RESUMEN
Owing to their pleiotropic metabolic benefits, glucagon-like peptide-1 receptor (GLP-1R) agonists have been successfully utilized for treating metabolic diseases, such as type 2 diabetes and obesity. As part of our efforts in developing long-acting peptide therapeutics, we have previously reported a peptide engineering strategy that combines peptide side chain stapling with covalent integration of a serum protein-binding motif in a single step. Herein, we have used this strategy to develop a second generation extendin-4 analog rigidified with a symmetrical staple, which exhibits an excellent in vivo efficacy in an animal model of diabetes and obesity. To simplify the scale-up manufacturing of the lead GLP-1R agonist, a semisynthesis protocol was successfully developed, which involves recombinant expression of the linear peptide followed by attachment of a polyethylene glycol (PEG)-fatty acid staple in a subsequent chemical reaction step.
Asunto(s)
Exenatida/análogos & derivados , Exenatida/metabolismo , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Animales , Diabetes Mellitus Tipo 2 , Exenatida/química , Ácidos Grasos/química , Masculino , Ratones , Estructura Molecular , Obesidad , Péptidos/química , Péptidos/metabolismo , Polietilenglicoles/químicaRESUMEN
Oxyntomodulin (OXM) is an intestinal peptide hormone that activates both glucagon-like peptide-1 (GLP-1) and glucagon (GCG) receptors. The natural peptide reduces body weight in obese subjects and exhibits direct acute glucoregulatory effects in patients with type II diabetes. However, the clinical utility of OXM is limited due to its lower in vitro potency and short in vivo half-life. To overcome these issues, we developed stapled, long-acting, and highly potent OXM analogs with balanced activities at both GLP-1 and GCG receptors. The lead molecule O14 exhibits potent and long-lasting effects on glucose control, body weight loss, and reduction of hepatic fat reduction in DIO mice. Importantly, O14 significantly reversed hepatic steatosis; reduced liver weight, total cholesterol, and hepatic triglycerides; and improved markers of liver function in a nonalcoholic steatohepatitis (NASH) mouse model. A symmetrical version of the peptide was also shown to be more efficacious and long-lasting in controlling glucose than semaglutide and the clinical candidate cotadutide in wild-type mice, highlighting the utility of our designs of the dual agonist as a potential new therapy for diabetes and liver diseases.
Asunto(s)
Peso Corporal/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Oxintomodulina/farmacología , Oxintomodulina/farmacocinética , Animales , Glucemia/metabolismo , Colesterol/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Enfermedad del Hígado Graso no Alcohólico/sangre , Oxintomodulina/uso terapéutico , Triglicéridos/metabolismoRESUMEN
Anorexigenic peptides offer promise as potential therapies targeting the escalating global obesity epidemic. Prolactin-releasing peptide (PrRP), a novel member of the RFamide family secreted by the hypothalamus, shows therapeutic potential by decreasing food intake and body weight in rodent models via GPR10 activation. Here we describe the design of a long-acting PrRP using our recently developed novel multiple ethylene glycol-fatty acid (MEG-FA) stapling platform. By incorporating serum albumin binding fatty acids onto a covalent side chain staple, we have generated a series of MEG-FA stapled PrRP analogs with enhanced serum stability and in vivo half-life. Our lead compound 18-S4 exhibits good in vitro potency and selectivity against GPR10, improved serum stability, and extended in vivo half-life (7.8 h) in mouse. Furthermore, 18-S4 demonstrates a potent body weight reduction effect in a diet-induced obesity (DIO) mouse model, representing a promising long-acting PrRP analog for further evaluation in the chronic obesity setting.
RESUMEN
A panel of three lipid-modified, functionalized biphenyl cross-linkers (fBph) were synthesized and subsequently employed in the preparation of the stapled oxyntomodulin (OXM) analogs. In a luciferase-based reporter assay, these stapled OXM analogs showed varying degree of potency in activating GLP-1R and GCGR, presumably due to the disparate effect of the lipid chains on the local environment close to the ligand-receptor binding interface. In particular, the fBph-1 cross-linked peptide with the lipid chain attached to position-3 of the biphenyl cross-linker exhibited the highest dual agonist activity.
