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Epigenetic drug combination induces genome-wide demethylation and altered gene expression in neuro-ectodermal tumor-derived cell lines

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Abstract

Background

Epigenetic alterations are inherent to cancer cells, and epigenetic drugs are currently primarily used to treat hematological malignancies. Pediatric neuro-ectodermal tumors originate from neural crest cells and also exhibit epigenetic alterations involving e.g. apoptotic pathways, which suggests that these tumors may also be sensitive to epigenetic drugs. This notion prompted us to assess molecular and functional effects of low dosage epigenetic drugs in neuro-ectodermal tumor-derived cell lines of pediatric origin.

Results

In 17 neuroblastoma (NBL) and 5 peripheral primitive neuro-ectodermal tumor (PNET) cell lines a combination treatment of 5-aza-2′-deoxycytidine (DAC) and Trichostatin A (TSA) at nanomolar dosages was found to reduce proliferation and to induce wide-spread DNA demethylation, accompanied by major changes in gene expression profiles. Approximately half of the genes that were significantly up-regulated upon treatment exhibited a significant demethylation in their promoter regions. In the NBL cell lines, almost every cellular pathway (193/200) investigated showed expression alterations after treatment, especially a marked up-regulation of genes in the p53 pathway. The combination treatment also resulted in up-regulation of known epigenetically regulated genes such as X-chromosomal genes, tissue-specific genes and a limited number of imprinted genes, as well as known tumor suppressor genes and oncogenes.

Conclusions

Nanomolar dosages of epigenetic drugs have a dramatic impact on the genomes of neuro-ectodermal tumor-derived cell lines, including alterations in DNA methylation and concomitant alterations in gene expression.

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Abbreviations

DAC:

5-aza-2′-deoxycytidine

DMH:

differential methylation hybridization

HDACi:

histone deacetylase inhibitor

MDS:

myelodysplastic syndrome

NBL:

neuroblastoma

NMA:

MYCN amplified

PNET:

primitive neuro-ectodermal tumor

PRC2:

polycomb receptor complex 2

TSA:

trichostatin A

ZEB:

zebularine

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Acknowledgments

We would like to thank Judith Boer and Maarten van Iterson for their help in normalization of the CpG island methylation array data and their input in discussions on data analysis. This work was funded by the Dutch Neuroblastoma Foundation Villa Joep, the Dutch Cancer Society and the ODAS foundation (The Netherlands).

Conflict of interest

We have no conflict of interest or financial conflict to disclose.

Author information

Authors and Affiliations

  1. Department of Pediatric Oncology-Hematology, Erasmus MC-Sophia Children’s Hospital, Dr. Molewaterplein 60, 3015, GJ, Rotterdam, The Netherlands

    Floor A.M. Duijkers, Renee X. de Menezes, Inès J. Goossens-Beumer, Dominique J.P.M. Stumpel, Pieter Admiraal, Rob Pieters, Jules P.P. Meijerink & Max M. van Noesel

  2. Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands

    Renee X. de Menezes

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  1. Floor A.M. Duijkers
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Electronic supplementary materials

Below is the link to the electronic supplementary material.

Supplementary Figure 1

(Figure made available from previously published paper; Iterson et al. 2012 SAGMB, publisher De Gruyter) Schematic Differential methylation and Hybridization (DMH) protocol Protocol for enrichment of methylated DNA fragments, which also enriches for fragments without methylation sensitive restriction sites. (DOCX 1061 kb)

Supplementary Figure 2

(Figures made available from previously published paper; Iterson et al. 2012 SAGMB, publisher De Gruyter) MA-plots for six cell lines with weighted P-spline curves using different weights (invariant probes in black). The blue curve is a normal normalization obtained without any weights, the red curve is mainly based on the invariant probes with a high weight for the invariant probes (weight=10.000) versus all the other probes (w=1), and the yellow curve is an intermediate with an equal total weight of all the invariant probes (weight=250) versus all the other probes (weight =1). The yellow curve was chosen for normalization purposes because it also covers the high intensity range where no or little invariant probes are present. (DOCX 128 kb)

Supplementary Figure 3

(Figure made available from previously published paper; Iterson et al. 2012 SAGMB, publisher De Gruyter): MA-plots before and after normalization for a single cell line (treated and untreated, invariant probes in black). The upper panel shows raw data with normal P-spline and equally weighted P-spline curves respectively in green and red. The middle panels shows normal P-spline normalized data and the lower panels the weighted P-spline normalization data. The chosen equally weighted p-spline normalization method creates quite similarly-shaped curves compared with normal normalization methods. (DOCX 72 kb)

