in vivo biodistribution.
Assuntos
Neoplasias Encefálicas , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Tirosina , Animais , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Humanos , Camundongos , Tirosina/análogos & derivados , Tirosina/química , Linhagem Celular Tumoral , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/síntese química , Distribuição Tecidual , Radioisótopos de Flúor/química , Glioblastoma/diagnóstico por imagem , Glioblastoma/metabolismo , Camundongos Nus , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismoRESUMO
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.
Assuntos
Barreira Hematorretiniana , Células Endoteliais , Fatores de Transcrição Forkhead , Neovascularização Retiniana , Animais , Masculino , Camundongos , Angiogênese , Barreira Hematorretiniana/metabolismo , Células Endoteliais/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pericitos/metabolismo , Retina/metabolismo , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Vasos Retinianos/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Cadeia Pesada da Proteína-1 Reguladora de Fusão , Transportador 1 de Aminoácidos Neutros Grandes , Lipoilação , Proteínas de Membrana , Fosfatidiletanolaminas , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Fosfatidiletanolaminas/metabolismo , Lisossomos/metabolismo , Membrana Celular/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Células HEK293 , Multimerização Proteica , Ligação Proteica , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Concentração de Íons de HidrogênioRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Sobrevivência Celular , Gânglios Espinais , Compostos de Metilmercúrio , Células de Schwann , Animais , Gânglios Espinais/metabolismo , Gânglios Espinais/efeitos dos fármacos , Compostos de Metilmercúrio/toxicidade , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Nervos Periféricos/metabolismo , Nervos Periféricos/efeitos dos fármacos , Masculino , Ratos , Proteína 2 Associada à Farmacorresistência MúltiplaRESUMO
BACKGROUND: Large amino acid transporter type 1 (LAT1) provides cancer cells with essential amino acids for both protein synthesis and cell growth and may predict patient prognosis. Additionally, LAT1 inhibition can be a therapeutic target. This study aimed to examine the prognostic significance of LAT1 expression in lung cancer, paying special attention to adenocarcinoma subtypes. METHODS: Tissue microarrays (TMA) of 1,560 total cores obtained from surgically resected lung cancer specimens between 1995 and 2008 at our hospital were used. Overall, 795 cases of adenocarcinoma were identified, and 717 underwent further evaluation. Immunohistochemical staining of whole slides and TMA cores were assessed to set H-score cutoff value.. Immunohistochemical expression of LAT1 was examined based on the subtypes of adenocarcinoma. Statistical analyses explored the prognostic significance of LAT1. RESULTS: Adenocarcinoma accounted for 71.8% of all cases (n = 795), and 216 cases (27.1%) expressed LAT1. The 795 cases were categorized into five subtypes: lepidic (n = 29, 3.6%), papillary (n = 601, 75.6%), acinar (n = 58, 7.3%), and solid (n = 9, 1.1%); 717 of the 795 cases were further assessed according to the exclusion criteria. The LAT1-positive ratio increased as the architectural grade increased. Notably, in papillary adenocarcinoma, the LAT1-positive group had significantly lower overall survival compared to the negative group (10-year survival: 45.6% vs. 60.8%, p < 0.001). CONCLUSION: LAT1 expression was higher in high-grade subtypes of pulmonary adenocarcinoma. Moreover, LAT1 expression is useful for predicting prognosis, particularly in papillary adenocarcinoma, facilitating prognostic stratification of papillary adenocarcinoma.
Assuntos
Adenocarcinoma de Pulmão , Transportador 1 de Aminoácidos Neutros Grandes , Neoplasias Pulmonares , Análise Serial de Tecidos , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Feminino , Masculino , Prognóstico , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/mortalidade , Pessoa de Meia-Idade , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/diagnóstico , Idoso , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/análise , AdultoRESUMO
L-type amino acid transporter 1 (LAT1) is specifically expressed in many malignancies, contributes to the transport of essential amino acids, such as leucine, and regulates the mammalian target of rapamycin (mTOR) signaling pathway. We investigated the expression profile and functional role of LAT1 in prostate cancer using JPH203, a specific inhibitor of LAT1. LAT1 was highly expressed in castration-resistant prostate cancer (CRPC) cells, including C4-2 and PC-3 cells, but its expression level was low in castration-sensitive LNCaP cells. JPH203 significantly inhibited [14C] leucine uptake in CRPC cells but had no effect in LNCaP cells. JPH203 inhibited the proliferation, migration, and invasion of CRPC cells but not of LNCaP cells. In C4-2 cells, Cluster of differentiation (CD) 24 was identified by RNA sequencing as a novel downstream target of JPH203. CD24 was downregulated in a JPH203 concentration-dependent manner and suppressed activation of the Wnt/ß-catenin signaling pathway. Furthermore, an in vivo study showed that JPH203 inhibited the proliferation of C4-2 cells in a castration environment. The results of this study indicate that JPH203 may exert its antitumor effect in CRPC cells via mTOR and CD24.
