Ian M. Wilson

Epigenomics: mapping the methylome.

DNA methylation is integral to normal development and disease processes. However, the genomic distribution of methylated sequences – the methylome – is poorly understood. We have recently developed a platform technology for rapid assessment of methylation status throughout the human genome in a high-resolution, high-throughput manner. This is achieved by coupling a methylated DNA immunoprecipitation (MeDIP) method for isolating methyl cytosine rich fragments with array-based comparative genomic hybridization (array CGH). Using a combination of whole genome tiling path BAC arrays and CpG island microarrays, DNA methylation profiles are obtained simultaneously at both genome-wide and locus-specific levels. A comparison between male and female DNA using MeDIP-array CGH revealed unexpected hypomethylation of the inactive x-chromosome in gene-poor regions. Furthermore, comparisons between cancer and non-cancer cell types yielded differential methylation patterns that link genetic and epigenetic instability offering a new approach to decipher misregulation in cancer. Finally, we provide new data showing epigenomic instability in lung cancer cells with concurrent regions of genetic and epigenetic alterations harbouring known oncogenes.

DNA Methylation Is Globally Disrupted and Associated with Expression Changes in Chronic Obstructive Pulmonary Disease Small Airways

DNA methylation is an epigenetic modification that is highly disrupted in response to cigarette smoke and involved in a wide spectrum of malignant and nonmalignant diseases, but surprisingly not previously assessed in small airways of patients with chronic obstructive pulmonary disease (COPD). Small airways are the primary sites of airflow obstruction in COPD. We sought to determine whether DNA methylation patterns are disrupted in small airway epithelia of patients with COPD, and evaluate whether changes in gene expression are associated with these disruptions. Genome-wide methylation and gene expression analysis were performed on small airway epithelial DNA and RNA obtained from the same patient during bronchoscopy, using Illuminas Infinium HM27 and Affymetrixs Genechip Human Gene 1.0 ST arrays. To control for known effects of cigarette smoking on DNA methylation, methylation and gene expression profiles were compared between former smokers with and without COPD matched for age, pack-years, and years of smoking cessation. Our results indicate that aberrant DNA methylation is (1) a genome-wide phenomenon in small airways of patients with COPD, and (2) associated with altered expression of genes and pathways important to COPD, such as the NF-E2-related factor 2 oxidative response pathway. DNA methylation is likely an important mechanism contributing to modulation of genes important to COPD pathology. Because these methylation events may underlie disease-specific gene expression changes, their characterization is a critical first step toward the development of epigenetic markers and an opportunity for developing novel epigenetic therapeutic interventions for COPD.

Integrating the multiple dimensions of genomic and epigenomic landscapes of cancer

Advances in high-throughput, genome-wide profiling technologies have allowed for an unprecedented view of the cancer genome landscape. Specifically, high-density microarrays and sequencing-based strategies have been widely utilized to identify genetic (such as gene dosage, allelic status, and mutations in gene sequence) and epigenetic (such as DNA methylation, histone modification, and microRNA) aberrations in cancer. Although the application of these profiling technologies in unidimensional analyses has been instrumental in cancer gene discovery, genes affected by low-frequency events are often overlooked. The integrative approach of analyzing parallel dimensions has enabled the identification of (a) genes that are often disrupted by multiple mechanisms but at low frequencies by any one mechanism and (b) pathways that are often disrupted at multiple components but at low frequencies at individual components. These benefits of using an integrative approach illustrate the concept that the whole is greater than the sum of its parts. As efforts have now turned toward parallel and integrative multidimensional approaches for studying the cancer genome landscape in hopes of obtaining a more insightful understanding of the key genes and pathways driving cancer cells, this review describes key findings disseminating from such high-throughput, integrative analyses, including contributions to our understanding of causative genetic events in cancer cell biology.

