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Exogenous overexpression of the metastasis suppressor gene Nm23-H1 reduces the metastatic potential of multiple types of cancer cells and suppresses in vitro tumor cell motility and invasion. Mutational analysis of Nm23-H1 revealed that substitution mutants P96S and S120G did not inhibit motility and invasion. To elucidate the molecular mechanism of Nm23-H1 motility suppression, expression microarray analysis of an MDA-MB-435 cancer cell line overexpressing wild-type Nm23-H1 was done and cross-compared with expression profiles from lines expressing the P96S and S120G mutants. Nine genes, MET, PTN, SMO, FZD1, L1CAM, MMP2, NETO2, CTGF, and EDG2, were down-regulated by wild-type but not by mutant Nm23-H1 expression. Reduced expression of these genes coincident with elevated Nm23-H1 expression was observed in human breast tumor cohorts, a panel of breast carcinoma cell lines, and hepatocellular carcinomas from control versus Nm23-M1 knockout mice. The functional significance of the down-regulated genes was assessed by transfection and in vitro motility assays. Only EDG2 overexpression significantly restored motility to Nm23-H1–suppressed cancer cells, enhancing motility by 60-fold in these cells. In addition, silencing EDG2 expression with small interfering RNA reduced the motile phenotype of metastatic breast cancer cells. These data suggest that Nm23-H1 suppresses metastasis, at least in part, through down-regulation of EDG2 expression. [Cancer Res 2007;67(15):7238–46]

Loss of NM23-H1 expression correlates with the degree of metastasis and with unfavorable clinical prognosis in several types of human carcinoma. However, the mechanistic basis for the metastasis suppressor function of NM23-H1 is obscure. We silenced NM23-H1 expression in human hepatoma and colon carcinoma cells and methodologically investigated effects on cell-cell adhesion, migration, invasion, and signaling linked to cancer progression. NM23-H1 silencing disrupted cell-cell adhesion mediated by E-cadherin, resulting in β-catenin nuclear translocation and T-cell factor/lymphoid-enhancing factor-1 transactivation. Further, NM23-H1 silencing promoted cellular scattering, motility, and extracellular matrix invasion by promoting invadopodia formation and upregulating several matrix metalloproteinases (MMP), including membrane type 1 MMP. In contrast, silencing the related NM23-H2 gene was ineffective at promoting invasion. NM23-H1 silencing activated proinvasive signaling pathways involving Rac1, mitogen-activated protein kinases, phosphatidylinositol 3-kinase (PI3K)/Akt, and src kinase. Conversely, NM23-H1 was dispensable for cancer cell proliferation in vitro and liver regeneration in NM23-M1 null mice, instead inducing cellular resistance to chemotherapeutic drugs in vitro. Analysis of NM23-H1 expression in clinical specimens revealed high expression in premalignant lesions (liver cirrhosis and colon adenoma) and the central body of primary liver or colon tumors, but downregulation at the invasive front of tumors. Our findings reveal that NM23-H1 is critical for control of cell-cell adhesion and cell migration at early stages of the invasive program in epithelial cancers, orchestrating a barrier against conversion of in situ carcinoma into invasive malignancy. Cancer Res; 70(19); 7710–22. ©2010 AACR.