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1.
Proc Natl Acad Sci U S A ; 115(3): E428-E437, 2018 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-29282319

RESUMEN

Fibrous dysplasia (FD) is a disease caused by postzygotic activating mutations of GNAS (R201C and R201H) that encode the α-subunit of the Gs stimulatory protein. FD is characterized by the development of areas of abnormal fibroosseous tissue in the bones, resulting in skeletal deformities, fractures, and pain. Despite the well-defined genetic alterations underlying FD, whether GNAS activation is sufficient for FD initiation and the molecular and cellular consequences of GNAS mutations remains largely unresolved, and there are no currently available targeted therapeutic options for FD. Here, we have developed a conditional tetracycline (Tet)-inducible animal model expressing the GαsR201C in the skeletal stem cell (SSC) lineage (Tet-GαsR201C/Prrx1-Cre/LSL-rtTA-IRES-GFP mice), which develops typical FD bone lesions in both embryos and adult mice in less than 2 weeks following doxycycline (Dox) administration. Conditional GαsR201C expression promoted PKA activation and proliferation of SSCs along the osteogenic lineage but halted their differentiation to mature osteoblasts. Rather, as is seen clinically, areas of woven bone admixed with fibrous tissue were formed. GαsR201C caused the concomitant expression of receptor activator of nuclear factor kappa-B ligand (Rankl) that led to marked osteoclastogenesis and bone resorption. GαsR201C expression ablation by Dox withdrawal resulted in FD-like lesion regression, supporting the rationale for Gαs-targeted drugs to attempt FD cure. This model, which develops FD-like lesions that can form rapidly and revert on cessation of mutant Gαs expression, provides an opportunity to identify the molecular mechanism underlying FD initiation and progression and accelerate the development of new treatment options.


Asunto(s)
Displasia Fibrosa Ósea/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Células Madre Mesenquimatosas/metabolismo , Animales , Antibacterianos/toxicidad , Desarrollo Óseo/efectos de los fármacos , Huesos/patología , Diferenciación Celular , Doxiciclina/toxicidad , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Ratones , Mutación
2.
Gastroenterology ; 155(5): 1593-1607.e12, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30142336

RESUMEN

BACKGROUND & AIMS: Mutations at hotspots in GNAS, which encodes stimulatory G-protein, α subunits, are detected in approximately 60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. We generated mice with KRAS-induced IPMNs that also express a constitutively active form of GNAS in pancreas and studied tumor development. METHODS: We generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras;Gnas mice); pancreatic tissues of these mice express activated KRAS and also express a mutant form of GNAS (GNASR201C) upon doxycycline administration. Mice that were not given doxycycline were used as controls, and survival times were compared by Kaplan-Meier analysis. Pancreata were collected at different time points after doxycycline administration and analyzed by histology. Pancreatic ductal adenocarcinomas (PDACs) were isolated from mice and used to generate cell lines, which were analyzed by reverse transcription polymerase chain reaction, immunoblotting, immunohistochemistry, and colony formation and invasion assays. Full-length and mutant forms of yes-associated protein (YAP) were expressed in PDAC cells. IPMN specimens were obtained from 13 patients with IPMN undergoing surgery and analyzed by immunohistochemistry. RESULTS: All Kras;Gnas mice developed pancreatic cystic lesions that resemble human IPMNs; the grade of epithelial dysplasia increased with time. None of the control mice developed cystic lesions. Approximately one third of Kras;Gnas mice developed PDACs at a median of 30 weeks after doxycycline administration, whereas 33% of control mice developed PDACs. Expression of GNASR201C did not accelerate the development of PDACs compared with control mice. However, the neoplasms observed in Kras;Gnas mice were more differentiated, and expressed more genes associated with ductal phenotypes, than in control mice. PDACs isolated from Kras;Gnas mice had activation of the Hippo pathway; in cells from these tumors, phosphorylated YAP1 was sequestered in the cytoplasm, and this was also observed in human IPMNs with GNAS mutations. Sequestration of YAP1 was not observed in PDAC cells from control mice. CONCLUSIONS: In mice that express activated KRAS in the pancreas, we found expression of GNASR201C to cause development of more differentiated tumors, with gene expression pattern associated with the ductal phenotype. Expression of mutant GNAS caused phosphorylated YAP1 to be sequestered in the cytoplasm, altering tumor progression.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Carcinoma Ductal Pancreático/etiología , Cromograninas/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Mutación , Neoplasias Quísticas, Mucinosas y Serosas/etiología , Neoplasias Pancreáticas/etiología , Fosfoproteínas/fisiología , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Animales , Proteínas de Ciclo Celular , Línea Celular Tumoral , Cromograninas/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gs/fisiología , Humanos , Ratones , Fosfoproteínas/análisis , Fosfoproteínas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Proteínas Señalizadoras YAP
3.
Anal Chem ; 88(22): 10775-10784, 2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27732780

