Diminished expression of the metastasis suppressor protein RKIP was previously reported in a number of cancers. The underlying mechanism remains unknown. Here, we show that the expression of RKIP negatively correlates with that of Snail zinc-transcriptional repressor, a key modulator of normal and neoplastic epithelial-mesenchymal transition (EMT) program. With a combination of loss-of-function and gain-of-function approaches, we showed that Snail repressed the expression of RKIP in metastatic prostate cancer cell lines. The effect of Snail on RKIP was on the level of transcriptional initiation and mediated by a proximal E-box on the RKIP promoter. (Chatterjee et al., 2004;Park et al., 2005). Consistent with its demonstrated inhibitory effect on Raf and NF-kB signaling, we and others have shown that the expression levels of RKIP are downregulated in a number of tumors, including highly metastatic prostate, breast and colon cancer, hepatocellular carcinoma, melanomas and insulinomas (Fu et al., 2003;Chatterjee et al., 2004;Schuierer et al., 2004Schuierer et al., , 2006Zhang et al., 2004;Hegan et al., 2005;Al-Mulla et al., 2006;Lee et al., 2006). The importance of RKIP in metastases was highlighted by the finding that restoration of RKIP expression inhibits prostate cancer metastasis in a murine model (Fu et al., 2003(Fu et al., , 2005. More recent studies have shown that RKIP is also a good prognostic marker of the pathogenesis of human prostate cancer (Fu et al., 2005) and a prognostic indicator for overall survival and disease-free survival in colorectal cancer (Al-Mulla et al., 2006). Collectively, these studies suggest that RKIP is a novel cancer metastasis suppressor and an effector of signal transduction pathways leading to apoptosis. In spite of the abundance of experimental evidence on the deleterious consequences of reduced RKIP expression in tumors, the mechanisms responsible for the downregulation of RKIP in cancer are not completely understood.To set up a system to study the transcription regulation of RKIP, we examined RKIP expression levels in cancer cell lines with different metastatic capacity. In accordance with clinical tumor studies, we observed that expression levels of RKIP proteins progressively decreased in breast and prostate cancer cell lines of increasing metastatic potential. The expression of RKIP is low in invasive and metastatic breast (MB231, MB435 and 578T) and prostate (DU145 and PC3) cell lines and high in noninvasive cell lines like MCF7, BT20 and LNCaP (Figure 1a). Notably, RKIP protein levels correlated well with those of the intercellular adhesion protein E-cadherin (E-cad). E-cad is a well-documented tumor metastasis suppressor protein that is regulated by the Snail and closely related Slug transcription factors (Peinado et al., 2007). Quantitation of RKIP transcript levels in the different cancer cell lines by qRT-PCR demonstrated that they correlated with the levels of the protein, (Figure 1b), suggesting that RKIP expression is downregulated at the RNA level, via changes...

Epigenetic modifications such as histone methylation play an important role in human cancer metastasis. Enhancer of zeste homolog 2 (EZH2), which encodes the histone methyltransferase component of the polycomb repressive complex 2 (PRC2), is overexpressed widely in breast and prostate cancers and epigenetically silences tumor suppressor genes. Expression levels of the novel tumor and metastasis suppressor Raf-1 kinase inhibitor protein (RKIP) have been shown to correlate negatively with those of EZH2 in breast and prostate cell lines as well as in clinical cancer tissues. Here, we show that the RKIP/EZH2 ratio significantly decreases with the severity of disease and is negatively associated with relapse-free survival in breast cancer. Using a combination of loss-and gain-of-function approaches, we found that EZH2 negatively regulated RKIP transcription through repressionassociated histone modifications. Direct recruitment of EZH2 and suppressor of zeste 12 (Suz12) to the proximal E-boxes of the RKIP promoter was accompanied by H3-K27-me3 and H3-K9-me3 modifications. The repressing activity of EZH2 on RKIP expression was dependent on histone deacetylase promoter recruitment and was negatively regulated upstream by miR-101. Together, our findings indicate that EZH2 accelerates cancer cell invasion, in part, via RKIP inhibition. These data also implicate EZH2 in the regulation of RKIP transcription, suggesting a potential mechanism by which EZH2 promotes tumor progression and metastasis.