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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 Illumina’s Infinium HM27 and Affymetrix’s 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.

Molecular mechanisms responsible for lymphoma resistance to apoptosis often involve the bcl-2 pathway. In this study, we investigated the cell signaling pathways activated in bcl-2-overexpressing human mantle cell lymphoma cell lines (JVM-2 and Z-138) that have been treated with oblimersen, a molecular gene silencing strategy that effectively suppresses bcl-2 in vitro and in vivo. Z-138 cells expressed higher levels of bcl-2 and were more sensitive to the effects of bcl-2 silencing, mediated by oblimersen or bcl-2 small interfering RNA, in vitro. Tumors derived following injection of Z-138 cells were sensitive to oblimersen as judged by decreases in tumor growth rate and decreases in cell proliferation (as measured by Ki-67). Immunohistochemistry and Western blot analysis of oblimersen-treated Z-138 tumors revealed a dose-dependent decrease in bcl-2 levels and an associated increase in the proapoptotic proteins caspase-3 and caspase-9. Silencing bcl-2 in Z-138 xenografts revealed an associated dose-dependent suppression of bax, a decrease in nuclear factor-κB and phospho-nuclear factor-κB, and transient loss of p53 levels. Coimmunoprecipitation studies suggest that the latter observation is mediated by an association between bcl-2 and phospho-mdm2. Bcl-2 silencing also led to p27 down-regulation and coimmunoprecipitation studies point to a role for bcl-2 in regulation of p27 localization/degradation. Bcl-2 silencing was also correlated with loss of cyclin D1a protein levels but not cyclin D1b levels. Coimmunoprecipitation studies indicate that bcl-2 may mediate its effects on cyclin D1a via interaction with p38 mitogen-activated protein kinase as well as a previously unreported interaction between bcl-2 and cyclin D1a. [Mol Cancer Ther 2008;7(4):749–58]

Cancer is a disease characterized by genomic instability. Comparative genomic hybridization (CGH) is a technique designed for detecting segmental genomic alterations. Recent advances in array-based CGH technology have enabled examination of chromosomal regions in unprecedented detail, revolutionizing our understanding of tumour genomes. A number of array-based technologies have been developed, aiming to improve the resolution of CGH, enabling researchers to refine and define regions in the genome that may be causal to cancer, and facilitating gene discovery at a rapid rate. This article reviews the various array CGH platforms and their use in the study of cancer genomes. In addition, the need for high-resolution analysis is discussed as well as the importance of studying early-stage disease to discover genetic alterations that may be causal to cancer progression and aetiology.

Inpatient facilities in palliative care units are generally considered to be mainly for cancer patients. We present and discuss the results of a survey that attempted to estimate the number of noncancer patients requiring inpatient palliative care. Questionnaires sent to all general practices in the Thames Valley area asked about the diagnosis and the number of bed-days that would have been required for each noncancer patient in the practice dying in the last year or still in their care. The replies suggest that about 11 noncancer patients per practice per year were in need of respite or continuing care. For the Thames Valley area this would amount to at least 66 000 bed-days per year for noncancer patients, compared with the current provision, mainly for cancer patients, of about 40 000 bed-days per year. The diagnoses involved and the reasons why our figures may overestimate need, are discussed. There can be no doubt that, if the need is to be met, current facilities will be inadequate and additional beds and services will be required.

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.

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.

Background: Nuclear factor kappa B (NF-κB) signaling is essential for lung cancer development, and therefore, may serve as a target for intervention. However, the genetic mechanisms responsible for its activation are not fully understood. Kelch-like ECH-associated protein 1 (KEAP1) binds protein substrates to the Cullin-3 (CUL3)/Ring box protein-1 (RBX1) E3 ubiquitin ligase complex where ubiquitination signals substrates for proteosomal degradation. Recently, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), an activator of NF-κB, was shown to be a substrate of KEAP1, implicating KEAP1's involvement in regulating NF-κB signaling. Loss of function of KEAP1 leads to IKKβ accumulation and NF-κB activation. We hypothesized that DNA deletions of the other E3 complex components (CUL3 and RBX1) are frequent alterations that disrupt complex function and contribute to NF-κB activation in lung cancer. Methods: We screened DNA copy number profiles of 261 non-small cell lung cancer (NSCLC) tumors for DNA alterations at the KEAP1, CUL3, RBX1, and IKK loci. Profiles were generated by array comparative genomic hybridization on the SMRT array (sub-megabase resolution tiling) platform. We also analyzed mRNA expression of these genes and 9 well annotated NF-κB target genes, using gene expression profiles generated with Agilent gene expression microarrays for a subset (n=48) of the tumors. Results: Our investigation of genetic disruption to the E3 ubiquitin ligase complex components revealed 54% of tumors harbored DNA copy number loss of at least one complex component (KEAP1, CUL3, or RBX1) or gain of IKKβ. Moreover, at the expression level, 81% of tumors analyzed had aberrant expression of one of these genes (underexpression of complex components or overexpression of IKKβ). Interestingly, the copy number alterations identified appeared to segregate with adenocarcinoma (AC) or squamous cell carcinoma (SCC) histology; KEAP1 loss was more prevalent in AC while CUL3 loss and IKKβ gain were more frequent in SCC. When NF-κB target gene expression was analyzed, we observed higher expression of 5/9 genes in tumors with underexpression of an E3 ubiquitin ligase complex component relative to matched non-malignant tissue from the same individual. Conclusions: The presence and strikingly high frequency of genetic disruption and aberrant expression of the E3 ubiquitin ligase complex components (KEAP1, CUL3, and RBX1) revealed in this study provides evidence of its importance in lung cancer. These data suggest that DNA level alterations to this complex may represent a novel mechanism of NF-κB activation in lung cancer. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A14.

