Nm23-H1 suppresses tumor cell motility by downregulating the LPA receptor

Exogenous overexpression of the metastasis suppressor gene Nm23-H1 reduces the metastatic potential of multiple types of cancer cells. In addition, Nm23-H1 expression suppresses in vitro tumor cell motility and invasion, important correlates of metastasis. The molecular mechanism by which Nm23-H1 suppresses metastasis, motility and invasion has yet to be defined. However, mutational analysis of Nm23-H1 has revealed that substitution mutant P96S and S120G are unable to inhibit cell motility. To elucidate the molecular mechanism of Nm23-H1 motility suppression, we performed expression microarray analysis of an MDA-MB- 435 carcinoma cell line overexpressing wild-type Nm23-H1 and cross-compared the expression profile to lines expressing 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 (p<0.05). 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 and Nm23-M1 knockout mice. The functional significance of down regulated genes was assessed by transfection and in vitro motility assays. Only EDG2 overexpression significantly restored motility to Nm23-H1- suppressed cancer cells and it enhanced motility by 60-fold in these cells. In addition, silencing EDG2 expression with siRNA, reduced the motile phenotype of metastatic breast cancer cells. These data suggest that Nm23-H1 suppresses metastasis, at least in part, through downregulation of EDG2 expression and that compounds directed toward EDG2 may be useful for treating aggressive, low Nm23-H1-expressing breast tumors. Keywords: Human MDA-MB-435 Breast cancer derived cell lines Total RNA was extracted from confluent MDA-MB- 435-carcinoma cell lines H1-177 overexpressing wild type Nm23-H1, S-22 (P96S mutant) and I-205 (S120G mutant). The RNA was purified using the Trizol reagent from Invitrogen, Inc., following the manufacturer’s suggested protocol. Triplicate sample hybridizations were done for comparing H1-177 to C-100 (including one dye swap), Four replicates hybridizations were done for comparing H1-177 to S-22 (including two dye swap) and four replicates hybridizations were done for comparing H1-177 to I-205 (including two dye swap). The raw data for the dye swap experiment was reversed so that the data match the other two replicates.