Assuntos
Antígeno CD24 , Movimento Celular , Proliferação de Células , Transportador 1 de Aminoácidos Neutros Grandes , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Linhagem Celular Tumoral , Animais , Proliferação de Células/efeitos dos fármacos , Antígeno CD24/metabolismo , Camundongos , Movimento Celular/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Benzoxazóis/farmacologia , Leucina/farmacologia , Leucina/análogos & derivados , Camundongos Nus , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Tirosina/análogos & derivadosRESUMO
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.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma Pulmonar de Células não Pequenas , Proliferação de Células , Exossomos , Glutamina , Neoplasias Pulmonares , Metiltransferases , Invasividade Neoplásica , Animais , Humanos , Camundongos , Adenosina/análogos & derivados , Adenosina/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Exossomos/metabolismo , Exossomos/genética , Expressão Gênica/genética , Glutamina/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/fisiologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Metiltransferases/metabolismo , Metiltransferases/genética , Metiltransferases/fisiologia , Invasividade Neoplásica/genética , Microambiente Tumoral/genéticaRESUMO
Metabolic rewiring is a key feature of cancer cells to support the demands of growth and proliferation. The metabolism of amino acids is altered in many cancers, including pancreatic cancer. The cellular uptake of amino acids is regulated by amino acid transporters, such as L-type amino acid transporter 1 (LAT1). Accumulating evidence suggests that LAT1 is overexpressed in pancreatic cancer and confers a poor prognosis. Here we discuss the prospects of utilizing LAT1 as a novel target for pancreatic cancer therapy.
Assuntos
Transportador 1 de Aminoácidos Neutros Grandes , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Terapia de Alvo Molecular/métodosRESUMO
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.
Assuntos
Sistemas de Transporte de Aminoácidos , Transportador 1 de Aminoácidos Neutros Grandes , Animais , Camundongos , Diferenciação Celular/genética , Proliferação de Células/genética , Transportador 1 de Aminoácidos Neutros Grandes/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genéticaRESUMO
OBJECTIVE: To detect the expression of L-type amino acid transporter 1 (LAT1) in non-Hodgkin's lymphoma (NHL) tissues, and analyze its effect on clinicopathological characteristics and prognosis of patients. METHODS: A total of 92 NHL patients who were treated in our hospital from January 2017 to April 2019 were collected. The expression of LAT1 in NHL tissue was detected by immunohistochemistry and compared between patients with different pathological features (including sex, Ann Arbor stage, extranodal infiltration, Ki-67). The risk factors affecting mortality were analyzed using univariate and multivariate Cox proportional hazards regression. Receiver operating characteristic (ROC) curve was used to detect the predictive value of percentage of LAT1-positive cells in NHL tissue for patient mortality, and analyzing the effect of percentage of LAT1-positive cells on survival rate. RESULTS: LAT1 was positively expressed in NHL tissue. The high expression rate of LAT1 in Ann Arbor stage III and IV groups were higher than that in Ann Arbor stage I group, that in extranodal infiltration group was higher than non-extranodal infiltration group, and that in Ki-67 positive expression group was higher than Ki-67 negative expression group (all P < 0.05). The remission rate after 3 courses of treatment in high-LAT1 expression group was 70.7%, which was lower than 91.2% in low-LAT1 expression group (P < 0.05). Ann Arbor stage III and IV, extranodal invasion, Ki-67 positive expression and increased expression of LAT1 (LAT1-positive cell percentage score ≥2) were risk factors for mortality. The cut-off value of percentage of LAT1-positive cells for predicting NHL death was 45.6%, and the area under the ROC curve was 0.905 (95%CI: 0.897-0.924). The 3-year survival rate of high-LAT1 level group (the percentage of LAT1-positive cells≥45.6%) was 50.00%, which was lower than 78.26% of low-LAT1 level group (P < 0.05). CONCLUSION: The expression level of LAT1 in NHL tissue increases, which affects Ann Arbor stage and extranodal infiltration of patients. LAT1 is a risk factor for death.
Assuntos
Transportador 1 de Aminoácidos Neutros Grandes , Linfoma não Hodgkin , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Linfoma não Hodgkin/metabolismo , Linfoma não Hodgkin/patologia , Prognóstico , Masculino , Feminino , Fatores de Risco , Taxa de Sobrevida , Estadiamento de Neoplasias , Curva ROC , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Células Endoteliais , Transfusão de Eritrócitos , Eritrócitos , Transportador 1 de Aminoácidos Neutros Grandes , S-Nitrosotióis , Animais , Eritrócitos/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Células Endoteliais/metabolismo , S-Nitrosotióis/metabolismo , Camundongos , Humanos , Pulmão/metabolismo , Óxido Nítrico/metabolismo , Camundongos Endogâmicos C57BL , Adesão CelularRESUMO
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.