RESUMEN
There is considerable inter-individual variability in susceptibility to weight gain despite an equally obesogenic environment in large parts of the world. Whereas many studies have focused on identifying the genetic susceptibility to obesity, we performed a GWAS on metabolically healthy thin individuals (lowest 6th percentile of the population-wide BMI spectrum) in a uniquely phenotyped Estonian cohort. We discovered anaplastic lymphoma kinase (ALK) as a candidate thinness gene. In Drosophila, RNAi mediated knockdown of Alk led to decreased triglyceride levels. In mice, genetic deletion of Alk resulted in thin animals with marked resistance to diet- and leptin-mutation-induced obesity. Mechanistically, we found that ALK expression in hypothalamic neurons controls energy expenditure via sympathetic control of adipose tissue lipolysis. Our genetic and mechanistic experiments identify ALK as a thinness gene, which is involved in the resistance to weight gain.
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Quinasa de Linfoma Anaplásico/genética , Delgadez/genética , Tejido Adiposo/metabolismo , Adulto , Animales , Línea Celular , Estudios de Cohortes , Drosophila/genética , Estonia , Femenino , Humanos , Leptina/genética , Lipólisis/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/genética , Interferencia de ARN/fisiología , Adulto JovenRESUMEN
Lung cancer is the leading cause of cancer deaths. Besides smoking, epidemiological studies have linked female sex hormones to lung cancer in women; however, the underlying mechanisms remain unclear. Here we report that the receptor activator of nuclear factor-kB (RANK), the key regulator of osteoclastogenesis, is frequently expressed in primary lung tumors, an active RANK pathway correlates with decreased survival, and pharmacologic RANK inhibition reduces tumor growth in patient-derived lung cancer xenografts. Clonal genetic inactivation of KRasG12D in mouse lung epithelial cells markedly impairs the progression of KRasG12D -driven lung cancer, resulting in a significant survival advantage. Mechanistically, RANK rewires energy homeostasis in human and murine lung cancer cells and promotes expansion of lung cancer stem-like cells, which is blocked by inhibiting mitochondrial respiration. Our data also indicate survival differences in KRasG12D -driven lung cancer between male and female mice, and we show that female sex hormones can promote lung cancer progression via the RANK pathway. These data uncover a direct role for RANK in lung cancer and may explain why female sex hormones accelerate lung cancer development. Inhibition of RANK using the approved drug denosumab may be a therapeutic drug candidate for primary lung cancer.
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Neoplasias Pulmonares/metabolismo , Receptor Activador del Factor Nuclear kappa-B/fisiología , Células Epiteliales Alveolares/metabolismo , Animales , Respiración de la Célula , Células Cultivadas , Metabolismo Energético , Femenino , Hormonas Esteroides Gonadales/fisiología , Homeostasis , Humanos , Pulmón/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Ratones , Mitocondrias/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptor Activador del Factor Nuclear kappa-B/antagonistas & inhibidores , Receptor Activador del Factor Nuclear kappa-B/genética , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Mucosa Respiratoria/metabolismoRESUMEN
Bone metastases are a frequent complication of many cancers that result in severe disease burden and pain. Since the late nineteenth century, it has been thought that the microenvironment of the local host tissue actively participates in the propensity of certain cancers to metastasize to specific organs, and that bone provides an especially fertile 'soil'. In the case of breast cancers, the local chemokine milieu is now emerging as an explanation for why these tumours preferentially metastasize to certain organs. However, as the inhibition of chemokine receptors in vivo only partially blocks metastatic behaviour, other factors must exist that regulate the preferential metastasis of breast cancer cells. Here we show that the cytokine RANKL (receptor activator of NF-kappaB ligand) triggers migration of human epithelial cancer cells and melanoma cells that express the receptor RANK. RANK is expressed on cancer cell lines and breast cancer cells in patients. In a mouse model of melanoma metastasis, in vivo neutralization of RANKL by osteoprotegerin results in complete protection from paralysis and a marked reduction in tumour burden in bones but not in other organs. Our data show that local differentiation factors such as RANKL have an important role in cell migration and the tissue-specific metastatic behaviour of cancer cells.
