RESUMEN

Rac and Cdc42, are homologous GTPases that regulate cell migration, invasion, and cell cycle progression; thus, representing key targets for metastasis therapy. We previously reported on the efficacy of MBQ-167, which blocks both Rac1 and Cdc42 in breast cancer cells and mouse models of metastasis. To identify compounds with increased activity, a panel of MBQ-167 derivatives was synthesized, maintaining its 9-ethyl-3-(1H-1,2,3-triazol-1-yl)-9H-carbazole core. Similar to MBQ-167, MBQ-168 and EHop-097, inhibit activation of Rac and Rac1B splice variant and breast cancer cell viability, and induce apoptosis. MBQ-167 and MBQ-168 inhibit Rac and Cdc42 by interfering with guanine nucleotide binding, and MBQ-168 is a more effective inhibitor of PAK (1,2,3) activation. EHop-097 acts via a different mechanism by inhibiting the interaction of the guanine nucleotide exchange factor (GEF) Vav with Rac. MBQ-168 and EHop-097 inhibit metastatic breast cancer cell migration, and MBQ-168 promotes loss of cancer cell polarity to result in disorganization of the actin cytoskeleton and detachment from the substratum. In lung cancer cells, MBQ-168 is more effective than MBQ-167 or EHop-097 at reducing ruffle formation in response to EGF. Comparable to MBQ-167, MBQ-168 significantly inhibits HER2+ tumor growth and metastasis to lung, liver, and spleen. Both MBQ-167 and MBQ-168 inhibit the cytochrome P450 (CYP) enzymes 3A4, 2C9, and 2C19. However, MBQ-168 is ~10X less potent than MBQ-167 at inhibiting CYP3A4, thus demonstrating its utility in relevant combination therapies. In conclusion, the MBQ-167 derivatives MBQ-168 and EHop-097 are additional promising anti metastatic cancer compounds with similar and distinct mechanisms.

Asunto(s)

Proteínas de Unión al GTP , Proteínas de Unión al GTP rac , Ratones , Animales , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Movimiento Celular , División Celular

RESUMEN

BACKGROUND: Acute myeloid leukemia (AML) is the most common type of acute leukemia and biologically heterogeneous diseases with poor prognosis. Thus, we aimed to identify prognostic markers to effectively predict the prognosis of AML patients and eventually guide treatment. METHODS: Prognosis-associated genes were determined by Kaplan-Meier and multivariate analyses using the expression and clinical data of 173 AML patients from The Cancer Genome Atlas database and validated in an independent Oregon Health and Science University dataset. A prognostic risk score was computed based on a linear combination of 5-gene expression levels using the regression coefficients derived from the multivariate logistic regression model. The classification of AML was established by unsupervised hierarchical clustering of CALCRL, DOCK1, PLA2G4A, FCHO2 and LRCH4 expression levels. RESULTS: High FCHO2 and LRCH4 expression was related to decreased mortality. While high CALCRL, DOCK1, PLA2G4A expression was associated with increased mortality. The risk score was predictive of increased mortality rate in AML patients. Hierarchical clustering analysis of the five genes discovered three clusters of AML patients. The cluster1 AML patients were associated with lower cytogenetics risk than cluster2 or 3 patients, and better prognosis than cluster3 patients (P values < 0.05 for all cases, fisher exact test or log-rank test). CONCLUSION: The gene panel comprising CALCRL, DOCK1, PLA2G4A, FCHO2 and LRCH4 as well as the risk score may offer novel prognostic biomarkers and classification of AML patients to significantly improve outcome prediction.

