Global Identification of Genes Targeted by Dnmt3b for Epigenetic Silencing in Lung Cancer

ORIGINAL ARTICLE Global identiﬁcation of genes targeted by DNMT3b for epigenetic silencing in lung cancer

I Teneng, CS Tellez, MA Picchi, DM Klinge, CM Yingling, AM Snider, Y Liu and SA Belinsky

The maintenance cytosine DNA methyltransferase DNMT1 and de novo methyltransferase DNMT3b cooperate to establish aberrant DNA methylation and chromatin complexes to repress gene transcription during cancer development. The expression of DNMT3b was constitutively increased 5–20-fold in hTERT/CDK4-immortalized human bronchial epithelial cells (HBECs) before treatment with low doses of tobacco carcinogens. Overexpression of DNMT3b increased and accelerated carcinogen-induced transformation. Genome-wide proﬁling of transformed HBECs identiﬁed 143 DNMT3b-target genes, many of which were transcriptionally regulated by the polycomb repressive complex 2(PRC2) complex and silenced through aberrant methylation in non-small-cell lung cancer cell lines. Two genes studied in detail, MAL and OLIG2, were silenced during transformation, initially through enrichment for H3K27me3 and H3K9me2, commonly methylated in lung cancer, and exert tumor suppressor effects in vivo through modulating cancer-related pathways. Re-expression of MAL and OLIG2 to physiological levels dramatically reduced the growth of lung tumor xenografts. Our results identify a key role for DNMT3b in the earliest stages of initiation and provide a comprehensive catalog of genes targeted for silencing by this methyltransferase in non-small-cell lung cancer.

Oncogene (2015) 34, 621–630; doi:10.1038/onc.2013.580; published online 27 January 2014 Keywords: DNMT3b; lung cancer; DNA methylation; MAL; OLIG2

INTRODUCTION and progression.10,15–17 In contrast, deletion of DNMT3a using a Lung cancer is responsible for over 1.5 million deaths globally, conditional mouse tumor model suggests this methyltransferase with non-small-cell lung cancers (NSCLCs) comprising adenocarci- through deficiency may promote tumor growth and progression, noma (AdC) and squamous cell carcinoma (SCC) accounting for but not initiation.17 DNMT3b can functionally interact with DNMT1 470% of cases.1,2 Epigenetic silencing of cell regulatory and to initiate new patterns of gene silencing during cancer.18 tumor suppressor genes through chromatin remodeling and Simultaneous disruption of DNMT1 and DNMT3b in HCT116 cells cytosine methylation are critical for the initiation and progression (no effect on DNMT3a expression) resulted in ablation of 99% of of lung cancer.3,4 Candidate gene studies and genome-wide methyltransferase activity, an effect that surpassed the additive approaches from our group and others have identified hundreds effect of targeting either gene individually and resulted in of epigenetically silenced genes in NSCLCs.5–8 Most recently, The re-expression of p16, TIMP3 and IGF2.19 DNMTs also participate Cancer Genome Atlas (TCGA) has used the Infinium Human in cytosine methylation-independent transcriptional silencing by methylation 450K array (HM450K) to map the methylation state of recruiting transcriptionally repressive complexes that include cytosines across the genome of more than a hundred each of methyl-CpG binding domain proteins and polycomb proteins to AdCs and SCCs, providing a rich resource for validating findings the nucleosome.20 Thus, DNMT1 and DNMT3b appear to play a from hypothesis-driven studies such as those investigating factors major cooperative role in establishing aberrant DNA methylation underlying the reprogramming of the epigenome.9 and chromatin complexes to repress transcription. Although this Three cytosine DNA methyltransferase enzymes (DNMTs) important role for DNMT3b in cancer development has been catalyze DNA methylation at the 50 cytosine of CpG sites. DNMT1 established, only five genes have been identified as targets for is the most abundant, and functions as a maintenance silencing by this methyltransferase.21–23 Our group has identified methyltransferase to establish normal methylation patterns key molecular changes driving transformation and clonal during embryogenesis, and to reproduce these patterns during outgrowth of pre-neoplastic cells during exposure to replication, but can also initiate de novo methylation following carcinogens in tobacco smoke using an in vitro model of hTERT/ DNA damage.10–12 DNMT1 expression is increased during CDK4-immortalized human bronchial epithelial cells (HBECs).24 premalignancy and in cancers including lung (up to 200-fold).13 Low-dose weekly treatment (12 weeks) of HBEC lines with DNMT3a and DNMT3b function as de novo methyltransferases to genotoxic but not cytotoxic doses of the carcinogens N-nitroso- establish new methylation patterns by targeting normally N-methylurea (MNU) and benzo(a)pyrene-diolepoxide 1 (BPDE) unmethylated CpG sites on gene promoters.14 This function of induced transformation as seen by colony formation in soft agar; DNMT3a and DNMT3b could contribute to the reprogramming of however, the transformed cells did not grow as xenografts in nude the epigenome seen in lung and other cancers. This supposition is mice.24 Levels of DNMT1 protein increased during carcinogen supported for DNMT3b whose expression is increased in exposure and in transformed clones, whereas levels of DNMT3b hepatocellular, breast, oral and lung cancer to affect initiation were only increased in the transformed cells. Moreover,

