RESUMO
INTRODUCTION: Colorectal cancer (CRC) is driven by a small set of oncogenic and tumour suppressor mutations. However, different combinations of mutations often lead to poor tumour responses to individual anticancer drugs. We have investigated the antiproliferative and in vitro cytotoxic activity of pair-wise combinations of inhibitors which target specific signalling pathways in colon cancer cells. OBJECTIVES: To target specific signaling pathways pairwise with inhibitors in order to kill colon cancer cells. METHODS: The effects of different concentrations of two inhibitors on the proliferation and viability of colon cancer cell lines were measured using cell titre glow and cytotoxic assays in 2D and 3D cell micro-cultures. One successful drug combination was used to treat a colon cancer cell line growing as a xenograft in nude mice. RESULTS: Colon cancer cells in non-adherent cultures were killed more effectively by combinations of pyrvinium pamoate (a Wnt pathway inhibitor) and ABT263 (a pro-apoptotic Bcl-2 family inhibitor) or Ly29004 (a PI3kinase inhibitor). However, in a mouse xenograft model, the formulation and toxicity of the ABT737/PP combination prevent the use of these drugs for treatment of tumours. Fortunately, oral analogues of PP (pyrvinium phosphate, PPh) and ABT737(ABT263) have equivalent activity and can be used for treatment of mice carrying SW620 colorectal cancer xenografts. The PPh/ABT263 induced SW620 tumour cell apoptosis and reduced the rate of SW620 tumour growth. CONCLUSION: By combining a Wnt signaling inhibitor (pyrvinium phosphate) and a pro-survival inhibitor (ABT263) colon cancer cells can be killed. Combinations of Wnt signalling inhibitors with an inhibitor of the Bcl pro-survival protein family should be considered for the treatment of patients with precancerous colon adenomas or advanced colorectal cancers with APC mutations.
Assuntos
Antineoplásicos , Neoplasias do Colo , Neoplasias Colorretais , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Sistemas de Liberação de Medicamentos , Humanos , Camundongos , Camundongos Nus , Fosfatos/farmacologia , Via de Sinalização Wnt/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismoRESUMO
Targeting cell division by chemotherapy is a highly effective strategy to treat a wide range of cancers. However, there are limitations of many standard-of-care chemotherapies: undesirable drug toxicity, side-effects, resistance and high cost. New small molecules which kill a wide range of cancer subtypes, with good therapeutic window in vivo, have the potential to complement the current arsenal of anti-cancer agents and deliver improved safety profiles for cancer patients. We describe results with a new anti-cancer small molecule, WEHI-7326, which causes cell cycle arrest in G2/M, cell death in vitro, and displays efficacious anti-tumor activity in vivo. WEHI-7326 induces cell death in a broad range of cancer cell lines, including taxane-resistant cells, and inhibits growth of human colon, brain, lung, prostate and breast tumors in mice xenografts. Importantly, the compound elicits tumor responses as a single agent in patient-derived xenografts of clinically aggressive, treatment-refractory neuroblastoma, breast, lung and ovarian cancer. In combination with standard-of-care, WEHI-7326 induces a remarkable complete response in a mouse model of high-risk neuroblastoma. WEHI-7326 is mechanistically distinct from known microtubule-targeting agents and blocks cells early in mitosis to inhibit cell division, ultimately leading to apoptotic cell death. The compound is simple to produce and possesses favorable pharmacokinetic and toxicity profiles in rodents. It represents a novel class of anti-cancer therapeutics with excellent potential for further development due to the ease of synthesis, simple formulation, moderate side effects and potent in vivo activity. WEHI-7326 has the potential to complement current frontline anti-cancer drugs and to overcome drug resistance in a wide range of cancers.