Asunto(s)

Proteína Similar al Receptor de Calcitonina/genética , Proteínas de Unión a Ácidos Grasos/genética , Expresión Génica , Fosfolipasas A2 Grupo IV/genética , Leucemia Mieloide Aguda/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al GTP rac/genética , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/mortalidad , Análisis Multivariante , Pronóstico , Factores de Riesgo , Resultado del Tratamiento

RESUMEN

Celiac disease (CeD) is a common immune-mediated disease of the small intestine that is triggered by exposure to dietary gluten. While the HLA locus plays a major role in disease susceptibility, 39 non-HLA loci were also identified in a study of 24,269 individuals. We now build on this earlier study by adding 4125 additional Caucasian samples including an Argentinian cohort. In doing so, we not only confirm the previous associations, we also identify two novel independent genome-wide significant associations at loci: 12p13.31 and 22q13.1. By applying a genomics approach and differential expression analysis in CeD intestinal biopsies, we prioritize potential causal genes at these novel loci, including LTBR, CYTH4, and RAC2. Nineteen prioritized causal genes are overlapping known drug targets. Pathway enrichment analysis and expression of these genes in CeD biopsies suggest that they have roles in regulating multiple pathways such as the tumor necrosis factor (TNF) mediated signaling pathway and positive regulation of I-κB kinase/NF-κB signaling.

Asunto(s)

Enfermedad Celíaca/genética , Sitios Genéticos , Polimorfismo de Nucleótido Simple , Argentina , Enfermedad Celíaca/patología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 22/genética , Europa (Continente) , Estudio de Asociación del Genoma Completo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Receptor beta de Linfotoxina/genética , Receptor beta de Linfotoxina/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteína RCA2 de Unión a GTP

RESUMEN

Several lines of indirect evidence, such as mutations or dysregulated expression of genes related to cytoskeleton, have suggested that cytoskeletal dynamics, a process essential for axons and dendrites development, is compromised in autism spectrum disorders (ASD). However, no study has yet examined whether cytoskeleton dynamics is functionally altered in cells from ASD patients. Here we investigated the regulation of actin cytoskeleton dynamics in stem cells from human exfoliated deciduous teeth (SHEDs) of 13 ASD patients and 8 control individuals by inducing actin filament depolymerization and then measuing their reconstruction upon activation of the RhoGTPases Rac, Cdc42 or RhoA. We observed that stem cells from seven ASD individuals (53%) presented altered dymanics of filament reconstruction, including a patient recently studied by our group whose iPSC-derived neuronal cells show shorten and less arborized neurites. We also report potentially pathogenic genetic variants that might be related to the alterations in actin repolymerization dynamics observed in some patient-derived cells. Our results suggest that, at least for a subgroup of ASD patients, the dynamics of actin polymerization is impaired, which might be ultimately leading to neuronal abnormalities.

Asunto(s)

Citoesqueleto de Actina/química , Actinas/química , Trastorno del Espectro Autista/genética , Neuronas/química , Citoesqueleto de Actina/genética , Actinas/genética , Animales , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/patología , Regulación de la Expresión Génica/genética , Humanos , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Neuronas/patología , Exfoliación Dental , Proteína de Unión al GTP cdc42/genética , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rhoA/genética

RESUMEN

RAC3 is an oncogene naturally overexpressed in several tumors. Besides its role as coactivator, it can exert several protumoral cytoplasmic actions. Autophagy was found to act either as a tumor suppressor during the early stages of tumor development, or as a protector of the tumor cell in later stages under hypoxic conditions. We found that RAC3 overexpression inhibits autophagy when induced by starvation or rapamycin and involves RAC3 nuclear translocation-dependent and -independent mechanisms. Moreover, hypoxia inhibits the RAC3 gene expression leading to the autophagy process, allowing tumor cells to survive until angiogenesis occurs. The interplay between RAC3, hypoxia, and autophagy could be an important mechanism for tumor progression and a good target for a future anticancer therapy.