Supplementary Figure 4

Methylation ratios all cell lines before and after normalization. Box and whisker plots displaying the distribution of methylation levels (2log ratios) of all neuro-ectodermal cell line pairs. On the x-axis, untreated (U) and treated (T) cell line pairs are displayed. Panel A and B show the unnormalized data in which a clear shift towards lower methylation levels for treated cell lines is observed in each treatment pair. Panel C and D show the lowess normalized data that results in the loss of demethylating effects due to signal scaling. Panel E and F show normalized data using the weighed P-spline normalization method that is based on methylation independent control probes as used throughout the further article (Iterson 2012, SAGMB). This method removed the technical noise, while retaining the demethylating treatment effect. X-axis: cell line pairs from left to right NMB, SK-N-MC, CHP-100, SH-SY5Y, LAN-5, KCNR, TR-14, IMR-32, SK-N-DW, SJNB-8, AMC-106c, TC-32, SK-N-SH, SJNB-1, SH-EP-21N/tet2/N, SK-N-BE, SK-N-AS, NGP-C4, SH-EP-2/tet2, GI-M-EN, SJNB-10, CB-AGPN. (DOCX 623 kb)

Supplementary Figure 5

Upregulation of TNFRSF25 mRNA expression after epigenetic treatment. Light grey: non-MYCN amplified (NMA-) cell line SK-N-AS. Dark grey: NMA+ cell line IMR-32. On theX-axis it is indicated which single or combination drug treatment was used. The Y-axis shows the fold of expression change of TNFRSF25 compared with the untreated control. Mi = methylation inhibitor; HDACi = histone deacetylase inhibitor. Concentrations drugs used: Decitabine 7.5 μM in SK-N-AS (+/- IC50) and 50nM in IMR-32 (+/- IC50). Zebularine 60 μM (+/- IC50), FK228 1.5nM (+/- IC50), TSA 100 nM (+/- IC50). (DOCX 64 kb)

Supplementary Figure 6

Limiting dilution approach for identification of the optimal concentration of DAC and TSA. TAQMAN analysis for the TNFRSF25 gene was performed in 7 NBL and 1 PNET line with increasing concentrations of DAC and TSA. For each drug combination, numbers refer to the fold upregulation of TNFRSF25 gene expression compared with the untreated cell line or lowest treatment concentration if the value of the untreated sample is missing. Whereas colors refer to an estimation of cell viability as deduced from absolute GAPDH levels. (DOCX 660 kb)

Supplementary Figure 7

Upregulation of TNFRSF10C (A), TNFRSF10D (B) and RASSF1A (C) mRNA expression after epigenetic treatment. On the x-axis the different cell lines with in white, the MYCN amplified (NMA) NBL cell lines, in dark grey the non-NMA cell lines and in light grey the PNET cell lines. On the y-axis the fold change of mRNA expression after treatment with DAC and TSA. Most consistently upregulated cell lines across the different genes: SH-EP-21N, SH-SY5Y, NMB, SK-N-AS. (DOCX 65 kb)

Supplementary Figure 8

Cell cycle distribution in four cell lines. A. IMR-32, B. NMB, C. GI-M-EN, D. CHP-100)) with and without DAC 30nM and TSA 25nM pre-treatment. The PI signal is shown on the x-axis and the cell count on the y-axis. In CHP-100 a small shift from G2 to G0-G1 stage has been shown without changes in the other cell lines. Cell line SK-N-MC missing due to a lack of material. (DOCX 183 kb)

Supplementary Figure 9

PI-Annexin measurement of apoptosis in all cell lines. On the Y-axis the signal for annexin IV (apoptosis marker), on the Y-axis PI staining (marker necrosis/membrane permeability). No large shifts noted. (DOCX 376 kb)

Supplementary Figure 10

Cell viability (triplicate measurements) in response to Vincristin and Doxorubicin with and without DAC 30nM and TSA 25nM pre-treatment. On the X- axis accumulating dosages of drugs (10log scale), on the y-axis cell viability. Vincristin sensitivity seems to be enhanced at low concentrations after DAC and TSA pre-treatment in NMB, no other effects noted. (DOCX 106 kb)

Supplementary Figure 11

Principle component analysis based on expression array probesets. Unsupervised principal component analyses of expression array data using all probe sets (left panel) and most variant probe sets (right panel, n=236 with variance >3). Green, PNETs; Blue, NBL non-NMA; Red, NBL NMA; Purple, SHEP-21N (MYCN transvectant). (DOCX 81 kb)

Supplementary Figure 12

Validation of upregulation of epigenetically regulated genes after DAC and TSA. Confirmation array expression changes tissue specific genes (DDR1 and DAZL) and X-chromosomal genes (XIST and ACRC) by RT-QPCR. The cell lines that did not show upregulation of XIST (within oval) were all female cell lines. These cell lines already had high XIST expression before treatment. Cluster 2 consists of non-MYCN amplified NBL cell lines GI-M-EN, SK-N-AS, SH-EP-2, and SH-EP-21N, while cluster 3 consists of all other NBL cell lines, which are mostly MYCN amplified. (DOCX 61 kb)

Supplementary Figure 13

Upregulation TP53 after DAC and TSA treatment. Western blot p53 protein expression, showing upregulated expression of p53 after DAC and TSA treatment for NMB, SJNB8 (in short SJ8) and AMC106c (in short AMC). U, untreated; T, treated. (DOCX 36 kb)

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Duijkers, F.A., de Menezes, R.X., Goossens-Beumer, I.J. et al. Epigenetic drug combination induces genome-wide demethylation and altered gene expression in neuro-ectodermal tumor-derived cell lines. Cell Oncol. 36, 351–362 (2013). https://doi.org/10.1007/s13402-013-0140-x

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