Assuntos
Antimitóticos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Animais , Antimitóticos/farmacocinética , Antimitóticos/toxicidade , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células Hep G2 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Mitose/efeitos dos fármacos , Neoplasias/patologia , Células PC-3 , Ratos Sprague-Dawley , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ten strains representing four lineages of the Pseudomonas fluorescens group (P. chlororaphis, P. corrugata, P. koreensis, and P. fluorescens subgroups) were evaluated for toxicity to the tobacco hornworm Manduca sexta and the common fruit fly Drosophila melanogaster. The three strains within the P. chlororaphis subgroup exhibited both oral and injectable toxicity to the lepidopteran M. sexta. All three strains have the gene cluster encoding the FitD insect toxin and a ΔfitD mutant of P. protegens strain Pf-5 exhibited diminished oral toxicity compared to the wildtype strain. Only one of the three strains, P. protegens Pf-5, exhibited substantial levels of oral toxicity against the dipteran D. melanogaster. Three strains in the P. fluorescens subgroup, which lack fitD, consistently showed significant levels of injectable toxicity against M. sexta. In contrast, the oral toxicity of these strains against D. melanogaster was variable between experiments, with only one strain, Pseudomonas sp. BG33R, causing significant levels of mortality in repeated experiments. Toxin complex (Tc) gene clusters, which encode insecticidal properties in Photorhabdus luminescens, were identified in the genomes of seven of the ten strains evaluated in this study. Within those seven genomes, six types of Tc gene clusters were identified, distinguished by gene content, organization and genomic location, but no correlation was observed between the presence of Tc genes and insect toxicity of the evaluated strains. Our results demonstrate that members of the P. fluorescens group have the capacity to kill insects by both FitD-dependent and independent mechanisms.
Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Família Multigênica , Pseudomonas fluorescens/genética , Animais , Drosophila melanogaster , ManducaRESUMO
Pseudomonas protegens strain Pf-5 is a soil bacterium that was first described for its capacity to suppress plant diseases and has since been shown to be lethal to certain insects. Among these is the common fruit fly Drosophila melanogaster, a well-established model organism for studies evaluating the molecular and cellular basis of the immune response to bacterial challenge. Pf-5 produces the insect toxin FitD, but a ΔfitD mutant of Pf-5 retained full toxicity against D. melanogaster in a noninvasive feeding assay, indicating that FitD is not a major determinant of Pf-5's oral toxicity against this insect. Pf-5 also produces a broad spectrum of exoenzymes and natural products with antibiotic activity, whereas a mutant with a deletion in the global regulatory gene gacA produces none of these exoproducts and also lacks toxicity to D. melanogaster. In this study, we made use of a panel of Pf-5 mutants having single or multiple mutations in the biosynthetic gene clusters for seven natural products and two exoenzymes that are produced by the bacterium under the control of gacA. Our results demonstrate that the production of rhizoxin analogs, orfamide A, and chitinase are required for full oral toxicity of Pf-5 against D. melanogaster, with rhizoxins being the primary determinant.
Assuntos
Proteínas de Bactérias/metabolismo , Quitinases/metabolismo , Drosophila melanogaster/microbiologia , Lipopeptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , Pseudomonas/metabolismo , Animais , Proteínas de Bactérias/genética , Quitinases/genética , Drosophila melanogaster/efeitos dos fármacos , Genes Reguladores , Lipopeptídeos/toxicidade , Mutação , Peptídeos Cíclicos/toxicidade , Pseudomonas/enzimologia , Pseudomonas/genética , Pseudomonas/patogenicidade , VirulênciaRESUMO
We have expressed and purified three soluble fragments of the human LRIG1-ECD (extracellular domain): the LRIG1-LRR (leucine-rich repeat) domain, the LRIG1-3Ig (immunoglobulin-like) domain, and the LRIG1-LRR-1Ig fragment using baculovirus vectors in insect cells. The two LRIG1 domains crystallised so that we have been able to determine the three-dimensional structures at 2.3Å resolution. We developed a three-dimensional structure for the LRIG1-ECD using homology modelling based on the LINGO-1 structure. The LRIG1-LRR domain and the LRIG1-LRR-1Ig fragment are monomers in solution, whereas the LRIG1-3Ig domain appears to be dimeric. We could not detect any binding of the LRIG1 domains or the LRIG1-LRR-1Ig fragment to the EGF receptor (EGFR), either in solution using biosensor analysis or when the EGFR was expressed on the cell surface. The FLAG-tagged LRIG1-LRR-1Ig fragment binds weakly to colon cancer cells regardless of the presence of EGFRs. Similarly, neither the soluble LRIG1-LRR nor the LRIG1-3Ig domains nor the full-length LRIG1 co-expressed in HEK293 cells inhibited ligand-stimulated activation of cell-surface EGFR.