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Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Proteínas Portadoras/metabolismo , Movimiento Celular , Glicoproteínas de Membrana/metabolismo , Metástasis de la Neoplasia/patología , Animales , Neoplasias Óseas/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Portadoras/genética , Muerte Celular , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Células Epiteliales/patología , Femenino , Humanos , Melanoma/metabolismo , Melanoma/patología , Glicoproteínas de Membrana/genética , Ratones , Especificidad de Órganos , Parálisis , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Transducción de SeñalRESUMEN
Oncogenic mutations in the small GTPase RAS contribute to ~30% of human cancers. In a Drosophila genetic screen, we identified novel and evolutionary conserved cancer genes that affect Ras-driven tumorigenesis and metastasis in Drosophila including confirmation of the tetraspanin Tsp29Fb. However, it was not known whether the mammalian Tsp29Fb orthologue, TSPAN6, has any role in RAS-driven human epithelial tumors. Here we show that TSPAN6 suppressed tumor growth and metastatic dissemination of human RAS activating mutant pancreatic cancer xenografts. Whole-body knockout as well as tumor cell autonomous inactivation using floxed alleles of Tspan6 in mice enhanced KrasG12D-driven lung tumor initiation and malignant progression. Mechanistically, TSPAN6 binds to the EGFR and blocks EGFR-induced RAS activation. Moreover, we show that inactivation of TSPAN6 induces an epithelial-to-mesenchymal transition and inhibits cell migration in vitro and in vivo. Finally, low TSPAN6 expression correlates with poor prognosis of patients with lung and pancreatic cancers with mesenchymal morphology. Our results uncover TSPAN6 as a novel tumor suppressor receptor that controls epithelial cell identify and restrains RAS-driven epithelial cancer.
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Oncogenes , Neoplasias Pancreáticas , Tetraspaninas , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Genes ras , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Ratones , Mutación , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Tetraspaninas/genética , Tetraspaninas/metabolismoRESUMEN
The recent emergence of multiple SARS-CoV-2 variants has caused considerable concern due to both reduced vaccine efficacy and escape from neutralizing antibody therapeutics. It is, therefore, paramount to develop therapeutic strategies that inhibit all known and future SARS-CoV-2 variants. Here, we report that all SARS-CoV-2 variants analyzed, including variants of concern (VOC) Alpha, Beta, Gamma, Delta, and Omicron, exhibit enhanced binding affinity to clinical grade and phase 2 tested recombinant human soluble ACE2 (APN01). Importantly, soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by all current VOC strains with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. Effective inhibition of infections with SARS-CoV-2 variants was validated and confirmed in two independent laboratories. These data show that SARS-CoV-2 variants that have emerged around the world, including current VOC and several variants of interest, can be inhibited by soluble ACE2, providing proof of principle of a pan-SARS-CoV-2 therapeutic.
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Enzima Convertidora de Angiotensina 2 , Tratamiento Farmacológico de COVID-19 , Humanos , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2RESUMEN
BACKGROUND: Hundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase. RESULTS: The C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC50 values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation. CONCLUSIONS: Identification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.
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Cryptosporidium parvum/enzimología , Proteínas Quinasas/análisis , Proteínas Protozoarias/análisis , Cryptosporidium parvum/genética , Bases de Datos de Proteínas , Plasmodium falciparum/enzimología , Proteínas Quinasas/clasificación , Proteínas Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , Toxoplasma/enzimologíaRESUMEN
During the development of multicellular organisms, concerted actions of molecular signalling networks determine whether cells undergo proliferation, differentiation, death or ageing. Here we show that genetic inactivation of the stress signalling kinase, MKK7, a direct activator of JNKs in mice, results in embryonic lethality and impaired proliferation of hepatocytes. Beginning at passage 4-5, mkk7(-/-) mouse embryonic fibroblasts (MEFs) display impaired proliferation, premature senescence and G2/M cell cycle arrest. Similarly, loss of c-Jun or expression of a c-JunAA mutant in which the JNK phosphorylation sites were replaced with alanine results in a G2/M cell-cycle block. The G2/M cell-cycle kinase CDC2 was identified as a target for the MKK7-JNK-c-Jun pathway. These data show that the MKK7-JNK-c-Jun signalling pathway couples developmental and environmental cues to CDC2 expression, G2/M cell cycle progression and cellular senescence in fibroblasts.
