RESUMEN
The Alanine-Serine-Cysteine transporter (SLC1A5, ASCT2), is a neutral amino acid exchanger involved in the intracellular homeostasis of amino acids in peripheral tissues. Given its role in supplying glutamine to rapidly proliferating cancer cells in several tumor types such as triple-negative breast cancer and melanoma, ASCT2 has been identified as a key drug target. Here we use a range of computational methods, including homology modeling and ligand docking, in combination with cell-based assays, to develop hypotheses for structure-function relationships in ASCT2. We perform a phylogenetic analysis of the SLC1 family and its prokaryotic homologs to develop a useful multiple sequence alignment for this protein family. We then generate homology models of ASCT2 in two different conformations, based on the human EAAT1 structures. Using ligand enrichment calculations, the ASCT2 models are then compared to crystal structures of various homologs for their utility in discovering ASCT2 inhibitors. We use virtual screening, cellular uptake and electrophysiology experiments to identify a non-amino acid ASCT2 inhibitor that is predicted to interact with the ASCT2 substrate binding site. Our results provide insights into the structural basis of substrate specificity in the SLC1 family, as well as a framework for the design of future selective and potent ASCT2 inhibitors as cancer therapeutics.
RESUMEN
l-type amino acid transporters (LAT1-4) are expressed in various cancer types and are involved in the uptake of essential amino acids such as leucine. Here we investigated the expression of LAT1-4 in endometrial adenocarcinoma and evaluated the contribution of LATs to endometrial cancer cell growth. Analysis of human gene expression data showed that all four LAT family members are expressed in endometrial adenocarcinomas. LAT1 was the most highly expressed, and showed a significant increase in both serous and endometrioid subtypes compared to normal endometrium. Endometrioid patients with the highest LAT1 levels exhibited the lowest disease-free survival. The pan-LAT inhibitor BCH led to a significant decrease in cell growth and spheroid area in four endometrial cancer cell lines tested in vitro. Knockdown of LAT1 by shRNA inhibited cell growth in HEC1A and Ishikawa cells, as well as inhibiting spheroid area in HEC1A cells. These data show that LAT1 plays an important role in regulating the uptake of essential amino acids such as leucine into endometrial cancer cells. Increased ability of BCH compared to LAT1 shRNA at inhibiting Ishikawa spheroid area suggests that other LAT family members may also contribute to cell growth. LAT1 inhibition may offer an effective therapeutic strategy in endometrial cancer patients whose tumours exhibit high LAT1 expression.
Asunto(s)
Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/terapia , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/terapia , Transportador de Aminoácidos Neutros Grandes 1/biosíntesis , Aminoácidos Cíclicos/farmacología , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Transportador de Aminoácidos Neutros Grandes 1/genética , Terapia Molecular Dirigida , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Esferoides CelularesRESUMEN
The Alanine-Serine-Cysteine transporter ASCT2 (SLC1A5) is a membrane protein that transports neutral amino acids into cells in exchange for outward movement of intracellular amino acids. ASCT2 is highly expressed in peripheral tissues such as the lung and intestines where it contributes to the homeostasis of intracellular concentrations of neutral amino acids. ASCT2 also plays an important role in the development of a variety of cancers such as melanoma by transporting amino acid nutrients such as glutamine into the proliferating tumors. Therefore, ASCT2 is a key drug target with potentially great pharmacological importance. Here, we identify seven ASCT2 ligands by computational modeling and experimental testing. In particular, we construct homology models based on crystallographic structures of the aspartate transporter GltPh in two different conformations. Optimization of the models' binding sites for protein-ligand complementarity reveals new putative pockets that can be targeted via structure-based drug design. Virtual screening of drugs, metabolites, fragments-like, and lead-like molecules from the ZINC database, followed by experimental testing of 14 top hits with functional measurements using electrophysiological methods reveals seven ligands, including five activators and two inhibitors. For example, aminooxetane-3-carboxylate is a more efficient activator than any other known ASCT2 natural or unnatural substrate. Furthermore, two of the hits inhibited ASCT2 mediated glutamine uptake and proliferation of a melanoma cancer cell line. Our results improve our understanding of how substrate specificity is determined in amino acid transporters, as well as provide novel scaffolds for developing chemical tools targeting ASCT2, an emerging therapeutic target for cancer and neurological disorders.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/química , Sistema de Transporte de Aminoácidos ASC/ultraestructura , Evaluación Preclínica de Medicamentos/métodos , Modelos Químicos , Simulación del Acoplamiento Molecular , Mapeo de Interacción de Proteínas/métodos , Algoritmos , Secuencia de Aminoácidos , Sitios de Unión , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Unión Proteica , Análisis de Secuencia de Proteína/métodos , Homología de Secuencia de AminoácidoRESUMEN
