Weiguo Han

Gene promoter methylation assayed in exhaled breath, with differences in smokers and lung cancer patients.

BackgroundThere is a need for new, noninvasive risk assessment tools for use in lung cancer population screening and prevention programs.MethodsTo investigate the technical feasibility of determining DNA methylation in exhaled breath condensate, we applied our previously-developed method for tag-adapted bisulfite genomic DNA sequencing (tBGS) for mapping of DNA methylation, and adapted it to exhaled breath condensate (EBC) from lung cancer cases and non-cancer controls. Promoter methylation patterns were analyzed in DAPK, RASSF1A and PAX5β promoters in EBC samples from 54 individuals, comprised of 37 controls [current- (n = 19), former- (n = 10), and never-smokers (n = 8)] and 17 lung cancer cases [current- (n = 5), former- (n = 11), and never-smokers (n = 1)].ResultsWe found: (1) Wide inter-individual variability in methylation density and spatial distribution for DAPK, PAX5β and RASSF1A. (2) Methylation patterns from paired exhaled breath condensate and mouth rinse specimens were completely divergent. (3) For smoking status, the methylation density of RASSF1A was statistically different (p = 0.0285); pair-wise comparisons showed that the former smokers had higher methylation density versus never smokers and current smokers (p = 0.019 and p = 0.031). For DAPK and PAX5β, there was no such significant smoking-related difference. Underlying lung disease did not impact on methylation density for this geneset. (4) In case-control comparisons, CpG at -63 of DAPK promoter and +52 of PAX5β promoter were significantly associated with lung cancer status (p = 0.0042 and 0.0093, respectively). After adjusting for multiple testing, both loci were of borderline significance (padj = 0.054 and 0.031). (5) The DAPK gene had a regional methylation pattern with two blocks (1)~-215~-113 and (2) -84 ~+26); while similar in block 1, there was a significant case-control difference in methylation density in block 2 (p = 0.045); (6)Tumor stage and histology did not impact on the methylation density among the cases. (7) The results of qMSP applied to EBC correlated with the corresponding tBGS sequencing map loci.ConclusionOur results show that DNA methylation in exhaled breath condensate is detectable and is likely of lung origin. Suggestive correlations with smoking and lung cancer case-control status depend on individual gene and CpG site examined.

Candidate Dietary Phytochemicals Modulate Expression of Phase II Enzymes GSTP1 and NQO1 in Human Lung Cells

Many phytochemicals possess cancer-preventive properties, some putatively through phase II metabolism-mediated mutagen/oxidant quenching. We applied human lung cells in vitro to investigate the effects of several candidate phytopreventive agents, including green tea extracts (GTE), broccoli sprout extracts (BSE), epigallocatechin gallate (EGCG), sulforaphane (SFN), phenethyl isothiocyanate (PEITC), and benzyl isothiocyanate (BITC), on inducing phase II enzymes glutathione S-transferase P1 (GSTP1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) at mRNA and protein levels. Primary normal human bronchial epithelial cells (NHBE), immortalized human bronchial epithelial cells (HBEC), and lung adenocarcinoma cells (A549) were exposed to diet-achievable levels of GTE and BSE (0.5, 1.0, 2.0 mg/L), or individual index components EGCG, SFN, PEITC, BITC (0.5, 1.0, 2.0 micromol/L) for 24 h, 48 h, and 6 d, respectively. mRNA assays employed RNA-specific quantitative RT-PCR and protein assays employed Western blotting. We found that in NHBE cells, while GSTP1 mRNA levels were slightly but significantly increased after exposure to GTE or BSE, NQO1 mRNA increased to 2- to 4-fold that of control when exposed to GTE, BSE, or SFN. Effects on NQO1 mRNA expression in HBEC cells were similar. NQO1 protein expression increased up to 11.8-fold in SFN-treated NHBE cells. Both GSTP1 and NQO1 protein expression in A549 cells were constitutively high but not induced under any condition. Our results suggest that NQO1 is more responsive to the studied chemopreventive agents than GSTP1 in human lung cells and there is discordance between single agent and complex mixture effects. We conclude that modulation of lung cell phase II metabolism by chemopreventive agents requires cell- and agent-specific discovery and testing.

