The primary objective of this proposal is to elucidate new mechanisms by which the multifunctional cytokine TGF- affects tumor metastasis. TGF- plays important roles in controlling many types of cellular functions in the regulation of development and homeostasis. During tumorigenesis, TGF- is known to inhibit tumor formation in the early stage, but act as a potent promoter for late stages of the process, including metastasis. While the framework of the primary signaling pathway through which TGF- acts to regulate a wide range of biological processes has been established, the mechanisms by which TGF- functions to determine specific outcomes of those biological processes remain to be fully explored. Many microRNAs (miRNAs) are found to be down-regulated in cancer cells, leading to the up-regulation of their target genes to affect tumorigenesis. We intend to test the hypothesis that specific miRNAs are mediators of TGF- to regulate various cellular functions in the context of tumor metastasis. In preliminary studies, we found that the expression level of a set of miRNAs in breast and liver cancers was decreased upon treatment of TGF-. Among them, miR-34a and miR-126 were chosen for further analyses because of their putative target genes, CCL22 and SDF1, are involved in the recruitment of regulatory T cells (Treg) and mesenchymal stem cells (MSCs), respectively. Based on those results, we further hypothesize that TGF- enhances tumor metastasis by changing the tumor microenvironment through two new mechanisms: the inhibition of immune function via the repression of miR-34a to induce the production of CCL22, which in turn stimulates the accumulation of Treg cells in the areas where metastatic colonization occurs, and recruitment of MSCs to support the growth of metastatic cells. We propose two aims to test our hypothesis, determining the functions of miR-34a and miR-126 as mediators of TGF- to affect metastasis in vitro and in vivo, and elucidating the mechanism by which TGF- regulates the expression/processing of these miRNAs. Ultimately, characterization of these novel mechanisms by which TGF- promotes metastasis will provide potential new targets for metastatic cancer therapy.