RESUMEN
Targeting the function of membrane transporters in cancer stemlike cells is a potential new therapeutic approach. Cystine-glutamate antiporter xCT expressed in CD44 variant (CD44v)-expressing cancer cells contributes to the resistance to oxidative stress as well as cancer therapy through promoting glutathione (GSH)-mediated antioxidant defense. Amino acid transport by xCT might, thus, be a promising target for cancer treatment, whereas the determination factors for cancer cell sensitivity to xCT-targeted therapy remain unclear. Here, we demonstrate that high expression of xCT and glutamine transporter ASCT2 is correlated with undifferentiated status and diminished along with cell differentiation in head and neck squamous cell carcinoma (HNSCC). The cytotoxicity of the xCT inhibitor sulfasalazine relies on ASCT2-dependent glutamine uptake and glutamate dehydrogenase (GLUD)-mediated α-ketoglutarate (α-KG) production. Metabolome analysis revealed that sulfasalazine treatment triggers the increase of glutamate-derived tricarboxylic acid cycle intermediate α-KG, in addition to the decrease of cysteine and GSH content. Furthermore, ablation of GLUD markedly reduced the sulfasalazine cytotoxicity in CD44v-expressing stemlike HNSCC cells. Thus, xCT inhibition by sulfasalazine leads to the impairment of GSH synthesis and enhancement of mitochondrial metabolism, leading to reactive oxygen species (ROS) generation and, thereby, triggers oxidative damage. Our findings establish a rationale for the use of glutamine metabolism (glutaminolysis)-related genes, including ASCT2 and GLUD, as biomarkers to predict the efficacy of xCT-targeted therapy for heterogeneous HNSCC tumors.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Glutatión/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Terapia Molecular Dirigida/métodos , Células Madre Neoplásicas/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Sistema de Transporte de Aminoácidos ASC/genética , Sistema de Transporte de Aminoácidos y+/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/farmacología , Adhesión Celular , Diferenciación Celular , Línea Celular Tumoral , Cisplatino/farmacología , Glutamato Deshidrogenasa/metabolismo , Glutamina/metabolismo , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Receptores de Hialuranos/análisis , Receptores de Hialuranos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Metaboloma , Ratones , Ratones Desnudos , Antígenos de Histocompatibilidad Menor/genética , Mitocondrias/metabolismo , Oxidación-Reducción , Estrés Oxidativo , ARN Mensajero/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Sulfasalazina/farmacologíaRESUMEN
KRAS mutations are detected in numerous human cancers, but there are few effective drugs for KRAS-mutated cancers. Transporters for amino acids and glucose are highly expressed on cancer cells, possibly to maintain rapid cell growth and metabolism. Alanine-serine-cysteine transporter 2 (ASCT2) is a primary transporter for glutamine in cancer cells. In this study, we developed a novel monoclonal antibody (mAb) recognizing the extracellular domain of human ASCT2, and investigated whether ASCT2 can be a therapeutic target for KRAS-mutated cancers. Rats were immunized with RH7777 rat hepatoma cells expressing human ASCT2 fused to green fluorescent protein (GFP). Splenocytes from the immunized rats were fused with P3X63Ag8.653 mouse myeloma cells, and selected and cloned hybridoma cells secreting Ab3-8 mAb were established. This mAb reacted with RH7777 transfectants expressing ASCT2-GFP proteins in a GFP intensity-dependent manner. Ab3-8 reacted with various human cancer cells, but not with non-cancer breast epithelial cells or ASCT2-knocked out HEK293 and SW1116 cells. In SW1116 and HCT116 human colon cancer cells with KRAS mutations, treatment with Ab3-8 reduced intracellular glutamine transport, phosphorylation of AKT and ERK, and inhibited in vivo tumor growth of these cells in athymic mice. Inhibition of in vivo tumor growth by Ab3-8 was not observed in HT29 colon and HeLa uterus cancer cells with wild-type KRAS. These results suggest that ASCT2 is an excellent therapeutic target for KRAS-mutated cancers.