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1.
Cancer Discov ; 14(2): 274-289, 2024 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-37982575

RESUMEN

Fulvestrant is used to treat patients with hormone receptor-positive advanced breast cancer, but acquired resistance is poorly understood. PlasmaMATCH Cohort A (NCT03182634) investigated the activity of fulvestrant in patients with activating ESR1 mutations in circulating tumor DNA (ctDNA). Baseline ESR1 mutations Y537S are associated with poor outcomes and Y537C with good outcomes. Sequencing of baseline and EOT ctDNA samples (n = 69) revealed 3/69 (4%) patients acquired novel ESR1 F404 mutations (F404L, F404I, and F404V), in cis with activating mutations. In silico modeling revealed that ESR1 F404 contributes to fulvestrant binding to estrogen receptor-alpha (ERα) through a pi-stacking bond, with mutations disrupting this bond. In vitro analysis demonstrated that single F404L, E380Q, and D538G models were less sensitive to fulvestrant, whereas compound mutations D538G + F404L and E380Q + F404L were resistant. Several oral ERα degraders were active against compound mutant models. We have identified a resistance mechanism specific to fulvestrant that can be targeted by treatments in clinical development. SIGNIFICANCE: Novel F404 ESR1 mutations may be acquired to cause overt resistance to fulvestrant when combined with preexisting activating ESR1 mutations. Novel combinations of mutations in the ER ligand binding domain may cause drug-specific resistance, emphasizing the potential of similar drug-specific mutations to impact the efficacy of oral ER degraders in development. This article is featured in Selected Articles from This Issue, p. 201.


Asunto(s)
Neoplasias de la Mama , ADN Tumoral Circulante , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Fulvestrant/farmacología , Fulvestrant/uso terapéutico , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , ADN Tumoral Circulante/genética , Mutación
2.
BMC Biol ; 9: 54, 2011 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-21834987

RESUMEN

BACKGROUND: Cell migration is essential during development and in human disease progression including cancer. Most cell migration studies concentrate on known or predicted components of migration pathways. RESULTS: Here we use data from a genome-wide RNAi morphology screen in Drosophila melanogaster cells together with bioinformatics to identify 26 new regulators of morphology and cytoskeletal organization in human cells. These include genes previously implicated in a wide range of functions, from mental retardation, Down syndrome and Huntington's disease to RNA and DNA-binding genes. We classify these genes into seven groups according to phenotype and identify those that affect cell migration. We further characterize a subset of seven genes, FAM40A, FAM40B, ARC, FMNL3, FNBP3/FBP11, LIMD1 and ZRANB1, each of which has a different effect on cell shape, actin filament distribution and cell migration. Interestingly, in several instances closely related isoforms with a single Drosophila homologue have distinct phenotypes. For example, FAM40B depletion induces cell elongation and tail retraction defects, whereas FAM40A depletion reduces cell spreading. CONCLUSIONS: Our results identify multiple regulators of cell migration and cytoskeletal signalling that are highly conserved between Drosophila and humans, and show that closely related paralogues can have very different functions in these processes.


Asunto(s)
Citoesqueleto de Actina/genética , Movimiento Celular/genética , Forma de la Célula/genética , Secuencia Conservada/genética , Proteínas/genética , Actinas/metabolismo , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Genes de Insecto/genética , Pruebas Genéticas , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Fenotipo , Proteínas/metabolismo , ARN Interferente Pequeño/metabolismo , Cicatrización de Heridas/genética
3.
Nat Commun ; 13(1): 5258, 2022 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-36071033

RESUMEN

CDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89). We also identified heterozygous RB1 loss as biomarker of acquired resistance and poor clinical outcome. Combination of the CDK4/6 inhibitor ribociclib with the PI3K inhibitor alpelisib showed antitumor activity in estrogen receptor-positive non-basal-like breast cancer patient-derived xenografts, independently of PIK3CA, ESR1 or RB1 mutation, also in drug de-escalation experiments or omitting endocrine therapy. Our results offer insights into predicting primary/acquired resistance to CDK4/6 inhibitors and post-progression therapeutic strategies.


