Nucleoside Drugs Induce Cellular Differentiation by Caspase-Dependent Degradation of Stem Cell Factors

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 2'-deoxy-5-azacytidine (decitabine), also induced a decrease in global DNA methylation. Nevertheless, only decitabine and 1beta-arabinofuranosylcytosine (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

DNA (de)methylation in embryonic stem cells controls CTCF-dependent chromatin boundaries

Coordinated changes of DNA (de)methylation, nucleosome positioning and chromatin binding of the architectural protein CTCF play an important role for establishing cell type specific chromatin states during differentiation. To elucidate molecular mechanisms that link these processes we studied the perturbed DNA modification landscape in mouse embryonic stem cells (ESCs) carrying a double knockout (DKO) of the TET1 and TET2 dioxygenases. These enzymes are responsible for the conversion of 5-methylcytosine (5mC) into its hydroxymethylated (5hmC), formylated (5fC) or carboxylated (5caC) forms. We determined changes in nucleosome positioning, CTCF binding, DNA methylation and gene expression in DKO ESCs, and developed biophysical models to predict differential CTCF binding. Methylation-sensitive nucleosome repositioning accounted for a significant portion of CTCF binding loss in DKO ESCs, while unmethylated and nucleosome-depleted CpG islands were enriched for CTCF sites that remained occupied. A number of CTCF sites also displayed direct correlations with the CpG modification state: CTCF was preferentially lost from sites that were marked with 5hmC in wild type cells but not from 5fC enriched sites. In addition, we found that some CTCF sites can act as bifurcation points defining the differential methylation landscape. CTCF loss from such sites, e.g. at promoters, boundaries of chromatin loops and topologically associated domains (TADs), was correlated with DNA methylation/demethylation spreading and can be linked to downregulation of neighbouring genes. Our results reveal a hierarchical interplay between cytosine modifications, nucleosome positions and DNA sequence that determines differential CTCF binding and regulates gene expression

Embryonic Carcinoma Cells Show Specific Dielectric Resistance Profiles during Induced Differentiation

<div><p>Induction of differentiation in cancer stem cells by drug treatment represents an important approach for cancer therapy. The understanding of the mechanisms that regulate such a forced exit from malignant pluripotency is fundamental to enhance our knowledge of tumour stability. Certain nucleoside analogues, such as 2′-deoxy-5-azacytidine and 1β-arabinofuranosylcytosine, can induce the differentiation of the embryonic cancer stem cell line NTERA 2 D1 (NT2). Such induced differentiation is associated with drug-dependent DNA-damage, cellular stress and the proteolytic depletion of stem cell factors. In order to further elucidate the mode of action of these nucleoside drugs, we monitored differentiation-specific changes of the dielectric properties of growing NT2 cultures using electric cell-substrate impedance sensing (ECIS). We measured resistance values of untreated and retinoic acid treated NT2 cells in real-time and compared their impedance profiles to those of cell populations triggered to differentiate with several established substances, including nucleoside drugs. Here we show that treatment with retinoic acid and differentiation-inducing drugs can trigger specific, concentration-dependent changes in dielectric resistance of NT2 cultures, which can be observed as early as 24 hours after treatment. Further, low concentrations of nucleoside drugs induce differentiation-dependent impedance values comparable to those obtained after retinoic acid treatment, whereas higher concentrations induce proliferation defects. Finally, we show that impedance profiles of substance-induced NT2 cells and those triggered to differentiate by depletion of the stem cell factor OCT4 are very similar, suggesting that reduction of OCT4 levels has a dominant function for differentiation induced by nucleoside drugs and retinoic acid. The data presented show that NT2 cells have specific dielectric properties, which allow the early identification of differentiating cultures and real-time label-free monitoring of differentiation processes. This work might provide a basis for further analyses of drug candidates for differentiation therapy of cancers.</p> </div

Induced differentiation by a defined panel of drugs.

<p>(<b>A</b>) Impedance profiles comparing NT2 cells treated with retinoic acid (RA, 10 µM), hexamethylene bisacetamide (HMBA, 5 mM), 5-azacytidine (AZA, 1 µM), deoxycytidine (dC, 1 µM), fibroblast growth factor 2 (FGF, 50 µM), 2′-deoxy-5-azacytidine (DAC, 1 µM) and 1β-arabinofuranosylcytosine (araC, 1 µM) during a 4 day period. Measurements were executed at 45 kHz in 5-minute intervals for 96 hours. The mean of three independent experiments is shown. Standard deviations are not shown to avoid crowding of the diagram. For single diagrams including standard deviations and statistical tests for these data sets see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059895#pone.0059895.s002" target="_blank">Fig. S2</a>. (<b>B</b>) Average cell numbers of three replicates of untreated and treated NT2 cells after 24 and 96 hours. Nucleoside drugs are cytotoxic at the concentrations used and show significant growth inhibition. Standard deviations are indicated by error bars. (<b>C</b>) Microscopic images (10× magnification) of NT2 control cells and NT2 cells treated with the various substances mentioned in (A) after 24 and 96 hours of treatment. (<b>D</b>) qRT-PCR expression analysis of stem cell factors <i>NANOG</i>, <i>OCT4</i> and the differentiation markers <i>NESTIN</i>, <i>SNAP25</i> and <i>TUBB3</i> in treated and NT2 control cells after 24 and 96 hours of treatment. All qRT-PCR measurements were repeated at least three times and internally normalised to the corresponding <i>β-actin</i> values. Standard deviations are indicated by error bars. Treatments showing significant differences comparing expression levels at 24 hours with those at 96 hours are marked with an asterisk (two-tailed student’s t-test; p<0.05).</p

Induced concentration-dependent differentiation of NT2 cells by RA.

