RESUMEN
The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A165b. Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy.
Asunto(s)
Empalme Alternativo , Bevacizumab , Neoplasias del Colon/metabolismo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Neovascularización Patológica/metabolismo , Proteínas de Unión a Poli(A)/biosíntesis , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Animales , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Células HEK293 , Células HeLa , Xenoinjertos , Humanos , Ratones Desnudos , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Proteínas de Unión a Poli(A)/genética , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Antígeno Intracelular 1 de las Células T , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Tumour growth is dependent on angiogenesis, the key mediator of which is vascular endothelial growth factor-A (VEGF-A). VEGF-A exists as two families of alternatively spliced isoforms - pro-angiogenic VEGF(xxx) generated by proximal, and anti-angiogenic VEGF(xxx)b by distal splicing of exon 8. VEGF(165)b inhibits angiogenesis and is downregulated in tumours. Here, we show for the first time that administration of recombinant human VEGF(165)b inhibits colon carcinoma tumour growth and tumour vessel density in nude mice, with a terminal plasma half-life of 6.2h and directly inhibited angiogenic parameters (endothelial sprouting, orientation and structure formation) in vitro. Intravenous injection of (125)I-VEGF(165)b demonstrated significant tumour uptake lasting at least 24h. No adverse effects on liver function or haemodynamics were observed. These results indicate that injected VEGF(165)b was taken up into the tumour as an effective anti-angiogenic cancer therapy, and provide proof of principle for the development of this anti-angiogenic growth factor splice isoform as a novel cancer therapy.