RESUMO
Priming of the organ-specific premetastatic sites is thought to be an important yet incompletely understood step during metastasis. In this study, we show that the metastatic tumors we examined overexpress granulocyte-colony stimulating factor (G-CSF), which expands and mobilizes Ly6G+Ly6C+ granulocytes and facilitates their subsequent homing at distant organs even before the arrival of tumor cells. Moreover, G-CSF-mobilized Ly6G+Ly6C+ cells produce the Bv8 protein, which has been implicated in angiogenesis and mobilization of myeloid cells. Anti-G-CSF or anti-Bv8 antibodies significantly reduced lung metastasis. Transplantation of Bv8 null fetal liver cells into lethally irradiated hosts also reduced metastasis. We identified an unexpected role for Bv8: the ability to stimulate tumor cell migration through activation of one of the Bv8 receptors, prokineticin receptor (PKR)-1. Finally, we show that administration of recombinant G-CSF is sufficient to increase the numbers of Ly6G+Ly6C+ cells in organ-specific metastatic sites and results in enhanced metastatic ability of several tumors.
Assuntos
Antígenos Ly/imunologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Granulócitos/efeitos dos fármacos , Granulócitos/imunologia , Neoplasias Pulmonares/patologia , Metástase Neoplásica , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Feminino , Perfilação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/genética , Granulócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos SCID , Análise em Microsséries , Transplante de Neoplasias , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
PURPOSE: Multiple endocrine neoplasia type 1 (MEN1) is defined clinically by the combined occurrence of multiple tumors, typically of the parathyroid glands, pancreatic islet cells, and anterior pituitary gland. A mouse model with a heterozygous deletion of the Men1 gene recapitulates the tumorigenesis of MEN1. We wished to determine the role of vascular endothelial growth factor (VEGF)-A in the vascularization and growth of MEN1-associated tumors, with an emphasis on pituitary adenomas. EXPERIMENTAL DESIGN: To investigate whether tumor growth in Men1(+/-) mice is mediated by VEGF-A dependent angiogenesis, we carried out a monotherapy with the anti-VEGF-A monoclonal antibody (mAb) G6-31. We evaluated tumor growth by magnetic resonance imaging and assessed vascular density in tissue sections. We also measured hormone levels in the serum. RESULTS: During the treatment with mAb G6-31, a significant inhibition of the pituitary adenoma growth was observed, leading to an increased mean tumor doubling-free survival compared with mice treated with a control antibody. Similarly, the growth of s.c. pituitary adenoma transplants was effectively inhibited by administration of anti-VEGF-A mAb. Serum prolactin was lowered by mAb G6-31 treatment but not by control antibody, potentially providing a new therapeutic approach for treating the hormonal excess in MEN1 patients. Additionally, the vascular density in pancreatic islet tumors was significantly reduced by the treatment. CONCLUSIONS: These results suggest that VEGF-A blockade may represent a nonsurgical treatment for benign tumors of the endocrine system.
Assuntos
Adenoma/metabolismo , Neoplasia Endócrina Múltipla Tipo 1/patologia , Neoplasias Hipofisárias/patologia , Prolactina/sangue , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Adenoma/sangue , Adenoma/patologia , Animais , Anticorpos Monoclonais/farmacologia , Modelos Animais de Doenças , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Camundongos , Camundongos Mutantes , Camundongos Nus , Neoplasia Endócrina Múltipla Tipo 1/sangue , Neoplasia Endócrina Múltipla Tipo 1/metabolismo , Neovascularização Patológica , Neoplasias Hipofisárias/sangue , Neoplasias Hipofisárias/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacosRESUMO
The histidine triad (HIT) protein Hint has been found to associate with mammalian Cdk7, as well as to interact both physically and genetically with the budding yeast Cdk7 homologue Kin28. To study the function of Hint and to explore its possible role in modulating Cdk7 activity in vivo, we have characterized the expression pattern of murine Hint and generated Hint-deficient (Hint(-/-)) mice. Hint was widely expressed during mouse development, with pronounced expression in several neuronal ganglia, epithelia, hearts, and testes from embryonic day 15 onward. Despite this widespread expression, disruption of Hint did not impair murine development. Moreover, Hint-deficient mice had a normal life span and were apparently healthy. Histological examination of tissues with high Hint expression in wild-type animals did not show signs of abnormal pathology in Hint(-/-) mice. Functional redundancy within the HIT family was addressed by crossing Hint(-/-) mice with mice lacking the related HIT protein, Fhit, and by assaying the expression levels of the HIT protein gene family members Hint2 and Hint3 in Hint(+/+) and Hint(-/-) tissues. Finally, Cdk7 kinase activity and cell cycle kinetics were found to be comparable in wild-type and Hint(-/-) mouse embryonic fibroblasts, suggesting that Hint may not be a key regulator of Cdk7 activity.