Asunto(s)
Antineoplásicos , Neoplasias de la Mama , Inhibidores de Proteínas Quinasas , Antineoplásicos/uso terapéutico , Biomarcadores , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores de Estrógenos/metabolismo , Proteínas de Unión a Retinoblastoma/genética , Proteínas de Unión a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo
4.
Cancer Discov ; 11(1): 92-107, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32958578

RESUMEN

Cyclin-dependent kinase 4/6 (CDK4/6) and PI3K inhibitors synergize in PIK3CA-mutant ER-positive HER2-negative breast cancer models. We conducted a phase Ib trial investigating the safety and efficacy of doublet CDK4/6 inhibitor palbociclib plus selective PI3K inhibitor taselisib in advanced solid tumors, and triplet palbociclib plus taselisib plus fulvestrant in 25 patients with PIK3CA-mutant, ER-positive HER2-negative advanced breast cancer. The triplet therapy response rate in PIK3CA-mutant, ER-positive HER2-negative cancer was 37.5% [95% confidence interval (CI), 18.8-59.4]. Durable disease control was observed in PIK3CA-mutant ER-negative breast cancer and other solid tumors with doublet therapy. Both combinations were well tolerated at pharmacodynamically active doses. In the triplet group, high baseline cyclin E1 expression associated with shorter progression-free survival (PFS; HR = 4.2; 95% CI, 1.3-13.1; P = 0.02). Early circulating tumor DNA (ctDNA) dynamics demonstrated high on-treatment ctDNA association with shorter PFS (HR = 5.2; 95% CI, 1.4-19.4; P = 0.04). Longitudinal plasma ctDNA sequencing provided genomic evolution evidence during triplet therapy. SIGNIFICANCE: The triplet of palbociclib, taselisib, and fulvestrant has promising efficacy in patients with heavily pretreated PIK3CA-mutant ER-positive HER2-negative advanced breast cancer. A subset of patients with PIK3CA-mutant triple-negative breast cancer derived clinical benefit from palbociclib and taselisib doublet, suggesting a potential nonchemotherapy targeted approach for this population.This article is highlighted in the In This Issue feature, p. 1.


Asunto(s)
Neoplasias de la Mama , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Fulvestrant , Humanos , Imidazoles , Oxazepinas , Fosfatidilinositol 3-Quinasas , Piperazinas , Piridinas , Receptor ErbB-2/genética
5.
J Biol Chem ; 284(31): 20763-72, 2009 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-19497848

RESUMEN

Calcium (Ca2+) signaling by the pro-inflammatory cytokine interleukin-1 (IL-1) is dependent on focal adhesions, which contain diverse structural and signaling proteins including protein phosphatases. We examined here the role of protein-tyrosine phosphatase (PTP) alpha in regulating IL-1-induced Ca2+ signaling in fibroblasts. IL-1 promoted recruitment of PTPalpha to focal adhesions and endoplasmic reticulum (ER) fractions, as well as tyrosine phosphorylation of the ER Ca2+ release channel IP3R. In response to IL-1, catalytically active PTPalpha was required for Ca2+ release from the ER, Src-dependent phosphorylation of IP3R1 and accumulation of IP3R1 in focal adhesions. In pulldown assays and immunoprecipitations PTPalpha was required for the association of PTPalpha with IP3R1 and c-Src, and this association was increased by IL-1. Collectively, these data indicate that PTPalpha acts as an adaptor to mediate functional links between focal adhesions and the ER that enable IL-1-induced Ca2+ signaling.


