RESUMEN
A kinesin family member 5b (KIF5B)-MET proto-oncogene, receptor tyrosine kinase (MET) rearrangement was reported in patients with lung adenocarcinoma but its oncogenic function was not fully evaluated. We used one-step reverse transcription-polymerase chain reaction for RNA samples to screen for the KIF5B-MET fusion in 206 lung adenocarcinoma and 28 pulmonary sarcomatoid carcinoma patients. Genomic breakpoints of KIF5B-MET were determined by targeted next-generation sequencing. Soft agar colony formation assays, proliferation assays, and a xenograft mouse model were used to investigate its oncogenic activity. In addition, specific MET inhibitors were administered to evaluate their anti-tumor activities. A KIF5B-MET fusion variant in a patient with a mixed-type adenocarcinoma and sarcomatoid tumor was identified, and another case was found in a pulmonary sarcomatoid carcinoma patient. Both cases carried the same chimeric gene, a fusion between exons 1-24 of KIF5B and exons 15-21 of MET. KIF5B-MET-overexpressing cells exhibited significantly increased proliferation and colony-forming ability. Xenograft tumors harboring the fusion gene demonstrated significantly elevated tumor growth. Ectopic expression of the fusion gene stimulated the phosphorylation of KIF5B-MET as well as downstream STAT3, AKT, and ERK1/2 signaling pathways. The MET inhibitors significantly repressed cell proliferation; phosphorylation of downstream STAT3, AKT, and ERK1/2; and xenograft tumorigenicity. In conclusion, the KIF5B-MET variant was demonstrated to have an oncogenic function in cancer cells. These findings have immediate clinical implications for the targeted therapy of subgroups of non-small cell lung cancer patients.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Variación Genética/genética , Cinesinas/genética , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/genética , Oncogenes/genética , Proteínas Proto-Oncogénicas c-met/genética , Adenocarcinoma/genética , Adenocarcinoma del Pulmón , Animales , Carcinogénesis/genética , Línea Celular , Proliferación Celular/genética , Exones/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proto-Oncogenes Mas , Translocación Genética/genéticaRESUMEN
Upon nutrient deprivation, cells are thought to suppress biosynthesis but activate catabolic pathways to provide alternative energy sources and nutrients. However, here we provide evidence that in adult male C. elegans, both biosynthesis and degradation activities, including ribosome biogenesis and turnover, are enhanced during early starvation and appear to depend on the availability of intestinal lipid stores. Upon depletion of the intestinal lipids, further food deprivation results in a significant reduction in metabolic activity in the starved male worms. Our data show that adult C. elegans exhibits a two-phase metabolic response to starvation stress: an initial phase with enhanced metabolic activity that rapidly exhausts the lipid stores, followed by a phase with low metabolic activity, which outlasts the life of fed control worms. DAF-2 insulin/IGF-1 receptor signaling to the RAS pathway is required for the starvation-induced ribosome biogenesis and rapid lipid depletion in the initial phase of starvation.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Insulina/metabolismo , Mucosa Intestinal/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Privación de Alimentos , Factor I del Crecimiento Similar a la Insulina/genética , Metabolismo de los Lípidos/genética , Longevidad/genética , Masculino , Actividad Motora , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor IGF Tipo 1/genética , Receptor de Insulina/genética , Ribosomas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
2,5,7,8-Tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), the water-soluble metabolite of alpha-tocopherol (alpha-TOH) with a shortened side chain but an intact hydroxychroman structure, has been identified in human urine and are thought to be produced in significant amount at excess intake of alpha-TOH. In previous studies, CEHCs in biological specimens were measured by HPLC, GC-MS or LC-MS, preceded by a hydrolysis procedure using either enzyme or methanolic HCl. In an attempt to analyze alpha-CEHC in rat urine accordingly, we observed that enzyme hydrolysis was relatively inefficient in releasing alpha-CEHC compared to high concentrations of HCl. The HCl releasable alpha-CEHC conjugate was isolated and chemically identified as 6-O-sulfated alpha-CEHC (alpha-CEHC sulfate). Using the synthetic alpha-CEHC sulfate standard, it was found that sulfatase could not hydrolyze to a significant extent. On the other hand, pretreatment with HCl at 60 degrees C in the presence of ascorbate, followed by a one-step ether extraction, not only hydrolyzed the sulfate conjugate completely but also extracted alpha-CEHC with high recovery. The inclusion of ascorbate minimized the conversion of alpha-CEHC to alpha-tocopheronolactone in the HCl pretreatment. A complete procedure for the quantitative analysis of alpha-CEHC including HCl hydrolysis, ether extraction and reverse phase isocratic HPLC-ECD was thus established. In conclusion, alpha-CEHC sulfate was isolated and identified as the HCl-releasable conjugate of alpha-CEHC in rat urine. A rapid and sensitive method with high reproducibility for the determination of free, conjugated and total alpha-CEHC is then established.