Convergence of cytokines and hypoxia on colorectal cancer angiogenesis

Abstract

Over-expression of Epidermal Growth Factor (EGF) and hypoxia are features of colorectal cancer (CRC). Such observations have helped develop novel therapies such as cetuximab, a recombinant monoclonal EGF receptor antibody antagonist. Recent studies uncovered a pro-angiogenic role for EGF but it is unclear whether this is mediated by Vascular Endothelial Growth Factor (VEGF) in CRC. Moreover, it is unknown whether hypoxia synergises with EGF to accentuate their individual pro-angiogenic effects. The aim of the study was to investigate the angiogenic effect promoted by EGF in combination with hypoxia in CRC. CRC cell lines Colo 201 and Caco-2 were stimulated with EGF and/or exposed to 1% O2 or dimethyloxalylglycine (DMOG), a hypoxia-mimetic. Gene and protein expression of hypoxia inducible factor (HIF)-1α, HIF-2α and VEGF were assessed. Gene signatures of CRC cells following EGF, hypoxia and DMOG stimulations were assessed by angiogenesis PCR arrays. The functional significance was assessed by human umbilical vein endothelial cell (HUVEC) migration assay co-cultured with Caco-2. HIF-1α and VEGF protein were upregulated in Caco-2 with EGF and DMOG stimulation. However, Colo 201 demonstrated an increase in VEGF mRNA and protein only under hypoxia but not with EGF. The discrepancy in responses to EGF was attributed to differences in ERK kinase signalling between the two cell types. PCR angiogenesis arrays also identified angiopoietin-like 4 and ephrin A3 as novel genes which were upregulated by HIF overexpression, and stabilin-1, a gene specifically downregulated by EGFR-activation. Functional relevance was confirmed by HUVEC migration assay, demonstrating similar numbers of migrated HUVEC cell numbers in co-cultures with Caco-2 pre-stimulated with EGF and DMOG compared to unstimulated controls. In conclusion, EGF receptor activation plays a role in CRC angiogenesis by increasing VEGF and HIF-1α protein expression, although such effect is dependent upon functional ERK MAPK signalling. However, hypoxia and DMOG consistently exerted an over-riding stimulus in significantly promoting expression of angiogenic mediators to drive endothelial migration and angiogenesis in CRC.