RESUMEN
Peptide hormone relaxin-2, a member of the insulin family of peptides, plays a key role in hemodynamics and renal function and has shown preclinical efficacy in multiple disease models, including acute heart failure, fibrosis, preeclampsia, and corneal wound healing. Recently, serelaxin, a recombinant version of relaxin-2, has been studied in a large phase 3 clinical trial (RELAX-AHF-2) for acute decompensated heart failure patients with disappointing outcome. The poor in vivo half-life of relaxin-2 may have limited its therapeutic efficacy and long-term cardiovascular benefit. Herein, we have developed a semisynthetic methodology and generated potent, fatty acid-conjugated relaxin analogs with long-acting pharmacokinetic (PK) profile in rodents. The enhanced PK properties translated into improved and long-lasting pharmacodynamic effect in pubic ligament elongation (PLE) studies. The resultant novel relaxin analog, R9-13, represents the first long-acting relaxin-2 analog and could potentially improve the clinical efficacy and outcome for this important peptide hormone. This semisynthetic methodology could also be applied to other cysteine-rich peptides and proteins for half-life extension.
Asunto(s)
Diseño de Fármacos , Lípidos/química , Relaxina/química , Relaxina/uso terapéutico , Secuencia de Aminoácidos , Animales , Semivida , Insuficiencia Cardíaca/tratamiento farmacológico , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Relaxina/farmacocinéticaRESUMEN
Glucagon-like peptide 2 (GLP-2) is a hormone that has been shown to stimulate intestinal growth and attenuate intestinal inflammation. Despite being efficacious in a variety of animal models of disease, its therapeutic potential is hampered by the short half-life in vivo. We now describe a highly potent, stapled long-acting GLP-2 analog, peptide 10, that has a more than 10-fold longer half-life than teduglutide and improved intestinotrophic and anti-inflammatory effects in mouse models of DSS-induced colitis.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Colitis/tratamiento farmacológico , Fármacos Gastrointestinales/farmacología , Péptido 2 Similar al Glucagón/farmacología , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/farmacocinética , Colitis/inducido químicamente , Reactivos de Enlaces Cruzados , AMP Cíclico/biosíntesis , Sulfato de Dextran , Diseño de Fármacos , Femenino , Fármacos Gastrointestinales/síntesis química , Fármacos Gastrointestinales/farmacocinética , Péptido 2 Similar al Glucagón/síntesis química , Péptido 2 Similar al Glucagón/farmacocinética , Semivida , Intestinos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Conformación Molecular , Péptidos/farmacocinética , Péptidos/farmacologíaRESUMEN
Elevated triglyceride (TG) levels are well-correlated with the risk for cardiovascular disease (CVD). Apolipoprotein CIII (ApoC-III) is a key regulator of plasma TG levels through regulation of lipolysis and lipid synthesis. To identify novel regulators of TG levels, we carried out a high throughput screen (HTS) using an ApoC-III homogenous time resolved fluorescence (HTRF) assay. We identified several retinoic acid receptor (RAR) agonists that reduced secreted ApoC-III levels in human hepatic cell lines. The RARα specific agonist AM580 inhibited secreted ApoC-III by >80% in Hep3B cells with an EC50 ~2.9 nM. In high-fat diet induced fatty-liver mice, AM580 reduced ApoC-III levels in liver as well as in plasma (~60%). In addition, AM580 treatment effectively reduced body weight, hepatic and plasma TG, and total cholesterol (TC) levels. Mechanistically, AM580 suppresses ApoC-III synthesis by downregulation of HNF4α and upregulation of SHP1 expression. Collectively, these studies suggest that an RARα specific agonist may afford a new strategy for lipid-lowering and CVD risk reduction.