Lung Cancer Program, Lovelace Respiratory Research Institute, Albuquerque, NM, USA. Correspondence: Dr SA Belinsky, Lung Cancer, Lovelace Respiratory Research Institute, LRRI, 2425 Ridgecrest Drive SE, Albuquerque, 87108 NM, USA. E-mail: [email protected] Received 16 October 2013; revised 22 November 2013; accepted 10 December 2013; published online 27 January 2014 Genes targeted by DNMT3b for epigenetic silencing I Teneng et al 622 knockdown of DNMT1, but not DNMT3a or DNMT3b, prevented weeks of carcinogen treatment. Although no colonies were carcinogen-induced transformation and methylation of key tumor observed after 4 weeks of carcinogen exposure, colonies were suppressor genes such as E-cadherin.24 Thus, this premalignancy observed in the HBEC2 clones overexpressing DNMT3b at week 8, model can identify key molecular changes that drive cell but not in controls, indicating that elevated DNMT3b expression transformation and the likely clonal outgrowth of preneoplastic not only increases transformation efficiency, but also accelerates lung epithelial cells that occurs in the chronic smoker. transformation (Figures 1d and e, week 8). After 4 weeks of The purpose of the current study was to assess the effect of carcinogen exposure, HBECs had acquired fibroblast-like constitutively increasing the expression of DNMT3b in HBECs mesenchyme-like morphology and gene and microRNA expres- before treatment with low doses of tobacco carcinogens on sion signatures that persisted throughout the 12 weeks and in transformation and malignancy and to use this model to facilitate transformed cells (not shown), consistent with our previous the comprehensive identification of methylated genes regulated work.25 Transformed DNMT3b-expressing HBEC2 clones by DNMT3b. The prevalence for methylation of novel regulated recovered from soft agar did not form tumors when inoculated DNMT3b genes was evaluated in primary tumors, validated into nude mice (not shown). through the TCGA, and the function and tumor suppressing properties of selected genes were characterized. Overexpression of DNMT3b in HBEC2 targets specific genes for methylation during carcinogen-induced transformation RESULTS The HM450K array that interrogated 14 155 genes (27% of which are polycomb repressive complex 2(PRC2) regulated) was used to Expression of DNMT3b is increased in NSCLC cell lines and primary lung tumors comprehensively identify methylated DNMT3b-regulated genes resulting from transformation. The methylation profile of trans- The expression of DNMT3b was quantified in NSCLCs and in formed HBEC2 transfected with empty plasmid (HBEC2-pCDNA4- primary lung tumors to provide a physiological range to use when MBT) was compared with transformed HBECs overexpressing selecting the stable HBEC2 clones overexpressing this gene and to DNMT3b (designated HBEC2-DNMT3b5-MBT and HBEC2- extend the limited literature on the change in expression for this DNMT3b12-MBT). Genes methylated in nontransformed (sham) gene in lung cancer. Of the 22 cancer cell lines, 20 (91%) showed and in transformed HBEC2 transfected with empty plasmid or significantly increased DNMT3b expression (Po0.05) ranging DNMT3b were excluded, resulting in 143 genes that were from 2 to 25-fold compared with the nontransformed HBEC2 methylated exclusively in one or both DNMT3b-overexpressing (Figure 1a). DNMT3b expression was determined in 23 AdC and 18 transformed HBEC2 (Supplementary Figure S2A and SCC, respectively, in comparison with paired normal tissue Supplementary Table S1). This list was greatly enriched (55%) for (Figure 1b). DNMT3b expression was increased 1.34–29-fold in genes that are regulated by the PRC2 proteins that include EZH2, 65% of AdC (mean ¼ 3.5-fold, s.d. ¼ 1.2, P ¼ 0.03) relative to EED and SUZ12 responsible for catalyzing the trimethylation at normal tissue, and 1.5–37-fold in 89% of SCC (mean ¼ 5.6-fold, Lysine27 of histone H3 that contributes to a repressive chromatin s.d. ¼ 2.1, P ¼ 0.03). state.26 The involvement of these genes for development of malignant lung cancer was assessed by determining their Generation and characterization of HBEC2 clones overexpressing prevalence in 17 NSCLC cell lines interrogated previously for DNMT3b genome-wide methylation using the HM450K array Twelve HBEC2 clones were characterized following transfection (Supplementary Table S2). Of the 143 DNMT3b-target genes, 133 with a DNMT3b expression plasmid and selection with zeocin. Two (93%) were methylated in multiple tumor lines (at least 2–18 cell clones, designated HBEC2-DNMT3b5 and HBEC2-DNMT3b12, lines per gene). Ingenuity Pathway Analysis revealed that these showed 5- and 20-fold increased expression of DNMT3b compared 143 DNMT3b-methylated genes are involved in regulating with the control plasmid, levels that reflected the range of important molecular and cellular functions disrupted in cancer expression seen in primary tumors (Figures 1b and c). Damiani that include movement, growth, development, proliferation, et al.24 showed an association between DNA repair capacity (DRC) morphology, death and survival (Supplementary Table S3). and transformation efficiency of HBECs following carcinogen exposure, and therefore we examined whether constitutive DNMT3b-target genes are commonly methylated in primary lung overexpression of DNMT3b would modify DRC. HBEC2s were AdC and SCC treated with BPDE for 1 h and cytochalasin for 27 h and DRC was Six DNMT3b-methylated genes were selected for additional assessed by the cytokinesis-block micronucleus assay that validation in primary NSCLCs based on being silenced in multiple quantifies the number of chromosome breaks. Expression of tumor cell lines, showing no previous evidence for alteration in lung DNMT3b in HBEC2 did not affect DRC (Supplementary Figure S1A). cancer, and having distinct proposed or validated functions such as Overexpression of DNMT3b in HBEC2 did not affect DNMT1 regulating WNT signaling and stem cell differentiation (Table 1).27,28 expression (Supplementary Figure S1B), and did not induce The selected genes were Frizzled-related protein-b (FRZB), cadherin- spontaneous transformation based on lack of colony formation 7(CDH7), oligodendrocyte lineage transcription factor 2 (OLIG2), in soft agar (not shown). homeobox B13 (HOXB13), myelin and lymphocyte-associated protein (MAL)andtropomyosina-1 (TPM1). HOXB13 has Overexpression of DNMT3b accelerates and increases previously been reported as a DNMT3b-regulated gene in carcinogen-induced transformation efficiency colorectal cancer.22 Combined bisulfite-restriction analysis The two HBEC2 clones overexpressing DNMT3b were treated with (COBRA) was first used to confirm the methylation state of these MNU and BPDE, once a week for 1 h for 12 weeks in conjunction genes in the transformed DNMT3b-overexpressing cells compared with HBEC2 transfected with empty plasmid (HBEC2-pCDNA4), to with untreated and transformed HBEC2 containing empty plasmid. determine the effect of this de novo methyltransferase on The results confirmed exclusive or dramatically increased extent of carcinogen-induced transformation efficiency. HBEC2-DNMT3b5 methylation of all six genes in the transformed DNMT3b- and HBEC2-DNMT3b12 had a 2.8- and 2.2-fold increase (Po0.05) in overexpressing HBEC2 lines (Supplementary Figure S2B). The transformation efficiency, respectively, after 12 weeks of treatment prevalence of methylation was determined by methylation-specific compared with the empty plasmid control (Figures 1d and e, PCR (MSP) in AdC (n ¼ 20 each from smokers and never smokers) week 12). The effect of DNMT3b expression on the time to and SCC from smokers (n ¼ 20). Methylation prevalence was transformation was assessed by soft agar assays after 4 and 8 25–95% in tumors from smokers and 9–70% in tumors from never