Divergent Genomic and Epigenomic Landscapes of Lung Cancer Subtypes Underscore the Selection of Different Oncogenic Pathways during Tumor Development

For therapeutic purposes, non-small cell lung cancer (NSCLC) has traditionally been regarded as a single disease. However, recent evidence suggest that the two major subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SqCC) respond differently to both molecular targeted and new generation chemotherapies. Therefore, identifying the molecular differences between these tumor types may impact novel treatment strategy. We performed the first large-scale analysis of 261 primary NSCLC tumors (169 AC and 92 SqCC), integrating genome-wide DNA copy number, methylation and gene expression profiles to identify subtype-specific molecular alterations relevant to new agent design and choice of therapy. Comparison of AC and SqCC genomic and epigenomic landscapes revealed 778 altered genes with corresponding expression changes that are selected during tumor development in a subtype-specific manner. Analysis of >200 additional NSCLCs confirmed that these genes are responsible for driving the differential development and resulting phenotypes of AC and SqCC. Importantly, we identified key oncogenic pathways disrupted in each subtype that likely serve as the basis for their differential tumor biology and clinical outcomes. Downregulation of HNF4α target genes was the most common pathway specific to AC, while SqCC demonstrated disruption of numerous histone modifying enzymes as well as the transcription factor E2F1. In silico screening of candidate therapeutic compounds using subtype-specific pathway components identified HDAC and PI3K inhibitors as potential treatments tailored to lung SqCC. Together, our findings suggest that AC and SqCC develop through distinct pathogenetic pathways that have significant implication in our approach to the clinical management of NSCLC.

Genetic Disruption of KEAP1/CUL3 E3 Ubiquitin Ligase Complex Components is a Key Mechanism of NF-KappaB Pathway Activation in Lung Cancer

Introduction: Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) (IKK-&bgr;/IKK-2), which activates NF-&kgr;B, is a substrate of the KEAP1-CUL3-RBX1 E3-ubiquitin ligase complex, implicating this complex in NF-&kgr;B pathway regulation. We investigated complex component gene disruption as a novel genetic mechanism of NF-&kgr;B activation in non-small cell lung cancer. Methods: A total of 644 tumor- and 90 cell-line genomes were analyzed for gene dosage status of the individual complex components and IKBKB. Gene expression of these genes and NF-&kgr;B target genes were analyzed in 48 tumors. IKBKB protein levels were assessed in tumors with and without complex or IKBKB genetic disruption. Complex component knockdown was performed to assess effects of the E3-ligase complex on IKBKB and NF-&kgr;B levels, and phenotypic importance of IKBKB expression was measured by pharmacological inhibition. Results: We observed strikingly frequent genetic disruption (42%) and aberrant expression (63%) of the E3-ligase complex and IKBKB in the samples examined. Although both adenocarcinomas and squamous cell carcinomas showed complex disruption, the patterns of gene disruption differed. IKBKB levels were elevated with complex disruption, knockdown of complex components increased activated forms of IKBKB and NF-&kgr;B proteins, and IKBKB inhibition detriments cell viability, highlighting the biological significance of complex disruption. NF-&kgr;B target genes were overexpressed in samples with complex disruption, further demonstrating the effect of complex disruption on NF-&kgr;B activity. Conclusions: Gene dosage alteration is a prominent mechanism that disrupts each component of the KEAP1-CUL3-RBX1 complex and its NF-&kgr;B stimulating substrate, IKBKB. Herein, we show that, multiple component disruption of this complex represents a novel mechanism of NF-&kgr;B activation in non-small cell lung cancer.

Transcriptome Profiles of Carcinoma-in-Situ and Invasive Non-Small Cell Lung Cancer as Revealed by SAGE

Background Non-small cell lung cancer (NSCLC) presents as a progressive disease spanning precancerous, preinvasive, locally invasive, and metastatic lesions. Identification of biological pathways reflective of these progressive stages, and aberrantly expressed genes associated with these pathways, would conceivably enhance therapeutic approaches to this devastating disease. Methodology/Principal Findings Through the construction and analysis of SAGE libraries, we have determined transcriptome profiles for preinvasive carcinoma-in-situ (CIS) and invasive squamous cell carcinoma (SCC) of the lung, and compared these with expression profiles generated from both bronchial epithelium, and precancerous metaplastic and dysplastic lesions using Ingenuity Pathway Analysis. Expression of genes associated with epidermal development, and loss of expression of genes associated with mucociliary biology, are predominant features of CIS, largely shared with precancerous lesions. Additionally, expression of genes associated with xenobiotic metabolism/detoxification is a notable feature of CIS, and is largely maintained in invasive cancer. Genes related to tissue fibrosis and acute phase immune response are characteristic of the invasive SCC phenotype. Moreover, the data presented here suggests that tissue remodeling/fibrosis is initiated at the early stages of CIS. Additionally, this study indicates that alteration in copy-number status represents a plausible mechanism for differential gene expression in CIS and invasive SCC. Conclusions/Significance This study is the first report of large-scale expression profiling of CIS of the lung. Unbiased expression profiling of these preinvasive and invasive lesions provides a platform for further investigations into the molecular genetic events relevant to early stages of squamous NSCLC development. Additionally, up-regulated genes detected at extreme differences between CIS and invasive cancer may have potential to serve as biomarkers for early detection.

