Tumor Necrosis Factor-α and Apoptosis Induction in Melanoma Cells through Histone Modification by 3-Deazaneplanocin A
2013; Elsevier BV; Volume: 134; Issue: 5 Linguagem: Inglês
10.1038/jid.2013.489
ISSN1523-1747
AutoresRyo Tanaka, Nicholas Donovan, Qiang Yu, Reiko F. Irie, Dave S.�B. Hoon,
Tópico(s)Immune responses and vaccinations
Resumochromatin immunoprecipitation 3-deazaneplanocin A enhancer of zeste homolog 2 methylation-specific PCR assay polycomb-repressive complex 2 propidium iodide quantitative PCR quantitative real-time reverse-transcriptase–PCR reverse transcriptase–PCR suppressor of zeste 12 homolog (Drosophila) tumor necrosis factor-α trichostatin A Malignant melanoma is a highly aggressive form of skin cancer that can be difficult to manage once metastasis has occurred. Tumor necrosis factor-α (TNF-α) is a cytokine that influences the tumor microenvironment, activates tumor inflammation, and induces cell death (Balkwill, 2009Balkwill F. Tumour necrosis factor and cancer.Nat Rev Cancer. 2009; 9: 361-371Crossref PubMed Scopus (1295) Google Scholar). TNF-α induces the signal-transduction pathways associated with cell survival through NF-κB or caspase 8. Melanoma cells can produce TNF-α (Landsberg et al., 2012Landsberg J. Kohlmeyer J. Renn M. et al.Melanomas resist T-cell therapy through inflammation-induced reversible dedifferentiation.Nature. 2012; 490: 412-416Crossref PubMed Scopus (397) Google Scholar); however, its expression is heterogenous and the regulation of tumor cell TNF-α production is poorly understood. Epigenetic deregulation has an important role in aberrant gene expression and melanoma progression (Tanemura et al., 2009Tanemura A. Terando A.M. Sim M.S. et al.CpG island methylator phenotype predicts progression of malignant melanoma.Clin Cancer Res. 2009; 15: 1801-1807Crossref PubMed Scopus (150) Google Scholar). Several tumor-related genes are consistently aberrantly hypermethylated during melanoma progression (Tanemura et al., 2009Tanemura A. Terando A.M. Sim M.S. et al.CpG island methylator phenotype predicts progression of malignant melanoma.Clin Cancer Res. 2009; 15: 1801-1807Crossref PubMed Scopus (150) Google Scholar; Greenberg et al., 2012Greenberg E.S. Chong K.K. Huynh K.T. et al.Epigenetic biomarkers in skin cancer.Cancer Lett. 2012; 12: 371-382Google Scholar; Hoshimoto et al., 2012Hoshimoto S. Kuo C.T. Chong K.K. et al.AIM1 and LINE-1 epigenetic aberrations in tumor and serum relate to melanoma progression and disease outcome.J Invest Dermatol. 2012; 132: 1689-1697Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Similarly, histone modification has also been shown to regulate gene expression (Kouzarides, 2007Kouzarides T. Chromatin modifications and their function.Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8059) Google Scholar) by affecting both the initiation and the progression of cancer by various mechanisms. An important repressive histone marker, H3K27me3, is induced by enhancer of zeste homolog 2 (EZH2) (Chang and Hung, 2011Chang C.J. Hung M.C. The role of EZH2 in tumour progression.Br J Cancer. 2011; 106: 243-247Crossref PubMed Scopus (269) Google Scholar). 3-Deazaneplanocin A (DZNep) is a potent S-adenosylhomocysteine hydrolase inhibitor, which can indirectly inhibit S-adenosyl-methionine–dependent reactions related to various methyltransferases (Miranda et al., 2009Miranda T.B. Cortez C.C. Yoo C.B. et al.DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation.Mol Cancer Ther. 2009; 8: 1579-1588Crossref PubMed Scopus (461) Google Scholar). It has been shown to inhibit the histone methyltransferase polycomb-repressive complex 2 (PRC2) components EZH2, suppressor of zeste 12 homolog (SUZ12), and embryonic ectoderm development (Tan et al., 2007Tan J. Yang X. Zhuang L. et al.Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells.Genes Dev. 2007; 21: 1050-1063Crossref PubMed Scopus (751) Google Scholar). DZNep acts as an epigenetic modifying agent that represses H3K27me3 (Tan et al., 2007Tan J. Yang X. Zhuang L. et al.Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells.Genes Dev. 2007; 21: 1050-1063Crossref PubMed Scopus (751) Google Scholar). DZNep can induce apoptosis in breast cancer cells, but not in normal cells (Tan et al., 2007Tan J. Yang X. Zhuang L. et al.Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells.Genes Dev. 2007; 21: 1050-1063Crossref PubMed Scopus (751) Google Scholar). Trichostatin A (TSA), a histone deacetylase inhibitor, can block histone hypoacetylation to restore expression levels of several tumor suppressor genes and induce apoptosis in human cancer cells such as melanoma. Recently, we showed that the combination of DZNep and TSA can induce the repression of H3K27me3 and the elevation of H3K9ac in prostate cancer cells in order to significantly amplify tissue inhibitor of metalloproteinase-3 expression (Shinojima et al., 2012Shinojima T. Yu Q. Huang S.K. et al.Heterogeneous epigenetic regulation of TIMP3 in prostate cancer.Epigenetics. 