RESUMEN
Spontaneous tumor regression has been documented in a small proportion of human cancer patients, but the specific mechanisms underlying tumor regression without treatment are not well understood. Tasmanian devils are threatened with extinction from a transmissible cancer due to universal susceptibility and a near 100% case fatality rate. In over 10,000 cases, <20 instances of natural tumor regression have been detected. Previous work in this system has focused on Tasmanian devil genetic variation associated with the regression phenotype. Here, we used comparative and functional genomics to identify tumor genetic variation associated with tumor regression. We show that a single point mutation in the 5' untranslated region of the putative tumor suppressor RASL11A significantly contributes to tumor regression. RASL11A was expressed in regressed tumors but silenced in wild-type, nonregressed tumors, consistent with RASL11A downregulation in human cancers. Induced RASL11A expression significantly reduced tumor cell proliferation in vitro The RAS pathway is frequently altered in human cancers, and RASL11A activation may provide a therapeutic treatment option for Tasmanian devils as well as a general mechanism for tumor inhibition.
Asunto(s)
Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Marsupiales/fisiología , Proteínas de Unión al GTP Monoméricas/metabolismo , Regresión Neoplásica Espontánea , Neoplasias/veterinaria , Animales , Femenino , Proteínas de Unión al GTP Monoméricas/genética , Neoplasias/genética , Neoplasias/patología , Células Tumorales CultivadasRESUMEN
Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Apoptosis/genética , Biología Computacional , Humanos , Neoplasias/patología , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/químicaRESUMEN
Patients with type 2 diabetes often present with cardiovascular complications; however, it is not clear how diabetes promotes cardiac dysfunction. In murine models, deletion of the gene encoding aryl hydrocarbon nuclear translocator (ARNT, also known as HIF1ß) in the liver or pancreas leads to a diabetic phenotype; however, the role of ARNT in cardiac metabolism is unknown. Here, we determined that cardiac-specific deletion of Arnt in adult mice results in rapid development of cardiomyopathy (CM) that is characterized by accumulation of lipid droplets. Compared with hearts from ARNT-expressing mice, ex vivo analysis of ARNT-deficient hearts revealed a 2-fold increase in fatty acid (FA) oxidation as well as a substantial increase in the expression of PPARα and its target genes. Furthermore, deletion of both Arnt and Ppara preserved cardiac function, improved survival, and completely reversed the FA accumulation phenotype, indicating that PPARα mediates the detrimental effects of Arnt deletion in the heart. Finally, we determined that ARNT directly regulates Ppara expression by binding to its promoter and forming a complex with HIF2α. Together, these findings suggest that ARNT is a critical regulator of myocardial FA metabolism and that its deletion leads to CM and an increase in triglyceride accumulation through PPARα.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Cardiomiopatías Diabéticas/genética , Metabolismo de los Lípidos , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Cardiomiopatías Diabéticas/metabolismo , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Ratones Noqueados , Ratones Obesos , Miocardio/metabolismo , Oxidación-Reducción , PPAR alfa/genética , PPAR alfa/metabolismo , Ratas Sprague-Dawley , Transcripción Genética , Triglicéridos/metabolismo , Remodelación VentricularRESUMEN
Hexokinase-II (HKII) is highly expressed in the heart and can bind to the mitochondrial outer membrane. Since cardiac hypertrophy is associated with a substrate switch from fatty acid to glucose, we hypothesized that a reduction in HKII would decrease cardiac hypertrophy after pressure overload. Contrary to our hypothesis, heterozygous HKII-deficient (HKII(+/-)) mice displayed increased hypertrophy and fibrosis in response to pressure overload. The mechanism behind this phenomenon involves increased levels of reactive oxygen species (ROS), as HKII knockdown increased ROS accumulation, and treatment with the antioxidant N-acetylcysteine (NAC) abrogated the exaggerated response. HKII mitochondrial binding is also important for the hypertrophic effects, as HKII dissociation from the mitochondria resulted in de novo hypertrophy, which was also attenuated by NAC. Further studies showed that the increase in ROS levels in response to HKII knockdown or mitochondrial dissociation is mediated through increased mitochondrial permeability and not by a significant change in antioxidant defenses. Overall, these data suggest that HKII and its mitochondrial binding negatively regulate cardiac hypertrophy by decreasing ROS production via mitochondrial permeability.
Asunto(s)
Cardiomegalia/metabolismo , Hexoquinasa/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Cardiomegalia/patología , Células Cultivadas , Fibrosis , Heterocigoto , Hexoquinasa/genética , Hexoquinasa/metabolismo , Masculino , Ratones , Mitocondrias/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Presión , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
RATIONALE: Cardiomyocytes switch substrate utilization from fatty acid to glucose under ischemic conditions; however, it is unknown how perturbations in glycolytic enzymes affect cardiac response to ischemia/reperfusion (I/R). Hexokinase (HK)II is a HK isoform that is expressed in the heart and can bind to the mitochondrial outer membrane. OBJECTIVE: We sought to define how HKII and its binding to mitochondria play a role in cardiac response and remodeling after I/R. METHODS AND RESULTS: We first showed that HKII levels and its binding to mitochondria are reduced 2 days after I/R. We then subjected the hearts of wild-type and heterozygote HKII knockout (HKII(+/)â») mice to I/R by coronary ligation. At baseline, HKII(+/)â» mice have normal cardiac function; however, they display lower systolic function after I/R compared to wild-type animals. The mechanism appears to be through an increase in cardiomyocyte death and fibrosis and a reduction in angiogenesis; the latter is through a decrease in hypoxia-inducible factor-dependent pathway signaling in cardiomyocytes. HKII mitochondrial binding is also critical for cardiomyocyte survival, because its displacement in tissue culture with a synthetic peptide increases cell death. Our results also suggest that HKII may be important for the remodeling of the viable cardiac tissue because its modulation in vitro alters cellular energy levels, O2 consumption, and contractility. CONCLUSIONS: These results suggest that reduction in HKII levels causes altered remodeling of the heart in I/R by increasing cell death and fibrosis and reducing angiogenesis and that mitochondrial binding is needed for protection of cardiomyocytes.