Oncogene (2015) 621 – 630 & 2015 Macmillan Publishers Limited Genes targeted by DNMT3b for epigenetic silencing I Teneng et al 623

Figure 1. DNMT3b is overexpressed in lung cancer and increases transformation efficiency of HBECs. (a, b) DNMT3b expression is increased in NSCLC cell lines (a) and in lung tumor/normal pairs (b). Quantitative real-time PCR (RT–qPCR) was performed using complementary DNA (cDNA) from RNA isolated from NSCLC cell lines or in paired tumor/normal samples from lung cancer patients. Expression is normalized to b-actin. Expression was significantly increased in 20 of 22 cell lines (Po0.05). DNMT3b expression was increased 1.34–29-fold in 65% of AdC (*Po0.05) relative to normal tissue, and 1.5–37-fold in 89% of SCC (*Po0.05). (c) Generation of DNMT3b-overexpressing HBECs. HBECs were transfected with DNMT3b or empty pCNA4 plasmids and stable clones selected and expression verified by RT–qPCR and western blot. (d) DNMT3b overexpression increases transformation efficiency and accelerates transformation in carcinogen-treated HBECs. HBECs expressing DNMT3b or pCNA4 plasmid were treated with MNU/BPDE for 12 weeks (WK12) and seeded in soft agar to assess colony formation. DNMT3b increases transformation efficiency 2.2–2.8-fold and accelerates transformation, as colonies were visible after 8 weeks (WK8) only in HBECs with DNMT3b-expressing plasmids (d, e). Statistical values were calculated using a paired, two-tailed t-test. Error bars indicate mean±s.d. smokers (Table 2). No significant difference by tumor histology was more than three times higher in AdC from smokers compared with seen. HOXB13, MAL and FRZB showed 2–3-fold increased pre- never smokers and this significant difference (P ¼ 0.02) remained valence for methylation in smokers compared with nonsmoker after approximately doubling the samples sizes interrogated for AdCs. Interestingly, the prevalence for methylation of MAL was methylation of this gene.

