RESUMO
The Controlling Cancer Summit, London, UK, 17-19 May 2016 The Controlling Cancer Summit is an intimate informal meeting that annually gathers international academic and clinical researchers to network and debate the current advancements and challenges of oncology research. This year, it focused not only on diagnostic/prognostic biomarkers and genetic influences in cancer but also novel and sometimes unconventional therapeutic interventions. This report will summarize the meeting highlights that contribute to our comprehension of cancer biology and new innovative ways to target this disease.
Assuntos
Neoplasias/terapia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Dano ao DNA , Reparo do DNA , Exossomos/metabolismo , Humanos , Nanomedicina/métodos , Neoplasias/diagnóstico , Neoplasias/etiologia , Neoplasias/metabolismoRESUMO
INTRODUCTION: Most breast cancer-related deaths result from metastasis, a process involving dynamic regulation of tumour cell adhesion and migration. The adhesion protein CD44, a key regulator of cell migration, is enriched in cholesterol-enriched membrane microdomains termed lipid rafts. We recently reported that raft affiliation of CD44 negatively regulates interactions with its migratory binding partner ezrin. Since raft affiliation is regulated by post-translational modifications including palmitoylation, we sought to establish the contribution of CD44 palmitoylation and lipid raft affiliation to cell migration. METHODS: Recovery of CD44 and its binding partners from raft versus non-raft membrane microdomains was profiled in non-migrating and migrating breast cancer cell lines. Site-directed mutagenesis was used to introduce single or double point mutations into both CD44 palmitoylation sites (Cys286 and Cys295), whereupon the implications for lipid raft recovery, phenotype, ezrin co-precipitation and migratory behaviour was assessed. Finally CD44 palmitoylation status and lipid raft affiliation was assessed in primary cultures from a small panel of breast cancer patients. RESULTS: CD44 raft affiliation was increased during migration of non-invasive breast cell lines, but decreased during migration of highly-invasive breast cells. The latter was paralleled by increased CD44 recovery in non-raft fractions, and exclusive non-raft recovery of its binding partners. Point mutation of CD44 palmitoylation sites reduced CD44 raft affiliation in invasive MDA-MB-231 cells, increased CD44-ezrin co-precipitation and accordingly enhanced cell migration. Expression of palmitoylation-impaired (raft-excluded) CD44 mutants in non-invasive MCF-10a cells was sufficient to reversibly induce the phenotypic appearance of epithelial-to-mesenchymal transition and to increase cell motility. Interestingly, cell migration was associated with temporal reductions in CD44 palmitoylation in wild-type breast cells. Finally, the relevance of these findings is underscored by the fact that levels of palmitoylated CD44 were lower in primary cultures from invasive ductal carcinomas relative to non-tumour tissue, while CD44 co-localisation with a lipid raft marker was less in invasive ductal carcinoma relative to ductal carcinoma in situ cultures. CONCLUSION: Our results support a novel mechanism whereby CD44 palmitoylation and consequent lipid raft affiliation inversely regulate breast cancer cell migration, and may act as a new therapeutic target in breast cancer metastasis.
Assuntos
Neoplasias da Mama/patologia , Receptores de Hialuronatos/metabolismo , Lipoilação , Microdomínios da Membrana/metabolismo , Invasividade Neoplásica/patologia , Mama/citologia , Mama/fisiologia , Carcinoma Ductal de Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Humanos , Receptores de Hialuronatos/genética , Mutagênese Sítio-Dirigida , Ligação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Cancer cell migration is an early event in metastasis, the main cause of breast cancer-related deaths. Cholesterol-enriched membrane domains called lipid rafts influence the function of many molecules, including the raft-associated protein CD44. We describe a novel mechanism whereby rafts regulate interactions between CD44 and its binding partner ezrin in migrating breast cancer cells. Specifically, in nonmigrating cells, CD44 and ezrin localized to different membranous compartments: CD44 predominantly in rafts, and ezrin in nonraft compartments. After the induction of migration (either nonspecific or CD44-driven), CD44 affiliation with lipid rafts was decreased. This was accompanied by increased coprecipitation of CD44 and active (threonine-phosphorylated) ezrin-radixin-moesin (ERM) proteins in nonraft compartments and increased colocalization of CD44 with the nonraft protein, transferrin receptor. Pharmacological raft disruption using methyl-ß-cyclodextrin also increased CD44-ezrin coprecipitation and colocalization, further suggesting that CD44 interacts with ezrin outside rafts during migration. Conversely, promoting CD44 retention inside lipid rafts by pharmacological inhibition of depalmitoylation virtually abolished CD44-ezrin interactions. However, transient single or double knockdown of flotillin-1 or caveolin-1 was not sufficient to increase cell migration over a short time course, suggesting complex crosstalk mechanisms. We propose a new model for CD44-dependent breast cancer cell migration, where CD44 must relocalize outside lipid rafts to drive cell migration. This could have implications for rafts as pharmacological targets to down-regulate cancer cell migration.