Asunto(s)

Autofagia , Proteínas de Unión al GTP rac/metabolismo , Hipoxia de la Célula , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Expresión Génica , Genes Supresores de Tumor , Células HEK293 , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Neoplasias/genética , Coactivadores de Receptor Nuclear/genética , Coactivadores de Receptor Nuclear/metabolismo , Proteínas de Unión al GTP rac/genética

RESUMEN

The retinoblastoma protein (pRb) is a cell cycle regulator inactivated in most human cancers. Loss of pRb function results from mutations in the gene coding for pRb or for any of its upstream regulators. Although pRb is predominantly known as a cell cycle repressor, our data point to additional pRb functions in cell adhesion. Our data show that pRb regulates the expression of a wide repertoire of cell adhesion genes and regulates the assembly of the adherens junctions required for cell adhesion. We conducted our studies in osteoblasts, which depend on both pRb and on cell-to-cell contacts for their differentiation and function. We generated knockout mice in which the RB gene was excised specifically in osteoblasts using the cre-lox P system and found that osteoblasts from pRb knockout mice did not assemble adherens junction at their membranes. pRb depletion in wild type osteoblasts using RNAi also disrupted adherens junctions. Microarrays comparing pRb-expressing and pRb-deficient osteoblasts showed that pRb controls the expression of a number of cell adhesion genes, including cadherins. Furthermore, pRb knockout mice showed bone abnormalities consistent with osteoblast adhesion defects. We also found that pRb controls the function of merlin, a well-known regulator of adherens junction assembly, by repressing Rac1 and its effector Pak1. Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss. Our data support a pRb function in cell adhesion while elucidating the mechanism for this function. Our work suggests that in some tumor types pRb inactivation results in both a loss of cell cycle control that promotes initial tumor growth as well as in a loss of cell-to-cell contacts, which contributes to later stages of metastasis.

Asunto(s)

Osteoblastos/metabolismo , Proteína de Retinoblastoma/metabolismo , Células 3T3 , Uniones Adherentes/genética , Uniones Adherentes/fisiología , Animales , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular/genética , Adhesión Celular/fisiología , Comunicación Celular/genética , Comunicación Celular/fisiología , Proliferación Celular , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones SCID , Modelos Biológicos , Neuropéptidos/genética , Neuropéptidos/metabolismo , Osteoblastos/citología , Osteogénesis/genética , Osteogénesis/fisiología , Osteosarcoma/genética , Osteosarcoma/metabolismo , Interferencia de ARN , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cráneo/embriología , Cráneo/metabolismo , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1

RESUMEN

The p160 nuclear receptor co-activators represent a family of molecules, which are recruited by steroid nuclear receptors as well as other transcription factors that are overexpressed in several tumors. We investigated the role of one member of this family on the sensitivity of cells to apoptosis. We observed that overexpression of the RAC3 (receptor-associated co-activator-3) p160 co-activator inhibits hydrogen peroxide-induced cell death in human embryonic kidney 293 (HEK293) cells. The mechanism involves the activation of anti-apoptotic pathways mediated through enhanced nuclear factor kappa B (NF-kappaB) activity, inhibition of caspase-9 activation, diminished apoptotic-inducing factor (AIF) nuclear localization and a change in the activation pattern of several kinases, including an increase in both AKT and p38 kinase activities, and inhibition of ERK2. Moreover, RAC3 has been found associated with a protein complex containing AIF, Hsp90 and dynein, suggesting a role for the co-activator in the cytoplasmatic nuclear transport of these proteins associated with cytoskeleton. These results demonstrate that there are several molecular pathways that could be affected by their overexpression, including those not restricted to steroid regulation or the nuclear action of co-activators, which results in diminished sensitivity to apoptosis. Furthermore, this could represent one mechanism by which co-activators contribute to tumor development.

Asunto(s)

Apoptosis , Citosol/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Transporte Activo de Núcleo Celular , Apoptosis/efectos de los fármacos , Factor Inductor de la Apoptosis/metabolismo , Caspasa 9/metabolismo , Línea Celular , Dineínas/metabolismo , Activación Enzimática , Regulación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , FN-kappa B/metabolismo , Unión Proteica , Proteínas Quinasas/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Proteínas de Unión al GTP rac/genética