& 2015 Macmillan Publishers Limited Oncogene (2015) 621 – 630 Genes targeted by DNMT3b for epigenetic silencing I Teneng et al 624 CpGs within the promoter of the MAL and OLIG2 are selectively mark and de novo cytosine methylation.33 Thus, the timing for methylated in TCGA lung tumors compared with normal samples enrichment of H3K27me3 and another common repressive MAL, because of its selectivity for methylation in tumors from chromatin mark, H3K9me2, with respect to loss of gene smokers, and OLIG2, whose tumor suppressor function to date has expression and methylation of MAL and OLIG2 was evaluated been limited to the nervous system, were selected for more after 4 weeks of carcinogen treatment and in transformed clones extensive characterization.29–32 HM450K arrays generated by TCGA compared with nontransformed HBEC2. In addition, because the are publically available at https://tcga-data.nci.nih.gov for 369 lung PRC2 complex can also recruit DNMT3b and these genes were tumors and 74 corresponding normal lung samples; however, identified based on overexpression of this methyltransferase, smoking history is not annotated. Four and eight CG oligonucleotide chromatin immunoprecipitation (ChIP) for this gene was also probes reside in the region interrogated above for methylation state performed. ChIP followed by quantitative real-time PCR was used of the MAL and OLIG2 genes, respectively. Average b values and to define the enrichment over À 100 bp and À 1000 bp upstream range for each probe in normal tissue were compared with values in of the transcriptional start site of the MAL and OLIG2 promoter the tumors. Normal lung tissue contains more than 40 cell types regions. Maximum enrichments for H3K27Me3, H3K9Me2 and (B70% are not epithelial) and the average b value ranged from 0.7 DNMT3b were localized to À 464 to À 386 bp and À 932 bp to to0.24andfrom0.09to0.17forOLIG2 and MAL (Tables 3 and 4). À 833 bp upstream of the transcriptional start site for MAL and Thus, as matched normal tissue was not available for most tumors OLIG2, respectively (Figure 2). DNMT3b was enriched 2- to 6-fold at and with the cell heterogeneity in normal tissue, we elected to set both gene promoters in the DNMT3b-overexpressing HBEC lines at conservative cutoffs for classification of methylation using b X0.45 4 weeks and in transformed cells, whereas only modest for OLIG2 and MAL to estimate prevalence for methylation of each enrichment was seen in HBEC2 containing the empty plasmid probe. Using these criteria, highly significant differences were seen (Figure 2a). GATA2, a gene not identified as being regulated for each probe regarding prevalence of methylation and when by DNMT3b in our screen, but regulated by PRC2, was used as averaged across probes, approximate to that seen by MSP in a control and was not enriched for DNMT3b (Supplementary primary tumors (Tables 2–4). No significant differences were Figure S3). observed between AdC and SCC (not shown). H3K27me3 was enriched at the MAL and OLIG2 promoters of HBEC2 empty plasmid at week 4 (1.5–5-fold) and in the corresponding transformed cells (4–30-fold; Figure 2b). DNMT3b- Silencing of MAL and OLIG2 is initiated through methylation of histone H3 Tails during carcinogen-induced transformation overexpressing cells had further increased enrichment (up to 30-fold) for H3K27me3 compared with control cells (1.3–4-fold) at MAL and OLIG2 are PRC2-target genes that as a group are enriched week 4 that generally persisted in transformed cells for these for silencing in many cancers through acquiring the H3K27me3 genes. H3K9me2 was also enriched at the MAL and OLIG2 promoters of HBEC2 empty plasmid at week 4 (0.5–5 fold) and in transformed cells (0.2–2-fold), again with higher levels of Table 1. Prevalence for methylation of DNMT3b-target genes in NSCLC enrichment observed in the DNMT3b-overexpressing cell lines cell lines (Figure 2c). Interestingly, enrichment of H3K9me2 persisted at the Genes NSCLC cell lines (n ¼ 17) MAL promoter in transformed cells, but was dramatically reduced at the OLIG2 promoter in the transformed cells. Expression of MAL and OLIG2 was not significantly reduced in HBEC2 with empty Ma b-value plasmid at week 4, whereas a modest reduction (38%) was seen in transformed cells (Figure 2d). In contrast, 40–60% reduction was n (%) Mean±s.d. Min–max seen at 4 weeks for both genes in the DNMT3b-overexpressing CDH7 9 (53) 0.53±0.30 0.08–0.93 lines and almost complete silencing (80–100%) was observed in FRZB 16 (94) 0.78±0.18 0.27–0.92 the transformed clones (Figure 2d). COBRA analyses showed no HOXB13 5 (29) 0.40±0.37 0.05–0.95 methylation at week 4 for either gene (Supplementary Figure S4), OLIG2 10 (59) 0.50± 0.30 0.03–0.91 supporting chromatin remodeling as the mechanism for initiating MAL 4 (24) 0.29±0.39 0.02–0.97 TPM1 2 (11) 0.23±0.24 0.08–0.96 the reduced transcription. Consistent with silencing of these genes through chromatin remodeling and promoter methylation in the Abbreviations: M, methylated; NSCLC, non-small-cell lung cancer. The cell DNMT3b-overexpressing transformed cells, modest and strong lines studied were Calu6, Calu3, H23, H358, H1229, H1434, H1568, H1975, re-expression was seen following TSA (Trichostatin A) and DAC H1993, H2023, H2085, H2170, H2228, SKMES1, SW900, HCC827 and PC9. treatment (Supplementary Figure S4). In the Calu6 and H358 aProbes with bX0.45 were considered methylated. tumor lines where methylation of MAL and OLIG2 is even more

Table 2. Prevalence for methylation of DNMT3b-target genes in primary lung adenocarcinomas and squamous cell carcinomas

Gene Adenocarcinoma Squamous cell carcinoma

(Never smokers) (Smokers) (Smokers)

Prevalence Percent Prevalence Percent Prevalence Percent

CDH7 14/20 70% 15/20 75% 19/20 95% FRZB 6/20 30% 12/20 60% 12/20 60% HOXB13 3/20 15% 9/20 45% 5/20 25% OLIG2 11/20 55% 11/20 55% 9/20 45% MALa 3/34 9% 13/39 33% 10/40 25% TPM1 10/20 50% 9/20 45% 14/20 75%

aP ¼ 0.02 when comparing methylation in adenocarcinomas from smokers and never smokers.