Asunto(s)
Apolipoproteína C-III/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Receptor alfa de Ácido Retinoico/agonistas , Animales , Apolipoproteína C-III/sangre , Apolipoproteína C-III/genética , Línea Celular Tumoral , Dieta Alta en Grasa , Regulación de la Expresión Génica/efectos de los fármacos , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Hígado/metabolismo , Ratones , Triglicéridos/sangreRESUMEN
Recent studies have suggested that modulation of two or more signaling pathways can achieve substantial weight loss and glycemic stability. We have developed an approach to the generation of bifunctional antibody agonists that activate leptin receptor and GLP-1 receptor. Leptin was fused into the complementarity determining region 3 loop of the light chain alone, or in combination with exendin-4 (EX4) fused at the N-terminus of the heavy chain of Herceptin. The antibody fusions exhibit similar or increased in vitro activities on their cognate receptors, but 50-100-fold longer circulating half-lives in rodents compared to the corresponding native peptides/proteins. The efficacy of the leptin/EX4 dual antibody fusion on weight loss, especially fat mass loss, was enhanced in ob/ob mice and DIO mice compared to the antibody fusion of either EX4 or leptin alone. This work demonstrates the versatility of this combinatorial fusion strategy for generating dual antibody agonists with long half-lives.
Asunto(s)
Anticuerpos/química , Hormonas/uso terapéutico , Animales , Semivida , Hormonas/química , Hormonas/farmacocinética , RatonesRESUMEN
Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), glucagon (GCG) receptor (GCGR), and glucose-dependent insulinotropic polypeptide (GIP, also known as gastric inhibitory polypeptide) receptor (GIPR), are three metabolically related peptide hormone receptors. A novel approach to the generation of multifunctional antibody agonists that activate these receptors has been developed. Native or engineered peptide agonists for GLP-1R, GCGR, and GIPR were fused to the N-terminus of the heavy chain or light chain of an antibody, either alone or in pairwise combinations. The fusion proteins have similar inâ vitro biological activities on the cognate receptors as the corresponding peptides, but circa 100-fold longer plasma half-lives. The GLP-1R mono agonist and GLP-1R/GCGR dual agonist antibodies both exhibit potent effects on glucose control and body weight reduction in mice, with the dual agonist antibody showing enhanced activity in the latter.
Asunto(s)
Anticuerpos/inmunología , Péptido 1 Similar al Glucagón/agonistas , Glucagón/agonistas , Receptores de la Hormona Gastrointestinal/agonistas , Animales , Anticuerpos/genética , Anticuerpos/metabolismo , Peso Corporal/efectos de los fármacos , Femenino , Glucagón/inmunología , Péptido 1 Similar al Glucagón/inmunología , Células HEK293 , Semivida , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/metabolismo , Ratones , Ratones Obesos , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Ingeniería de Proteínas , Ratas , Ratas Sprague-Dawley , Receptores de la Hormona Gastrointestinal/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Antidiabetic treatments aiming to reduce body weight are currently gaining increased interest. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist administered twice daily via s.c. injection, improves glycemic control, often with associated weight reduction. To further improve the therapeutic efficacy of exendin-4, we have developed a novel peptide engineering strategy that incorporates a serum protein binding motif onto a covalent side-chain staple and applied to the peptide to enhance its helicity and, as a consequence, its potency and serum half-life. We demonstrated that one of the resulting peptides, E6, has significantly improved half-life and glucose tolerance in an oral glucose tolerance test in rodents. Chronic treatment of E6 significantly decreased body weight and fasting blood glucose, improved lipid metabolism, and also reduced hepatic steatosis in diet-induced obese mice. Moreover, the high potency of E6 allowed us to administer this peptide using a dissolvable microstructure-based transdermal delivery system. Pharmacokinetic and pharmacodynamic studies in guinea pigs showed that a single 5-min application of a microstructure system containing E6 significantly improved glucose tolerance for 96 h. This delivery strategy may offer an effective and patient-friendly alternative to currently marketed GLP-1 injectables and can likely be extended to other peptide hormones.