Asunto(s)
Señalización del Calcio/efectos de los fármacos , Retículo Endoplásmico/enzimología , Adhesiones Focales/enzimología , Interleucina-1/farmacología , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/metabolismo , Familia-src Quinasas/metabolismo , Animales , Células Cultivadas , Retículo Endoplásmico/efectos de los fármacos , Adhesiones Focales/efectos de los fármacos , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratones , Fosfotirosina/metabolismo , Unión Proteica/efectos de los fármacos , Ratas
6.
Anesthesiology ; 110(6): 1341-7, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19417614

RESUMEN

BACKGROUND: Previous experimental studies of ventilator-induced lung injury have shown that positive end-expiratory pressure (PEEP) is protective. The authors hypothesized that the application of PEEP during volume-controlled ventilation with a moderately high tidal volume (VT) in previously healthy in vivo rats does not attenuate ventilator-induced lung injury if the peak airway pressure markedly increases during the application of PEEP. METHODS: Sixty healthy, male Sprague-Dawley rats were anesthetized and randomized to be mechanically ventilated for 4 h at (1) VT of 6 ml/kg, (2) VT of 20 ml/kg, or (3) VT of 20 ml/kg plus 10 cm H2O of PEEP. Peak airway pressures, gas exchange, histologic evaluation, mortality, total lung tissue cytokine gene expression, and serum cytokine concentrations were analyzed. RESULTS: Peak airway pressures exceeded 30 cm H2O with high VT plus PEEP. All lungs ventilated with high VT had perivascular edema and inflammatory infiltrates. In addition, those ventilated with PEEP had small hemorrhages foci. Five animals from the high VT plus PEEP group died (P = 0.020). Animals ventilated with high VT (with or without PEEP) had a substantial increase in serum interleukin-6 (P = 0.0002), and those ventilated with high VT plus PEEP had a 5.5-fold increase in systemic levels of tumor necrosis factor-alpha (P = 0.007). CONCLUSIONS: In contrast to previous reports, PEEP exacerbated lung damage and contributed to fatal outcome in an in vivo, mild overdistension model of ventilator-induced lung injury in previously healthy rats. That is, the addition of high PEEP to a constant large VT causes injury in previously healthy animals.


Asunto(s)
Lesión Pulmonar/etiología , Respiración con Presión Positiva/efectos adversos , Ventiladores Mecánicos/efectos adversos , Presión del Aire , Animales , Citocinas/biosíntesis , Citocinas/sangre , Lesión Pulmonar/patología , Masculino , ARN/biosíntesis , ARN/genética , Ratas , Ratas Sprague-Dawley , Pruebas de Función Respiratoria , Mecánica Respiratoria/fisiología , Volumen de Ventilación Pulmonar/fisiología
7.
Clin Cancer Res ; 23(18): 5561-5572, 2017 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-28606920

RESUMEN

Purpose: Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer that is associated with a poor prognosis. We evaluated the activity of CDK4/6 inhibitors across the TNBC subtypes and investigated mechanisms of sensitivity.Experimental Design: A panel of cell lines representative of TNBC was tested for in vitro and in vivo sensitivity to CDK4/6 inhibition. A fluorescent CDK2 activity reporter was used for single-cell analysis in conjunction with time-lapse imaging.Results: The luminal androgen receptor (LAR) subtype of TNBC was highly sensitive to CDK4/6 inhibition both in vitro (P < 0.001 LAR vs. basal-like) and in vivo in MDA-MB-453 LAR cell line xenografts. Single-cell analysis of CDK2 activity demonstrated differences in cell-cycle dynamics between LAR and basal-like cells. Palbociclib-sensitive LAR cells exit mitosis with low levels of CDK2 activity, into a quiescent state that requires CDK4/6 activity for cell-cycle reentry. Palbociclib-resistant basal-like cells exit mitosis directly into a proliferative state, with high levels of CDK2 activity, bypassing the restriction point and the requirement for CDK4/6 activity. High CDK2 activity after mitosis is driven by temporal deregulation of cyclin E1 expression. CDK4/6 inhibitors were synergistic with PI3 kinase inhibitors in PIK3CA-mutant TNBC cell lines, extending CDK4/6 inhibitor sensitivity to additional TNBC subtypes.Conclusions: Cell-cycle dynamics determine the response to CDK4/6 inhibition in TNBC. CDK4/6 inhibitors, alone and in combination, are a novel therapeutic strategy for specific subgroups of TNBC. Clin Cancer Res; 23(18); 5561-72. ©2017 AACR.