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Senescencia Celular/genética , Fase G2/genética , Hepatocitos/enzimología , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Mitosis/genética , Estrés Fisiológico/enzimología , Animales , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Feto , Fibroblastos/citología , Fibroblastos/enzimología , Genes Letales/genética , Hepatocitos/citología , Proteínas Quinasas JNK Activadas por Mitógenos , Hígado/anomalías , Hígado/patología , MAP Quinasa Quinasa 7 , Sistema de Señalización de MAP Quinasas/genética , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación/genética , Fosforilación , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/genética , Estrés Fisiológico/genéticaRESUMEN
Protein-based affinity reagents (like antibodies or alternative binding scaffolds) offer wide-ranging applications for basic research and therapeutic approaches. However, whereas small chemical molecules efficiently reach intracellular targets, the delivery of macromolecules into the cytosol of cells remains a major challenge; thus cytosolic applications of protein-based reagents are rather limited. Some pathogenic bacteria have evolved a conserved type III secretion system (T3SS) which allows the delivery of effector proteins into eukaryotic cells. Here, we enhance the T3SS of an avirulent strain of Salmonella typhimurium to reproducibly deliver multiple classes of recombinant proteins into eukaryotic cells. The efficacy of the system is probed with both DARPins and monobodies to functionally inhibit the paradigmatic and largely undruggable RAS signaling pathway. Thus, we develop a bacterial secretion system for potent cytosolic delivery of therapeutic macromolecules.
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Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Citosol/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Células HCT116 , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Proteínas Recombinantes/genética , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrollo , Sistemas de Secreción Tipo III/genéticaRESUMEN
Breast cancer risk is reduced by number of pregnancies and breastfeeding duration, however studies of breast changes during or after pregnancy are rare. Breast volume changes - although not linked to breast cancer risk - might be an interesting phenotype in this context for correlative studies, as changes of breast volume vary between pregnant women. Serum receptor activator of nuclear factor kappa B ligand (RANKL) and its antagonist osteoprotegerin (OPG) were measured prospectively before gestational week 12, and three-dimensional breast volume assessments were performed. A linear regression model including breast volume at the start of pregnancy, RANKL, OPG, and other factors was used to predict breast volume at term. The mean breast volume was 413 mL at gestational week 12, increasing by a mean of 99 mL up to gestational week 40. In addition to body mass index and breast volume at the beginning of pregnancy, RANKL and OPG appeared to influence breast volume with a mean increase by 32 mL (P = 0.04) and a mean reduction by 27 mL (P = 0.04), respectively. Linking the RANKL/RANK/OPG pathway with breast volume changes supports further studies aiming at analysing breast changes during pregnancy with regard to breast cancer risk.
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Mama/metabolismo , Osteoprotegerina/metabolismo , Embarazo/metabolismo , Ligando RANK/metabolismo , Adulto , Mama/anatomía & histología , Femenino , Humanos , Estudios Prospectivos , Salud de la MujerRESUMEN
OBJECTIVE: We aim to characterize the causality and molecular and functional underpinnings of HACE1 deficiency in a mouse model of a recessive neurodevelopmental syndrome called spastic paraplegia and psychomotor retardation with or without seizures (SPPRS). METHODS: By exome sequencing, we identified 2 novel homozygous truncating mutations in HACE1 in 3 patients from 2 families, p.Q209* and p.R332*. Furthermore, we performed detailed molecular and phenotypic analyses of Hace1 knock-out (KO) mice and SPPRS patient fibroblasts. RESULTS: We show that Hace1 KO mice display many clinical features of SPPRS including enlarged ventricles, hypoplastic corpus callosum, as well as locomotion and learning deficiencies. Mechanistically, loss of HACE1 results in altered levels and activity of the small guanosine triphosphate (GTP)ase, RAC1. In addition, HACE1 deficiency results in reduction in synaptic puncta number and long-term potentiation in the hippocampus. Similarly, in SPPRS patient-derived fibroblasts, carrying a disruptive HACE1 mutation resembling loss of HACE1 in KO mice, we observed marked upregulation of the total and active, GTP-bound, form of RAC1, along with an induction of RAC1-regulated downstream pathways. CONCLUSIONS: Our results provide a first animal model to dissect this complex human disease syndrome, establishing the first causal proof that a HACE1 deficiency results in decreased synapse number and structural and behavioral neuropathologic features that resemble SPPRS patients.
