ARTICLE ABSTRACTProgression of breast cancer to metastatic bone disease is linked to deregulated expression of the transcription factor Runx2. Therefore, our goal was to evaluate the potential for clinical use of Runx2-targeting miRNAs to reduce tumor growth and bone metastatic burden. Expression analysis of a panel of miRNAs regulating Runx2 revealed a reciprocal relationship between the abundance of Runx2 protein and two miRNAs, miR-135 and miR-203. These miRNAs are highly expressed in normal breast epithelial cells where Runx2 is not detected, and absent in metastatic breast cancer cells and tissue biopsies that express Runx2. Reconstituting metastatic MDA-MB-231-luc cells with miR-135 and miR-203 reduced the abundance of Runx2 and expression of the metastasis-promoting Runx2 target genes IL11, MMP-13, and PTHrP. In addition, tumor cell viability was decreased and migration suppressed in vitro. Orthotopic implantation of MDA-MB-231-luc cells delivered with miR-135 or miR-203, followed by an intratumoral administration of the synthetic miRNAs, reduced the tumor growth and spontaneous metastasis to bone. Furthermore, intratibial injection of these miRNA-delivered cells impaired tumor growth in the bone environment and inhibited bone resorption. Importantly, reconstitution of Runx2 in MDA-MB-231-luc cells delivered with miR-135 and miR-203 reversed the inhibitory effect of the miRNAs on tumor growth and metastasis. Thus, we have identified that aberrant expression of Runx2 in aggressive tumor cells is related to the loss of specific Runx2-targeting miRNAs and that a clinically relevant replacement strategy by delivery of synthetic miRNAs is a candidate for a therapeutic approach to prevent metastatic bone disease by this route. Cancer Res; 75(7); 1433–44. ©2015 AACR.