RESUMEN
PURPOSE: The activator protein (AP)-2alpha transcription factor plays a crucial role in the progression of several human tumors, including malignant melanoma, prostate, and breast cancer. Loss of AP-2alpha results in deregulation of several genes with AP-2alpha binding motifs such as E-cadherin, p21WAF1, MMP-2, MCAM/MUC18, VEGF, and c-KIT. The purpose of our study was to determine AP-2alpha expression distribution among grades of gliomas and any possible effect on prognosis. EXPERIMENTAL DESIGN: A tissue microarray was assembled from all surgical glioma cases with available tissue samples at M.D. Anderson Cancer Center since 1986 to include 72 glioblastomas, 49 anaplastic astrocytomas, 9 low-grade astrocytoma, 37 oligodendrogliomas, 37 anaplastic oligodendrogliomas, 15 mixed oligoastrocytomas, 20 anaplastic mixed oligoastrocytomas, and 7 gliosarcomas. The microarray included normal brain tissue, and AP-2alpha expression was determined by immunohistochemistry. RESULTS: AP-2alpha expression was lost on 99% (P < 0.001) and 98% (P < 0.001) of glioblastomas and anaplastic astrocytomas, respectively, compared with grade 2 astrocytomas and normal brain, all of which (100%) maintained expression of AP-2alpha. The loss of AP-2alpha was a negative prognostic indicator within the overall category of gliomas by univariate analysis (rate ratio, 4.30; 95% confidence interval, 2.60-7.10; P < 0.001). However, there was no significant effect of loss of AP-2alpha expression on survival observed after adjustment for patient age, Karnofsky Performance Scale score, tumor grade, and extent of resection (rate ratio, 1.2; 95% confidence interval, 0.6-2.2; P = 0.6). CONCLUSIONS: AP-2alpha expression correlates inversely with glioma grade, suggesting a direct role in glioma tumorigenicity, possibly through subsequent deregulation of target genes. Of all the previously characterized markers of progression, the loss of AP-2alpha would be the most common (96.2%) molecular marker as an astrocytic tumor evolves from grade 2 to 3.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/fisiología , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/fisiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencias de Aminoácidos , Antígenos CD/biosíntesis , Astrocitoma/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/mortalidad , Antígeno CD146 , Proteínas de Ciclo Celular/biosíntesis , Línea Celular Tumoral , Niño , Preescolar , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Glioma/diagnóstico , Glioma/mortalidad , Humanos , Inmunohistoquímica , Metaloproteinasa 2 de la Matriz/biosíntesis , Persona de Mediana Edad , Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Oligodendroglioma/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Proto-Oncogénicas c-kit/biosíntesis , Factores de Tiempo , Factor de Transcripción AP-2 , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
Activator protein-2alpha (AP-2) is a transcription factor that regulates proliferation and differentiation in mammalian cells. We have shown previously that although AP-2 is expressed highly in normal prostatic epithelium, its expression is lost in high-grade prostatic intraepithelial neoplasia and prostate cancer, suggesting that loss of AP-2 plays a role in prostate cancer development. We demonstrate that forced AP-2 expression in the prostate cancer cell line LNCaP-LN3 (AP-2 negative) inhibited dramatically tumor incidence in nude mice. To identify the genes that might have been responsible for this effect, we used microchip expression array. We found several genes known to be involved in malignancy were deregulated, including the vascular endothelial growth factor (VEGF) gene. Because VEGF was down-regulated by 14.7-fold in the AP-2-transfected cells and because it is a major angiogenic factor in prostate cancer development and progression, we chose to examine the AP-2-VEGF interaction. Our evidence suggests that AP-2 repressed transcriptionally the VEGF promoter by competing with the transcriptional activator Sp3. Loss of AP-2 in prostate cancer cells reduced the AP-2:Sp3 ratio and activated VEGF expression. AP-2 acts as a tumor-suppressor gene in prostate cancer. Elucidating the molecular events resulting from loss of AP-2 in the prostate epithelium has implications for the understanding and prevention of the onset of prostate cancer.
Asunto(s)
Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Endotelio Vascular/patología , Neovascularización Patológica/prevención & control , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/patología , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Cromatina/genética , Cromatina/aislamiento & purificación , Clonación Molecular , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Desnudos , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/metabolismo , Factor de Transcripción AP-2 , Factores de Transcripción/genética , Transfección , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
PURPOSE: In recent years, the incidence of cutaneous melanoma has increased more than that of any other cancer. Dacarbazine is considered the gold standard for treatment, having a response rate of 15% to 20%, but most responses are not sustained. Previously, we have shown that short exposure of primary cutaneous melanoma cells to dacarbazine resulted in the upregulation of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF). The purpose of the present study was to determine how long-term exposure of melanoma cells to dacarbazine would affect their tumorigenic and metastatic potential in vivo. MATERIALS AND METHODS: The primary cutaneous melanoma cell lines SB2 and MeWo were repeatedly exposed in vitro to increasing concentrations of dacarbazine, and dacarbazine-resistant cell lines SB2-D and MeWo-D were selected and examined for their ability to grow and metastasize in nude mice. RESULTS: The dacarbazine-resistant cell lines SB2-D and MeWo-D exhibited increased tumor growth and metastatic behavior in vivo. This increase could be explained by the activation of RAF, MEK, and ERK, which led to the upregulation of IL-8 and VEGF. More IL-8, VEGF, matrix metalloproteinase-2 (MMP-2), and microvessel density (CD-31) were found in tumors produced by SB2-D and MeWo-D in vivo than in those produced by their parental counterparts. No mutations were observed in BRAF. CONCLUSION: Our results have significant clinical implications. Treatment of melanoma patients with dacarbazine could select for a more aggressive melanoma phenotype. We propose that combination treatment with anti-VEGF/IL-8 or MEK inhibitors may potentiate the therapeutic effects of dacarbazine.