Oncogene (2015) 621 – 630 & 2015 Macmillan Publishers Limited Genes targeted by DNMT3b for epigenetic silencing I Teneng et al 625 Table 3. Comparison of methylated probes within the MAL promoter CpG island between lung tumor vs normal tissue from NSCLC patients

HM450K probe ID Normal tissue (n ¼ 74) NSCLC (n ¼ 368) P-value (t-test)

Ma b-value M b-value

n (%) Mean±s.d. Min–max n (%) Mean±s.d. Min–max

cg05142617 0 (0) 0.24±0.05 0.14–0.39 250 (68) 0.50±0.18 0.04–0.87 8.68E À 30 cg21245652 0 (0) 0.27± 0.02 0.23–0.27 215 (58) 0.47±0.10 0.16–0.75 1.97E À 44 cg09983051 0 (0) 0.13±0.03 0.08–0.22 26 (7) 0.24±0.12 0.04–0.62 6.24E À 15 cg04804539 0 (0) 0.08±0.01 0.03–0.16 21 (6) 0.19±0.13 0.03–0.64 5.18E À 12 Abbreviations: M, methylated; NSCLC, non-small-cell lung cancer. aProbes with bZ0.45 were considered methylated.

Table 4. Comparison of methylated probes within the OLIG2 promoter CpG island between lung tumor vs normal tissue from NSCLC patients

HM450K probe ID Normal tissue (n ¼ 74) NSCLC (n ¼ 368) P-value (t-test)

Ma b-value M b-value

n (%) Mean±s.d. Min–max n (%) Mean±s.d. Min–max

cg08729810 0 (0) 0.18±0.04 0.11–0.28 28 (8) 0.24±0.11 0.05–0.63 8.34E À 06 cg27254482 0 (0) 0.04±0.02 0.01–0.11 105 (29) 0.30±0.19 0.01–0.84 3.07E À 27 cg03696345 0 (0) 0.07±0.01 0.01–0.15 29 (9) 0.20±0.14 0.04–0.86 1.94E À 12 cg15299832 0 (0) 0.02±0.01 0.01–0.04 26 (7) 0.11±0.15 0.01–0.79 3.56E À 08 cg08870743 0 (0) 0.02±0.01 0.01–0.08 77 (21) 0.23±0.21 0.01–0.91 4.75E À 17 cg23253569 0 (0) 0.02±0.01 0.01–0.01 73 (20) 0.22±0.21 0.01–0.90 2.75E À 16 cg09793121 0 (0) 0.09±0.04 0.02–0.25 208 (57) 0.41±0.19 0.01–0.78 7.98E À 39 cg22869726 0 (0) 0.09±0.05 0.02–0.27 220 (60) 0.43±0.22 0.01–0.86 1.32E À 36 Abbreviations: M, methylated; NSCLC, non-small-cell lung cancer. aProbes with bX0.45 were considered methylated. extensive (b-values between 0.75 and 0.97), only DAC treatment MBT), Calu6 and H358 cells with or without stable MAL or OLIG2 was effective in inducing gene re-expression (Supplementary expression were compared to identify pathways regulated in part Figure S5). Knocking down DNMT3b in Calu6, H358 or in by MAL or OLIG2. Re-expression of MAL resulted in increased transformed DNMT3b-overexpressing HBEC2s did not induce re- expression (Z50%) of 393, 1592, 947 and 396 genes, whereas 93, expression of MAL or OLIG2 (not shown), consistent with the role 708, 236 and 124 genes were decreased in Calu6, H358, HBEC2- of DNMT1 role in maintaining promoter hypermethylation. DNMT3b5-MBT and HBEC2-DNMT3b12-MBT, respectively (Supplementary Table S4). Re-expression of OLIG2 increased expression (X50%) of 178, 1495, 648 and 689 genes, whereas Restoring MAL and OLIG2 expression dramatically suppresses soft agar colony formation and tumor growth in nude mice 38, 676, 107 and 214 genes were decreased in Calu6, H358, HBEC2-DNMT3b5-MBT and HBEC2-DNMT3b12-MBT, respectively MAL and OLIG2 were re-expressed through stable integration of (Supplementary Table S4). Ingenuity Pathway Analyses imputing expression plasmids in DNMT3b-overexpressing transformed all the top genes affected by re-expression of MAL or OLIG2 HBEC2 clones, Calu6 and H358. Expression of MAL and OLIG2 revealed several important cell functions affected by re-expression were undetectable in parental Calu6 and H358, whereas only faint of either gene in all cell lines. The affected pathways included bands were visible in transformed HBECs (not shown). The clones cell movement, growth, proliferation, cell cycle, death, survival, selected for study exhibited expression levels of these genes that tumor maintenance and cell-to-cell interactions (Supplementary reflected that seen in parental HBEC2 and normal bronchial Figures S6 and S7). epithelial cells (Figure 3a). Re-expression of MAL and OLIG2 significantly reduced soft agar colony formation (Po0.005) in transformed DNMT3b-overexpressing HBEC2s, Calu6 and H358 by DISCUSSION 40–87% (Figure 3b). The tumor sizes and weights at the time of killing of mouse containing H358 and Calu6 xenografts over- This study demonstrates a likely critical role for DNMT3b in the clonal expansion of pre-neoplastic cells. Increased expression of expressing these genes were significantly (Po0.05) reduced compared with control cells (Figures 3c–f). Histopathology findings DNMT3b at levels seen in primary lung tumors increased and showed increased apoptosis and necrosis that was most evident accelerated transformation of HBECs. This premalignancy model in the H358 lines expressing either MAL or OLIG2 (Figure 3g). has enabled a comprehensive identification of the DNMT3b- regulated epigenome that has uncovered more than a hundred genes, many of which are regulated transcriptionally by PRC2. Re-expression of MAL and OLIG2 modulates multiple cancer- Most important, two of the genes studied in detail, MAL and associated pathways OLIG2, are silenced early in premalignancy, commonly methylated Genome-wide transcription patterns of transformed DNMT3b- in NSCLC and exert tumor suppressor effects in vivo through expressing HBEC2s (HBEC2-DNMT3b5-MBT and HBEC2-DNMT3b12- modulating multiple cancer-related pathways.