Asunto(s)
Péptido 1 Similar al Glucagón/química , Ingeniería de Proteínas , Administración Cutánea , Secuencia de Aminoácidos , Peso Corporal , Dicroismo Circular , AMP Cíclico/biosíntesis , Péptido 1 Similar al Glucagón/administración & dosificación , Péptido 1 Similar al Glucagón/farmacocinética , Prueba de Tolerancia a la Glucosa , Células HEK293 , HumanosRESUMEN
Incretin-based peptides are effective therapeutics for treating type 2 diabetes mellitus (T2DM). Oxyntomodulin (OXM), a dual agonist of GLP-1R and GCGR, has shown superior weight loss and glucose lowering effects, compared to single GLP-1R agonists. To overcome the short half-life and rapid renal clearance of OXM, which limit its therapeutic potential, both lipid and PEG modified OXM analogs have been reported. However, these approaches often result in reduced potency or PEG-associated toxicity. Herein, we report a new class of cross-linked OXM analogs that show increased plasma stability and higher potency in activating both GLP-1R and GCGR. Moreover, the extended in vivo half-life results in superior antihyperglycemic activity in mice compared to the wild-type OXM.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Oxintomodulina/química , Oxintomodulina/farmacología , Receptores de Glucagón/agonistas , Secuencia de Aminoácidos , Animales , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/farmacocinética , Reactivos de Enlaces Cruzados/farmacología , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Células HEK293 , Humanos , Hipoglucemiantes/sangre , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Oxintomodulina/sangre , Proteolisis , Receptores de Glucagón/metabolismoRESUMEN
Bovine antibody BLV1H12 possesses a unique "stalk-knob" architecture in its ultralong heavy chain CDR3, allowing substitutions of the "knob" domain with protein agonists to generate functional antibody chimeras. We have generated a humanized glucagon-like peptide-1 (GLP-1) receptor agonist antibody by first introducing a coiled-coil "stalk" into CDR3H of the antibody herceptin. Exendin-4 (Ex-4), a GLP-1 receptor agonist, was then fused to the engineered stalk with flexible linkers, and a Factor Xa cleavage site was inserted immediately in front of Ex-4 to allow release of the N-terminus of the fused peptide. The resulting clipped herceptin-Ex-4 fusion protein is more potent inâ vitro in activating GLP-1 receptors than the Ex-4 peptide. The clipped herceptin-Ex-4 has an extended plasma half-life of approximately four days and sustained control of blood glucose levels for more than a week in mice. This work provides a novel approach to the development of human or humanized agonist antibodies as therapeutics.
Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Péptidos/inmunología , Receptores de Glucagón/agonistas , Receptores de Glucagón/inmunología , Proteínas Recombinantes de Fusión/inmunología , Ponzoñas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/farmacología , Bovinos , Exenatida , Receptor del Péptido 1 Similar al Glucagón , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Péptidos/farmacología , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Trastuzumab , Ponzoñas/química , Ponzoñas/genética , Ponzoñas/farmacologíaRESUMEN
Oxidative stress can induce premature cellular senescence. Senescent cells secrete various growth factors and cytokines, such as IL-6, that can signal to the tumor microenvironment and promote cancer cell growth. Sirtuin 1 (Sirt1) is a class III histone deacetylase that regulates a variety of physiological processes, including senescence. We found that caveolin-1, a structural protein component of caveolar membranes, is a direct binding partner of Sirt1, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82-101) to the caveolin-binding domain of Sirt1 (amino acids 310-317). Our data show that oxidative stress promotes the sequestration of Sirt1 into caveolar membranes and the interaction of Sirt1 with caveolin-1, which lead to inhibition of Sirt1 activity. Reactive oxygen species stimulation promotes acetylation of p53 and premature senescence in wild-type but not caveolin-1 null mouse embryonic fibroblasts (MEFs). Either down-regulation of Sirt1 expression or re-expression of caveolin-1 in caveolin-1 null MEFs restores reactive oxygen species-induced acetylation of p53 and premature senescence. In addition, overexpression of caveolin-1 induces stress induced premature senescence in p53 wild-type but not p53 knockout MEFs. Phosphorylation of caveolin-1 on tyrosine 14 promotes the sequestration of Sirt1 into caveolar membranes and activates p53/senescence signaling. We also identified IL-6 as a caveolin-1-specific cytokine that is secreted by senescent fibroblasts following the caveolin-1-mediated inhibition of Sirt1. The caveolin-1-mediated secretion of IL-6 by senescent fibroblasts stimulates the growth of cancer cells. Therefore, by inhibiting Sirt1, caveolin-1 links free radicals to the activation of the p53/senescence pathway and the protumorigenic properties of IL-6.
