RESUMEN
Integrin-linked kinase (ILK) is a mediator of aggressive phenotype in pancreatic cancer. On the basis of our finding that knockdown of either KRAS or ILK has a reciprocal effect on the other's expression, we hypothesized the presence of an ILK-KRAS regulatory loop that enables pancreatic cancer cells to regulate KRAS expression. This study aimed to elucidate the mechanism by which this regulatory circuitry is regulated and to investigate the translational potential of targeting ILK to suppress oncogenic KRAS signaling in pancreatic cancer. Interplay between KRAS and ILK and the roles of E2F1, c-Myc and heterogeneous nuclear ribonucleoprotein as intermediary effectors in this feedback loop was interrogated by genetic manipulations through small interfering RNA/short hairpin RNA knockdown and ectopic expression, western blotting, PCR, promoter-luciferase reporter assays, chromatin immunoprecipitation and pull-down analyses. In vivo efficacy of ILK inhibition was evaluated in two murine xenograft models. Our data show that KRAS regulated the expression of ILK through E2F1-mediated transcriptional activation, which, in turn, controlled KRAS gene expression via hnRNPA1-mediated destabilization of the G-quadruplex on the KRAS promoter. Moreover, ILK inhibition blocked KRAS-driven epithelial-mesenchymal transition and growth factor-stimulated KRAS expression. The knockdown or pharmacological inhibition of ILK suppressed pancreatic tumor growth, in part, by suppressing KRAS signaling. These studies suggest that this KRAS-E2F1-ILK-hnRNPA1 regulatory loop enables pancreatic cancer cells to promote oncogenic KRAS signaling and to interact with the tumor microenvironment to promote aggressive phenotypes. This regulatory loop provides a mechanistic rationale for targeting ILK to suppress oncogenic KRAS signaling, which might foster new therapeutic strategies for pancreatic cancer.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/fisiología , Neoplasias Pancreáticas/patología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Transducción de Señal/fisiología , Animales , Línea Celular Tumoral , Factor de Transcripción E2F1/fisiología , Transición Epitelial-Mesenquimal , Ribonucleoproteína Nuclear Heterogénea A1 , Humanos , Ratones , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas Proto-Oncogénicas p21(ras)/genética , Activación TranscripcionalRESUMEN
BACKGROUND: Arginase is a metabolic enzyme for the amino acid arginine that participates in the immune response to trauma. We hypothesize that surgical trauma induces arginase expression and activity in the human immune system. METHODS: Peripheral mononuclear cell (MNC) arginase activity and expression and plasma nitric oxide metabolites and interleukin (IL)-10 were measured in patients undergoing elective general surgery. Twenty-two healthy volunteers served as a comparison population. RESULTS: MNC arginase activity increased within 6 hours of surgery (p < 0.05) and coincided with increased arginase I protein expression. Plasma nitric oxide metabolites decreased significantly postoperatively (p < 0.05). Patients lacking an elevation in IL-10 failed to demonstrate increased MNC arginase activity. CONCLUSION: Increased MNC arginase expression may contribute to postsurgical immune dysfunction by affecting arginine use and availability and nitric oxide metabolism in the immune system. Plasma IL-10 may play a role in regulating MNC arginase activity.
Asunto(s)
Arginasa/metabolismo , Sistema Inmunológico/enzimología , Leucocitos Mononucleares/enzimología , Procedimientos Quirúrgicos Operativos , Adulto , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Estudios de Casos y Controles , Femenino , Humanos , Sistema Inmunológico/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Leucocitos Mononucleares/metabolismo , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Óxido Nítrico/sangre , Óxido Nítrico/metabolismoRESUMEN
Arginine is the sole substrate for nitric oxide (NO) synthesis by NO synthases (NOS) and promotes the proliferation and maturation of human T-cells. Arginine is also metabolized by the enzyme arginase, producing urea and ornithine, the precursor for polyamine production. We sought to determine the molecular mechanisms regulating arginase and NOS in splenic immune cells after trauma. C3H/HeN mice underwent laparotomy as simulated moderate trauma or anesthesia alone (n = 24 per group). Six, 12, 24, or 48 h later, 6 animals from each group were sacrificed, and splenectomy was performed and plasma collected. Six separate animals had neither surgery nor anesthesia and were sacrificed to provide resting values (t = 0 h). Spleen arginase I and II and iNOS mRNA abundance, arginase I protein expression, and arginase activity were determined. Plasma NO metabolites (nitrite + nitrate) were also measured. Trauma increased spleen arginase I protein expression and activity (P = 0.01) within 12 and for at least 48 h after injury and coincided with up-regulated arginase I mRNA abundance at 24 h. Neither arginase II nor iNOS mRNA abundance in the spleen was significantly increased by trauma at 24 h. Plasma nitrite + nitrate was decreased in animals 48 h post-injury compared to anesthesia controls (P < 0.05). Trauma induces up-regulation of arginase I gene expression in splenic immune cells within 24 h of injury. Arginase II is not significantly up-regulated at that time point. Arginase I, rather than iNOS appears to be the dominant route for arginine metabolism in splenic immune cells 24 h after trauma.