& 2015 Macmillan Publishers Limited Oncogene (2015) 621 – 630 Genes targeted by DNMT3b for epigenetic silencing I Teneng et al 626

Figure 2. Reduced expression and enrichment of DNMT3b and chromatin marks at the MAL and OLIG2 promoters during carcinogen exposure. (a–d) DNMT3b is recruited to the promoters of MAL and OLIG2 after 4 weeks (WK4) and in transformed cells (a), concurrent with enrichment for H3K27Me3 (b) and H3K9Me2 (Po0.05). ChIP data are plotted as fold enrichment in treated cells (WK4 or transformed MBT) relative to untreated cells for each cell line. (d) Reduced expression of MAL and OLIG2 at WK4 and transformed cells (MBT) relative to untreated cells (Po0.05). Statistical values were calculated using a paired, two-tailed t-test. Error bars indicate mean±s.d.

The striking acceleration and increase in transformation promoters.34,35 This scenario was also seen in our studies in which efficiency observed in the HBEC2 cells overexpressing DNMT3b is DNMT3b enrichment coincided with enrichment of H3K27me3 at likely because of the approximate threefold increase in the the MAL and OLIG2 promoters before methylation. Moreover, we number of genes epigenetically silenced during transformation have previously shown that GATA2, a PRC2-regulated gene, was compared with the empty plasmid control. The regulation of more enriched for H3K27me3 at its promoter, but current studies did than half of the DNMT3b genes by the PRC2 suggests many of not detect enrichment of DNMT3b, thus supporting the selectivity these genes will be silenced early in lung cancer development by which the PRC2 may recruit DNMTs to participate in the through chromatin remodeling mediated in part by enrichment initiation of transcriptional repression.36 Together, this work for H3K27me3. This is consistent with previous studies supporting reinforces previous studies suggesting that DNMT3b is required the hypothesis of a stem cell signature in cancer in which for tumor development and cancer cell survival.22,37 differentiated cells undergoing transformation reacquire stem cell The DNMT3b-regulated genes HOXB13, FRZB, MAL, OLIG2 and characteristics through a process of dedifferentiation thereby CDH7 identified and further characterized in this study showed acquiring the H3K27me3 mark and de novo cytosine methyla- some potential tumor suppressor functions and silencing by tion.25,33 Our past and now current studies provide support for this methylation in cancers other than lung.22,28,38–40 For example, premise by showing previously that exposure of HBECs to tobacco re-expression of HOXB13 abolished tumor growth in nude mice, carcinogens induced a persistent, irreversible and multifaceted and was silenced in colorectal cancer at a prevalence (B45%) dedifferentiation program marked by epithelilial-to-mesenchymal similar to that observed in our study of primary lung AdC.22 transition and the emergence of stem cell-like properties.25 The Methylation of FRZB is seen in 30% of high-grade, noninvasive epithelilial-to-mesenchymal transition seen and recapitulated in bladder cancer.41 This gene functions as a negative regulator of the current studies (not shown) was epigenetically driven, initially Wnt signaling and its enforced expression in an androgen- by chromatin remodeling through H3K27me3 enrichment and independent prostate cancer model decreased colony formation later by ensuing DNA methylation to sustain silencing of tumor- and tumor growth in a xenograft mouse model.28 The Wnt suppressive microRNAs, miR-200b, miR-200c and miR-205. The proteins are a large family of secreted glycoproteins that activate PRC2 can recruit the DNMTs and this may play an important role signal transduction pathways that include cell proliferation and in tumor-specific targeting of de novo methylation at specific gene migration.42 Thus, the common silencing of FRZB in NSCLCs and