Assuntos
Receptores ErbB/química , Receptores ErbB/metabolismo , Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Técnicas Biossensoriais , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cristalografia por Raios X , Células HEK293 , Humanos , Proteínas de Repetições Ricas em Leucina , Ligantes , Microscopia de Fluorescência , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/metabolismo , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Células Tumorais CultivadasRESUMO
Most colon cancers arise from somatic mutations in the tumor suppressor gene APC (adenomatous polyposis coli), and these mutations cause constitutive activation of the Wnt-to-ß-catenin pathway in the intestinal epithelium. Because Wnt-ß-catenin signaling is required for homeostasis and regeneration of the adult intestinal epithelium, therapeutic targeting of this pathway is challenging. We found that genetic activation of the cytokine-stimulated pathway mediated by the receptor gp130, the associated Jak (Janus kinase) kinases, and the transcription factor Stat3 (signal transducer and activator of transcription 3) was required for intestinal regeneration in response to irradiation-induced damage in wild-type mice and for tumorigenesis in Apc-mutant mice. Systemic pharmacological or partial genetic inhibition of gp130-Jak-Stat3 signaling suppressed intestinal regeneration, the growth of tumors in Apc-mutant mice, and the growth of colon cancer xenografts. The growth of Apc-mutant tumors depended on gp130-Jak-Stat3 signaling for induction of the polycomb repressor Bmi-1, and the associated repression of genes encoding the cell cycle inhibitors p16 and p21. However, suppression of gp130-Jak-Stat3 signaling did not affect Wnt-ß-catenin signaling or homeostasis in the intestine. Thus, these data not only suggest a molecular mechanism for how the gp130-Jak-Stat3 pathway can promote cancer but also provide a rationale for therapeutic inhibition of Jak in colon cancer.
Assuntos
Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes APC/fisiologia , Mucosa Intestinal/fisiologia , Regeneração/fisiologia , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Neoplasias do Colo/genética , Receptor gp130 de Citocina/metabolismo , Primers do DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Técnicas Histológicas , Imuno-Histoquímica , Janus Quinase 1/metabolismo , Luciferases , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
Mutations in the APC or ß-catenin genes are well-established initiators of colorectal cancer, yet modifiers that facilitate the survival and progression of nascent tumor cells are not well defined. Using genetic and pharmacologic approaches in mouse colorectal cancer and human colorectal cancer xenograft models, we show that incipient intestinal tumor cells activate CDC42, an APC-interacting small GTPase, as a crucial step in malignant progression. In the mouse, Cdc42 ablation attenuated the tumorigenicity of mutant intestinal cells carrying single APC or ß-catenin mutations. Similarly, human colorectal cancer with relatively higher levels of CDC42 activity was particularly sensitive to CDC42 blockade. Mechanistic studies suggested that Cdc42 may be activated at different levels, including at the level of transcriptional activation of the stem cell-enriched Rho family exchange factor Arhgef4. Our results indicate that early-stage mutant intestinal epithelial cells must recruit the pleiotropic functions of Cdc42 for malignant progression, suggesting its relevance as a biomarker and therapeutic target for selective colorectal cancer intervention.