EYA4 is inactivated biallelically at a high frequency in sporadic lung cancer and is associated with familial lung cancer risk

In an effort to identify novel biallelically inactivated tumor suppressor genes (TSGs) in sporadic invasive and preinvasive non-small-cell lung cancer (NSCLC) genomes, we applied a comprehensive integrated multiple ‘omics’ approach to investigate patient-matched, paired NSCLC tumor and non-malignant parenchymal tissues. By surveying lung tumor genomes for genes concomitantly inactivated within individual tumors by multiple mechanisms, and by the frequency of disruption in tumors across multiple cohorts, we have identified a putative lung cancer TSG, Eyes Absent 4 (EYA4). EYA4 is frequently and concomitantly deleted, hypermethylated and underexpressed in multiple independent lung tumor data sets, in both major NSCLC subtypes and in the earliest stages of lung cancer. We found that decreased EYA4 expression is not only associated with poor survival in sporadic lung cancers but also that EYA4 single-nucleotide polymorphisms are associated with increased familial cancer risk, consistent with EYA4s proximity to the previously reported lung cancer susceptibility locus on 6q. Functionally, we found that EYA4 displays TSG-like properties with a role in modulating apoptosis and DNA repair. Cross-examination of EYA4 expression across multiple tumor types suggests a cell-type-specific tumorigenic role for EYA4, consistent with a tumor suppressor function in cancers of epithelial origin. This work shows a clear role for EYA4 as a putative TSG in NSCLC.

Abstract LB-346: A novel lung tumor suppressor implicated in somatic and familial cancers

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Lung cancer (LC) is the most common cause of cancer death worldwide. Previous familial linkage studies have identified a tumor suppressor locus on 6q23-25. However, no single gene has yet been implicated within this 30 Mb region. Discovering the genetic and epigenetic events that affect LC risk and development will lead to better methods for risk assessment, early detection and treatment. Methods: Genome-wide genes disrupted by two-hit inactivation were identified by combining gene dosage, DNA methylation, and gene expression assays for a group of lung adenocarcinomas (AC) and adjacent non-malignant tissues. Gene expression, DNA hypermethylation and/or copy number aberrations were validated in data from AC, squamous cell carcinoma (SqCC), and pre-malignant lesions by querying other cohorts using gene-specific and whole-genome approaches. The role of DNA methylation in gene silencing was assessed using inhibition of DNMT by 5′-azacytidine. The association of allelic variants with LC risk was investigated in 193 familial LC cases and 213 controls collected by the Genetic Epidemiology of Lung Cancer Consortium (GELCC) using a Cochrane-Armitage trend test. The association of gene expression with prognosis was performed on public data using a Mantel-Cox log test. Stable mRNA knock-downs were generated using lentiviral delivery of a gene-specific shRNA, and apoptotic cells were counted using Annexin5/propidium iodide staining. Results: Integration of AC gene dosage, DNA methylation and mRNA expression showed EYA4 to be frequently affected by two-hits and significantly down-regulated. Quantitative PCR techniques confirmed that EYA4 was hypermethylated (46%) and down-regulated (72%), validating our microarray results. A direct link between EYA4 methylation and expression was verified by restoration of expression after 5′-azacytidine treatment in methylated cell lines. Congruent with EYA family member function, in vitro assays revealed that EYA4 knock-down cells displayed a decrease in the number of apoptotic cells - a hallmark of cancer. Further investigations led to the discovery of frequent EYA4 disruption in SqCC and pre-neoplastic tissue. The GELCC dataset was examined to assess EYA4 allelotype association with familial risk. Doing so revealed that numerous EYA4 variants are associated with increased risk. Finally, the association of EYA4 expression with survival was investigated along with other somatically altered genes at 6q23-25. Of these genes, low EYA4 expression was found to be the most significantly associated with poor prognosis. Conclusions: EYA4 is a frequently disrupted gene that maps to a locus previously associated with cancer risk. It is implicated in somatic as well as familial cancers, and is likely a tumor suppressor gene with apoptotic functions. The direct association of EYA4 with risk and survival underscores its relevance on a clinical level. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-346.

