PRL-3 and PRL-1 Promote Cell Migration, Invasion, and Metastasis1,2

We demonstrate here that Chinese hamster ovary cells stably express- ing PRL-3, a Mr 20,000 prenylated protein tyrosine phosphatase, or its relative, PRL-1, exhibit enhanced motility and invasive activity. A cata- lytically inactive PRL-3 mutant has significantly reduced migration- promoting activity. We observe that PRL-3 is associated with diverse membrane structures involved in cell movement. Furthermore, we show that PRL-3- and -1-expressing cells, but not control cells, induce meta- static tumor formation in mice. Thus, our results deliver the first evidence for a causative role of PRL-3 and -1 in promoting cell motility, invasion activity, and metastasis. for elevated PRL-3 transcripts in the other metastases. This study suggests the possibility that an excess of PRL-3 phosphatase is a key alteration contributing to the acquisition of metastatic properties of the tumor cells, but cell biological and mechanistic evidence supporting such a role is lacking. In this study we provide evidence to support a causal role of PRL-3 and -1 in tumor metastasis. Materials and Methods Generation of Stable CHO Cell Lines Expressing Myc-PRL-1, -3, and -Gal. To introduce the Myc epitope (10 amino acid residues) at the NH2 termini of PRL-1 and -3, a PCR-based approach was used. Forward primer A incorporating the Myc epitope (italicized; 5-gc gaattc acc atg gag cag aag ctg atc tcc gag gag gac ctcgct cga atg aac cgc cct gct c-3) and reverse primer B (5-gt ggatcc tta ttg aat aca aca gtt g-3) were used to amplify PRL-1 cDNA. Forward primer E incorporating the Myc epitope (5-gc gaattc acc atg gag cag aag ctg atc tcc gag gag gac ctc gcc cgc atg aac cgg cct gcg cct g-3) and reverse primer F (5-ct ggatcc cta cat gac gca gca tct ggt c-3) were used to amplify PRL-3 cDNA. The PCR fragments were digested with EcoRI and BamHI, and inserted into the inducible expression vector pStar (7). CHO-K1 cells (American Type Culture Collection, Manassas, VA) were used to gen- erate CHO cell lines stably expressing Myc-PRL-1 (clone 9), Myc-PRL-3 (clone 36), or -gal (clone 8 and 13), as described previously (3, 7). Briefly, the cells were transfected with the above pSTAR-Myc-PRL-1, Myc-PRL-3, or -gal plasmids, and cultured in RPMI 1640 supplemented with 10% FBS and selected in 1 mg/ml of neomycin. The c-Myc antibody (9E10) was from Santa Cruz Biotechnology (Santa Cruz, CA).