To further examine the prometastasis role of microRNA-200c, we also enhanced its function in the melanoma cell line B16F1 that has low metastatic propensity. Similar to our observations in the L1-R2-435-GFP xenograft model, BYL719 treatment of B16F1 cells with microRNA-200c mimics resulted in significantly more macroscopic lung metastases than the control mimics-treated cells in a syngeneic mouse model. The average number of surface lung metastases per mouse was 2.8 versus 20.3 at 2 weeks for controls and microRNA-200c mimics respectively. These results demonstrate significant increases in lung colonization efficiency due to enhancement of microRNA- 200c function. To determine the specificity of microRNA-200c in mediating the observed phenotype switch, we examined messenger RNA expression of Zeb1 and Zeb2 by Taqman RT-PCR in tail vein injected L2-R2 cells that were treated with microRNA- 200c mimics. These two genes are validated microRNA 200c targets. In microRNA-200c mimics treated L1-R2 cells, the expression Zeb1 and Zeb2 was decreased by 53% and 23%, respectively compared to the control mimics-treated cells confirming target specificity. Since one mechanism by which ZEB promotes EMT state is through transcriptional suppression of Ecadherin expression, and L1-R2-435-GFP cell lines were negative for E-cadherin and positive for vimentin, we searched for additional putative microRNA-200c gene VE-821 targets that are validated regulators of EMT or metastasis. We computationally prioritized putative, functional microRNA- 200c gene targets in the L1- and L1Mic-435-GFP models by combining the 681 sequence alignment predicted targets of microRNA-200c from TargetScan with microRNA and gene expression analysis of putative gene targets expressed in the lung derivative oligo- or polymetastatic cell lines as well as xenograft lung metastases. Of the 681 putative targets from TargetScan, 180 showed anti-correlation with microRNA-200c expression. Only three of these genes were significantly and differentially expressed between oligo and polymetastatic cell lines: FGD1 and USP25 from xenograft lung metastases and NEDD4L from lung cell lines. We chose NEDD4 and FGD1 for validation of microRNA-200c targeting based on their reported role in regulating EMT via TGF? signaling and Rho signaling, respectively. Shown in Fig. 6c, NEDD4 and FGD1 each contain a putative binding site for the microRNA-200 family members including microRNA- 200c. As expected, the expression of these two genes in microRNA-200c mimics-treated L1-R2 cells was inhibited by 47% and 50%, respectively compared with that in control-mimics treated cells. In contrast, the expression of vimentin, a non-putative microRNA gene target, was not significantly altered.