Asunto(s)
Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Ratones , Mitosis/efectos de los fármacos , Imagen Molecular , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Transducción de Señal/efectos de los fármacos , Análisis de la Célula Individual , Imagen de Lapso de Tiempo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología
9.
Cancer Res ; 76(8): 2301-13, 2016 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-27020857

RESUMEN

Small-molecule inhibitors of the CDK4/6 cell-cycle kinases have shown clinical efficacy in estrogen receptor (ER)-positive metastatic breast cancer, although their cytostatic effects are limited by primary and acquired resistance. Here we report that ER-positive breast cancer cells can adapt quickly to CDK4/6 inhibition and evade cytostasis, in part, via noncanonical cyclin D1-CDK2-mediated S-phase entry. This adaptation was prevented by cotreatment with hormone therapies or PI3K inhibitors, which reduced the levels of cyclin D1 (CCND1) and other G1-S cyclins, abolished pRb phosphorylation, and inhibited activation of S-phase transcriptional programs. Combined targeting of both CDK4/6 and PI3K triggered cancer cell apoptosis in vitro and in patient-derived tumor xenograft (PDX) models, resulting in tumor regression and improved disease control. Furthermore, a triple combination of endocrine therapy, CDK4/6, and PI3K inhibition was more effective than paired combinations, provoking rapid tumor regressions in a PDX model. Mechanistic investigations showed that acquired resistance to CDK4/6 inhibition resulted from bypass of cyclin D1-CDK4/6 dependency through selection of CCNE1 amplification or RB1 loss. Notably, although PI3K inhibitors could prevent resistance to CDK4/6 inhibitors, they failed to resensitize cells once resistance had been acquired. However, we found that cells acquiring resistance to CDK4/6 inhibitors due to CCNE1 amplification could be resensitized by targeting CDK2. Overall, our results illustrate convergent mechanisms of early adaptation and acquired resistance to CDK4/6 inhibitors that enable alternate means of S-phase entry, highlighting strategies to prevent the acquisition of therapeutic resistance to these agents. Cancer Res; 76(8); 2301-13. ©2016 AACR.


Asunto(s)
Neoplasias de la Mama/enzimología , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Receptores de Estrógenos/metabolismo , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Xenoinjertos , Humanos , Ratones , Piperazinas/uso terapéutico , Piridinas/uso terapéutico
10.
Cancer Discov ; 6(8): 838-851, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27179038

RESUMEN

UNLABELLED: FGFR1 and FGFR2 are amplified in many tumor types, yet what determines response to FGFR inhibition in amplified cancers is unknown. In a translational clinical trial, we show that gastric cancers with high-level clonal FGFR2 amplification have a high response rate to the selective FGFR inhibitor AZD4547, whereas cancers with subclonal or low-level amplification did not respond. Using cell lines and patient-derived xenograft models, we show that high-level FGFR2 amplification initiates a distinct oncogene addiction phenotype, characterized by FGFR2-mediated transactivation of alternative receptor kinases, bringing PI3K/mTOR signaling under FGFR control. Signaling in low-level FGFR1-amplified cancers is more restricted to MAPK signaling, limiting sensitivity to FGFR inhibition. Finally, we show that circulating tumor DNA screening can identify high-level clonally amplified cancers. Our data provide a mechanistic understanding of the distinct pattern of oncogene addiction seen in highly amplified cancers and demonstrate the importance of clonality in predicting response to targeted therapy. SIGNIFICANCE: Robust single-agent response to FGFR inhibition is seen only in high-level FGFR-amplified cancers, with copy-number level dictating response to FGFR inhibition in vitro, in vivo, and in the clinic. High-level amplification of FGFR2 is relatively rare in gastric and breast cancers, and we show that screening for amplification in circulating tumor DNA may present a viable strategy to screen patients. Cancer Discov; 6(8); 838-51. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.


Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Evolución Clonal/genética , Amplificación de Genes , Piperazinas/farmacología , Pirazoles/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/genética , Animales , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Terapia Molecular Dirigida , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Tomografía de Emisión de Positrones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Taquicininas/metabolismo , Tomografía Computarizada por Rayos X , Ensayos Antitumor por Modelo de Xenoinjerto
11.
Cancer Discov ; 3(9): 1058-71, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23744832

RESUMEN

UNLABELLED: Activation of fibroblast growth factor receptors (FGFR) is a common oncogenic event. Little is known about the determinants of sensitivity to FGFR inhibition and how these may vary between different oncogenic FGFRs. Using parallel RNA interference (RNAi) genetic screens, we show that the EGF receptor (EGFR) limits sensitivity to FGFR inhibition in FGFR3-mutant and -translocated cell lines, but not in other FGFR-driven cell lines. We also identify two distinct mechanisms through which EGFR limits sensitivity. In partially FGFR3-dependent lines, inhibition of FGFR3 results in transient downregulation of mitogen-activated protein kinase signaling that is rescued by rapid upregulation of EGFR signaling. In cell lines that are intrinsically resistant to FGFR inhibition, EGFR dominates signaling via repression of FGFR3, with EGFR inhibition rescued by delayed upregulation of FGFR3 expression. Importantly, combinations of FGFR and EGFR inhibitors overcome these resistance mechanisms in vitro and in vivo. Our results illustrate the power of parallel RNAi screens in identifying common resistance mechanisms to targeted therapies. SIGNIFICANCE: Our data identify a novel therapeutic approach to the treatment of FGFR3-mutant cancer, emphasizing the potential of combination approaches targeting both FGFR3 and EGFR. Our data extend the role of EGFR in mediating resistance to inhibitors targeting a mutant oncogene, showing that EGFR signaling can repress mutant FGFR3 to induce intrinsic resistance to FGFR targeting.


Asunto(s)
Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Neoplasias/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Dasatinib , Receptores ErbB/genética , Gefitinib , Xenoinjertos , Ensayos Analíticos de Alto Rendimiento , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Trasplante de Neoplasias , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Quinazolinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal/genética , Tiazoles/farmacología
12.
Mol Biol Cell ; 22(1): 105-16, 2011 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-21118992

RESUMEN

The docking protein Gab2 is overexpressed in several human malignancies, including breast cancer, and is associated with increased metastatic potential. Here we report that Gab2 overexpression in MCF-10A mammary epithelial cells led to delayed cell spreading, a decrease in stress fibers and mature focal adhesions, and enhanced cell migration. Expression of a Gab2 mutant uncoupled from 14-3-3-mediated negative feedback (Gab2(2xA)) led to a more mesenchymal morphology and acquisition of invasive potential. Expression of either Gab2 or Gab2(2xA) led to decreased activation of RhoA, but only the latter increased levels of Rac-GTP. Expression of constitutively active RhoA in MCF-10A/Gab2 cells restored stress fibers and focal adhesions, indicating that Gab2 signals upstream of RhoA to suppress these structures. Mutation of the two Shp2-binding sites to phenylalanine (Gab2(ΔShp2)) markedly reduced the effects of Gab2 on cellular phenotype and RhoA activation. Expression of Gab2 or Gab2(2xA), but not Gab2(ΔShp2), promoted Vav2 phosphorylation and plasma membrane recruitment of p190A RhoGAP. Knockdown of p190A RhoGAP reversed Gab2-mediated effects on stress fibers and focal adhesions. The identification of a novel pathway downstream of Gab2 involving negative regulation of RhoA by p190A RhoGAP sheds new light on the role of Gab2 in cancer progression.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Mama/citología , Movimiento Celular , Proteína de Unión al GTP rhoA/metabolismo , Mama/ultraestructura , Neoplasias de la Mama/patología , Línea Celular , Proliferación Celular , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Femenino , Adhesiones Focales , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Expresión Génica , Humanos , Immunoblotting , Microscopía Fluorescente , Mutación , Invasividad Neoplásica , Metástasis de la Neoplasia , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , ARN Interferente Pequeño , Fibras de Estrés/metabolismo , Fibras de Estrés/ultraestructura , Proteína de Unión al GTP rhoA/genética
13.
Am J Physiol Cell Physiol ; 294(4): C931-44, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18216165