RESUMEN
Cancer is a major and still increasing cause of death in humans. Most cancer cells have a fundamentally different metabolic profile from that of normal tissue. This shift away from mitochondrial ATP synthesis via oxidative phosphorylation towards a high rate of glycolysis, termed Warburg effect, has long been recognized as a paradigmatic hallmark of cancer, supporting the increased biosynthetic demands of tumor cells. Here we show that deletion of apoptosis-inducing factor (AIF) in a KrasG12D-driven mouse lung cancer model resulted in a marked survival advantage, with delayed tumor onset and decreased malignant progression. Mechanistically, Aif deletion leads to oxidative phosphorylation (OXPHOS) deficiency and a switch in cellular metabolism towards glycolysis in non-transformed pneumocytes and at early stages of tumor development. Paradoxically, although Aif-deficient cells exhibited a metabolic Warburg profile, this bioenergetic change resulted in a growth disadvantage of KrasG12D-driven as well as Kras wild-type lung cancer cells. Cell-autonomous re-expression of both wild-type and mutant AIF (displaying an intact mitochondrial, but abrogated apoptotic function) in Aif-knockout KrasG12D mice restored OXPHOS and reduced animal survival to the same level as AIF wild-type mice. In patients with non-small cell lung cancer, high AIF expression was associated with poor prognosis. These data show that AIF-regulated mitochondrial respiration and OXPHOS drive the progression of lung cancer.
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Factor Inductor de la Apoptosis/fisiología , Carcinogénesis/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Conjuntos de Datos como Asunto , Progresión de la Enfermedad , Glucólisis , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Fosforilación OxidativaRESUMEN
Parasites from the protozoan phylum Apicomplexa are responsible for diseases, such as malaria, toxoplasmosis and cryptosporidiosis, all of which have significantly higher rates of mortality and morbidity in economically underdeveloped regions of the world. Advances in vaccine development and drug discovery are urgently needed to control these diseases and can be facilitated by production of purified recombinant proteins from Apicomplexan genomes and determination of their 3D structures. To date, both heterologous expression and crystallization of Apicomplexan proteins have seen only limited success. In an effort to explore the effectiveness of producing and crystallizing proteins on a genome-scale using a standardized methodology, over 400 distinct Plasmodium falciparum target genes were chosen representing different cellular classes, along with select orthologues from four other Plasmodium species as well as Cryptosporidium parvum and Toxoplasma gondii. From a total of 1008 genes from the seven genomes, 304 (30.2%) produced purified soluble proteins and 97 (9.6%) crystallized, culminating in 36 crystal structures. These results demonstrate that, contrary to previous findings, a standardized platform using Escherichia coli can be effective for genome-scale production and crystallography of Apicomplexan proteins. Predictably, orthologous proteins from different Apicomplexan genomes behaved differently in expression, purification and crystallization, although the overall success rates of Plasmodium orthologues do not differ significantly. Their differences were effectively exploited to elevate the overall productivity to levels comparable to the most successful ongoing structural genomics projects: 229 of the 468 target genes produced purified soluble protein from one or more organisms, with 80 and 32 of the purified targets, respectively, leading to crystals and ultimately structures from one or more orthologues.
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Genoma de Protozoos/genética , Plasmodium falciparum/química , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Animales , Cristalización , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Modelos Moleculares , Plasmodium falciparum/genética , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , SolubilidadRESUMEN
Fungal infections claim an estimated 1.5 million lives each year. Mechanisms that protect from fungal infections are still elusive. Recognition of fungal pathogens relies on C-type lectin receptors (CLRs) and their downstream signaling kinase SYK. Here we report that the E3 ubiquitin ligase CBLB controls proximal CLR signaling in macrophages and dendritic cells. We show that CBLB associates with SYK and ubiquitinates SYK, dectin-1, and dectin-2 after fungal recognition. Functionally, CBLB deficiency results in increased inflammasome activation, enhanced reactive oxygen species production, and increased fungal killing. Genetic deletion of Cblb protects mice from morbidity caused by cutaneous infection and markedly improves survival after a lethal systemic infection with Candida albicans. On the basis of these findings, we engineered a cell-permeable CBLB inhibitory peptide that protects mice from lethal C. albicans infections. We thus describe a key role for Cblb in the regulation of innate antifungal immunity and establish a novel paradigm for the treatment of fungal sepsis.