<p>(<b>A</b>) Impedance profiles comparing induction profiles of different RA concentrations during a 4 day period. Measurements were executed at 45 kHz in 5-minute intervals for 96 hours. The mean of three independent experiments is shown. Standard deviations are not shown to avoid crowding of the diagram. For single diagrams including standard deviations and statistical tests for these data sets see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059895#pone.0059895.s001" target="_blank">Fig. S1</a>. (<b>B</b>) qRT-PCR expression analysis of stem cell factors <i>NANOG</i>, <i>OCT4</i> and the differentiation markers <i>HOXA1</i> and <i>SNAP25</i> and in RA- treated and control NT2 cells after 96 hours of treatment. The concentration of RA employed correlates negatively with the expression of stem cell factors, but positively with the expression of differentiation markers. All qRT-PCR measurements were repeated at least three times and internally normalised to the corresponding <i>β-actin</i> values. Standard deviations are indicated by error bars.</p

Retinoic acid induced neuronal differentiation of NT2 EC cells.

<p>(<b>A</b>) Impedance profiles comparing RA-induced (10 µM - red) and untreated NT2 cells (blue) during a 4 day period. The mean of three independent experiments is shown. Standard deviations are indicated by error bars every four hours. Measurements were executed at 45 kHz in 5-minute intervals for 96 hours. Normalised resistance values were compared by two-tailed Student’s t-test. After 20 hours of RA treatment differences in impedance values start to become statistically significant (*p<0.05, **p<0.005). Black lines show regions with significant differences in respect to the untreated cell control. (<b>B</b>) Average cell numbers of three replicates of untreated and RA-treated NT2 cells after 24 and 96 hours do not differ significantly. Standard deviations are indicated by error bars. (<b>C</b>) Microscopic images (10× magnification) of NT2 control cells and NT2 cells treated with RA for 24 and 96 hours. No clear differentiation phenotype becomes apparent for the RA treatment. (<b>D</b>) qRT-PCR expression analysis of stem cell factors <i>NANOG</i>, <i>OCT4</i> and the differentiation markers <i>NESTIN</i>, <i>SNAP25</i> and <i>HOXA1</i> in RA- treated and control cells after 24 and 96 hours of treatment. Data is shown in logarithmic scale. Only <i>HOXA1</i> is prominently induced by retinoic acid at both time points. The stemness genes are only found reduced after 96 hours of RA treatment. All qRT-PCR measurements were repeated at least three times and internally normalised to the corresponding <i>β-actin</i> values. Standard deviations are indicated by error bars. Two-tailed student’s t-test showed significant differences when comparing expression levels of <i>OCT4</i>, <i>NANOG</i> and <i>HOXA1</i> at 24 hours with the expression levels at 96 hours. (*p<0.05, **p<0.005).</p

Induced differentiation by RNAi-mediated depletion of OCT4.

<p>Impedance profiles of control NT2 cells (blue - scrambled knock down) and NT2 cells depleted for OCT4 (red) during a 6 day period. Measurements were executed at 45 kHz in 5-minute intervals for 6 days. The mean of three independent experiments is shown. Standard deviations are indicated by error bars every four hours. Student’s t-test was used for statistical analysis. Differences between control and knock down experiments in the indicated regions have been found to be statistically significant (*p<0.05, **p<0.005, ***p<0.001).</p

Epigenome changes in active and inactive Polycomb-group-controlled regions

The Polycomb group (PcG) of proteins conveys epigenetic inheritance of repressed transcriptional states. In Drosophila, the Polycomb repressive complex 1 (PRC1) maintains the silent state by inhibiting the transcription machinery and chromatin remodelling at core promoters. Using immunoprecipitation of in vivo formaldehyde-fixed chromatin in phenotypically diverse cultured cell lines, we have mapped PRC1 components, the histone methyl transferase (HMT) Enhancer of zeste (E(z)) and histone H3 modifications in active and inactive PcG-controlled regions. We show that PRC1 components are present in both cases, but at different levels. In particular, active target promoters are nearly devoid of E(z) and Polycomb. Moreover, repressed regions are trimethylated at lysines 9 and 27, suggesting that these histone modifications represent a mark for inactive PcG-controlled regions. These PcG-specific repressive marks are maintained by the action of the E(z) HMT, an enzyme that has an important role not only in establishing but also in maintaining PcG repression