RESUMEN

We characterized the role of protein tyrosine phosphatase (PTP)-alpha in focal adhesion (FA) formation and remodeling using wild-type and PTPalpha-deficient (PTPalpha(-/-)) cells. Compared with wild-type cells, spreading PTPalpha(-/-) fibroblasts displayed fewer leading edges and formed elongated alpha-actinin-enriched FA at the cell periphery. These features suggest the presence of slowly remodeling cell adhesions and were phenocopied in human fibroblasts in which PTPalpha was knocked down using short interfering RNA (siRNA) or in NIH-3T3 fibroblasts expressing catalytically inactive (C433S/C723S) PTPalpha. Fluorescence recovery after photobleaching showed slower green fluorescence protein-alpha-actinin recovery in the FA of PTPalpha(-/-) than wild-type cells. These alterations correlated with reduced cell spreading, adhesion, and polarization and retarded contraction of extracellular matrices in PTPalpha(-/-) fibroblasts. Activation of Rac1 and its recruitment to FA during spreading were diminished in cells expressing C433S/C723S PTPalpha. Rac1(-/-) cells also displayed abnormally elongated and peripherally distributed FA that failed to remodel. Conversely, expression of constitutively active Rac1 restored normal FA remodeling in PTPalpha(-/-) cells. We conclude that PTPalpha is required for remodeling of FA during cell spreading via a pathway involving Rac1.


Asunto(s)
Adhesiones Focales/metabolismo , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Actinina/metabolismo , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica , Humanos , Potenciales de la Membrana , Paxillin/metabolismo , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/genética , Proteína de Unión al GTP rac1/genética
14.
EMBO J ; 27(17): 2305-16, 2008 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-19172738

RESUMEN

Grb2-associated binder (Gab)2 functions downstream of a variety of receptor and cytoplasmic tyrosine kinases as a docking platform for specific signal transducers and performs important functions in both normal physiology and oncogenesis. Gab2 signalling is promoted by its association with specific receptors through the adaptor Grb2. However, the molecular mechanisms that attenuate Gab2 signals have remained unclear. We now demonstrate that growth factor-induced phosphorylation of Gab2 on two residues, S210 and T391, leads to recruitment of 14-3-3 proteins. Together, these events mediate negative-feedback regulation, as Gab2(S210A/T391A) exhibits sustained receptor association and signalling and promotes cell proliferation and transformation. Importantly, introduction of constitutive 14-3-3-binding sites into Gab2 renders it refractory to receptor activation, demonstrating that site-selective binding of 14-3-3 proteins is sufficient to terminate Gab2 signalling. Furthermore, this is associated with reduced binding of Grb2. This leads to a model where signal attenuation occurs because 14-3-3 promotes dissociation of Gab2 from Grb2, and thereby uncouples Gab2 from the receptor complex. This represents a novel regulatory mechanism with implications for diverse tyrosine kinase signalling systems.


Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas 14-3-3/química , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Línea Celular , Retroalimentación Fisiológica , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal
15.
J Biol Chem ; 281(41): 31093-105, 2006 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-16905534