Asunto(s)
Caveolina 1/metabolismo , Senescencia Celular , Interleucina-6/metabolismo , Neoplasias/metabolismo , Estrés Oxidativo , Sirtuina 1/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Animales , Western Blotting , Caveolas , Caveolina 1/genética , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Inmunoprecipitación , Ratones , Ratones Noqueados , Células 3T3 NIH , Neoplasias/genética , Neoplasias/patología , Fosforilación , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/genéticaRESUMEN
Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth and metabolism. Its activity is controlled by various types of signals, including growth factors, nutrients, and stresses. In this study, we show that changes in expression levels of two antiapoptotic proteins, Bcl-2 and Bcl-XL, also affect mTORC1 signaling activity. In cells overexpressing Bcl-XL, mTORC1 activity is increased and becomes less sensitive to growth factor or nutrient conditions. In contrast, reduction in expression levels of the two antiapoptotic proteins inhibits mTORC1 signaling activity. Our results suggest that the effect of Bcl-2 and Bcl-XL on mTORC1 is mediated by FKBP38, an inhibitor of mTORC1. The two proteins compete with mTORC1 for FKBP38 binding and hence alter mTORC1 activity. This study reveals a novel cross-talk between Bcl-2/XL and mTORC1 signaling, which is likely to contribute to cancer development.
Asunto(s)
Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína bcl-X/metabolismo , Apoptosis , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Mitocondrias/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
Caveolin-1, the structural protein component of caveolae, acts as a scaffolding protein that functionally regulates signaling molecules. We show that knockdown of caveolin-1 protein expression enhances chemotherapeutic drug-induced apoptosis and inhibits long-term survival of colon cancer cells. In vitro studies demonstrate that caveolin-1 is a novel Ku70-binding protein, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82-101) to the caveolin-binding domain (CBD) of Ku70 (amino acids 471-478). Cell culture data show that caveolin-1 binds Ku70 after treatment with chemotherapeutic drugs. Mechanistically, we found that binding of caveolin-1 to Ku70 inhibits the chemotherapeutic drug-induced release of Bax from Ku70, activation of Bax, translocation of Bax to mitochondria and apoptosis. Potentiation of apoptosis by knockdown of caveolin-1 protein expression is greatly reduced in the absence of Bax expression. Finally, we found that overexpression of wild type Ku70, but not a mutant form of Ku70 that cannot bind to caveolin-1 (Ku70 ΦâA), limits the chemotherapeutic drug-induced Ku70/Bax dissociation and apoptosis. Thus, caveolin-1 acts as an anti-apoptotic protein in colon cancer cells by binding to Ku70 and inhibiting Bax-dependent cell death.