Abstract A21: Global DNA methylation analysis of bronchial epithelia of former smokers with COPD, with and without lung cancer

Introduction: Emerging evidence suggests that aberrant epigenetic regulation is involved in the development and progression of malignant and nonmalignant respiratory diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. Moreover, patients with COPD have an increased risk of developing lung cancer. Besides similar risk factors such as tobacco smoke exposure, little is known about the shared biology between COPD and lung cancer. Smoking causes aberrations in airway and lung parenchyma at both the genomic and epigenomic levels, resulting in global changes to gene expression. In this study, we hypothesize that alterations at the level of DNA methylation in airway epithelia of former smokers (FS) with COPD with and without non-small cell lung cancer (NSCLC) may be used to identify genes involved in the pathogenesis of these respiratory diseases, independent of the effects of active smoking. Methods: Bronchial epithelial cells were obtained from brushings of small airways ( Results: COPD patients are distinguished from non-COPD patients based on airway methylation profiles. Genes differentially methylated in airways between COPD and non-COPD patients include several modulators of aryl hydrocarbon receptor and IL6 signaling, as well as genes previously implicated in COPD, including immune chemotaxis regulators (CXCL11, CCR8) and GABA receptor signaling (GABRA5). Airway epithelial DNA from COPD patients with NSCLC compared to those without NSCLC was differentially methylated at sites encoding multiple key regulators of xenobiotic metabolism, regulators of free radical savaging/detoxification and retinol metabolic pathway components including several alcohol dehydrogenase, glutathione S transferase, and UDP glucoronosyltransferase genes. Conclusion: Our preliminary results suggest a role for DNA methylation in the deregulation of previously identified COPD-related genes, and specifically highlight differences in airways of COPD patients with/without NSCLC corresponding to well-known smoking-related metabolomic processes. Knowledge of DNA methylation disruption will further our understanding of the etiological role of COPD in the development of lung cancer, and contribute to the development of chemo-prevention strategies targeting the biology of both COPD and NSCLC. Supported by CIHR. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A21.

Abstract A16: Reversibly expressed and differentially methylated genes in airways of current and former smokers

Background: Smoking-related disease, including chronic obstructive pulmonary disease (COPD) and lung cancer, account for the third greatest cause of mortality and the number one cause of cancer-related death worldwide. Former smokers (FS) remain at an elevated risk for both diseases and are the fastest growing population of newly diagnosed lung cancer patients, emphasizing the need for greater understanding of molecular mechanisms associated with smoking and smoking cessation. Cigarette smoke induces DNA damage in airway and lung tissues at the genomic and epigenomic levels, where it is associated with changes to gene expression. Studies on current (CS), FS, and never smokers have identified reversible and irreversible changes in gene expression that occur upon smoking cessation. Since methylation is a reversible gene regulatory mark that is also aberrantly affected by cigarette smoke, we hypothesize that the reversible nature of genes differentially expressed in bronchial epithelial cells in the airways of CS and FS, may be due to changes in DNA methylation. Methods: Bronchial epithelial cells were obtained from brushings of small airways ( Results: Methylation and expression analysis identified 9 genes overexpressed and hypomethylated in CS relative to FS, notably GPX2 and GSTA2 both involved in pathways previously shown to be upregulated in CS. These pathways include aryl hydrocarbon receptor signaling; a central metabolic pathway activated in response to halogenated and polycyclic aromatic hydrocarbons, and the NRF2 mediated oxidative stress response pathway, involved in the detoxification of reactive oxygen and intermediates. Additionally, 3 genes were found to be underexpressed and hypermethylated in CS relative to FS, notably SYF2 and CXCL6, involved in cell cycle regulation and inflammatory disease (including COPD) respectively. Conclusion: Our data reveal a panel of genes whose change in gene expression upon smoking cessation may be regulated by DNA methylation, corresponding to well known genes involved in smoking metabolism and oxidative stress response. The identification of differentially methylated and expressed genes between CS and FS may provide insight into the mechanism of smoking related disease. As methylation is a reversible DNA modification, this knowledge may lead to the application of preventative epigenetic therapeutics for the growing population of FS and the immense health burden and mortality associated with cigarette smoke. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A16.