RESUMEN

The cars we drive, the homes we live in, the restaurants we visit, and the laboratories and offices we work in are all a part of the modern human habitat. Remarkably, little is known about the diversity of chemicals present in these environments and to what degree molecules from our bodies influence the built environment that surrounds us and vice versa. We therefore set out to visualize the chemical diversity of five built human habitats together with their occupants, to provide a snapshot of the various molecules to which humans are exposed on a daily basis. The molecular inventory was obtained through untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of samples from each human habitat and from the people that occupy those habitats. Mapping MS-derived data onto 3D models of the environments showed that frequently touched surfaces, such as handles (e.g., door, bicycle), resemble the molecular fingerprint of the human skin more closely than other surfaces that are less frequently in direct contact with humans (e.g., wall, bicycle frame). Approximately 50% of the MS/MS spectra detected were shared between people and the environment. Personal care products, plasticizers, cleaning supplies, food, food additives, and even medications that were found to be a part of the human habitat. The annotations indicate that significant transfer of chemicals takes place between us and our built environment. The workflows applied here will lay the foundation for future studies of molecular distributions in medical, forensic, architectural, space exploration, and environmental applications.


Asunto(s)
Ecosistema , Espectrometría de Masas , Compuestos Orgánicos/análisis , Compuestos Orgánicos/química , Cromatografía Liquida , Humanos , Iones/análisis , Espectrometría de Masas en Tándem
4.
Pharm Res ; 31(11): 3106-19, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24848339

RESUMEN

PURPOSE: Although Cu complexes have been investigated as anticancer agents, there has been no description of Cu itself as a cancer killing agent. A stealth liposomal Cu formulation (LpCu) was studied in vitro and in vivo. METHODS: LpCu was evaluated in prostate cancer origin PC-3 cells by a metabolic cytotoxicity assay, by monitoring ROS, and by flow cytometry. LpCu efficacy was evaluated in vivo using intratumoral and intravenous injections into mice bearing PC-3 xenograft tumors. Toxicology was assessed by performing hematological and blood biochemistry assays, and tissue histology and Cu distribution was investigated by elemental analysis. RESULTS: LpCu and free Cu salts displayed similar levels of cell metabolic toxicity and ROS. Flow cytometry indicated that the mechanisms of cell death were both apoptosis and necrosis. Animals injected i.t. with 3.5 mg/kg or i.v. with 3.5 and 7.0 mg/kg LpCu exhibited significant tumor growth inhibition. Kidney and eye were the main organs affected by Cu-mediated toxicities, but spleen and liver were the major organs of Cu deposition. CONCLUSIONS: LpCu was effective at reducing tumor burden in the xenograft prostate cancer model. There was histological evidence of Cu toxicity in kidneys and eyes of animals treated at the maximum tolerated dose of LpCu 7.0 mg/kg.


Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Cobre/farmacología , Cobre/farmacocinética , Liposomas/farmacología , Liposomas/farmacocinética , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Química Farmacéutica/métodos , Modelos Animales de Enfermedad , Xenoinjertos/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Desnudos , Próstata/efectos de los fármacos , Distribución Tisular/fisiología , Trasplante Heterólogo/métodos , Ensayos Antitumor por Modelo de Xenoinjerto/métodos
5.
J Pharm Pharm Sci ; 17(4): 583-602, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25579435

RESUMEN

PURPOSE: The formulation of histone deacetylase inhibitors (HDACi) is challenging due to poor water solubility and rapid elimination of drugs in vivo. This study investigated the effects of complexing iron (Fe3+) to the HDACi suberoylanilide hydroxamic acid (SAHA) and LAQ824 (LAQ) prior to their encapsulation into PEGylated liposomes, and investigated whether this technique could improve drug solubility, in vitro release and in vivo pharmacokinetic (PK) properties. METHODS. The reaction stoichiometry, binding constants and solubility were measured for Fe complexes of SAHA and LAQ. The complexes were passively encapsulated into PEGylated liposomes and characterized by size distribution, zeta-potential, encapsulation efficiency (EE), and in vitro drug release studies. PC-3 cells were used to verify the in vitro anticancer activity of the formulations. In vivo pharmacokinetic properties of liposomal LAQ-Fe (L-LAQ-Fe) was evaluated in rats. RESULTS. SAHA and LAQ form complexes with Fe at 1:1 stoichiometric ratio, with a binding constant on the order of 104 M-1. Fe complexation improved the aqueous solubility and the liposomal encapsulation efficiency of SAHA and LAQ (29-35% EE, final drug concentration > 1 mM). Liposomal encapsulated complexes (L-HDACi-Fe) exhibited sustained in vitro release properties compared to L-HDACi but cytotoxicity on PC-3 cells was comparable to free drugs. The PK of L-LAQ-Fe revealed 15-fold improvement in the plasma t1/2 (12.11 h)and 211-fold improvement in the AUC∞ (105.7 µg·h/ml) compared to free LAQ (0.79 h, 0.5 µg·h/ml). Similarly, the plasma t1/2 of Fe was determined to be 11.83 h in a separate experiment using radioactive Fe-59. The majority of Fe-59 activity was found in liver and spleen of rats and correlates with liposomal uptake by the mononuclear phagocyte system. CONCLUSIONS. We have demonstrated that encapsulation of Fe complexes of HDACi into PEGylated liposomes can improve overall drug aqueous solubility, in vitro release and in vivo pharmacokinetic properties.