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Receptores de Hialuronatos/metabolismo , Microdomínios da Membrana/metabolismo , Caveolina 1/metabolismo , Compartimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Ácido Hialurônico/farmacologia , Proteínas de Membrana/metabolismo , Modelos Biológicos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Frações Subcelulares/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
Intestinal epithelial barrier disruption is a feature of inflammatory bowel disease (IBD), but whether barrier disruption precedes or merely accompanies inflammation remains controversial. Tight junction (TJ) adhesion complexes control epithelial barrier integrity. Since some TJ proteins reside in cholesterol-enriched regions of the cell membrane termed lipid rafts, we sought to elucidate the relationship between rafts and intestinal epithelial barrier function. Lipid rafts were isolated from Caco-2 intestinal epithelial cells primed with the proinflammatory cytokine interferon-γ (IFN-γ) or treated with methyl-ß-cyclodextrin as a positive control for raft disruption. Rafts were also isolated from the ilea of mice in which colitis had been induced in conjunction with in vivo intestinal permeability measurements, and lastly from intestinal biopsies of ulcerative colitis (UC) patients with predominantly mild or quiescent disease. Raft distribution was analyzed by measuring activity of the raft-associated enzyme alkaline phosphatase and by performing Western blot analysis for flotillin-1. Epithelial barrier integrity was estimated by measuring transepithelial resistance in cytokine-treated cells or in vivo permeability to fluorescent dextran in colitic mice. Raft and nonraft fractions were analyzed by Western blotting for the TJ proteins occludin and zonula occludens-1 (ZO-1). Our results revealed that lipid rafts were disrupted in IFN-γ-treated cells, in the ilea of mice with subclinical colitis, and in UC patients with quiescent inflammation. This was not associated with a clear pattern of occludin or ZO-1 relocalization from raft to nonraft fractions. Significantly, a time-course study in colitic mice revealed that disruption of lipid rafts preceded the onset of increased intestinal permeability. Our data suggest for the first time that lipid raft disruption occurs early in the inflammatory cascade in murine and human colitis and, we speculate, may contribute to subsequent disruption of epithelial barrier function.
Assuntos
Enterite/patologia , Mucosa Intestinal/patologia , Intestinos/patologia , Microdomínios da Membrana/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biópsia , Western Blotting , Células CACO-2 , Centrifugação com Gradiente de Concentração , Colite Ulcerativa/patologia , Dieta , Eletroforese em Gel de Poliacrilamida , Enterite/induzido quimicamente , Enterite/genética , Feminino , Humanos , Interleucina-10/genética , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Pessoa de Meia-Idade , Permeabilidade , Junções Íntimas/patologiaRESUMO
Fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate numerous cellular processes. Deregulation of FGFR signalling is observed in a subset of many cancers, making activated FGFRs a highly promising potential therapeutic target supported by multiple preclinical studies. However, early-phase clinical trials have produced mixed results with FGFR-targeted cancer therapies, revealing substantial complexity to targeting aberrant FGFR signalling. In this Review, we discuss the increasing understanding of the differences between diverse mechanisms of oncogenic activation of FGFR, and the factors that determine response and resistance to FGFR targeting.
Assuntos
Neoplasias/tratamento farmacológico , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/administração & dosagem , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/genéticaRESUMO
FGFR2 genetic translocations are frequent in cholangiocarcinoma, yet despite initial sensitivity to FGFR inhibitors in clinic, patients quickly become resistant to targeted therapies. The work published by Goyal and colleagues demonstrates that acquisition of gatekeeper mutations in FGFR2 and intratumoral heterogeneity drive resistance in patients with FGFR2-translocated intrahepatic cholangiocarcinoma, which will have important implications for management of the disease in clinic. Cancer Discov; 7(3); 248-9. ©2017 AACR.See related article by Goyal et al., p. 252.
Assuntos
Colangiocarcinoma/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos , Humanos , Neoplasias Hepáticas , MutaçãoRESUMO
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
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.