P2-05-06: Role of Repulsive Guidance Molecule b (RGMb) in HGF Mediated Angiogenesis.

Abstract

Abstract Introduction: Hepatocyte Growth Factor (HGF) has been widely documented as playing a key role in enhancing the aggressive nature of cancer through its ability to promote cellular processes such as migration, invasion and angiogenesis. Development of a blood supply is vital to advanced tumour growth and increased metastatic potential. In the current study we identified RGMb (DRAGON), a member of the Repulsive Guidance Molecule family, as being upregulated by HGF and assessed its potential to contribute to HGF mediated proangiogenic traits. Methods: Expression of RGMb was identified as being upregulated in human endothelial HECV cells following 4 hour treatment with HGF (40ng/ml) using micro array analysis and Q-PCR. Subsequently, the expression of RGMb was targeted through the transfection of HECV cells with a plasmid containing a ribozyme transgene specifically targeted to RGMb. The role of RGMb in HGF mediated cellular migration and tubule formation in vitro was examined. Results: Significant increases in RGMb expression in HECV cells were observed on the mirco-array following 4 hour treatment with HGF (P = 0.004) and this trend was also identified using Q-PCR. Transfection with the ribozyme transgene brought about substantial reductions in RGMb expression at both transcript and protein levels as assessed using RT-PCR, QPCR and Western blot analysis. Knockout of RGMb brought about a significant increase in migration rates, compared to HECV cells transfected with a closed pEF6 plasmid only (HECVpEF6), following a 90minute period (p = 0.034 at 90 minute time point vs pEF6 control). Treatment of HGF enhanced migration rates of HECV control cells with significant differences between untreated and HGF treated pEF6 cells obvious following 60 minutes (p < 0.05 at 60 and 75 minutes, p = 0.002 at 90 minutes). In contrast to this, HECV cells transfected with the RGMb ribozyme transgene (HECVRGMbKO) were unaffected by HGF treatment, with no significant differences observed between treated or untreated HECVRGMbKO cells at any time points. A similar trend was observed in the angiogenic tubule formation assay, where treatment of HECVpEF6 cells with HGF could significantly enhance the levels of tubules formed (mean tubule perimeter, untreated 8087+/− 632 vs HGF treated 13131+/− 988, p = 0.001). Treatment of HECVRGMbKO cells with HGF did not significantly enhance levels of tubule formation (mean tubule perimeter, untreated 7523+/−2458 vs HGF treated 11050+/−1512, p = 0.21). Conclusions: Targeting of RGMb in endothelial cells appears to reduce their sensitivity to the promotional effect of HGF on cell migration and tubule formation, important traits in the angiogenic cascade. Our data suggests that RGMb may be one molecule involved in the process through which HGF enhances angiogenic potential and targeting this molecule may be a useful strategy in a number of cancer types to interfere with HGF promoted angiogenesis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-06.