Asunto(s)
Antineoplásicos Alquilantes/efectos adversos , Dacarbazina/efectos adversos , Resistencia a Antineoplásicos/efectos de los fármacos , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-8/metabolismo , Melanoma/metabolismo , Melanoma/secundario , Ratones , Ratones Desnudos , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The incidence of cutaneous malignant melanoma in the United States has increased more than any other cancer in recent years. Chemotherapy for metastatic melanoma is disappointing, there being anecdotal cases of complete remission. Dacarbazine (DTIC) is considered the gold standard for treatment, having a response rate of 15-20%, but most responses are not sustained. The mechanisms for the increased chemotherapeutic resistance of melanoma are unclear. The objective of this study was to determine the mechanisms by which melanoma cells escape the cytotoxic effect of DTIC. Here, we show that DTIC induced interleukin (IL)-8 and vascular endothelial growth factor (VEGF) protein overexpression and secretion via transcriptional up-regulation in the two melanoma cell lines SB-2 and MeWo. Luciferase activity driven by the IL-8 and VEGF promoters was up-regulated by 1.5-2- and 1.6-3.5-fold, respectively, in the SB2 and MeWo melanoma cell lines. The mitogen-activated protein kinase signal transduction pathway seemed to regulate at least partially the activation of IL-8, whereas it was not involved in VEGF promoter regulation. Electrophoretic mobility shift analysis analyses have revealed an increase in binding activity of activator protein 1 (c-Jun) and nuclear factor-kappaB after DTIC treatment for both melanoma cell lines. Metastatic melanoma cell lines secreting high levels of IL-8 and VEGF were more resistant to DTIC than early primary melanomas secreting low levels of the cytokines. In addition, transfection of the primary cutaneous melanoma SB-2 cells with the IL-8 gene rendered them resistant to the cytotoxic effect of the drug, whereas the addition of IL-8-neutralizing antibody to metastatic melanoma cells lowered their sensitivity to DTIC. Taken together, our data demonstrate that DTIC can cause melanoma cells to secrete IL-8 and VEGF, which might render them resistant to the cytotoxic effects of the drug. We propose that combination treatment with anti-VEGF/IL-8 agents may potentiate the therapeutic effects of DTIC.
Asunto(s)
Dacarbazina/farmacología , Interleucina-8/metabolismo , Melanoma/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Humanos , Interleucina-8/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Transfección , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Elevated expression of pro-angiogenic cytokines is associated with aggressive tumour growth and decreased survival of patients with breast cancer. In general, the breast cancer cell lines with high vascular endothelial growth factor (VEGF) expression also express high levels of interleukin-8 (IL-8). The consequence of inhibiting mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K), both implicated in regulation of these cytokines, was examined in four cell lines. Treatment with the mitogen-activated protein kinase/extracellular signal-related kinase (MEK) inhibitor U0126 reduced expression of VEGF and IL-8 in MDA-MB-231 cells, partially inhibited expression in MDA-MB-468 and Hs578T cells, with minimal effects in GI101A cells. Treatment with LY294002 reduced cytokine expression in GI101A and MDA-MB-468 cells, with partial reduction in Hs578T and less effect in MDA-MB-231 cells. Thus, IL-8 and VEGF were regulated by different signalling pathways in different cell lines; this suggests that inhibition of the dominantly active pathway can downregulate both angiogenic cytokines. Recognising which signalling pathway is active may identify targets for anti-angiogenic therapy of breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Interleucina-8/metabolismo , Proteínas de Neoplasias/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Comunicación Celular , Línea Celular Tumoral , ADN/metabolismo , Ensayo de Inmunoadsorción Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , FN-kappa B/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Reacción en Cadena de la Polimerasa/métodos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Factor de Transcripción AP-1/metabolismo , TransfecciónRESUMEN
Increasing evidence implicates the protease-activated receptor-1 (PAR-1) as a contributor to tumor invasion and metastasis of human melanoma. Here we demonstrate that the metastatic potential of human melanoma cells correlates with overexpression of PAR-1. We also provide evidence that an inverse correlation exists between the expression of activator protein-2alpha (AP-2) and the expression of PAR-1 in human melanoma cells. Reexpression of AP-2 in WM266-4 melanoma cells, which are AP-2-negative, resulted in decreased mRNA and protein expression of PAR-1. The promoter of the PAR-1 gene contains multiple putative consensus elements for the transcription factors AP-2 and specificity protein 1 (Sp1). Chromatin immunoprecipitation analysis of the PAR-1 promoter regions bp -365 to -329 (complex 1) and bp -206 to -180 (complex 2) demonstrated that Sp1 was predominantly bound to the PAR-1 promoter in metastatic cells, whereas AP-2 was bound to the PAR-1 promoter in nonmetastatic cells. In vitro analysis of complex 1 demonstrated that AP-2 and Sp1 bound to this region in a mutually exclusive manner. Transfection experiments with full-length and progressive deletions of the PAR-1 promoter luciferase constructs demonstrated that metastatic melanoma cells had increased PAR-1 promoter activity compared with low and nonmetastatic melanoma cells. Our data show that exogenous AP-2 expression decreased promoter activity, whereas transient expression of Sp1 further increased expression of the reporter gene. Mutational analysis of complex 1 within PAR-1 luciferase constructs further demonstrated that the regulation of PAR-1 was mediated through interactions with AP-2 and Sp1. Our data suggest that loss of AP-2 in metastatic cells alters the AP-2/Sp1 ratio, resulting in overexpression of PAR-1. Taken together, our results provide strong evidence that loss of AP-2 correlates with overexpression of PAR-1, which in turn contributes to the acquisition of the malignant phenotype of human melanoma.