Assuntos
Neoplasias Colorretais/patologia , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Progressão da Doença , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The activation of the epidermal growth factor receptor (EGFR) kinase requires ligand binding to the extracellular domain (ECD). Previous reports demonstrate that the EGFR-ECD can be crystallized in two conformations - a tethered monomer or, in the presence of ligand, an untethered back-to-back dimer. We use Biosensor analysis to demonstrate that even in the monomeric state different C-terminal extensions of both truncated (EGFR(1-501))-ECD and full-length EGFR(1-621)-ECD can change the conformation of the ligand-binding site. The binding of a monoclonal antibody mAb806, which recognizes the dimer interface, to the truncated EGFR(1-501)-Fc fusion protein is reduced in the presence of ligand, consistent with a change in conformation. On the cell surface, the presence of erythroblastosis B2 (erbB2) increases the binding of mAb806 to the EGFR. The conformation of the erbB2: EGFR heterodimer interface changes when the cells are treated with epidermal growth factor (EGF). We propose that ligand induces kinase-inactive, pre-formed EGFR dimers and heterodimers to change conformation leading to kinase-active tetramers, where kinase activation occurs via an asymmetric interaction between EGFR dimers.
Assuntos
Receptores ErbB/química , Ligantes , Animais , Anticorpos Monoclonais/química , Técnicas Biossensoriais , Linhagem Celular , Dimerização , Epitopos/química , Corantes Fluorescentes/química , Células HEK293 , Humanos , Cinética , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: LGR5 (Leucine-rich repeat-containing G-protein coupled receptor 5) is the most established marker for intestinal stem cells. Mouse models show that LGR5+ cells are the cells of origin of intestinal cancer, and LGR5 expression is elevated in human colorectal cancers, however very little is known about LGR5 function or its contribution to the stem cell phenotype and to colorectal cancer. PRINCIPAL FINDINGS: We have modulated the expression of LGR5 by RNAi (inhibitory RNAs) or overexpression in colorectal cancer cell lines. Paradoxically, ablation of LGR5 induces increased invasion and anchorage-independent growth, and enhances tumourigenicity in xenografts experiments. Conversely, overexpression of LGR5 augments cell adhesion, reduces clonogenicity and attenuates tumourigenicity. Expression profiling revealed enhanced wnt signalling and upregulation of EMT genes upon knockdown of LGR5, with opposite changes in LGR5 overexpressing cells. These findings suggest that LGR5 is important in restricting stem cells to their niche, and that loss of LGR5 concomitant with activated wnt signalling may contribute to the invasive phenotype of colorectal carcinomas.
Assuntos
Adesão Celular/fisiologia , Neoplasias Colorretais/patologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Movimento Celular , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteínas Wnt/genética , Cicatrização , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Studies employing mouse models have identified crypt base and position +4 cells as strong candidates for intestinal epithelial stem cells. Equivalent cell populations are thought to exist in the human intestine; however robust and specific protein markers are lacking. Here, we show that in the human small and large intestine, PHLDA1 is expressed in discrete crypt base and some position +4 cells. In small adenomas, PHLDA1 was expressed in a subset of undifferentiated and predominantly Ki-67-negative neoplastic cells, suggesting that a basic hierarchy of differentiation is retained in early tumorigenesis. In large adenomas, carcinomas, and metastases PHLDA1 expression became widespread, with increased expression and nuclear localization at invasive margins. siRNA-mediated suppression of PHLDA1 in colon cancer cells inhibited migration and anchorage-independent growth in vitro and tumor growth in vivo. The integrins ITGA2 and ITGA6 were downregulated in response to PHLDA1 suppression, and accordingly cell adhesion to laminin and collagen was significantly reduced. We conclude that PHLDA1 is a putative epithelial stem cell marker in the human small and large intestine and contributes to migration and proliferation in colon cancer cells.