Glutamine is conditionally essential in cancer cells, being utilized as a carbon and nitrogen source for macromolecule production, as well as for anaplerotic reactions fuelling the tricarboxylic acid (TCA) cycle. In this study, we demonstrated that the glutamine transporter ASCT2 (SLC1A5) is highly expressed in prostate cancer patient samples. Using LNCaP and PC-3 prostate cancer cell lines, we showed that chemical or shRNA-mediated inhibition of ASCT2 function in vitro decreases glutamine uptake, cell cycle progression through E2F transcription factors, mTORC1 pathway activation and cell growth. Chemical inhibition also reduces basal oxygen consumption and fatty acid synthesis, showing that downstream metabolic function is reliant on ASCT2-mediated glutamine uptake. Furthermore, shRNA knockdown of ASCT2 in PC-3 cell xenografts significantly inhibits tumour growth and metastasis in vivo, associated with the down-regulation of E2F cell cycle pathway proteins. In conclusion, ASCT2-mediated glutamine uptake is essential for multiple pathways regulating the cell cycle and cell growth, and is therefore a putative therapeutic target in prostate cancer.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/genética , Regulación Neoplásica de la Expresión Génica , Glutamina/metabolismo , Neoplasias de la Próstata/genética , Sistema de Transporte de Aminoácidos ASC/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos ASC/metabolismo , Animales , Transporte Biológico , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Ácidos Grasos/metabolismo , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Desnudos , Antígenos de Histocompatibilidad Menor , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Metástasis de la Neoplasia , Oxígeno/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/prevención & control , ARN Interferente Pequeño , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAF(WT) (C8161 and WM852) and BRAF(V600E) mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2-mediated glutamine transport is a potential therapeutic target for both BRAF(WT) and BRAF(V600E) melanoma.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/biosíntesis , Glutamina/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/biosíntesis , Melanoma/patología , Complejos Multiproteicos/antagonistas & inhibidores , Neoplasias Cutáneas/patología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos ASC/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos ASC/genética , Aminoácidos Cíclicos/farmacología , Compuestos de Bencilo/farmacología , Transporte Biológico , Proteína Quinasa CDC2/biosíntesis , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Humanos , Leucina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Melanoma/metabolismo , Antígenos de Histocompatibilidad Menor , Complejos Multiproteicos/genética , Proteínas Proto-Oncogénicas B-raf/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Serina/análogos & derivados , Serina/farmacología , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Esferoides Celulares , Serina-Treonina Quinasas TOR/genética , Células Tumorales Cultivadas , Enzimas Ubiquitina-Conjugadoras/biosíntesisRESUMEN
Combining immunotherapy with targeted therapy blocking oncogenic BRAFV600 may result in improved treatments for advanced melanoma. In this study, we developed a BRAFV600E-driven murine model of melanoma, SM1, which is syngeneic to fully immunocompetent mice. SM1 cells exposed to the BRAF inhibitor vemurafenib (PLX4032) showed partial in vitro and in vivo sensitivity resulting from the inhibition of MAPK pathway signaling. Combined treatment of vemurafenib plus adoptive cell transfer therapy with lymphocytes genetically modified with a T-cell receptor (TCR) recognizing chicken ovalbumin (OVA) expressed by SM1-OVA tumors or pmel-1 TCR transgenic lymphocytes recognizing gp100 endogenously expressed by SM1 resulted in superior antitumor responses compared with either therapy alone. T-cell analysis showed that vemurafenib did not significantly alter the expansion, distribution, or tumor accumulation of the adoptively transferred cells. However, vemurafenib paradoxically increased mitogen-activated protein kinase (MAPK) signaling, in vivo cytotoxic activity, and intratumoral cytokine secretion by adoptively transferred cells. Taken together, our findings, derived from 2 independent models combining BRAF-targeted therapy with immunotherapy, support the testing of this therapeutic combination in patients with BRAFV600 mutant metastatic melanoma.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Indoles/farmacología , Melanoma/terapia , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Sulfonamidas/farmacología , Animales , Terapia Combinada , Femenino , Humanos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Masculino , Melanoma/enzimología , Melanoma/inmunología , Melanoma Experimental/enzimología , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Linfocitos T/inmunología , Linfocitos T/patología , Células Tumorales Cultivadas , VemurafenibRESUMEN
BACKGROUND: Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors. METHODS: We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed. RESULTS: Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor. CONCLUSIONS: Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).