2012; 7: 1279-1289Crossref PubMed Scopus (31) Google Scholar). To investigate the effect of histone modification to DZNep on melanoma cells, we treated and assessed five melanoma cell lines. Four of the five DZNep-treated cell lines (M12, M15, M101, and M223) resulted in the repression of H3K27me3 and PRC2 components EZH2 and SUZ12. In addition, cleaved poly (ADP-ribose) polymerase was detected, indicating that DZNep could induce apoptosis in these lines (Figure 1). Interestingly, H3K4me2 was repressed in the M15, M101, and M223 lines, showing that DZNep could repress PRC2 and H3K27me3 in specific melanoma cell lines. To determine whether cell death was induced through apoptosis, two types of assays were performed on the M101 cell line, which was highly sensitive to the DZNep treatment, using the optimal non-toxic dose (5 μM) of DZNep based on a cell viability analysis. In propidium iodide (PI) staining and cell cycle analysis, the sub-G1 fraction was shown to be enhanced by DZNep. By flow cytometry analysis using annexin V-FITC and PI staining, DZNep exposure caused an increase in the percentage of cells gated for annexin V+/PI–, early apoptosis, and annexin V+/PI+, late apoptosis/necrosis (Supplementary Figure 1A–C online). These results, combined with the western blot results, suggested that DZNep induces apoptosis in M101, M12, and M223 cell lines. Download .pdf (.67 MB) Help with pdf files Supplementary Information We proceeded to further identify apoptosis-specific gene expression induced by DZNep using reverse transcriptase–PCR (RT–PCR)-based RT2 Profiler Human Apoptosis PCR array (Supplementary Table 1 online). mRNA expression of 10 genes was upregulated by ⩾2-fold after DZNep treatment; TNF-α mRNA was upregulated 6.3-fold. Therefore, we focused on TNF-α expression. We performed a chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) array using anti-H3K27me3 and anti-H3K4me2 antibody to identify DZNep-activated PRC2 target genes in melanoma cells. Fold enrichment by quantitative ChIP was calculated by the ratio to input DNA. Supplementary Table 2 online shows 13 genes associated with decreased percentage input of repressive histone H3K27me3 and increased H3K4me2 in DZNep-treated cells compared with DMSO-treated cells. The percentage input on TNF-α in H3K27me3 was decreased by 2.0X, whereas there was an increase by 7.0X in H3K4me2. To confirm the results of the TNF-α ChIP-qPCR array, quantitative real-time reverse-transcriptase–PCR (qRT-PCR) was performed on M101, M12, LF0023, and M223. It was shown that TNF-α was upregulated in M101 and M223 cells by DZNep treatment (Supplementary Figure 2A–C online). Induction of TNF-α upregulation in M101, LF0023, and M223 cell lines was demonstrated in 5-aza-2′-deoxycytidine treatment. The M12 line was only upregulated by the combined treatment of both DZNep and TSA (Supplementary Figure 2B online). For M12, H3K9ac was also strongly activated by TSA and DZNep, inducing TNF-α (Supplementary Figure 3 online). To confirm that TNF-α was regulated by histone modification, we assessed the TNF-α promoter region (-1,536 bp to +879 bp of transcriptional start site) (Supplementary Figure 4 online). On using ChIP analysis, the percentage input ratio between H3K27me3 and H3 on TNF-α in M101 was found to be significantly decreased (P<0.01) in DZNep-treated specimens compared with DMSO-treated specimens. For H3K4me2 and H3K9ac, there was no significant difference between DMSO- and DZNep-treated cells (Supplementary Figure 5A online). This trend in H3K27me3 was similar to the results of the ChIP-PCR array (Supplementary Table 2 online). To assess DNA methylation status of the TNF-α promoter region, we performed a methylation-specific PCR assay (MSP). Methylated DNA in DMSO-treated cells was not changed by DZNep treatment (Supplementary Figure 5B online). These results suggest that DZNep may affect the repressive histone H3K27me3, restoring TNF-α expression level. To assess whether DZNep modification of TNF-α histone status was related to apoptosis, a ChIP analysis was performed on M12 and M223. For M12, the percentage input ratio between H3K27me3 and H3 on TNF-α was not significantly decreased by DZNep and no DNA methylation was evident (Supplementary Figure 6A(i) and B online). The combined treatment with DZNep and TSA restored TNF-α expression, suggesting that H3K9ac was related to the regulation of TNF-α; however, H3K27me3 was not strongly associated with TNF-α repression. In M223, the percentage input ratio between H3K27me3 and H3 on TNF-α was significantly decreased by DZNep; DNA was also methylated (Supplementary Figure 6A(ii) and B online) and 5-aza-2′-deoxycytidine treatment restored TNF-α expression (Supplementary