Nucleoside Drugs Induce Cellular Differentiation by Caspase-Dependent Degradation of Stem Cell Factors Tanja Musch, Yuva O¨ z, Frank Lyko, Achim Breiling* Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany Abstract Background: Stem cell characteristics are an important feature of human cancer cells and play a major role in the therapy resistance of tumours. Strategies to target cancer stem cells are thus of major importance for cancer therapy. Differentiation therapy by nucleoside drugs represents an attractive approach for the elimination of cancer stem cells. However, even if it is generally assumed that the activity of these drugs is mediated by their ability to modulate epigenetic pathways, their precise mode of action remains to be established. We therefore analysed the potential of three nucleoside analogues to induce differentiation of the embryonic cancer stem cell line NTERA 2 D1 and compared their effect to the natural ligand retinoic acid. Methodology/Principal Findings: All nucleoside analogues analyzed, but not retinoic acid, triggered proteolytic degradation of the Polycomb group protein EZH2. Two of them, 3-Deazaneplanocin A (DZNep) and 29-deoxy-5-azacytidine (decitabine), also induced a decrease in global DNA methylation. Nevertheless, only decitabine and 1b-arabinofurano- sylcytosine (cytarabine) effectively triggered neuronal differentiation of NT2 cells. We show that drug-induced differentiation, in contrast to retinoic acid induction, is caused by caspase activation, which mediates depletion of the stem cell factors NANOG and OCT4. Consistent with this observation, protein degradation and differentiation could be counteracted by co-treatment with caspase inhibitors or by depletion of CASPASE-3 and CASPASE-7 through dsRNA interference. In agreement with this, OCT4 was found to be a direct in-vitro-target of CASPASE-7. Conclusions/Significance: We show that drug-induced differentiation is not a consequence of pharmacologic epigenetic modulation, but is induced by the degradation of stem-cell-specific proteins by caspases. Our results thus uncover a novel pathway that induces differentiation of embryonic cancer stem cells and is triggered by the established anticancer drugs cytarabine and decitabine. These findings suggest new approaches for directly targeting the stem cell fraction of human tumours. Citation: Musch T, O¨ z Y, Lyko F, Breiling A (2010) Nucleoside Drugs Induce Cellular Differentiation by Caspase-Dependent Degradation of Stem Cell Factors. PLoS ONE 5(5): e10726. doi:10.1371/journal.pone.0010726 Editor: Chun-Ming Wong, University of Hong Kong, Hong Kong Received October 26, 2009; Accepted April 29, 2010; Published May 19, 2010 Copyright: 2010 Musch et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the DKFZ Intramural Research Programme and the Deutsche Forschungsgemeinschaft (Priority Programme 1356). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: a.breiling@dkfz.de Introduction Cancer is widely considered a developmental disease, caused by the destabilisation of the balance of cellular proliferation and differentiation [1,2]. Tumours thus originate from cells with stem cell characteristics that have acquired aberrant gene expression patterns, mostly due to mutations and epimutations. These aberrant gene expression patterns lead to a block in differentiation and trigger uncontrolled proliferation. Loss of differentiation is thus an important characteristic of tumour cells and represents a defining feature of human cancers. As a consequence, differenti- ation therapy has been developed into an important approach for the treatment of cancers, particularly leukaemias [3,4]. Stem cell features are controlled by a small group of transcription factors, like NANOG and OCT4, that maintain the expression of stem cell genes and repress the induction of differentiation-specific genes by recruiting repressive complexes of the Polycomb Group (PcG) [5,6]. Differentiation-associated genes in mouse ES cells are repressed by PcG proteins and are often also marked by DNA methylation [7]. These repressive marks are successively lost during differentiation. A prominent example is the loss of PcG repression at the HOXA gene cluster during neuronal differentiation [8,9]. It has also been shown that promoters of lineage-specific genes become methylated during differentiation, suggesting context-dependent interactions between DNA methyl- ation and Polycomb repression [10]. A key component of PcG repression is the histone methyltransferase EZH2 (enhancer of zeste homolog 2), the enzymatic core component of the Polycomb repressive complex 2 (PRC2). This protein creates specific trimethylation patterns of lysine 27 of histone H3 (H3K27me3), which leads to concomitant transcriptional silencing [11,12]. The characterization of drugs that modulate epigenetic processes and induce differentiation in human cancer cells represents an important aspect in the development of epigenetic cancer therapies. Retinoic acid (RA), which induces differentiation in many stem cell populations, was among the first substances used for differentiation therapy [3]. The seminal finding that the differentiation-inducing cytosine analogue 29-deoxy-5-azacytidine PLoS ONE | www.plosone.org 1 May 2010 | Volume 5 | Issue 5 | e10726