H‐Ras increases release of sphingosine resulting in down‐regulation of TSP‐1 in non‐transformed cells

Summary Tumour progression is continuously driven by a sequence of genetic events. The presence of mutant or activated Ras proteins represents an interesting paradigm for the investigation of oncogene‐dependent induction of tumour angiogenesis. These genes are widely distributed in human cancers. Previously we have shown that cells harbouring mutant H‐Ras release soluble unidentified factor(s) associated with lowered expression of an angiogenesis inhibitor – Thrombospondin‐1 – (TSP‐1) in adjacent normal tissue. In this study, we have addressed the question as to whether or not introduction of the H‐ras oncogene leads to increased production of sphingosine. To assess the amount of sphingosine in conditioned media, we developed a technique based on sphingolipid isolation and GC‐MSMS detection of specific silylated sphingosine derivatives. Cells harbouring mutant H‐Ras, release significant amounts of sphingosine in contrast to normal isogenic cells or premalignant cells. Increased concentration of sphingosine in conditioned media was correlated with their ability to down‐regulate the expression of TSP‐1. Moreover, medium collected in the presence of U0126, an inhibitor of MAPK kinase (MEK), contained undetectable amounts of sphingosine and had no ability to down‐regulate TSP‐1 expression. Overall, our studies suggest a H‐Ras‐dependent mechanism of changing the equilibrium of angiogenic factors in favour of induction of angiogenesis, where a central role is played by sphingosine, a low molecular entity. This represents an example of how a mechanism of translating genetic changes within transformed cells could be amplified into a much larger effect involving the tumour parenchyma and stroma, and this could greatly in turn accelerate local tumour growth and metastasis.