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Proteínas Adaptadoras Transductoras de Señales/inmunología , Candidiasis Invasiva/inmunología , Células Dendríticas/inmunología , Lectinas Tipo C/metabolismo , Macrófagos/inmunología , Péptidos/farmacología , Fagocitosis/inmunología , Proteínas Proto-Oncogénicas c-cbl/inmunología , Especies Reactivas de Oxígeno/inmunología , Sepsis/inmunología , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Western Blotting , Candida albicans , Caspasa 8 , Citocinas/inmunología , Células Dendríticas/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Inmunoprecipitación , Riñón , Lectinas Tipo C/efectos de los fármacos , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Fagocitosis/efectos de los fármacos , Fagocitosis/genética , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-cbl/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-cbl/genética , UbiquitinaciónRESUMEN
The HECT domain E3 ligase HACE1 has been identified as a tumor suppressor in multiple cancers. Here, we report that HACE1 is a central gatekeeper of TNFR1-induced cell fate. Genetic inactivation of HACE1 inhibits TNF-stimulated NF-κB activation and TNFR1-NF-κB-dependent pathogen clearance in vivo. Moreover, TNF-induced apoptosis was impaired in hace1 mutant cells and knockout mice in vivo. Mechanistically, HACE1 is essential for the ubiquitylation of the adaptor protein TRAF2 and formation of the apoptotic caspase-8 effector complex. Intriguingly, loss of HACE1 does not impair TNFR1-mediated necroptotic cell fate via RIP1 and RIP3 kinases. Loss of HACE1 predisposes animals to colonic inflammation and carcinogenesis in vivo, which is markedly alleviated by genetic inactivation of RIP3 kinase and TNFR1. Thus, HACE1 controls TNF-elicited cell fate decisions and exerts tumor suppressor and anti-inflammatory activities via a TNFR1-RIP3 kinase-necroptosis pathway.
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Linaje de la Célula , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 8/metabolismo , Linaje de la Célula/efectos de los fármacos , Colitis/metabolismo , Colitis/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Sulfato de Dextran , Embrión de Mamíferos/citología , Activación Enzimática/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Eliminación de Gen , Ratones Endogámicos C57BL , Mutación/genética , FN-kappa B/metabolismo , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitinación/efectos de los fármacosRESUMEN
Breast cancer is the most common female cancer, affecting approximately one in eight women during their life-time. Besides environmental triggers and hormones, inherited mutations in the breast cancer 1 (BRCA1) or BRCA2 genes markedly increase the risk for the development of breast cancer. Here, using two different mouse models, we show that genetic inactivation of the key osteoclast differentiation factor RANK in the mammary epithelium markedly delayed onset, reduced incidence, and attenuated progression of Brca1;p53 mutation-driven mammary cancer. Long-term pharmacological inhibition of the RANK ligand RANKL in mice abolished the occurrence of Brca1 mutation-driven pre-neoplastic lesions. Mechanistically, genetic inactivation of Rank or RANKL/RANK blockade impaired proliferation and expansion of both murine Brca1;p53 mutant mammary stem cells and mammary progenitors from human BRCA1 mutation carriers. In addition, genome variations within the RANK locus were significantly associated with risk of developing breast cancer in women with BRCA1 mutations. Thus, RANKL/RANK control progenitor cell expansion and tumorigenesis in inherited breast cancer. These results present a viable strategy for the possible prevention of breast cancer in BRCA1 mutant patients.