Oncogene (2015) 621 – 630 & 2015 Macmillan Publishers Limited Genes targeted by DNMT3b for epigenetic silencing I Teneng et al 627

Figure 3. Stable re-expression of MAL and OLIG2 signiﬁcantly suppresses soft agar colony formation and reduces tumor growth in nude mice. (A) MAL and OLIG2 were stably re-expressed in DNMT3b-expressing transformed HBECs, Calu6 and H358. Expression was compared with parental HBEC2 and with normal bronchial epithelial cells (NBECs). (B) Re-expression of MAL and OLIG2 markedly reduced colony formation compared with transformed control cells in DNMT3b-expressing HBECs (Po0.05), Calu6 (Po0.005) and H358 (Po0.05). (C–F) Tumor size and weights were signiﬁcantly reduced in mice injected with MAL- and OLIG2-expressing H358 cell lines (Po0.05), whereas modest reductions (P ¼ 0.06) were observed in the more aggressive Calu6 cell line. (G) Hematoxylin and eosin staining of tumors reveal increased necrotic foci and apoptotic cell debris in tumors from H358 expressing MAL and OLIG2. The arrowhead points to foci of necrosis, and the arrows point to apoptotic cell debris. Statistical values were calculated using a paired, two-tailed t-test. Error bars indicate mean±s.d. and *po0.05.

& 2015 Macmillan Publishers Limited Oncogene (2015) 621 – 630 Genes targeted by DNMT3b for epigenetic silencing I Teneng et al 628 during transformation of HBECs could be an important contributor described.24 The number of micronuclei per 1000 binucleated cells was to lung cancer progression. scored and is inversely related to DRC. The function of MAL and OLIG2 in lung cancer was largely unknown before their characterization in this study. Re-expression Soft agar colony formation of MAL and OLIG2 in lung cancer cell lines reduced anchorage- Cells were plated in triplicate in soft agar at a density of 6 Â 103 cells per independent growth in soft agar and tumorigenicity in nude mice, well in six-well plates. The colonies were fixed with methanol, stained with suggesting they may function as tumor suppressor genes. The Trypan blue and counted after 14–21 days. pathways affected through re-expression of these genes that include cell growth, proliferation, death, survival, movement, cell Gene expression analysis cycle, tumor maintenance and cell-to-cell interactions support this conclusion. Originally, MAL, a 17-kDa vesicle integral protein Quantitative real-time PCR was carried out using TaqMan assays (Life Technologies, Grand Island, NY, USA) with gene expression normalized to present in epithelial cells, Schwann cells and oligodendrocytes, PCNA or to b-actin using the 2 À DDCt method. Gene expression of MAL and was shown to associate with glycosphingolipids and to be OLIG2 was performed by conventional PCR using primers spanning exon– involved with organization, transport and maintenance of glyco- exon boundaries to avoid DNA amplification. sphingolipid-enriched membranes.43,44 MAL gene silencing has been reported in esophageal, breast and gastric cancer, and our Methylation and expression arrays findings also implicate epigenetic regulation of this gene predominantly in smoking-associated NSCLC.38,45,46 OLIG2 is a Bisulfite-modified DNA (1 mg) isolated from transformed clones and 32-kDa basic loop helix loop transcription factor expressed in both controls were hybridized to the HM450K Beadchip (Illumina, San Diego, CA, USA) for methylation arrays. Total RNA was isolated, converted to cRNA developing and mature vertebrate central nervous systems where 27 and hybridized using the Illumina whole-genome expression Direct it can induce neuronal stem cell differentiation. Previously, hybridization Assay. reported tumor suppressor functions of OLIG2 were limited to the nervous system in glioblastomas and human glial tumors where it 29–32 Combined bisulfite-restriction analysis activates p27 and RhoA. Our work has identified multiple 5 additional pathways in which these genes likely interact to effect COBRA of genomic DNA was performed as described previously. CpG regulation of lung epithelial cells. islands of different genes were amplified with primers specific for bisulfite- converted DNA, and digested for 2 h with BstuI. Digested products were The HBEC in vitro model mimics the clonal outgrowth of run alongside undigested products on a 2% agarose gel. Primer sequences preneoplastic cells and continues to provide new insights under- are available upon request. lying epigenetic deregulation, genes targeted for silencing and the genes and pathways affected in the earliest stages of initiation and progression. The transformation of lung epithelial cells to full Methylation-specific PCR malignancy likely requires the acquisition of genetic alterations MSP was used to screen for promoter methylation of six DNMT3b- such as KRAS and/or p53 mutations that are generally late changes regulated genes in lung AdC and SCC. Genes were scored as methylated if in tumor development and not present yet in our model of a PCR product was detected. Methylation-specific primer sequences and PCR conditions are available upon request. premalignancy.47,48 Indeed, Sato et al.49 showed that introducing combinations of oncogenic changes (p53, KRAS and MYC) could lead to transformation of HBECs to full malignancy. However, our ChIP assays and ChIP–PCR elucidation of the DNMT3b-methylated epigenome, its recruitment Antibodies specific to DNMT1, DNMT3b (Imgenex, San Diego, CA, USA), by PRC2 and contribution to premalignancy provides new total H3, trimethyl H3K27 and dimethyl H3K9 (Abcam, Cambridge, MA, opportunities to evaluate the efficacy of emerging targeted USA) were used to capture protein–DNA complexes. Quantitative PCR was epigenetic inhibitors as a cancer prevention strategy for lung carried out in triplicate, using Power SYBR Green PCR Master Mix (Applied 35 Biosystems, Carlsbad, CA, USA). The primer sequences and conditions for cancer. quantitative PCR are available upon request.