Assuntos
Células Epiteliais/citologia , Regulação Neoplásica da Expressão Gênica , Mucosa Intestinal/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Neoplasias do Colo/metabolismo , Células HCT116 , Humanos , Integrina alfa2/metabolismo , Integrina alfa6/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Células-Tronco/citologiaRESUMO
BACKGROUND: The fruit fly, Drosophila melanogaster, is a well-established model organism for probing the molecular and cellular basis of physiological and immune system responses of adults or late stage larvae to bacterial challenge. However, very little is known about the consequences of bacterial infections that occur in earlier stages of development. We have infected mid-second instar larvae with strains of Pseudomonas fluorescens to determine how infection alters the ability of larvae to survive and complete development. METHODOLOGY/PRINCIPAL FINDINGS: We mimicked natural routes of infection using a non-invasive feeding procedure to study the toxicity of the three sequenced P. fluorescens strains (Pf0-1, SBW25, and Pf-5) to Drosophila melanogaster. Larvae fed with the three strains of P. fluorescens showed distinct differences in developmental trajectory and survival. Treatment with SBW25 caused a subset of insects to die concomitant with a systemic melanization reaction at larval, pupal or adult stages. Larvae fed with Pf-5 died in a dose-dependent manner with adult survivors showing eye and wing morphological defects. In addition, larvae in the Pf-5 treatment groups showed a dose-dependent delay in the onset of metamorphosis relative to control-, Pf0-1-, and SBW25-treated larvae. A functional gacA gene is required for the toxic properties of wild-type Pf-5 bacteria. CONCLUSIONS/SIGNIFICANCE: These experiments are the first to demonstrate that ingestion of P. fluorescens bacteria by D. melanogaster larvae causes both lethal and non-lethal phenotypes, including delay in the onset of metamorphosis and morphological defects in surviving adult flies, which can be decoupled.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/microbiologia , Larva/crescimento & desenvolvimento , Pseudomonas fluorescens/fisiologia , Animais , Drosophila melanogaster/fisiologia , Ingestão de Alimentos , Larva/genética , Larva/microbiologia , Metamorfose BiológicaRESUMO
BACKGROUND: Frameshift mutations in microsatellite instability high (MSI-High) colorectal cancers are a potential source of targetable neo-antigens. Many nonsense transcripts are subject to rapid degradation due to nonsense-mediated decay (NMD), but nonsense transcripts with a cMS in the last exon or near the last exon-exon junction have intrinsic resistance to nonsense-mediated decay (NMD). NMD-resistant transcripts are therefore a likely source of expressed mutant proteins in MSI-High tumours. METHODS: Using antibodies to the conserved N-termini of predicted mutant proteins, we analysed MSI-High colorectal cancer cell lines for examples of naturally expressed mutant proteins arising from frameshift mutations in coding microsatellites (cMS) by immunoprecipitation and Western Blot experiments. Detected mutant protein bands from NMD-resistant transcripts were further validated by gene-specific short-interfering RNA (siRNA) knockdown. A genome-wide search was performed to identify cMS-containing genes likely to generate NMD-resistant transcripts that could encode for antigenic expressed mutant proteins in MSI-High colon cancers. These genes were screened for cMS mutations in the MSI-High colon cancer cell lines. RESULTS: Mutant protein bands of expected molecular weight were detected in mutated MSI-High cell lines for NMD-resistant transcripts (CREBBP, EP300, TTK), but not NMD-sensitive transcripts (BAX, CASP5, MSH3). Expression of the mutant CREBBP and EP300 proteins was confirmed by siRNA knockdown. Five cMS-bearing genes identified from the genome-wide search and without existing mutation data (SFRS12IP1, MED8, ASXL1, FBXL3 and RGS12) were found to be mutated in at least 5 of 11 (45%) of the MSI-High cell lines tested. CONCLUSION: NMD-resistant transcripts can give rise to expressed mutant proteins in MSI-High colon cancer cells. If commonly expressed in primary MSI-High colon cancers, MSI-derived mutant proteins could be useful as cancer specific immunological targets in a vaccine targeting MSI-High colonic tumours.
