Abstract 1371: Delineating the function of single VEGF isoforms using both proteomic and molecular approaches

Abstract

Abstract Vascular endothelial growth factor A (VEGF), a potent regulator of tumour angiogenesis, is differentially spliced to give rise to the predominant pro-angiogenic isoforms, VEGF121/120, 165/164 and 189/188 in human and mouse respectively. All VEGF isoforms bind to two tyrosine kinase receptors, VEGFR-1 and VEGFR-2, but elicit different signalling outcomes. However, this is further complicated by more recent work describing the existence of human anti-angiogenic VEGF isoforms (VEGFxxxb), which are thought to counteract pro-angiogenic isoforms, inferring that it is the balance between these two families of VEGF isoforms that determines tumour angiogenic outcome. We have shown previously that mouse fibrosarcoma cells expressing all VEGF isoforms (wt) or VEGF188 only, give rise to tumours which establish more slowly, exhibit higher rates of apoptosis, and exhibit more mature vasculature invested with pericytes, compared to VEGF164 or VEGF120 expressing tumours. Importantly this translated into resistance to the vascular disrupting agent (VDA) combretastatin A-4 phosphate (CA4P) (Cancer Res 2008: 68:2301). To address the hypothesis that differences in signalling pathways between VEGF isoforms contributes to the tumour vascular characteristics described above, we performed proteomic profiling of wt and VEGF isoform-specific tumour cell lines and solid tumour lysates using commercial angiogenesis and phospho-receptor tyrosine kinase arrays (R&D) as well as iTRAQ technology to identify differentially expressed proteins. In addition, using an RT-PCR based approach we determined whether or not the potential existence of mouse anti-angiogenic isoforms could be contributing to our data. Although able to infrequently detect mouse VEGFxxxb isoforms, we show that these must be PCR artefacts since they were detected in lysates derived from cell lines obtained from transgenic mice whose genetic construction precludes VEGFxxxb splicing from occurring. In contrast, protein profiling has revealed a number of VEGF isoform-specific differentially expressed proteins. In particular, we found that fibroblast growth factor (FGF-2), neuropilin 1 (NRP1) and platelet-derived growth factor receptor ≤ (PDGFRα) were up-regulated in VEGF188-specific tumour cells, whilst Neuropilin 2 (NRP2) was down-regulated in the same tumour extracts relative to VEGF164 and VEGF120. Our data infer VEGF188-specific differentially expressed candidate proteins in tumours, which may impact on the efficacy of VDA treatment therapies. Further experiments aim to determine whether manipulating the expression of these candidate proteins in VEGF188-specific tumours reverses their resistance to CA4P, thus identifying additional targets for anti-angiogenic therapy approaches. This work, was funded by a Program Grant from Cancer Research UK Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1371. doi:1538-7445.AM2012-1371