The very process of deregulated oncogene expression during cancer development also sensitizes cancer cells to apoptotic signals (1-3). Deregulated oncoproteins such as E1a and c-Myc promote apoptosis by activating multiple downstream proapoptotic effector pathways (4, 5). Additional mechanisms of sensitizing cancer cells to apoptosis by an activated oncoprotein have been described (6, 7). For example, E2F sensitized cells to apoptosis through down-regulation of anti-apoptotic signals (7). Here we show that cancer cells can also be sensitized to apoptosis by up-regulating the expression levels of RKIP (Raf kinase inhibitor protein). RKIP was originally identified as an interacting partner of Raf-1 and a negative regulator of the mitogen-activated protein kinase cascade initiated by Raf-1 (8). RKIP also inhibits nuclear factor B (NF-B)1 signaling by negatively modulating the activating phosphorylation of IKK␣ and IKK via upstream kinases (9). Although the molecular mechanism by which RKIP inhibits the Raf and NF-B signaling pathways has been partially delineated, little is known about the biological relevance of the inhibition of these pathways by RKIP. In addition to these functions, we presently demonstrate the rapid up-regulation of RKIP during induction of chemotherapy-triggered apoptosis in human prostate and breast cancer cells. However, in DNA-damaging agent-resistant cancer cells, treatment with the drugs does not up-regulate RKIP expression. Ectopic expression of RKIP sensitizes DNA damage agentresistant cells to undergo apoptosis. Down-regulation of RKIP expression confers resistance to 9-nitrocamptothecin (9NC) by releasing its inhibitory constraint on two major survival pathways in cancer cells. Our studies suggest that RKIP represents a novel apoptotic marker in human cancer cells. MATERIALS AND METHODSCell Lines, Plasmid Constructs, and Chemicals-The human breast cell lines 578T and 578Bst were purchased from American Type Culture Collection (Manassas, VA). A human breast cancer MCF7 cell subline resistant to 9NC treatment was a gift from Dr. Ray Frackelton (Brown University). The human prostate cell lines LNCaP, DU145, and PC3 were purchased from American Type Culture Collection. Early (Ͻ30)-or late (Ͼ100)-passage DU145 cells were not used for this study. The 9NC-resistant DU145 cell subline, RC1, was established by continuous exposure of DU145 cells to 9NC (10). All cell lines were grown in conditions suggested by American Type Culture Collection. MCF7 and
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.
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