Genome Wide Methylome Alterations in Lung Cancer

Aberrant cytosine 5-methylation underlies many deregulated elements of cancer. Among paired non-small cell lung cancers (NSCLC), we sought to profile DNA 5-methyl-cytosine features which may underlie genome-wide deregulation. In one of the more dense interrogations of the methylome, we sampled 1.2 million CpG sites from twenty-four NSCLC tumor (T)–non-tumor (NT) pairs using a methylation-sensitive restriction enzyme- based HELP-microarray assay. We found 225,350 differentially methylated (DM) sites in adenocarcinomas versus adjacent non-tumor tissue that vary in frequency across genomic compartment, particularly notable in gene bodies (GB; p<2.2E-16). Further, when DM was coupled to differential transcriptome (DE) in the same samples, 37,056 differential loci in adenocarcinoma emerged. Approximately 90% of the DM-DE relationships were non-canonical; for example, promoter DM associated with DE in the same direction. Of the canonical changes noted, promoter (PR) DM loci with reciprocal changes in expression in adenocarcinomas included HBEGF, AGER, PTPRM, DPT, CST1, MELK; DM GB loci with concordant changes in expression included FOXM1, FERMT1, SLC7A5, and FAP genes. IPA analyses showed adenocarcinoma-specific promoter DMxDE overlay identified familiar lung cancer nodes [tP53, Akt] as well as less familiar nodes [HBEGF, NQO1, GRK5, VWF, HPGD, CDH5, CTNNAL1, PTPN13, DACH1, SMAD6, LAMA3, AR]. The unique findings from this study include the discovery of numerous candidate The unique findings from this study include the discovery of numerous candidate methylation sites in both PR and GB regions not previously identified in NSCLC, and many non-canonical relationships to gene expression. These DNA methylation features could potentially be developed as risk or diagnostic biomarkers, or as candidate targets for newer methylation locus-targeted preventive or therapeutic agents.

Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)(n) repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)(n) was n = 4 > 5 ≫ 6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.

Smoking-Related Gene Expression in Laser Capture-Microdissected Human Lung

Purpose: Interindividual differences in quantitative expression could underlie a propensity for lung cancer. To determine precise individual gene expression signatures on a lung compartment–specific basis, we investigated the expression of carcinogen metabolism genes encoding cytochromes P450 (CYP) 1B1, 2A13, GSTP1, and a tumor suppressor gene p16 in laser capture–microdissected samples of human alveolar compartment (AC) and bronchial epithelial compartment (BEC) lung tissue from 62 smokers and nonsmokers. Experimental Design: Tobacco exposure was determined by plasma nicotine, cotinine, and smoking history. Precise mRNA expression was determined using our RNA-specific qRT-PCR strategy, and correlated with detailed demographic and clinical characteristics. Results: Several correlations of mRNA expression included (a) CYP1B1 in AC (positively with plasma nicotine level, P = 0.008; plasma cotinine level, P = 0.001), (b) GSTP1 in AC (positively with plasma cotinine level, P = 0.003), and (c) GSTP1 in BEC (negatively with smoke dose, P = 0.043; occupational risk, P = 0.019). CYP2A13 was rarely expressed in AC and not expressed in BEC. p16 expression was not correlated with any measured factor. For each gene, subjects showed expression that was individually concordant between these compartments. No clear association of mRNA expression with lung cancer risk was observed in this pilot analysis. Conclusions: The association between lung mRNA expression and tobacco exposure implies that gene-tobacco interaction is a measurable quantitative trait, albeit with wide interindividual variation. Gene expression tends to be concordant for alveolar and bronchial compartments for these genes in an individual, controlling for proximate tobacco exposure. (Clin Cancer Res 2009;15(24):7562–70)

Site-specific methylated reporter constructs for functional analysis of DNA methylation

