RESUMEN
BACKGROUND & AIMS: The NKG2D system is a potent immunosurveillance mechanism in cancer, wherein the activating NK cell receptor (NKG2D) on immune cells recognises its cognate ligands on tumour cells. Herein, we evaluated the expression of NKG2D ligands in hepatocellular carcinoma (HCC), in both humans and mice, taking the genomic features of HCC tumours into account. METHODS: The expression of NKG2D ligands (MICA, MICB, ULBP1 and ULBP2) was analysed in large human HCC datasets by Fluidigm TaqMan and RNA-seq methods, and in 2 mouse models (mRNA and protein levels) reproducing the features of both major groups of human tumours. RESULTS: We provide compelling evidence that expression of the MICA and MICB ligands in human HCC is associated with tumour aggressiveness and poor patient outcome. We also found that the expression of ULBP1 and ULBP2 was associated with poor patient outcome, and was downregulated in CTNNB1-mutated HCCs displaying low levels of inflammation and associated with a better prognosis. We also found an inverse correlation between ULBP1/2 expression levels and the expression of ß-catenin target genes in patients with HCC, suggesting a role for ß-catenin signalling in inhibiting expression. We showed in HCC mouse models that ß-catenin signalling downregulated the expression of Rae-1 NKG2D ligands, orthologs of ULBPs, through TCF4 binding. CONCLUSIONS: We demonstrate that the expression of NKG2D ligands is associated with aggressive liver tumorigenesis and that the downregulation of these ligands by ß-catenin signalling may account for the less aggressive phenotype of CTNNB1-mutated HCC tumours. LAY SUMMARY: The NKG2D system is a potent immunosurveillance mechanism in cancer. However, its role in hepatocellular carcinoma development has not been widely investigated. Herein, we should that the expression of NKG2D ligands by tumour cells is associated with a more aggressive tumour subtype.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Hepáticas/metabolismo , Transducción de Señal/genética , beta Catenina/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Estudios de Cohortes , Modelos Animales de Enfermedad , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Pronóstico , Adulto JovenRESUMEN
Bioproduction of chemical compounds is of great interest for modern industries, as it reduces their production costs and ecological impact. With the use of synthetic biology, metabolic engineering and enzyme engineering tools, the yield of production can be improved to reach mass production and cost-effectiveness expectations. In this study, we explore the bioproduction of D-psicose, also known as D-allulose, a rare non-toxic sugar and a sweetener present in nature in low amounts. D-psicose has interesting properties and seemingly the ability to fight against obesity and type 2 diabetes. We developed a biosensor-based enzyme screening approach as a tool for enzyme selection that we benchmarked with the Clostridium cellulolyticum D-psicose 3-epimerase for the production of D-psicose from D-fructose. For this purpose, we constructed and characterized seven psicose responsive biosensors based on previously uncharacterized transcription factors and either their predicted promoters or an engineered promoter. In order to standardize our system, we created the Universal Biosensor Chassis, a construct with a highly modular architecture that allows rapid engineering of any transcription factor-based biosensor. Among the seven biosensors, we chose the one displaying the most linear behavior and the highest increase in fluorescence fold change. Next, we generated a library of D-psicose 3-epimerase mutants by error-prone PCR and screened it using the biosensor to select gain of function enzyme mutants, thus demonstrating the framework's efficiency.