Induction of epididymis specific G-protein coupled receptor-64 (GPR64) in Ewing Tumors supports invasiveness and metastatic spread

Metastatic spread in Ewing Tumors (ET) is hematogenous and malignant features have been shown to correlate with hypoxia and angiogenesis. We identified several Ewing tumor specific genes (Staege MS et al. Cancer Res. 2004;64:8213-21). Microarray analysis confirmed an endothelial signature of this tumor and revealed the G-protein coupled receptor-64 (GPR64), an orphan receptor with normal expression restricted to human epididymis, to be highly induced in ET. Down-regulation of GPR64 in ET lines by RNA interference did not reduce their proliferative capacity in vitro as measured by plastic adherence dependent proliferation or contact independent growth in colony forming assays. Of interest inhibition of GPR64 expression in ET cell lines resulted in impaired endothelial differentiation in tube formation assays. Furthermore, GPR64 suppression substantially inhibited tumor growth and metastatic spread in immunodeficient Rag2-/-gammaC-/- mice. Microarray analysis of ET after GPR64 knock down revealed a GPR64-mediated induction of VEGF receptor 1 ligand placental growth factor (PGF) in ET. PGF itself was induced by EWS-FLI1 in mesenchymal stem cells. Repression of PGF expression in ET cell lines resulted in a similar phenotype as observed after GPR64 knockdown. GPR64 as well as PGF knock down correlated with a reduced proteolytic activity of Matrix Metalloproteinase MMP1 and invasiveness in vitro. MMP1 specific knock down resulted in the abrogation of metastasis of ET in Rag2-/-gammaC-/- mice. We conclude, that GPR64 and subsequent PGF up-regulation in ET orchestrate and promote endothelial invasiveness and metastatic spread and play a pivotal role in the pathogenesis and aggressiveness of this tumor.

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