Nm23-H1 mediates metastasis suppression through the receptors Edg2 and c-Met in MDA-MB-435 cells

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Diminished expression of the metastasis suppressor Nm23-H1 significantly correlates with tumor aggressiveness in breast cancer. exogenous overexpression of Nm23-H1 in the metastatic breast carcinoma cell line MDA-MB-435 reduces the metastatic capacity of these cells as determined by in vivo mouse injection experiments and in vitro assays of cell motility and soft agar colonization. Mutational analysis of Nm23-H1 has revealed that substitution mutants P96S and S120G are unable to inhibit cell motility and colonization. To elucidate the molecular mechanism by which Nm23-H1 mediates metastasis suppression, we performed microarray expression analysis using a comprehensive oligonucleotide array and mRNA extracted from MDA-MB-435 cells stably overexpressing wild-type Nm23-H1 and the P96S and S120G mutants as compared to a control-transfected cell line. The data revealed that 84 genes are down-regulated upon overexpression of wild-type Nm23-H1 and 103 genes are up-regulated (p<0.05, log₁₀(median ratio) > 0.23). Approximately 70 genes are specifically regulated by wild-type Nm23-H1, but not by the mutants. Presumably, these genes are critical to metastatic suppression and they include the down-regulated genes c-MET, PTN, L1CAM, FZD1 and EDG2 and the up-regulated genes SPP1 and GA17. Using quantitative RT-PCR and immunoblot analysis, we have confirmed the observed patterns of microarray expression in approximately 90% of the genes tested. We have determined the relevance of the relationship between Nm23-H1 and its regulated genes in clinical breast cancer specimens through analysis of published microarray data of two tumor cohorts. Within these datasets, a significant correlation has been established between expression of the Nm23-H1 gene and expression of twenty genes that it regulates, including c-MET, PTN, FZD1 and EDG2 (p<0.05). Finally, we have established a functional connection between Nm23-H1 regulated genes and suppression of in vitro hallmarks of metastasis. Of nine genes tested, overexpression of EDG2 alone restored the motility phenotype to Nm23-H1-expressing MDA-MB-435 cells by 60- to 200-fold. EDG2 encodes for a G-protein coupled receptor that binds to LPA, the lipid mediator that is a significant component of serum and regulates cell proliferation, differentiation and motility. Transfection of c-MET, the gene coding for the hepatocyte growth factor receptor, into Nm23-H1-expressing MDA-MB-435 cells enhanced the capacity of these cells for anchorage-independent growth in soft agar by 50%. Therefore, Nm23-H1 suppresses metastasis, at least in part, through the coordinated regulation of the EDG2 and c-MET genes.