RESUMO
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.
Assuntos
Neoplasias da Mama , DNA Tumoral Circulante , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fulvestranto/farmacologia , Fulvestranto/uso terapêutico , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , DNA Tumoral Circulante/genética , MutaçãoRESUMO
Williams-Beuren syndrome (WBS) is a multisystem genetic disease caused by the deletion of a region of 1.5-1.8 Mb on chromosome 7q11.23. The elastin gene seems to account for several comorbidities and distinct clinical features such including cardiovascular disease, connective tissue abnormalities, growth retardation, and gastrointestinal (GI) symptoms. Increasing evidence points to alterations in gut microbiota composition as a primary or secondary cause of some GI or extra-intestinal characteristics. In this study, we performed the first exploratory analysis of gut microbiota in WBS patients compared to healthy subjects (CTRLs) using 16S rRNA amplicon sequencing, by investigating the gut dysbiosis in relation to diseases and comorbidities. We found that patients with WBS have significant dysbiosis compared to age-matched CTRLs, characterized by an increase in proinflammatory bacteria such as Pseudomonas, Gluconacetobacter and Eggerthella, and a reduction of anti-inflammatory bacteria including Akkermansia and Bifidobacterium. Microbial biomarkers associated with weight gain, GI symptoms and hypertension were identified. Gut microbiota profiling could represent a new tool that characterise intestinal dysbiosis to complement the clinical management of these patients. In particular, the administration of microbial-based treatments, alongside traditional therapies, could help in reducing or preventing the burden of these symptoms and improve the quality of life of these patients.
Assuntos
Gastroenteropatias , Microbioma Gastrointestinal , Síndrome de Williams , Humanos , Síndrome de Williams/genética , Síndrome de Williams/diagnóstico , Disbiose/microbiologia , RNA Ribossômico 16S/genética , Qualidade de Vida , Gastroenteropatias/complicaçõesRESUMO
Williams-Beuren syndrome (WBS) is a rare genetic neurodevelopmental disorder with multi-systemic manifestations. The evidence that most subjects with WBS face gastrointestinal (GI) comorbidities, have prompted us to carry out a metaproteomic investigation of their gut microbiota (GM) profile compared to age-matched healthy subjects (CTRLs). Metaproteomic analysis was carried out on fecal samples collected from 41 individuals with WBS, and compared with samples from 45 CTRLs. Stool were extracted for high yield in bacterial protein group (PG) content, trypsin-digested and analysed by nanoLiquid Chromatography-Mass Spectrometry. Label free quantification, taxonomic assignment by the lowest common ancestor (LCA) algorithm and functional annotations by COG and KEGG databases were performed. Data were statistically interpreted by multivariate and univariate analyses. A WBS GM functional dissimilarity respect to CTRLs, regardless age distribution, was reported. The alterations in function of WBSs GM was primarily based on bacterial pathways linked to carbohydrate transport and metabolism and energy production. Influence of diet, obesity, and GI symptoms was assessed, highlighting changes in GM biochemical patterns, according to WBS subsets' stratification. The LCA-derived ecology unveiled WBS-related functionally active bacterial signatures: Bacteroidetes related to over-expressed PGs, and Firmicutes, specifically the specie Faecalibacterium prausnitzii, linked to under-expressed PGs, suggesting a depletion of beneficial bacteria. These new evidences on WBS gut dysbiosis may offer novel targets for tailored interventions.
Assuntos
Microbioma Gastrointestinal , Síndrome de Williams , Humanos , Bactérias/genética , Firmicutes , Trato GastrointestinalRESUMO
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.
Assuntos
Citoesqueleto de Actina/genética , Movimento Celular/genética , Forma Celular/genética , Sequência Conservada/genética , Proteínas/genética , Actinas/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Genes de Insetos/genética , Testes Genéticos , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Fenótipo , Proteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Cicatrização/genéticaRESUMO
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.