RESUMEN

Interleukin-1 (IL-1)-induced Ca2+ signaling in fibroblasts is constrained by focal adhesions. This process involves the proteintyrosine phosphatase SHP-2, which is critical for IL-1-induced phosphorylation of phospholipase Cgamma1, thereby enhancing IL-1-induced Ca2+ release and ERK activation. Currently, the mechanisms by which SHP-2 modulates Ca2+ release from the endoplasmic reticulum are not defined. We used immunoprecipitation and fluorescence protein-tagged SHP-2 or endoplasmic reticulum (ER)-protein expression vectors, and an ER-specific calcium indicator, to examine the functional relationships between SHP-2, focal adhesions, and IL-1-induced Ca2+ release from the ER. By total internal reflection fluorescence microscopy to image subplasma membrane compartments, SHP-2 co-localized with the ER-associated proteins calnexin and calreticulin at sites of focal adhesion formation in fibroblasts. IL-1beta promoted time-dependent recruitment of SHP-2 and ER proteins to focal adhesions; this process was blocked in cells treated with small interfering RNA for SHP-2 and in cells expressing a Y542F SHP-2 mutant. IL-1 stimulated inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release from the ER subjacent to the plasma membrane that was tightly localized around fibronectin-coated beads and was reduced 4-fold in cells expressing Tyr-542 SHP-2 mutant. In subcellular fractions enriched for ER proteins, immunoprecipitation demonstrated that IL-1-enhanced association of SHP-2 with the type 1 inositol 1,4,5-trisphosphate receptor was dependent on Tyr-542 of SHP-2. We conclude that Tyr-542 of SHP-2 modulates IL-1-induced Ca2+ signals and association of the ER with focal adhesions.


Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Interleucina-1/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Tirosina Fosfatasas/química , Animales , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Adhesiones Focales/química , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosforilación , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Transducción de Señal , Fracciones Subcelulares/metabolismo
16.
J Cell Physiol ; 207(1): 132-43, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16250012

RESUMEN

Interleukin-1beta (IL-1beta) mediates destruction of matrix collagens in diverse inflammatory diseases including arthritis, periodontitis, and pulmonary fibrosis by activating fibroblasts, cells that interact with matrix proteins through integrin-based adhesions. In vitro, IL-1beta signaling is modulated by focal adhesions, supramolecular protein complexes that are enriched with tyrosine kinases and phosphatases. We assessed the importance of tyrosine phosphatases in regulating cell-matrix interactions and IL-1beta signaling. In human gingival fibroblasts plated on fibronectin, IL-1beta enhanced the maturation of focal adhesions as defined by morphology and enrichment with paxillin and alpha-actinin. IL-1beta also induced activation of ERK and recruitment of phospho-ERK to focal complexes/adhesions. Treatment with the potent tyrosine phosphatase inhibitor pervanadate, in the absence of IL-1beta, recapitulated many of these responses indicating the importance of tyrosine phosphatases. Immunoblotting of collagen bead-associated complexes revealed that the tyrosine phosphatase, SHP-2, was also enriched in focal complexes/adhesions. Depletion of SHP-2 by siRNA or by homologous recombination markedly altered IL-1beta-induced ERK activation and maturation of focal adhesions. IL-1beta-induced tyrosine phosphorylation of SHP-2 on residue Y542 promoted focal adhesion maturation. Association of Gab1 with SHP-2 in focal adhesions correlated temporally with activation of ERK and was abrogated in cells expressing mutant (Y542F) SHP-2. We conclude that IL-1beta mediated maturation of focal adhesions is dependent on tyrosine phosphorylation of SHP-2 at Y542, leading to recruitment of Gab1, a process that may influence the downstream activation of ERK.


Asunto(s)
Fibroblastos/metabolismo , Adhesiones Focales/metabolismo , Interleucina-1/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal/fisiología , Actinina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Células Cultivadas , Colágeno/farmacología , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/efectos de los fármacos , Fibronectinas/farmacología , Adhesiones Focales/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Mutación , Paxillin/metabolismo , Fosforilación/efectos de los fármacos , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/genética , ARN Interferente Pequeño/genética , Transfección , Tirosina/metabolismo , Vanadatos/farmacología
17.
J Biol Chem ; 280(9): 8397-406, 2005 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-15563458