Figure 2B online). These results suggest that both can have DNA methylation and H3K27me3 have a significant role in TNF-α regulation. To assess the association between TNF-α expression and epigenetic status in melanoma tissues, we performed RT-PCR, ChIP-qPCR assay, and MSP (Supplementary Figure 7A–C online) on melanoma tumors (n=4). The percentage input ratio between H3K27me3 and H3 was highest in PT4. TNF-α could not be detected by qRT-PCR (Supplementary Figure 7A and B(i) online). However, the percentage input ratio between H3K4me2 and H3 and the percentage input ratio between H3K9ac and H3 were highest in PT2, which also showed TNF-α expression (Supplementary Figure 7B(ii, iii) online). The analysis suggested that repressive and active histones were associated with TNF-α regulation. In contrast, DNA methylation was not strongly related to the repression of TNF-α expression (Supplementary Figure 7C online). Among the four specimens, TNF-α expression level was highest in PT1; however, the percentage input ratio between H3K27me3 and H3 was relatively high and the percentage input ratio between H3K4me2/H3K9ac and H3 was relatively low. In PT1, other mechanisms such as regulation of transcription, splicing, message turnover, or translation may have a dominant role in regulating TNF-α expression over epigenetic factors. In summary, we showed that DZNep treatment induced apoptosis in melanoma cells, suggesting a change in chromatin architecture to a heterochromatin phenotype. We showed that DZNep treatment induced repression of PRC2 and H3K27me3, thereby restoring TNF-α expression. In addition, we confirmed that H3K27me3 can bind to the promoter region of TNF-α. Furthermore, the combination of DZNep and TSA restored TNF-α expression, confirming a significant role of H3K9ac in TNF-α upregulation. We showed that the TNF-α promoter region was hypermethylated and mRNA expression of TNF-α was restored by 5-aza-2′-deoxycytidine treatment. In combination, these results suggest that both DNA methylation and histone modification are strongly associated with the regulation of TNF-α expression. TNF-α expression levels in melanoma lines are generally quite low. Previously, it was reported that repressive H3K27me3 was linked to de novo DNA methylation of PRC2 target genes (Schlesinger et al., 2007Schlesinger Y. Straussman R. Keshet I. et al.Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer.Nat Genet. 2007; 39: 232-236Crossref PubMed Scopus (948) Google Scholar). H3K9 histone methylation may also have an important role in DNA methylation (Ohm et al., 2007Ohm J.E. McGarvey K.M. Yu X. et al.A stem cell-like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing.Nat Genet. 2007; 39: 237-242Crossref PubMed Scopus (873) Google Scholar). In contrast, it has been shown that DNA methylation and repressive histone modification are independently correlated with the silencing of various genes (Kondo et al., 2008Kondo Y. Shen L. Cheng A.S. et al.Gene silencing in cancer by histone H3 lysine 27 trimethylation independent of promoter DNA methylation.Nat Genet. 2008; 40: 741-750Crossref PubMed Scopus (521) Google Scholar). Sullivan et al., 2007Sullivan K.E. Reddy A.B. Dietzmann K. et al.Epigenetic regulation of tumor necrosis factor alpha.Mol Cell Biol. 2007; 27: 5147-5160Crossref PubMed Scopus (173) Google Scholar reported the epigenetic regulation mechanism of TNF-α both in development and in acute stimulation in leukemia. Although the expression level of H3K9ac in western blot analysis was unchanged, H3K4me2 and H3k9ac may be associated with the restoration of TNF-α by DZNep treatment. In contrast, H3K27me3 was clearly repressed by DZNep. We confirmed that DZNep could release binding of H3K27me3 to the promoter region on TNF-α and restore its expression. As both 5-aza-2′-deoxycytidine and DZNep treatment alone activated TNF-α expression, methylation of both the gene promoter and H3K27 may be independently associated with TNF-α repression. EZH2 expression in metastatic melanoma was shown to be significantly higher than in situ primary melanomas and benign nevi (McHugh et al., 2007McHugh J.B. Fullen D.R. Ma L. et al.Expression of polycomb group protein EZH2 in nevi and melanoma.J Cutan Pathol. 2007; 34: 597-600Crossref PubMed Scopus (69) Google Scholar). Although DZNep may have other active mechanisms, its role in EZH2 inhibition may show potential for melanoma treatment. The indirect activation of TNF-α in melanoma may be a promising alternative treatment approach. This work was supported by the Dr Miriam and Sheldon G Adelson Medical Research Foundation, the Weil Family Foundation, the Leslie and Susan Gonda (Goldschmied) Foundation (Los Angeles, CA), and by NIH, NCI P0 CA029605 Project II, and Core C. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
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