Asunto(s)
Antígenos Nucleares/metabolismo , Caveolina 1/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Antígenos Nucleares/genética , Apoptosis/genética , Apoptosis/fisiología , Western Blotting , Caveolina 1/genética , Proteínas de Unión al ADN/genética , Células HCT116 , Células HT29 , Humanos , Autoantígeno Ku , Unión Proteica , ARN Interferente Pequeño/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
According to the "free radical theory" of aging, normal aging occurs as the result of tissue damages inflicted by reactive oxygen species (ROS) when ROS production exceeds the antioxidant capacity of the cell. ROS induce cellular dysfunctions such as stress-induced premature senescence (SIPS), which is believed to contribute to normal organismal aging and play a role in age-related diseases. Consistent with this hypothesis, increased oxidative damage of DNA, proteins, and lipids have been reported in aged animals and senescent cells accumulate in vivo with advancing age. Caveolin-1 acts as a scaffolding protein that concentrates and functionally regulates signaling molecules. Recently, great progress has been made toward understanding of the role of caveolin-1 in stress-induced premature senescence. Data show that caveolin-mediated signaling may contribute to explain, at the molecular level, how oxidative stress promotes the deleterious effects of cellular senescence such as aging and age-related diseases. In this review, we discuss the cellular mechanisms and functions of caveolin-1 in the context of SIPS and their relevance to the biology of aging.
Asunto(s)
Envejecimiento/metabolismo , Envejecimiento/patología , Caveolina 1/metabolismo , Senescencia Celular/fisiología , Animales , Aterosclerosis/etiología , Proliferación Celular , Fibrosis , Humanos , Infecciones/etiología , Degeneración del Disco Intervertebral/etiología , Modelos Biológicos , Osteoartritis/etiología , Estrés Oxidativo , Enfisema Pulmonar/etiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Microambiente Tumoral , Cicatrización de HeridasRESUMEN
FKBP38 is a member of the family of FK506-binding proteins that acts as an inhibitor of the mammalian target of rapamycin (mTOR). The inhibitory action of FKBP38 is antagonized by Rheb, an oncogenic small GTPase, which interacts with FKBP38 and prevents its association with mTOR. In addition to the role in mTOR regulation, FKBP38 is also involved in binding and recruiting Bcl-2 and Bcl-X(L), two anti-apoptotic proteins, to mitochondria. In this study, we investigated the possibility that Rheb controls apoptosis by regulating the interaction of FKBP38 with Bcl-2 and Bcl-X(L). We demonstrate in vitro that the interaction of FKBP38 with Bcl-2 is regulated by Rheb in a GTP-dependent manner. In cultured cells, the interaction is controlled by Rheb in response to changes in amino acid and growth factor conditions. Importantly, we found that the Rheb-dependent release of Bcl-X(L) from FKBP38 facilitates the association of this anti-apoptotic protein with the pro-apoptotic protein Bak. Consequently, when Rheb activity increases, cells become more resistant to apoptotic inducers. Our findings reveal a novel mechanism through which growth factors and amino acids control apoptosis.
Asunto(s)
Regulación de la Expresión Génica , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuropéptidos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Proteína bcl-X/metabolismo , Aminoácidos/química , Apoptosis , Proteínas de Unión al GTP/metabolismo , Regulación Neoplásica de la Expresión Génica , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Mitocondrias/metabolismo , Modelos Biológicos , Proteína Homóloga de Ras Enriquecida en el Cerebro , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Epidermal growth factor receptor (EGFR) is an activated oncogene in many cancers. It can be transactivated by ligands of G protein-coupled receptors (GPCRs). We show here that a novel gene, human rhomboid family-1 (RHBDF1), which was recently reported to have a pivotal role in epithelial cancer cell growth in culture and in xenograft tumors, participates in the modulation of GPCR-mediated EGFR transactivation. The RHBDF1 protein localizes mainly in the endoplasmic reticulum. Silencing the RHBDF1 gene in head and neck squamous cancer cell line 1483 cells with siRNA causes an inhibition of gastrin-releasing peptide (GRP) -induced phosphorylation of EGFR and EGFR-dependent signaling proteins p44/42 MAPK and AKT, accompanied by an inhibition of GRP-induced survival, proliferation, and invasion of the cells. The EGFR signaling pathway itself remains intact, however, as the cells remain responsive to exogenous EGF. In addition, RHBDF1 gene silencing disrupts GRP-stimulated secretion of EGFR ligand TGF-alpha, but not the production of latent TGF-alpha, whereas engineered overexpression of RHBDF1 markedly accelerates the secretion of TGF-alpha. These findings are consistent with the view that RHBDF1 is critically involved in a GPCR ligand-stimulated process leading to the activation of latent EGFR ligands.