RESUMEN

Estrogen and structurally related molecules play critical roles in breast cancer. We reported that resveratrol (50 microM), an estrogen-like phytosterol from grapes, acts in an antiestrogenic manner in breast cancer cells to reduce cell migration and to induce a global and sustained extension of actin structures called filopodia. Herein, we report that resveratrol-induced filopodia formation is time-dependent and concentration-dependent. In contrast to resveratrol at 50 microM, resveratrol at 5 microM acts in a manner similar to estrogen by increasing lamellipodia, as well as cell migration and invasion. Because Rho GTPases regulate the extension of actin structures, we investigated a role for Rac and Cdc42 in estrogen and resveratrol signaling. Our results demonstrate that 50 microM resveratrol decreases Rac and Cdc42 activity, whereas estrogen and 5 microM resveratrol increase Rac activity in breast cancer cells. MDA-MB-231 cells expressing dominant-negative Cdc42 or dominant-negative Rac retain filopodia response to 50 microM resveratrol. Lamellipodia response to 5 microM resveratrol, estrogen, or epidermal growth factor is inhibited in cells expressing dominant-negative Rac, indicating that Rac regulates estrogen and resveratrol (5 microM) signaling to the actin cytoskeleton. These results indicate that signaling to the actin cytoskeleton by low and high concentrations of resveratrol may be differentially regulated by Rac and Cdc42.

Asunto(s)

Actinas/metabolismo , Adenocarcinoma/patología , Neoplasias de la Mama/patología , Citoesqueleto/efectos de los fármacos , Estradiol/farmacología , Estrógenos , Neoplasias Hormono-Dependientes/patología , Estilbenos/farmacología , Proteína de Unión al GTP cdc42/fisiología , Proteínas de Unión al GTP rac/fisiología , Adenocarcinoma/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Línea Celular Tumoral/ultraestructura , Movimiento Celular/efectos de los fármacos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Relación Dosis-Respuesta a Droga , Factor de Crecimiento Epidérmico/farmacología , Femenino , Genes Dominantes , Humanos , Invasividad Neoplásica , Neoplasias Hormono-Dependientes/metabolismo , Seudópodos/efectos de los fármacos , Seudópodos/ultraestructura , Proteínas Recombinantes de Fusión/genética , Resveratrol , Transfección , Proteína de Unión al GTP cdc42/genética , Proteínas de Unión al GTP rac/genética

RESUMEN

Rho GTPases are critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to cytoskeleton. The Rho guanine nucleotide exchange factors (RhoGEFs) have been implicated in direct activation of these GTPases. Here, we describe a novel RhoGEF, denominated EhGEF3 from the parasite Entamoeba histolytica, which encodes a 110 kDa protein containing the domain arrangement of a Dbl homology domain in tandem with a pleckstrin homology domain, the DH domain of EhGEF3 is closely related with the one of the Vav3 protein. Biochemical analysis revealed that EhGEF3 is capable of stimulating nucleotide exchange on the E. histolytica EhRacA and EhRho1 GTPases in vitro, however only a partial GEF activity toward Cdc42 was observed. Conserved residue analysis showed that the N816 and L817 residues are critical for EhGEF3 activity. Cellular studies revealed that EhGEF3 colocalises with EhRacA in the rear of migrating cells, probably regulating the retraction of the uroid and promoting the activation of these GTPase during the chemotactic response toward fibronectin, and that EhGEF3 also regulates EhRacA activation during the capping of cell receptors. These results suggest that EhGEF3 should have a direct role in activating EhRacA, and in bringing the activated GTPase to specific target sites such as the uroid.

Asunto(s)

Quimiotaxis , Entamoeba histolytica/enzimología , Entamoeba histolytica/fisiología , Regulación de la Expresión Génica/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Análisis por Conglomerados , Entamoeba histolytica/genética , Activación Enzimática , Factores de Intercambio de Guanina Nucleótido/análisis , Factores de Intercambio de Guanina Nucleótido/química , Técnicas In Vitro , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rac/genética

