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PDF file - 47K, Differentially methylated genes with beta values (3KT vs carcinogen exposed).

PDF file - 65K, Pathway analysis of differentially methylated genes.

DNA methylation is an early event in bronchial carcinogenesis and increased DNA methyltransferase (DNMT)1 protein expression is a crucial step in the oncogenic transformation of epithelia. Here, we investigate the role of class I histone deacetylases (HDAC) 1 to 3 in the stabilization of DNMT1 protein and as a potential therapeutic target for lung cancer chemoprevention. Long-term exposure of immortalized bronchial epithelial cells (HBEC-3KT) to low doses of tobacco-related carcinogens led to oncogenic transformation, increased HDAC expression, cell-cycle independent increased DNMT1 stability, and DNA hypermethylation. Overexpression of HDACs was associated with increased DNMT1 stability and knockdown of HDACs reduced DNMT1 protein levels and induced DNMT1 acetylation. This suggests a causal relationship among increased class I HDACs levels, upregulation of DNMT1 protein, and subsequent promoter hypermethylation. Targeting of class I HDACs with valproic acid (VPA) was associated with reduced HDAC expression and a profound reduction of DNMT1 protein level. Treatment of transformed bronchial epithelial cells with VPA resulted in reduced colony formation, demethylation of the aberrantly methylated SFRP2 promoter, and derepression of SFRP2 transcription. These data suggest that inhibition of HDAC activity may reverse or prevent carcinogen-induced transformation. Finally, immunohistochemistry on human lung cancer specimens revealed a significant increase in DNMT1, HDAC1, HDAC2, and HDAC3 expression, supporting our hypotheses that class I HDACs are mediators of DNMT1 stability. In summary, our study provides evidence for an important role of class I HDACs in controlling the stability of DNMT1 and suggests that HDAC inhibition could be an attractive approach for lung cancer chemoprevention. Cancer Prev Res; 7(3); 351–61. ©2014 AACR.

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PDF file - 1302K, S1: Primary transcript qPCR indicates HDAC1 and 2 are regulated transcriptionally, HDAC3 is not. S2: Analysis of golden gate methylation array identifies 42/807 genes hypermethylated after long term carcinogen exposure. S3: Cycloheximide 2ug/ml final was added to cultures of 3kt or T31 cells for the indicated times (hours). Cycloheximide blocks the VPA induced degradation of DNMT1. S4: HEK293 cells transiently transfected with pDest51-V5tagged HDAC1, 2 demonstrates protective role of HDACs on DNMT1. % indicates percent fold change of DNMT1 expression over parental cell. S5: 3kt cells stably transfected with HDAC3 demonstrates protective role on DNMT1. % indicates percent fold change of DNMT1 expression over parental cell. S6: Overexpression of HDAC1 or HDAC3 do not increase cellular proliferation compared to empty vector control. S7: 6 tumor samples and matched resected histologically normal lungs were stained for PCNA. PCNA staining intensity is quantified by weighted index. S8: AKT phosphorylation (S473) was assessed with and without VPA exposure in several different passages of carcinogen exposure. VPA had no effect on AKT phosphorylation. S9: T31 wells were grown in triplicates in logarithmic phase and counted manually. VPA's effects on proliferation were assessed at 0.1 mM and 0.5 mM. No discernable effect of particularly the low dose of VPA on growth was observed. S10: carcinogen-transformed T31 cells were passaged for 28 days in the presence of VPA (0.5mM). Bisulfite sequencing of the RASSF1 locus was performed on cells derived from day 0 and day 28.

DNA methylation is an early event in bronchial carcinogenesis and increased DNA methyltransferase (DNMT)1 protein expression is a crucial step in the oncogenic transformation of epithelia. Here, we investigate the role of class I histone deacetylases (HDAC) 1 to 3 in the stabilization of DNMT1 protein and as a potential therapeutic target for lung cancer chemoprevention. Long-term exposure of immortalized bronchial epithelial cells (HBEC-3KT) to low doses of tobacco-related carcinogens led to oncogenic transformation, increased HDAC expression, cell-cycle independent increased DNMT1 stability, and DNA hypermethylation. Overexpression of HDACs was associated with increased DNMT1 stability and knockdown of HDACs reduced DNMT1 protein levels and induced DNMT1 acetylation. This suggests a causal relationship among increased class I HDACs levels, upregulation of DNMT1 protein, and subsequent promoter hypermethylation. Targeting of class I HDACs with valproic acid (VPA) was associated with reduced HDAC expression and a profound reduction of DNMT1 protein level. Treatment of transformed bronchial epithelial cells with VPA resulted in reduced colony formation, demethylation of the aberrantly methylated SFRP2 promoter, and derepression of SFRP2 transcription. These data suggest that inhibition of HDAC activity may reverse or prevent carcinogen-induced transformation. Finally, immunohistochemistry on human lung cancer specimens revealed a significant increase in DNMT1, HDAC1, HDAC2, and HDAC3 expression, supporting our hypotheses that class I HDACs are mediators of DNMT1 stability. In summary, our study provides evidence for an important role of class I HDACs in controlling the stability of DNMT1 and suggests that HDAC inhibition could be an attractive approach for lung cancer chemoprevention. Cancer Prev Res; 7(3); 351–61. ©2014 AACR.

Introduction: Epigenetic alterations have been identified as key events in the pathogenesis of non-small cell lung cancer (NSCLC). Previous studies have shown that smoke carcinogen exposure leads to increases in DNA-methyltransferase (DNMT1) protein expression, and subsequent epigenetic alteration - mediating early bronchial carcinogenesis. DNMT1 protein turnover is regulated by posttranslational modifications such as acetylation, phosphorylation and methylation. In lung cancer, increased levels of type I histone deacetylases (HDACs) HDAC1 and HDAC3 have been associated with poor outcomes. Clinically, HDAC inhibitors have shown promise for the treatment of advanced NSCLC. Methods: In this study we use carcinogen exposed bronchial epithelial cells as a model of early carcinogenesis. We assayed these cells using various methods including soft-agar assays, methylation specific PCR and methylation array, siRNA knockdown, immunoblotting, co-immunoprecipitation and qPCR. We also analyzed a cohort of patient samples for expression of DNMT1 and HDACs by IHC. Results: In this study, we describe that a close interaction between DNMT1 and class I histone deacetylases (HDAC)s after carcinogen exposure and in human lung cancer samples. We show that this interaction is associated with DNMT1 protein stabilization, can be disrupted with HDAC inhibition, and that HDAC inhibition leads to a loss of DNMT1 protein levels through an acetylation driven mechanism. Conclusions: HDAC inhibition acts to epigenetically reprogram carcinogen exposed epithelia in part by blocking HDAC-DNMT1 interaction, thereby forcing the degradation of DNMT1. This highlights a possible role of HDAC inhibitors for the chemoprevention of lung cancer. Citation Format: Seth A. Brodie, Ge Li, Adam El-Kommos, Suresh S. Ramalingam, Mahdusmita Behera, Jeanne Kowalski, Khanjan Gandhi, Fadlo H. Khuri, Paula Vertino, Johann C. Brandes. Class I HDACs are mediators of smoke-carcinogen induced stabilization of DNMT1 and serve as promising targets for chemoprevention of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B30.