Methods to experimentally alter and functionally evaluate cytosine methylation in a site-specific manner have proven elusive. We describe a site-specific DNA methylation method, using synthetically methylated primers and high fidelity PCR coupled with ligation of reporter constructs. We applied this method to introduce methylated cytosines into fragments of the respective DAPK and RASSF1A promoters that had been cloned into luciferase reporters. We found that methylation of 3–7 residue CpG clusters that were 5′ adjacent to the transcription start site (TSS) of the DAPK gene produced up to a 54% decrease in promoter activity (p < 0.01). Similarly, for RASSF1A promoter reporter constructs, the methylation of either of two clusters of four CpGs each, but not an intervening cluster, produced a 63% decrease in promoter activity (p < 0.01), suggesting that precise mCpG position is crucial, and factors other than simple proximity to the TSS are at play. Chromatin immunoprecipitation analysis of these reporter constructs demonstrated that transcription factor Oct-1 and Sp1 preferentially bound the unmethylated vs. methylated DAPK or RASSF1A promoter reporter constructs at the functional CpG sites. Histone H1, hnRNP1, and MeCP2 showed preferential binding to methylated sequence at functional sites in these reporter constructs, as well as highly preferential (>8–80-fold) binding to native methylated vs. unmethylated chromatin. These results suggest that: (1) site-specific, precision DNA methylation of a reporter construct can be used for functional analysis of commonly observed gene promoter methylation patterns; (2) the reporter system contains key elements of the endogenous chromatin machinery.

Haplotype-tagging single nucleotide polymorphisms in the GSTP1 gene promoter and susceptibility to lung cancer §

BACKGROUND Glutathione S-transferase (GST) P1 is a major phase II xenobiotic-metabolizing enzyme in the human lung. Our laboratory had previously identified nine single nucleotide polymorphisms (SNPs) in the GSTP1 gene promoter, which were then grouped into three main haplotypes (Hap1, Hap2, and Hap3) based on statistical inference. Hap3 was found to display a high expression phenotype. The main objective of the current study was to test the association between GSTP1 promoter haplotypes with the risk of lung cancer after determining the promoter haplotypes experimentally through cloning and sequencing. METHODS We conducted a case-control analysis of 150 subjects with lung cancer and 329 controls with no personal history of the disease. The three statistically inferred GSTP1 promoter haplotypes were confirmed experimentally through cloning and sequencing. Haplotype-tagging SNPs were selected and GSTP1 haplotypes were tested for genetic association to lung cancer using unconditional logistic regression after adjusting for confounders. Statistical interaction between GSTP1 promoter haplotypes with either cigarette smoking or dietary fruit and vegetable intake were tested using the likelihood ratio test. RESULTS We did not find protective effects of Hap3 against lung cancer, despite an adequately powered design for this main effect. Homozygous variants of tagSNPs -1738 T>A and -354 G>T, which tag Hap2, showed an increased (but statistically non-significant) risk of lung cancer among all subjects as well as among individuals with low fruit and vegetable intake, compared to homozygous wildtypes for these SNPs. We did not find significant interactions between Hap2 and dietary intake of fruits and vegetables. CONCLUSIONS Our results do not support significant main and modifying effects for GSTP1 promoter haplotypes on susceptibility to lung cancer in this population, but reinforce the protective effects of dietary intake of fruits and vegetables.

Global, integrated analysis of methylomes and transcriptomes from laser capture microdissected bronchial and alveolar cells in human lung

ABSTRACT Gene regulatory analysis of highly diverse human tissues in vivo is essentially constrained by the challenge of performing genome-wide, integrated epigenetic and transcriptomic analysis in small selected groups of specific cell types. Here we performed genome-wide bisulfite sequencing and RNA-seq from the same small groups of bronchial and alveolar cells isolated by laser capture microdissection from flash-frozen lung tissue of 12 donors and their peripheral blood T cells. Methylation and transcriptome patterns differed between alveolar and bronchial cells, while each of these epithelia showed more differences from mesodermally-derived T cells. Differentially methylated regions (DMRs) between alveolar and bronchial cells tended to locate at regulatory regions affecting promoters of 4,350 genes. A large number of pathways enriched for these DMRs including GTPase signal transduction, cell death, and skeletal muscle. Similar patterns of transcriptome differences were observed: 4,108 differentially expressed genes (DEGs) enriched in GTPase signal transduction, inflammation, cilium assembly, and others. Prioritizing using DMR-DEG regulatory network, we highlighted genes, e.g., ETS1, PPARG, and RXRG, at prominent alveolar vs. bronchial cell discriminant nodes. Our results show that multi-omic analysis of small, highly specific cells is feasible and yields unique physiologic loci distinguishing human lung cell types in situ.