Asunto(s)
Inhibidores de Histona Desacetilasas/farmacocinética , Ácidos Hidroxámicos/farmacocinética , Hierro/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Área Bajo la Curva , Línea Celular Tumoral , Liberación de Fármacos , Femenino , Semivida , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Liposomas , Masculino , Tamaño de la Partícula , Fagocitos/metabolismo , Polietilenglicoles/química , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Ratas , Ratas Sprague-Dawley , Solubilidad , Vorinostat
6.
Cancer Res ; 79(7): 1438-1450, 2019 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-30894372

RESUMEN

Aberrant activation of the PI3K-mTOR signaling pathway occurs in >80% of head and neck squamous cell carcinomas (HNSCC), and overreliance on this signaling circuit may in turn represent a cancer-specific vulnerability that can be exploited therapeutically. mTOR inhibitors (mTORi) promote tumor regression in genetically defined and chemically induced HNSCC animal models, and encouraging results have been recently reported. However, the mTOR-regulated targets contributing to the clinical response have not yet been identified. Here, we focused on EIF4E-BP1 (4E-BP1), a direct target of mTOR that serves as key effector for protein synthesis. A systematic analysis of genomic alterations in the PIK3CA-mTOR pathway in HNSCC revealed that 4E-BP1 is rarely mutated, but at least one 4E-BP1 gene copy is lost in over 35% of the patients with HNSCC, correlating with decreased 4E-BP1 protein expression. 4E-BP1 gene copy number loss correlated with poor disease-free and overall survival. Aligned with a tumor-suppressive role, 4e-bp1/2 knockout mice formed larger and more lesions in models of HNSCC carcinogenesis. mTORi treatment or conditional expression of a mutant 4E-BP1 that cannot be phosphorylated by mTOR was sufficient to disrupt the translation-initiation complex and prevent tumor growth. Furthermore, CRISPR/Cas9-targeted 4E-BP1 HNSCC cells resulted in reduced sensitivity to mTORi in vitro and in vivo. Overall, these findings indicate that in HNSCC, mTOR persistently restrains 4E-BP1 via phosphorylation and that mTORi can restore the tumor-suppressive function of 4E-BP1. Our findings also support 4E-BP1 expression and phosphorylation status as a mechanistic biomarker of mTORi sensitivity in patients with HNSCC. SIGNIFICANCE: These findings suggest that EIF4E-BP1 acts as a tumor suppressor in HNSCC and that 4E-BP1 dephosphorylation mediates the therapeutic response to mTORi, providing a mechanistic biomarker for future precision oncology trials.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteínas Supresoras de Tumor/metabolismo , Animales , Benzoxazoles/farmacología , Biomarcadores de Tumor/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular , Neoplasias de Cabeza y Cuello/patología , Humanos , Ratones , Ratones Noqueados , Fosforilación , Pronóstico , Pirimidinas/farmacología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología
7.
Sci Signal ; 10(484)2017 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-28634209

RESUMEN

The ß2-adrenergic receptor (ß2AR) has provided a paradigm to elucidate how G protein-coupled receptors (GPCRs) control intracellular signaling, including the discovery that ß-arrestins, which bind to ligand-activated GPCRs, are central for GPCR function. We used genome editing, conditional gene deletion, and small interfering RNAs (siRNAs) to determine the roles of ß-arrestin 1 (ß-arr1) and ß-arr2 in ß2AR internalization, trafficking, and signaling to ERK. We found that only ß-arr2 was essential for ß2AR internalization. Unexpectedly, ß-arr1 and ß-arr2 and receptor internalization were dispensable for ERK activation. Instead, ß2AR signaled through Gαs and Gßγ subunits through a pathway that involved the tyrosine kinase SRC, the adaptor protein SHC, the guanine nucleotide exchange factor SOS, the small GTPase RAS, and the kinases RAF and MEK, which led to ERK activation. These findings provide a molecular framework for ß2AR signaling through ß-arrestin-independent pathways in key physiological functions and under pathological conditions.


Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal , beta-Arrestina 1/metabolismo , Arrestina beta 2/metabolismo , Animales , Endocitosis , GTP Fosfohidrolasas/metabolismo , Células HEK293 , Humanos , Ligandos , Ratones , Ratones Noqueados , Fosforilación , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción
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