MATERIALS AND METHODS DAC and TSA treatment Cell culture, carcinogen exposure and tumor specimens Cells were treated with vehicle (ethanol), 300 nM TSA for 18 h or 500 nM DAC for 96 h with media containing drug changed daily. HBEC2 immortalized with hTERT and CDK4 was obtained from Drs Shay and Minna (Southwestern Medical Center, Dallas, TX, USA). Cell culture conditions have been described previously.24 HBEC2 was exposed to Nude mouse assay 1.0 mM MNU and 0.1 mM BPDE for 1 h once a week for 12 weeks. Cells were Female athymic NCr-nu/nu mice (5 per group) were injected subcuta- harvested after 4, 8 and 12 weeks for molecular analyses. NSCLC cell lines neously with 1 Â 106 cells and tumor size monitored weekly.50 After killing, were obtained from the American Type Culture Collection (Manassas, VA, tumors were weighed and subjected to histological analyses. USA). Primary lung tumors and distant normal tissue were obtained as described previously.5 Statistical analysis All data are presented as mean ±s.d. Statistical analyses were performed Expression plasmids and transfections using a paired two-tailed test. Because of the strong association between Complementary DNA encoding DNMT3b was cloned into pCDNA4 plasmid methylation of CpGs around the transcriptional start site and gene (Invitrogen, Carlsbad, CA, USA) to generate the pCDNA4-DNMT3b silencing, our analytic strategy for the HM450K arrays focused on this expression plasmid that was conﬁrmed by sequencing. MAL and OLIG2 region to assess the methylation status of 84 735 CpG oligonucleotide expression plasmids were obtained from GeneCopoeia (Rockville, MD, probes within 200 base pairs 50 of the transcriptional start site and USA). HBEC2 or tumor lines were transfected using the Neon Transfection extending through the 50 untranslated region. Average signal intensity System (Invitrogen). Stable clones were identiﬁed through selection with between the methylated and unmethylated probes was determined, and a zeocin or hygromycin depending on the expression plasmid. Plasmid- b-value from 0 to 1 (fully methylated) was calculated. Genes whose average driven gene expression was validated at the RNA and/or protein level. b-values were X0.2 across the interrogated region in the untransformed HBEC2 were excluded from further analysis. Average b-values X0.45 for genes across the entire region interrogated were scored as positive for DNA repair capacity methylation. Pathway analyses were carried out using Ingenuity (Ingenuity HBEC2 lines were treated with BPDE for 1 h and cytochalasin for 27 h. DRC Pathway Analyses Software Version 6 (Redwood City, CA, USA)) for genes was measured by the cytokinesis-block micronucleus assay as previously whose expression was increased or decreased by 50% in cell lines

Oncogene (2015) 621 – 630 & 2015 Macmillan Publishers Limited Genes targeted by DNMT3b for epigenetic silencing I Teneng et al 629 overexpressing MAL or OLIG2 compared with controls. The P-values were 19 Rhee I, Bachman KE, Park BH, Jair KW, Yen RW, Schuebel KE et al. DNMT1 and calculated using Fisher’s exact tests. DNMT3b cooperate to silence genes in human cancer cells. Nature 2002; 416: 552–556. 20 Fuks F, Burgers WA, Brehm A, Hughes-Davies L, Kouzarides T. DNA methyl- CONFLICT OF INTEREST transferase Dnmt1 associates with histone deacetylase activity. Nat Genet 2000; 24: 88–91. The authors declare no conflict of interest. 21 Fan H, Chen L, Zhang F, Quan Y, Su X, Qiu X et al. MTSS1, a novel target of DNA methyltransferase 3B, functions as a tumor suppressor in hepatocellular carcinoma. Oncogene 2012; 31: 2298–2308. ACKNOWLEDGEMENTS 22 Ghoshal K, Motiwala T, Claus R, Yan P, Kutay H, Datta J et al. HOXB13, Data generated by The Cancer Genome Atlas (TCGA) project established by the NCI a target of DNMT3B, is methylated at an upstream CpG island, and and NHGRI were used to validate part of our findings. The dbGaP accession number functions as a tumor suppressor in primary colorectal tumors. PLoS One 2010; 5: for TCGA data is phs000178.v8.p7. Information about TCGA and the investigators and e10338. institutions that constitute the TCGA research network can be found at http:// 23 Linhart HG, Lin H, Yamada Y, Moran E, Steine EJ, Gokhale S et al. Dnmt3b pro- cancergenome.nih.gov/. We acknowledge the following for technical support: Kieu motes tumorigenesis in vivo by gene-specific de novo methylation and tran- Do, Randall Willink, Christopher Dagucon and Daniel E Juri. We also thank Dr scriptional silencing. Genes Dev 2007; 21: 3110–3122. 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Oncogene (2015) 621 – 630 & 2015 Macmillan Publishers Limited