Abstract A38: Whole methylome explorations of paired lung tumor and non-tumor clinical samples

Background: Hypermethylation in specific candidate gene promoters has been found during progressive lung carcinogenesis. To explore common methylation events on a genome-wide scale in lung cancer, we analyzed the methylation profiles of paired NSCLC tumor and far adjacent non-tumor samples using the HELP-microarray assay, which yields information on 1.2 million fragments throughout the genome. Methods: The HELP (HpaII tiny fragment Enriched by Ligation mediated PCR) assay is based on the generation of restriction enzyme libraries generated by methylation sensitive (HpaII) and methylation insensitive (MspI) isoschizomers, in its second generation as a microarray platform. The assay lends itself to low starting amounts of DNA (3 ug) and robust assessment of methylation status by comparing ratios of HpaII- generated-fragments to MspI- generated fragments co-hybridized to a Nimbelgen custom high-density microarrays. The CCGG sites were weighted if neighboring CCGGs were methylated in same direction. Here, 24 pairs of tumor and adjacent non-tumor samples were analyzed using the HELP assay. Summary of results: At p = 5E-6, we identified 26,138 differentially methylated fragments (corresponding to 2 CpG sites each) in tumor versus non-tumor. The overall trend was consistent with genome-wide hypomethylation and locus specific hypermethylation (localized to CG-island containing promoters). We could identify both known and novel regions of the genome as well as specific gene-promoters that are hypermethylated in tumor versus non-tumor. Region # loci # signif loci T Hypomethyl T Hypermethyl Promoter 151,568 576 69% 31% Gene Body 551,628 9,817 62% 38% Intergenic 540,473 15,745 97% 3% Conclusion: An interrogation of methylation status of 1.2 million loci throughout the genome in paired lung tumor/non-tumor specimens reveals many more differential methylation events in gene bodies and intergenic regions than in promoters. That said, many previously unreported differentially-methylated gene promoters were identified. We are able to discover individual methylation events common across different clinical specimens. Based on a set of priors, we have narrowed down promoter-specific hypermethylation events for further validation using tagged Bisulfite Genomic Sequencing. We are also working on the integration of methylome date with other genome-wide epigenetic and expression data from the same clinical samples. [Funding source: NCI 1RC1 CA145422-01; 1K24-CA139054-01]

Abstract 3863: Exhaled microRNAs as potential biomarkers in lung cancer

Introduction: There is a need for non-invasive airway-based biomarkers in lung carcinogenesis for both diagnosis, and risk assessment. MicroRNAs are small, stable, noncoding RNAs that function in gene regulation. Recent studies have revealed that microRNAs play important regulatory roles in carcinogenesis. Exhaled breath condensate (EBC) contains airway lining fluid molecules, including nucleic acids, as we9ve previously reported for DNA methylation. We previously developed an RNA-specific microRNA-PCR, whereby PCR artifact can be detected. Here we pilot the detection of microRNAs in EBC of lung cancer patients and controls. Methods: MicroRNA expression profiling using RNA-specific RT-qPCR was performed in EBC from 28 subjects, choosing literature-derived microRNAs that segregate tumors and non-tumor tissue, and an ongoing discovery effort we have commenced using microRNA-seq for tumor versus non-tumor comparison. As of abstract deadline, the top 12 significantly different microRNAs between lung tumor and non-tumor were chosen and their qPCR primers were designed. Results: So far, we have detected several microRNAs in EBC including 21, 34a, 153, 193b, 200c, 221; case-control comparisons are pending. Topographic validation studies are pending. Conclusion: By comparing the differences of microRNA expression between EBC from lung cancer cases and EBC from non-cancer controls, potential exhaled microRNA biomarkers might be found and used in early detection of lung cancer. This work was supported by National Institutes of Health grants1RC1 CA145422-01, 1K24-CA139054-01. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3863. doi:10.1158/1538-7445.AM2011-3863