Assuntos
Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Antígenos HLA-A/genética , Instabilidade de Microssatélites , Mutação , Linhagem Celular Tumoral , Códon sem Sentido , Biologia Computacional/métodos , Reparo do DNA , Mapeamento de Epitopos , Mutação da Fase de Leitura , Genoma , Humanos , Repetições de Microssatélites , Estrutura Terciária de ProteínaRESUMO
Tumor-derived cell lines are indispensable tools for understanding the contribution of activated signaling pathways to the cancer phenotype and for the design and testing of targeted signal therapies. In our study, we characterize 10 colorectal carcinoma cell lines for the presence of mutations in the wnt, Ras/MAPK, PI3K and p53 pathways. The mutational spectrum found in this panel of cell lines is similar to that detected in primary CRC, albeit with higher frequency of mutation in the beta-catenin and B-Raf genes. We have monitored activation of the wnt and Ras/MAPK pathways in these cells and analyzed their sensitivity to selective signaling inhibitors. Using beta-catenin subcellular distribution as a marker, we show that cells harboring APC mutations have low-level activated wnt signaling, which can be blocked by the extracellular wnt inhibitor DKK-1, suggesting autocrine activation of this pathway; proliferation of these cells is also blocked by DKK-1. In contrast, cells with beta-catenin mutations are unresponsive to extracellular wnt inhibition. Constitutive phosphorylation of MAPK is present in the majority of the cell lines and correlates with B-Raf but not K-Ras mutations; correspondingly, the proliferation of cells harboring mutations in B-Raf, but not K-Ras, is exquisitely sensitive inhibition of the MAPK pathway. We find no correlation between PI3K mutation or loss of PTEN expression and increased sensitivity to PI3K inhibitors. Our study discloses clear-cut differences in responsiveness to signaling inhibitors between individual mutations within an activated signaling pathway and suggests likely targets for signal-directed therapy of colorectal carcinomas.
Assuntos
Proliferação de Células , Neoplasias Colorretais/patologia , Genes APC , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Sequência de Bases , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Humanos , Fosforilação , Reação em Cadeia da Polimerase , Transdução de SinaisRESUMO
Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR(287-302) complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFR(C271A/C283A) mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFR(C271A/C283A). Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Receptores ErbB/imunologia , Proteínas de Neoplasias/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/uso terapêutico , Complexo Antígeno-Anticorpo/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Cristalografia por Raios X , Epitopos , Humanos , Camundongos , Camundongos Nus , Conformação Proteica , Desnaturação Proteica/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mice defective in both granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have severely impaired neutrophil production and function, yet these mice respond to acute pathogen challenge with a significant neutrophil response. We have recently reported the development of an in vitro system to detect granulopoietic cytokines secreted from cells isolated from G-CSF, GM-CSF double knockout mice. The conditioned media produced by these cells after stimulation with lipopolysaccharide or Candida albicans supports the production and differentiation of granulocytes (ie, the conditioned media contains neutrophil promoting activity [NPA]). We now show that the NPA in the G-CSF(-/-)/GM-CSF(-/-) conditioned media requires interleukin-6 (IL6), is abolished by soluble gp130, and can be specifically immunodepleted by an anti-IL6R antibody. NPA effects on bone marrow cells are also mimicked by Hyper-IL6, and the soluble IL6R is present in NPA. These results show that the IL6/sIL6R complex is the major effector of NPA. NPA production by mice defective for both G-CSF and GM-CSF uncovers an alternative pathway to granulocyte production, which is activated after exposure to pathogens.
Assuntos
Fator Estimulador de Colônias de Granulócitos/deficiência , Fator Estimulador de Colônias de Granulócitos e Macrófagos/deficiência , Granulócitos/metabolismo , Interleucina-6/metabolismo , Receptores de Interleucina-6/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Contagem de Células , Meios de Cultivo Condicionados , Fibroblastos/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Granulócitos/citologia , Granulócitos/efeitos dos fármacos , Interleucina-6/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Knockout , Monócitos/citologia , Monócitos/efeitos dos fármacos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Solubilidade/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Taxa de SobrevidaRESUMO
G-CSF and GM-CSF play important roles in regulating neutrophil production, survival, differentiation, and function. However, we have shown previously that G-CSF/GM-CSF double-deficient [knockout (KO)] mice still develop a profound neutrophilia in bone marrow and blood after infection with Candida albicans. This finding suggests the existence of other systems, which can regulate emergency neutrophil production. We have now developed an "in vitro" technique to detect and characterize a neutrophil-promoting activity (NPA) in the media conditioned by mouse embryonic fibroblasts (MEFs) derived from G-CSF(-/-)/GM-CSF(-/-) mice. NPA is produced in vitro by the MEFs after stimulation with LPS or heat-inactivated C. albicans. Although M-CSF added directly to bone marrow cultures does not sustain granulocyte production, our studies indicate that production of NPA requires activation of the M-CSF receptor (c-fms). First, G-CSF(-/-)/GM-CSF(-/-) MEFs produce high levels of NPA after stimulation with LPS or C. albicans, and G-CSF/GM-CSF/M-CSF triple-KO MEFs do not. Second, the production of NPA by the G-CSF(-/-)/GM-CSF(-/-) MEFs is reduced significantly upon incubation with neutralizing antibodies to M-CSF or c-fms. Third, NPA production by G-CSF(-/-)/GM-CSF(-/-)/M-CSF(-/-) fibroblasts is enhanced by supplementing culture medium with M-CSF. Thus, stimulation of c-fms by M-CSF is a prerequisite for the production of NPA.
