RESUMEN
Early in cancer development, tumour cells express vascular endothelial growth factor (VEGF), a secreted molecule that is important in all stages of angiogenesis, an essential process that provides nutrients and oxygen to the nascent tumor and thereby enhances tumor-cell survival and facilitates growth. Survivin, another protein involved in angiogenesis, is strongly expressed in most human cancers, where it promotes tumor survival by reducing apoptosis as well as favoring endothelial cell proliferation and migration. The mechanisms by which cancer cells induce VEGF expression and angiogenesis upon survivin up-regulation remain to be fully established. Since the PI3K/Akt signalling and ß-catenin-Tcf/Lef dependent transcription have been implicated in the expression of many cancer-related genes, including survivin and VEGF, we evaluated whether survivin may favor VEGF expression, release from tumor cells and induction of angiogenesis in a PI3K/Akt-ß-catenin-Tcf/Lef-dependent manner. Here, we provide evidence linking survivin expression in tumor cells to increased ß-catenin protein levels, ß-catenin-Tcf/Lef transcriptional activity and expression of several target genes of this pathway, including survivin and VEGF, which accumulates in the culture medium. Alternatively, survivin downregulation reduced ß-catenin protein levels and ß-catenin-Tcf/Lef transcriptional activity. Also, using inhibitors of PI3K and the expression of dominant negative Akt, we show that survivin acts upstream in an amplification loop to promote VEGF expression. Moreover, survivin knock-down in B16F10 murine melanoma cells diminished the number of blood vessels and reduced VEGF expression in tumors formed in C57BL/6 mice. Finally, in the chick chorioallantoid membrane assay, survivin expression in tumor cells enhanced VEGF liberation and blood vessel formation. Importantly, the presence of neutralizing anti-VEGF antibodies precluded survivin-enhanced angiogenesis in this assay. These findings provide evidence for the existance of a posititve feedback loop connecting survivin expression in tumor cells to PI3K/Akt enhanced ß-catenin-Tcf/Lef-dependent transcription followed by secretion of VEGF and angiogenesis.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , Melanoma Experimental/irrigación sanguínea , Neovascularización Patológica/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , beta Catenina/metabolismo , Animales , Pollos , Membrana Corioalantoides/metabolismo , Regulación hacia Abajo , Femenino , Células HEK293 , Humanos , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Neovascularización Patológica/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Survivin , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Melanomas are highly lethal skin tumours that are frequently treated by surgical resection. However, the efficacy of such procedures is often limited by tumour recurrence and metastasis. Caveolin-1 (CAV1) has been attributed roles as a tumour suppressor, although in late-stage tumours, its presence is associated with enhanced metastasis. The expression of this protein in human melanoma development and particularly how the presence of CAV1 affects metastasis after surgery has not been defined. CAV1 expression in human melanocytes and melanomas increases with disease progression and is highest in metastatic melanomas. The effect of increased CAV1 expression can then be evaluated using B16F10 murine melanoma cells injected into syngenic immunocompetent C57BL/6 mice or human A375 melanoma cells injected into immunodeficient B6Rag1-/- mice. Augmented CAV1 expression suppresses tumour formation upon a subcutaneous injection, but enhances lung metastasis of cells injected into the tail vein in both models. A procedure was initially developed using B16F10 melanoma cells in C57BL/6 mice to mimic better the situation in patients undergoing surgery. Subcutaneous tumours of a defined size were removed surgically and local tumour recurrence and lung metastasis were evaluated after another 14 days. In this postsurgery setting, CAV1 presence in B16F10 melanomas favoured metastasis to the lung, although tumour suppression at the initial site was still evident. Similar results were obtained when evaluating A375 cells in B6Rag1-/- mice. These results implicate CAV1 expression in melanomas as a marker of poor prognosis for patients undergoing surgery as CAV1 expression promotes experimental lung metastasis in two different preclinical models.
Asunto(s)
Caveolina 1/biosíntesis , Melanoma Experimental/metabolismo , Melanoma Experimental/cirugía , Neoplasias Cutáneas/cirugía , Animales , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/secundario , Melanoma/metabolismo , Melanoma/patología , Melanoma/cirugía , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
Augmented expression of cyclooxygenase-2 (COX-2) and enhanced production of prostaglandin E(2) (PGE(2)) are associated with increased tumor cell survival and malignancy. Caveolin-1 is a scaffold protein that has been proposed to function as a tumor suppressor in human cancer cells, although mechanisms underlying this ability remain controversial. Intriguingly, the possibility that caveolin-1 regulates the expression of COX-2 has not been explored. Here we show that augmented caveolin-1 expression in cells with low basal levels of this protein, such as human colon cancer (HT29, DLD-1), breast cancer (ZR75), and embryonic kidney (HEK293T) cells reduced COX-2 mRNA and protein levels and beta-catenin-Tcf/Lef and COX-2 gene reporter activity, as well as the production of PGE(2) and cell proliferation. Moreover, COX-2 overexpression or PGE(2) supplementation increased levels of the inhibitor of apoptosis protein survivin by a transcriptional mechanism, as determined by PCR analysis, survivin gene reporter assays and Western blotting. Furthermore, addition of PGE(2) to the medium prevented effects attributed to caveolin-1-mediated inhibition of beta-catenin-Tcf/Lef-dependent transcription. Finally, PGE(2) reduced the coimmunoprecipitation of caveolin-1 with beta-catenin and their colocalization at the plasma membrane. Thus, by reducing COX-2 expression, caveolin-1 interrupts a feedback amplification loop involving PGE(2)-induced signaling events linked to beta-catenin/Tcf/Lef-dependent transcription of tumor survival genes including cox-2 itself and survivin.