Abstract LB-170: Genotoxic stress-dependent, p53-directed post-transcriptional controls of the VEGFR-1, FLT1 gene

Abstract

Abstract We recently established that a C&amp;gt;T SNP in the promoter of the VEGFR-1 gene, commonly known as FLT1, generates a half-site p53 response element (RE-T) that results in p53 responsiveness of the promoter. It was also shown that p53 is required but not sufficient for FLT1 transactivation and that there is cooperative interaction with ligand-bound Estrogen Receptors (ERs) via two ER half-site (EREs) located near the RE-T. ER levels, specific ligands and genotoxic stresses were shown to influence the coordinated regulation of the FLT1-T promoter. We have now asked whether activation of p53 by genotoxic stress could also regulate FLT1 at post-transcriptional levels. The FLT1 transcript can undergo alternative splicing resulting either in a membrane-bound- (mFLT1) or in a truncated soluble-form (sFLT1) that appears to act as decoy receptor, where a portion of intron 13 is retained in the mRNA introducing a premature STOP codon and an alternative 3′UTR. We measured by qPCR the expression levels of mFLT1 and sFLT1 transcripts in cell lines differing for the promoter SNP and p53 status: HCT116 (C/T, p53 wt+/+or p53 null−/−), GIMEN (C/T, p53 wt), MCF7 (C/C, p53 wt). Surprisingly, we observed that the relative abundance of mFLT1 and sFLT1 was differentially affected by doxorubicin treatment (doxo) in that mFLT1 was up-regulated in C/T heterozygous p53 wt cells, as expected, while sFLT1 levels did not change or were even reduced, depending on the cell line. Notably, ectopic expression of p53 in HCT116−/− led to the same specific increase in mFLT1/sFLT1 expression ratio. On the contrary, treatment with 5FU, while effective at activating p53, led to a selective strong upregulation of sFLT1. This effect was seen also in the MCF7 C/C cell line, but appeared to be dependent on the presence of p53 wt, based on results in HCT116−/−. As the mFLT1 and sFLT1 transcripts contain different 3′UTRs, we investigated using luciferase-based reporter vectors whether these regulatory sequences could underlie the differential response to p53 activation. Results indicated that doxo treatment in HCT116+/+ selectively increased mFLT1–3′UTR stability. 5FU had no differential impact in this assay. We also developed an FLT1 minigene containing exon 13, exon 14 and part of the intron 13 as intervening sequence in a dual luciferase vector. Interestingly, in HCT116+/+ cells transfected with the minigene treatment with 5FU but not doxo led to a shift in the splicing pattern toward sFLT1. Treatment with Nutlin, a molecule disrupting p53-MDM2 interaction, has a similar impact as 5FU. The effect of genotoxic stress-dependent, p53-directed changes in FLT1 transcripts' balance on FLT1-induced signalling is currently being evaluated, using the specific ligand PlGF. Collectively our emerging results are revealing that, in response to specific stimuli p53 can regulate not only FLT1 transcription, but also its mRNA maturation and stability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-170. doi:10.1158/1538-7445.AM2011-LB-170</jats:p