Assuntos
Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Fator Estimulador de Colônias de Granulócitos/deficiência , Células Precursoras de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/deficiência , Substâncias de Crescimento/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Neutrófilos/metabolismo , Animais , Anticorpos/farmacologia , Doenças da Medula Óssea/metabolismo , Doenças da Medula Óssea/patologia , Candida albicans , Candidíase/metabolismo , Candidíase/patologia , Células Cultivadas , Embrião de Mamíferos/patologia , Feminino , Fibroblastos/patologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Células Precursoras de Granulócitos/patologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Lipopolissacarídeos/farmacologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Neutrófilos/patologia , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismoRESUMO
The epidermal growth factor receptor (EGFR) has at least two fundamental conformations: an inactive tethered conformation and an active untethered, ligand-bound "back-to-back" dimer, which may be part of an oligomeric complex. Monoclonal antibody (mAb) 806 is an EGFR-specific antibody that only binds a transitional form of the receptor after it untethers but before forming the back-to-back, ligated, active oligomer. We have shown that AG1478, a tyrosine kinase inhibitor of the EGFR, synergistically inhibits the growth of tumors overexpressing EGFR when used in combination with mAb 806 but the mechanism for this was not elucidated (Johns, T. G., Luwor, R. B., Murone, C., Walker, F., Weinstock, J., Vitali, A. A., Perera, R. M., Jungbluth, A. A., Stockert, E., Old, L. J., Nice, E. C., Burgess, A. W., and Scott, A. M. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15871-15876). We now show that AG1478 increases binding of mAb 806 to the cell surface through two distinct mechanisms: an immediate effect on the conformation of EGFR and a longer term increase in cell surface under-glycosylated EGFR, an event known to increase mAb 806 reactivity. Cross-linking studies demonstrated the presence of spontaneously occurring mAb 806-reactive dimers on the surface of cells overexpressing EGFR, which are rapidly increased by AG1478. Because they react with mAb 806, these dimers must exist in a conformation distinct from the ligated back-to-back dimer. Indeed, we detected similar dimers in 293T cells expressing the EGFR lacking the small dimerization/activation arm essential to the formation of the back-to-back dimer. Thus, some of the EGFR on the cell surface of cancer cells must exist as an untethered dimer that adopts a previously unreported conformation that is inactive. This information was used to optimize the therapeutic synergy between mAb 806 and AG1478 in a xenograft model.
Assuntos
Receptores ErbB/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Tirfostinas/farmacologia , Tirfostinas/uso terapêutico , Animais , Anticorpos Monoclonais/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dimerização , Receptores ErbB/química , Citometria de Fluxo , Humanos , Cinética , Camundongos , Camundongos Nus , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinazolinas , Transplante HeterólogoRESUMO
The site of action of growth factors on mammalian cell cycle has been assigned to the boundary between the G1 and S phases. We show here that Epidermal Growth Factor (EGF) is also required for mitosis. BaF/3 cells expressing the EGFR (BaF/wtEGFR) synthesize DNA in response to EGF, but arrest in S-phase. We have generated a cell line (BaF/ERX) with defective downregulation of the EGFR and sustained activation of EGFR signalling pathways: these cells undergo mitosis in an EGF-dependent manner. The transit of BaF/ERX cells through G2/M strictly requires activation of EGFR and is abolished by AG1478. This phenotype is mimicked by co-expression of ErbB2 in BaF/wtEGFR cells, and abolished by inhibition of the EGFR kinase, suggesting that sustained signalling of the EGFR, through impaired downregulation of the EGFR or heterodimerization, is required for completion of the cycle. We have confirmed the role of EGFR signalling in the G2/M phase of the cell cycle using a human tumor cell line which overexpresses the EGFR and is dependent on EGFR signalling for growth. These findings unmask an EGF-sensitive checkpoint, helping to understand the link between sustained EGFR signalling, proliferation and the acquisition of a radioresistant phenotype in cancer cells.