RESUMEN

The molecular, biochemical, and cellular characterization of EhGEF1 protein is described. Complete cDNA sequence of 1890 bp revealed an open reading frame that encodes a protein of 69 kDa. EhGEF1 is constituted of Dbl homology domain, pleckstrin homology domain, and several putative regulation sites. Studies of guanine nucleotide exchange activity of EhGEF1 on several GTPases from Entamoeba histolytica and Homo sapiens showed preferential activation on EhRacG, suggesting that EhGEF1 protein could be involved in mechanisms related to actin cytoskeleton activation, cytokinesis, capping, and uroid formation in trophozoite. Confocal microscopy studies of pExEhNeo/HSV-tagged-EhGEF1-transfected cells showed that trophozoites stimulated with ConA, EhGEF1, and EhRacG were localized at plasma membrane. Cellular studies showed that F-actin content of pExEhNeo/HSV-tagged-EhGEF1-transfected trophozoites as well as cellular migration and cell damage capacity were significantly altered. The observations suggest that EhRacG was the principal target of EhGEF1 and that EhGEF1 may provide a link between F-actin dynamics and EhRacG signaling.

Asunto(s)

Entamoeba histolytica/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Concanavalina A/farmacología , Secuencia Conservada , ADN Complementario/química , ADN Complementario/aislamiento & purificación , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Entamoeba histolytica/genética , Expresión Génica/fisiología , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Microscopía Confocal , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Reacción en Cadena de la Polimerasa , Transfección , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/fisiología , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/fisiología

RESUMEN

The biological and functional properties of beta2-chimaerin, a novel phorbol ester/diacylglycerol receptor unrelated to protein kinase C isozymes, are largely unknown. It has previously been established that beta2-chimaerin accelerates the hydrolysis rate of GTP from Rac1 in vitro, leading to the inactivation of this GTPase, which plays important roles in the control of actin cytoskeleton organization, proliferation, motility, and invasiveness. To explore the potential role of beta2-chimaerin in invasion and metastasis, we generated stable transfectants for its catalytic domain (the beta-GAP domain) in F3II murine mammary carcinoma cells. Reduced Rac-GTP levels were observed upon stimulation with epidermal growth factor in the beta-GAP clones compared with control cells. Moreover, a marked alteration in actin polymerization in response to epidermal growth factor was observed in the beta-GAP clones, suggesting impairment of Rac-dependent responses. The beta-GAP transfectants also evidenced slower growth rates and a striking reduction in their migratory properties. Adenoviral delivery of the beta-GAP domain into F3II cells also led to reduced proliferative and migratory responses. Importantly, significant differences were found between beta-GAP transfectants and control cells regarding their tumorigenic and metastatic properties after s.c. inoculation in syngeneic BALB/c mice. Tumors originating from beta-GAP transfectants showed a significantly lower growth rate and reduced invasive ability; in addition, a lower incidence of spontaneous lung metastases was observed. Our results indicate that beta2-chimaerin impairs key steps in the metastatic cascade and provide evidence for a rational modulation of the Rac signaling pathway in cancer treatment.

Asunto(s)

Proteínas Activadoras de GTPasa/fisiología , Neoplasias Mamarias Experimentales/patología , Proteínas de Neoplasias/fisiología , Actinas/metabolismo , Animales , Adhesión Celular/fisiología , Citoesqueleto/metabolismo , Femenino , Proteínas Activadoras de GTPasa/biosíntesis , Proteínas Activadoras de GTPasa/genética , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Estructura Terciaria de Proteína , Transfección , Células Tumorales Cultivadas , Proteínas de Unión al GTP rac/biosíntesis , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/fisiología

RESUMEN

The case of an infant with multiple, rapidly progressive, soft-tissue infections is presented. Despite features suggesting a neutrophil disorder, results of screening tests of phagocyte function were normal. A novel, multifaceted leukocyte disorder-distinguished by defects in shape change, chemotaxis, ingestion, degranulation, superoxide anion production, and bactericidal activity-was established secondary to a defect in Rac2.

Asunto(s)

Neutrófilos/fisiología , Infecciones de los Tejidos Blandos/genética , Proteínas de Unión al GTP rac/genética , Actividad Bactericida de la Sangre , Quimiotaxis de Leucocito , Humanos , Recién Nacido , Masculino , Fagocitosis , Transducción de Señal , Infecciones de los Tejidos Blandos/inmunología , Superóxidos/metabolismo , Proteína RCA2 de Unión a GTP