Assuntos
Hidrolases Anidrido Ácido , Quinases Ciclina-Dependentes/metabolismo , Embrião de Mamíferos/fisiologia , Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião de Mamíferos/anatomia & histologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Hidrolases/química , Hidrolases/genética , Hibridização In Situ , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Família Multigênica , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Distribuição Tecidual , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Anti-VEGF-A monoclonal antibodies, in combination with chemotherapy, result in a survival benefit in patients with metastatic colorectal and non-small cell lung cancer, but little is known regarding the impact of anti-VEGF-A therapy on benign or premalignant tumors. The Apc+/min mice have been widely used as a model recapitulating early intestinal adenoma formation. To investigate whether tumor growth in Apc+/min mice is mediated by VEGF-A-dependent angiogenesis, we used two independent approaches to inhibit VEGF-A: monotherapy with a monoclonal antibody (Mab) targeting VEGF-A and genetic deletion of VEGF-A selectively in intestinal epithelial cells. Short-term (3 or 6 weeks) treatment with anti-VEGF-A Mab G6-31 resulted in a nearly complete suppression of adenoma growth throughout the small intestine. Growth inhibition by Mab G6-31 was associated with a decrease in vascular density. Long-term (up to 52 weeks) treatment with Mab G6-31 led to a substantial increase in median survival. Deletion of VEGF-A in intestinal epithelial cells of Apc+/min mice yielded a significant inhibition of tumor growth, albeit of lesser magnitude than that resulting from Mab G6-31 administration. These results establish that inhibition of VEGF-A signaling is sufficient for tumor growth cessation and confers a long-term survival benefit in an intestinal adenoma model. Therefore, VEGF-A inhibition may be a previously uncharacterized strategy for the prevention of the angiogenic switch and growth in intestinal adenomas.
Assuntos
Genes APC/fisiologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Adenoma/irrigação sanguínea , Adenoma/genética , Adenoma/imunologia , Adenoma/terapia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Deleção de Genes , Hibridização In Situ , Neoplasias Intestinais/irrigação sanguínea , Neoplasias Intestinais/genética , Neoplasias Intestinais/imunologia , Neoplasias Intestinais/terapia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologia , Análise de Sobrevida , Fatores de TempoRESUMO
The mammalian Mat1 protein has been implicated in cell cycle regulation as part of the Cdk activating kinase (CAK), and in regulation of transcription as a subunit of transcription factor TFIIH. To address the role of Mat1 in vivo, we have used a Cre/loxP system to conditionally ablate Mat1 in adult mitotic and post-mitotic lineages. We found that the mitotic cells of the germ lineage died rapidly upon disruption of Mat1 indicating an absolute requirement of Mat1 in these cells. By contrast, post-mitotic myelinating Schwann cells were able to attain a mature myelinated phenotype in the absence of Mat1. Moreover, mutant animals did not show morphological or physiological signs of Schwann cell dysfunction into early adulthood. Beyond 3 months of age, however, myelinated Schwann cells in the sciatic nerves acquired a severe hypomyelinating morphology with alterations ranging from cells undergoing degeneration to completely denuded axons. This phenotype was coupled to extensive proliferation and remyelination that our evidence suggests was undertaken by the non-myelinated Schwann cell pool. These results indicate that Mat1 is not essential for the transcriptional program underlying the myelination of peripheral axons by Schwann cells and suggest that the function of Mat1 in RNA polymerase II-mediated transcription in these cells is regulatory rather than essential.
Assuntos
Quinases Ciclina-Dependentes/deficiência , Genes Reguladores/genética , Bainha de Mielina/metabolismo , Nervos Periféricos/crescimento & desenvolvimento , Subunidades Proteicas/deficiência , Células de Schwann/metabolismo , Fatores de Transcrição TFII/metabolismo , Animais , Divisão Celular/genética , Linhagem da Célula/genética , Quinases Ciclina-Dependentes/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína 2 de Resposta de Crescimento Precoce , Feminino , Marcação de Genes , Células Germinativas/citologia , Células Germinativas/crescimento & desenvolvimento , Células Germinativas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Bainha de Mielina/genética , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Fenótipo , Subunidades Proteicas/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Células de Schwann/patologia , Células de Schwann/ultraestrutura , Nervo Isquiático/crescimento & desenvolvimento , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Fator de Transcrição TFIIH , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição TFII/genética , Degeneração Walleriana/genética , Degeneração Walleriana/metabolismo , Degeneração Walleriana/patologia , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Inactivating germ-line mutations of LKB1 lead to Peutz-Jeghers syndrome (PJS). We have generated mice heterozygous for a targeted inactivating allele of Lkb1 and found that they develop severe gastrointestinal polyposis. In all cases, the polyps arising in the Lkb1+/- mice were found to be hamartomas that were histologically indistinguishable from polyps resected from PJS patients, indicating that Lkb1+/- mice model human PJS polyposis. No evidence for inactivation of the remaining wild-type Lkb1 allele in Lkb1+/- -associated polyps was observed. Moreover, polyps and other tissues in heterozygote animals exhibited reduced Lkb1 levels and activity, indicating that Lkb1 was haploinsufficient for tumor suppression. Analysis of the molecular mechanisms characterizing Lkb1+/- polyposis revealed that cyclooxygenase-2 (COX-2) was highly up-regulated in murine polyps concomitantly with activation of the extracellular signal-regulated kinases 1 and 2 (Erk1/2). Subsequent examination of a large series of human PJS polyps revealed that COX-2 was also highly up-regulated in the majority of these polyps. These findings thereby identify COX-2 as a potential target for chemoprevention in PJS patients.