Asunto(s)
Receptores ErbB/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Células Escamosas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Activación Transcripcional , Línea Celular Tumoral , Proliferación Celular , Retículo Endoplásmico/metabolismo , Receptores ErbB/genética , Péptido Liberador de Gastrina/farmacología , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Ligandos , Proteínas de la Membrana , Invasividad Neoplásica/patología , Neoplasias de Células Escamosas/genética , Neoplasias de Células Escamosas/patología , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
The rhomboid family of genes carry out a wide range of important functions in a variety of organisms. Little is known, however, about the function of the human rhomboid family-1 gene (RHBDF1). We show here that RHBDF1 function is essential to epithelial cancer cell growth. RHBDF1 mRNA level is significantly elevated in clinical specimens of invasive ductal carcinoma of the breast, and the protein is readily detectable in human breast cancer or head and neck cancer cell lines. Silencing the RHBDF1 gene with short interfering RNA (siRNA) results in apoptosis in breast cancer MDA-MB-435 cells and autophagy in head and neck squamous cell cancer 1483 cells. The treatment also leads to significant down-modulation of activated AKT and extracellular signal-regulated kinase in the cells, suggesting that critically diminished strength of these growth signals may be the key attributes of the induction of cell death. Furthermore, silencing the RHBDF1 gene in MDA-MB-435 or 1483 xenograft tumors on athymic nude mice by using i.v. administered histidine-lysine polymer nanoparticle-encapsulated siRNA results in marked inhibition of tumor growth. Our findings indicate that RHBDF1 has a pivotal role in sustaining growth signals in epithelial cancer cells and thus may serve as a therapeutic target for treating epithelial cancers.
Asunto(s)
Apoptosis , Autofagia , Células Epiteliales/patología , Receptores ErbB/genética , Silenciador del Gen , Neoplasias/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Proteínas de la Membrana , Ratones , Ratones Desnudos , Nanopartículas , Neoplasias/genética , Neoplasias de Células Escamosas/genética , Neoplasias de Células Escamosas/patología , Polímeros , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
EVL belongs to the Ena/VASP (Enabled/vasodilator-stimulated phosphoprotein) family of proteins [Mena (mammalian Ena), VASP and EVL], which have a range of roles in regulating the actin cytoskeleton. Growing evidence suggests that Mena and VASP are involved in carcinogenesis, though little is known about the significance of EVL's function in cancer. This study examined the expression levels of EVL mRNA in paired breast cancer specimens using semi-quantitative and real-time RT-PCR. In a comparison between matched breast tumors and normal tissues, a significant increase in the level of EVL mRNA was found in 23 of the 35 (65.7%) tumors (P=0.032). Patients in the advanced stages more frequently exhibited an elevated EVL expression in tumor tissues. The EVL mRNA relative expression level significantly correlated with clinical stages (P=0.021). To begin elucidating the mechanism, we measured the ability of EVL to transform NIH3T3 cells and regulate the motility of MCF-7 cells in vitro by focus formation and modified Boyden chamber assays. The results indicated that overexpression of EVL was insufficient to transform NIH3T3 cells, although the motility of EVL transfected MCF-7 cells was markedly promoted. Collectively, EVL expression level was higher in breast tumors compared to normal tissues and its up-regulation was positively associated with the clinical stages of breast cancer. Additionally, EVL may be implicated in invasion and/or metastasis of human breast cancer.