Assuntos
Ciclo Celular , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Receptores ErbB/genética , Receptores ErbB/fisiologia , Camundongos , Mitose , Receptor ErbB-2/fisiologia , Transdução de Sinais , TransfecçãoRESUMO
We have investigated functional effects of glycosylation at N(579) of the epidermal growth factor receptor (EGFR). Our previous study showed that the population of cell-surface expressed EGFRs in A431 cells, a human epidermoid carcinoma cell line, is composed of two subpopulations that differ by glycosylation at N(579) [Zhen et al. (2003) Biochemistry 42, 5478-5492]. To characterize the subpopulation of receptors not glycosylated at N(579), we established a 32D cell line expressing a point mutant of the EGFR (N579Q), which cannot be glycosylated at this position. Analysis of epitope accessibility suggests that the lack of glycosylation at N(579) weakens auto-inhibitory tether interactions, and cross-linking experiments suggest a somewhat elevated level of preformed N579Q-EGFR dimers in the absence of ligand relative to wild-type EGFR (WT-EGFR). However, ligand drives the majority of N579Q-EGFR dimerization, suggesting that untethering, while necessary, is not sufficient to drive dimerization. Ligand-binding experiments reveal a much greater fraction of N579Q-EGFRs in a high-affinity state compared to the fraction of WT-EGFRs in a high-affinity state. However, differences in the kinetic association and dissociation rates indicate that the high-affinity states of the WT and the N579Q receptors are distinct. EGF-stimulated phosphorylation in cells expressing N579Q-EGFRs results in notable differences in the pattern of tyrosine phosphorylated proteins compared with that obtained in cells expressing WT-EGFRs. Moreover, although WT-EGFRs confer cell survival in 32D cells in the absence of interleukin-3 and EGF, we found that receptors lacking glycosylation at N(579) do not. This is the first study of which we are aware to show that selective glycosylation of a specific N-glycosylation site can produce two functionally distinct receptors.
Assuntos
Asparagina/metabolismo , Receptores ErbB/química , Anticorpos , Asparagina/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glicosilação , Humanos , Cinética , Ligantes , Mutação Puntual , Conformação ProteicaRESUMO
A systematic study using solid phase peptide synthesis has been undertaken to examine the role of the disulfide bonds in the structure and function of mEGF. A combination of one, two and three native disulfide pair analogues of an active truncated (4-48) form of mEGF have been synthesised by replacing specific cysteine residues with isosteric a-amino-n-butyric acid (Abu). Oxidation of the peptides was performed using either conventional aerobic oxidation at basic pH, in DMSO under acidic conditions or via selective disulfide formation using orthogonal protection of the cysteine pairs. The contribution of individual, or pairs of, disulfide bonds to EGF structure was evaluated by CD and (1)H-NMR spectroscopy. The mitogenic activity of each analogue was determined using Balb/c 3T3 mouse fibroblastsAs we have reported previously (Barnham et al. 1998), the disulfide bond between residues 6 and 20 can be removed with significant retention of biological activity (EC50 20-50 nM). The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. We now show that removal of any other disulfide bond, either singly or in pairs, results in a major disruption of the tertiary structure, and a large loss of activity (EC50>900 nM). Remarkably, the linear analogue appears to have greater activity (EC50 580 nM) than most one and two disulfide bond analogues although it does not have a definable tertiary structure.