Assuntos
Antineoplásicos , Neoplasias da Mama , Inibidores de Proteínas Quinases , Antineoplásicos/uso terapêutico , Biomarcadores , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Estrogênio/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Constipation is a heterogeneous symptom so an accurate diagnosis requires an appropriate approach. OBJECTIVE AND METHODS: To establish the clinical guidelines for diagnosis and treatment of chronic constipation in Mexico we have reviewed the diagnostic aspects of constipation according to the availability of resources in our country. In addition, evidence-based recommendations have been provided. RESULTS: The symptoms that best define constipation in our population are infrequent stool, hard stool expulsion, excessive effort and sensation of incomplete evacuation. Digital rectal examination is a useful diagnostic tool to discard organic diseases and pelvic floor dyssynergia. Patients with risk factors such as recent onset of constipation, family history of inflammatory bowel disease or colon cancer and those with alarm signs (unintentional weight loss and significant anemia, blood in stool) should undergo endoscopic diagnostic studies. If they are available, the most useful tests in evaluating patients with functional constipation are colonic transit with radiopaque markers and anorectal manometry with balloon expulsion test. CONCLUSIONS: Patients with symptoms of chronic constipation should undergo advanced colorectal physiological studies to try to establish the cause of constipation.
Assuntos
Constipação Intestinal/diagnóstico , Doença Crônica , Constipação Intestinal/etiologia , Defecação , Exame Retal Digital , Medicina Baseada em Evidências , Fezes , Humanos , México , Distúrbios do Assoalho Pélvico/complicaçõesRESUMO
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.
Assuntos
Neoplasias da Mama , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Fulvestranto , Humanos , Imidazóis , Oxazepinas , Fosfatidilinositol 3-Quinases , Piperazinas , Piridinas , Receptor ErbB-2/genéticaRESUMO
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.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Adesões Focais/enzimologia , Interleucina-1/farmacologia , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Quinases da Família src/metabolismo , Animais , Células Cultivadas , Retículo Endoplasmático/efeitos dos fármacos , Adesões Focais/efeitos dos fármacos , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Fosfotirosina/metabolismo , Ligação Proteica/efeitos dos fármacos , RatosRESUMO
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.
Assuntos
Lesão Pulmonar/etiologia , Respiração com Pressão Positiva/efeitos adversos , Ventiladores Mecânicos/efeitos adversos , Pressão do Ar , Animais , Citocinas/biossíntese , Citocinas/sangue , Lesão Pulmonar/patologia , Masculino , RNA/biossíntese , RNA/genética , Ratos , Ratos Sprague-Dawley , Testes de Função Respiratória , Mecânica Respiratória/fisiologia , Volume de Ventilação Pulmonar/fisiologiaRESUMO
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.
Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Camundongos , Mitose/efeitos dos fármacos , Imagem Molecular , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única , Imagem com Lapso de Tempo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
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.
Assuntos
Neoplasias da Mama/enzimologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Xenoenxertos , Humanos , Camundongos , Piperazinas/uso terapêutico , Piridinas/uso terapêuticoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Evolução Clonal/genética , Amplificação de Genes , Piperazinas/farmacologia , Pirazóis/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/genética , Animais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Terapia de Alvo Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Tomografia por Emissão de Pósitrons , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Taquicininas/metabolismo , Tomografia Computadorizada por Raios X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Neoplasias/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Dasatinibe , Receptores ErbB/genética , Gefitinibe , Xenoenxertos , Ensaios de Triagem em Larga Escala , Humanos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Transplante de Neoplasias , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/genética , Tiazóis/farmacologiaRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Mama/citologia , Movimento Celular , Proteína rhoA de Ligação ao GTP/metabolismo , Mama/ultraestrutura , Neoplasias da Mama/patologia , Linhagem Celular , Proliferação de Células , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Feminino , Adesões Focais , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Expressão Gênica , Humanos , Immunoblotting , Microscopia de Fluorescência , Mutação , Invasividade Neoplásica , Metástase Neoplásica , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , RNA Interferente Pequeno , Fibras de Estresse/metabolismo , Fibras de Estresse/ultraestrutura , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
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.
Assuntos
Adesões Focais/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinina/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Humanos , Potenciais da Membrana , Paxilina/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
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.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas 14-3-3/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Linhagem Celular , Retroalimentação Fisiológica , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
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.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Interleucina-1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Tirosina Fosfatases/química , Animais , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Adesões Focais/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/metabolismo , Ratos , Transdução de Sinais , Frações Subcelulares/metabolismoRESUMO
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.
Assuntos
Fibroblastos/metabolismo , Adesões Focais/metabolismo , Interleucina-1/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Actinina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Colágeno/farmacologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibronectinas/farmacologia , Adesões Focais/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Mutação , Paxilina/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/genética , RNA Interferente Pequeno/genética , Transfecção , Tirosina/metabolismo , Vanadatos/farmacologiaRESUMO
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.