RESUMEN

Interleukin-1 (IL-1) signaling is dependent on focal adhesions, structures that are enriched with tyrosine kinases and phosphatases. Because the non-receptor tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is enriched in focal adhesions and IL-1-induced ERK activation requires increased Ca(2+), we determined whether SHP-2 modulates IL-1-induced Ca(2+) signaling. In SHP-2-deficient fibroblasts, IL-1-induced Ca(2+) signaling and ERK activation were markedly diminished compared with cells expressing SHP-2. IL-1-induced Ca(2+) release from the endoplasmic reticulum occurred in the vicinity of focal adhesions and was strongly inhibited by the blockage of phospholipase C (PLC) catalytic activity. Immunoprecipitation and immunostaining showed that SHP-2, the endoplasmic reticulum-specific protein calnexin, and PLCgamma1 were associated with focal adhesions; however, these associations and IL-1-induced ERK activation dissipated after cells were plated on non-integrin substrates. IL-1 promoted phosphorylation of SHP-2 and PLCgamma1. IL-1-induced phosphorylation of PLCgamma1 was diminished in SHP-2-deficient cells but was restored by stable transfection with SHP-2. BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)) blocked IL-1-induced phosphorylation of SHP-2 and PLCgamma1, indicating mutually dependent interactive roles for Ca(2+), SHP-2, and PLCgamma1 in IL-1 signaling. We conclude that SHP-2 is critical for IL-1-induced phosphorylation of PLCgamma1 and thereby enhances IL-1-induced Ca(2+) release and ERK activation. Focal adhesions co-localizing with the endoplasmic reticulum may provide molecular staging sites required for ERK activation.


Asunto(s)
Calcio/metabolismo , Ácido Egtácico/análogos & derivados , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Interleucina-1/metabolismo , Proteínas Tirosina Fosfatasas/fisiología , Fosfolipasas de Tipo C/metabolismo , Animales , Canales de Calcio/metabolismo , Calnexina/metabolismo , Células Cultivadas , Ácido Egtácico/farmacología , Retículo Endoplásmico/metabolismo , Activación Enzimática , Fibroblastos/metabolismo , Adhesiones Focales/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Receptores de Inositol 1,4,5-Trifosfato , Péptidos y Proteínas de Señalización Intracelular , Compuestos Macrocíclicos , Ratones , Microscopía Fluorescente , Oxazoles/farmacología , Fosfolipasa C gamma , Fosforilación , Polilisina/química , Proteína Fosfatasa 2 , Estructura Terciaria de Proteína , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección
18.
J Biol Chem ; 278(29): 27190-8, 2003 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-12721296

RESUMEN

Focal adhesion complexes are actin-rich, cytoskeletal structures that mediate cell adhesion to the substratum and also selectively regulate signal transduction pathways required for interleukin (IL)-1beta signaling to the MAP kinase, ERK. IL-1-induced ERK activation is markedly diminished in fibroblasts deprived of focal adhesions whereas activation of p38 and JNK is unaffected. While IL-1 signaling is known to involve the activity of protein and lipid kinases including MAP kinases, FAK, and PI3K, little is known about the role of phosphatases in the regulation of IL-1 signal generation and attenuation. Here we demonstrate that SHP-2, a protein tyrosine phosphatase present in focal adhesions, modulates IL-1-induced ERK activation and the transient actin stress fiber disorganization that occurs following IL-1 treatment in human gingival fibroblasts. Using a combination of immunoblotting, immunoprecipitation, and immunostaining we show that SHP-2 is present in nascent focal adhesions and undergoes phosphorylation on tyrosine 542 in response to IL-1 stimulation. Blocking anti-SHP-2 antibodies, electoporated into the cytosol of fibroblasts, inhibited IL-1-induced ERK activation, actin filament assembly, and cell contraction, indicating a role for SHP-2 in these processes. In summary, our data indicate that SHP-2, a focal adhesion-associated protein, participates in IL-1-induced ERK activation likely via an adaptor function.


Asunto(s)
Interleucina-1/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Células 3T3 , Actinas/metabolismo , Animales , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Adhesiones Focales/fisiología , Humanos , Interleucina-1/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Noqueados , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/deficiencia , Proteínas Tirosina Fosfatasas/genética
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