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Proteína BRCA1/genética , Neoplasias de la Mama/genética , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Animales , Proteína BRCA2/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Daño del ADN/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Receptor alfa de Estrógeno/metabolismo , Femenino , Genotipo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ligando RANK/antagonistas & inhibidores , Ligando RANK/genética , Receptor Activador del Factor Nuclear kappa-B/genética , Receptores de Progesterona/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Células Madre/citología , Células Madre/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: We have recently shown that genetic inactivation of phosphoinositide 3-kinase gamma (PI3Kgamma), the isoform linked to G-protein-coupled receptors, results in increased cardiac contractility with no effect on basal cell size. Signaling via the G-protein-coupled beta-adrenergic receptors has been implicated in cardiac hypertrophy and heart failure, suggesting that PI3Kgamma might play a role in the pathogenesis of heart disease. METHODS AND RESULTS: To determine the role for PI3Kgamma in hypertrophy induced by G-protein-coupled receptors and cardiomyopathy, we infused isoproterenol, a beta-adrenergic receptor agonist, into PI3Kgamma-deficient mice. Compared with controls, isoproterenol infusion in PI3Kgamma-deficient mice resulted in an attenuated cardiac hypertrophic response and markedly reduced interstitial fibrosis. Intriguingly, chronic beta-adrenergic receptor stimulation triggered impaired heart functions in wild-type mice, whereas PI3Kgamma-deficient mice retained their increased heart function and did not develop heart failure. The lack of PI3Kgamma attenuated the activation of Akt/protein kinase B and extracellular signal-regulated kinase 1/2 signaling pathways in cardiac myocytes in response to isoproterenol. beta1- and beta2-adrenergic receptor densities were decreased by similar amounts in PI3Kgamma-deficient and control mice, suggesting that PI3Kgamma isoform plays no role in the downregulation of beta-adrenergic receptors after chronic beta-adrenergic stimulation. CONCLUSIONS: Our data show that PI3Kgamma is critical for the induction of hypertrophy, fibrosis, and cardiac dysfunction function in response to beta-adrenergic receptor stimulation in vivo. Thus, PI3Kgamma may represent a novel therapeutic target for the treatment of decreased cardiac function in heart failure.
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Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/prevención & control , Isoproterenol , Fosfatidilinositol 3-Quinasas/deficiencia , Agonistas Adrenérgicos beta , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/enzimología , Cardiomegalia/prevención & control , Dominio Catalítico/genética , Modelos Animales de Enfermedad , Fibrosis/genética , Fibrosis/patología , Fibrosis/prevención & control , Corazón/efectos de los fármacos , Insuficiencia Cardíaca/inducido químicamente , Insuficiencia Cardíaca/patología , Masculino , Ratones , Ratones Noqueados , Miocardio/enzimología , Miocardio/patología , Fosfatidilinositol 3-Quinasas/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
PR homology domain-containing member 12 (PRDM12) belongs to a family of conserved transcription factors implicated in cell fate decisions. Here we show that PRDM12 is a key regulator of sensory neuronal specification in Xenopus. Modeling of human PRDM12 mutations that cause hereditary sensory and autonomic neuropathy (HSAN) revealed remarkable conservation of the mutated residues in evolution. Expression of wild-type human PRDM12 in Xenopus induced the expression of sensory neuronal markers, which was reduced using various human PRDM12 mutants. In Drosophila, we identified Hamlet as the functional PRDM12 homolog that controls nociceptive behavior in sensory neurons. Furthermore, expression analysis of human patient fibroblasts with PRDM12 mutations uncovered possible downstream target genes. Knockdown of several of these target genes including thyrotropin-releasing hormone degrading enzyme (TRHDE) in Drosophila sensory neurons resulted in altered cellular morphology and impaired nociception. These data show that PRDM12 and its functional fly homolog Hamlet are evolutionary conserved master regulators of sensory neuronal specification and play a critical role in pain perception. Our data also uncover novel pathways in multiple species that regulate evolutionary conserved nociception.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Neuronas/patología , Percepción del Dolor , Secuencia de Aminoácidos , Animales , Linaje de la Célula , Cristalografía por Rayos X , Drosophila , Femenino , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Humanos , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Mutación , Neurogénesis/genética , Neuronas/metabolismo , Estructura Terciaria de Proteína , Células Receptoras Sensoriales/metabolismo , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
Neutrophils are key innate immune effector cells that are essential to fighting bacterial and fungal pathogens. Here we report that mice carrying a hematopoietic lineage-specific deletion of Jagn1 (encoding Jagunal homolog 1) cannot mount an efficient neutrophil-dependent immune response to the human fungal pathogen Candida albicans. Global glycobiome analysis identified marked alterations in the glycosylation of proteins involved in cell adhesion and cytotoxicity in Jagn1-deficient neutrophils. Functional analysis confirmed marked defects in neutrophil migration in response to Candida albicans infection and impaired formation of cytotoxic granules, as well as defective myeloperoxidase release and killing of Candida albicans. Treatment with granulocyte/macrophage colony-stimulating factor (GM-CSF) protected mutant mice from increased weight loss and accelerated mortality after Candida albicans challenge. Notably, GM-CSF also restored the defective fungicidal activity of bone marrow cells from humans with JAGN1 mutations. These data directly identify Jagn1 (JAGN1 in humans) as a new regulator of neutrophil function in microbial pathogenesis and uncover a potential treatment option for humans.