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 (< 2 mm diameter) during bronchoscopy from FS with COPD (n=22), without COPD (n=22) and patients with COPD as well as previous surgical resection of Stage I NSCLC (n=6). Illumina's Infinium Methylation (HM27) assay was used to assess DNA methylation status of 27,578 CpG sites associated with 14,475 genes. 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.

Background: Previous familial linkage studies have identified a tumor suppressor locus at 6q23-25 in lung cancer (LC). However, no single gene has yet been implicated within this 30 Mb region. The identification of two-hit targets — genes disrupted by two mechanisms at the DNA level, such as copy number loss and hypermethylation — involved in LC risk and development will lead to better methods for risk assessment, early detection and treatment. Methods: EYA4 was identified as a two-hit target using multidimensional genome-wide analysis combining gene dosage, DNA methylation, and gene expression data in non-small cell lung cancer (NSCLC) and early pre-malignant lesions with matched adjacent non-malignant tissue. The association of allelic variants with LC risk was assessed in data collected by the Genetic Epidemiology of Lung Cancer Consortium (GELCC), and the association of gene expression with prognosis was investigated in public data using statistical analyses. Bronchial epithelial cells of LC patients and high risk individuals were examined for early hypermethylation of EYA4. The role of EYA4 in apoptosis, and the association of EYA4 with the DNA repair genes GADD45a and γ-H2AX was assayed using stable EYA4 mRNA knock-downs in lymphoblast cell lines. Cells were either serum-starved for 12 and 24 hours followed by Annexin5/propidium iodide staining and GADD45a qRT-PCR, or irradiated, and levels of γ-H2AX protein were measured over time. Results: Integration of NSCLC gene dosage, DNA methylation and mRNA expression showed EYA4 to be frequently affected by two-hits and significantly down-regulated. Numerous EYA4 variants were associated with familial LC, and low EYA4 expression was significantly associated with poor prognosis in multiple independent data sets. Congruent with EYA family member function, in vitro assays revealed that EYA4 knock-down cells displayed a decrease in the number of apoptotic cells following serum starvation. Further investigations led to the discovery of EYA4 hypermethylation not only in the bronchial epithelial brushings of NSCLC patients, but also in the brushings of individuals at a high risk of LC development. EYA4 knock-down led to attenuation of the GADD45a DNA damage repair response, as well as increased levels of γ-H2AX following DNA damage repair. Conclusions: EYA4 is a gene disrupted early and frequently 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 and DNA repair functions. The direct association of EYA4 with risk and survival as well as its early disruption underscores its relevance on a clinical level. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A29.

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 (< 2mm diameter) during bronchoscopy from 12 current smokers (CS) and 6 former smokers (FS). Methylation profiles were generated for 27,579 probes corresponding to 14,575 genes, using the Illumina Infinium Human Methylation27 platform. Methylation results were aligned to the set of reversibly expressed genes described in previous studies. Genes that become either hypermethylated and underexpressed or hypomethylated and overexpressed upon smoking cessation were selected for further study. 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.

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.

A27 Background Lung cancer is the most common cause of cancer death worldwide with more than 1.2 million people dying of the disease each year. Half of newly diagnosed lung cancer patients are former smokers. Understanding why former smokers develop lung cancer is clearly important to the development of early detection, prevention and treatment strategies for these people. The effects of cigarette smoke on the epigenome are widespread as both global DNA methylation and local DNA methylation have been identified, associated with genomic instability and tumour suppressor gene (TSG) silencing, respectively. In a cancer-specific context, upregulation of oncogenes and silencing of tumour suppressor genes both occur as a result of tobacco smoke exposure. Therefore, molecular studies examining tumors at genomic and epigenomic levels will likely identify causal genetic events involved in cancer development in former smokers. Objective The objective of this study is to determine the contribution of DNA methylation as a mechanism to lung cancer development in former smokers. Hypothesis As smoking induces methylation changes in bronchioepithelial cells, we hypothesize that this constitutes the first hit to TSG inactivation. In order to inactivate both alleles, these methylation changes would be maintained in tumors where a second hit would be found at the same gene loci. Materials and Methods Epithelial cells from former smokers (those with >10 years of smoking cessation) were collected from peripheral airways during routine bronchoscopy. Half of the cells were fixed in Cytolyt and the other half in RNAlater for DNA and RNA extraction, respectively. Copy number profiling was performed by array comparative genomic hybridization (aCGH) using whole-genome tiling path SMRT v2 BAC array. Methylation analysis was performed by coupling affinity based enrichment of methylated sequences with hybridization to the same aCGH platform described above. Expression status of genes was determined by gene expression microarray analysis using Agilent 44K expression arrays. RESULTS: Preliminary MeDIP aCGH of bronchial brush cells from eight former smokers who had previous surgical removal of Stage I NSCLC, and eight former smokers without NSCLC, of similar age (68±7 versus 62±6), revealed distinct differences in frequency of DNA methylation between cancer and non-cancer groups. For example, at 11p13 the cancer group shows hypermethylation at the WT1 locus, a known TSG. Analysis in 62 NSCLC tumors for gene dosage, showed a high frequency of loss at the WT1 locus. To examine downstream effects of these events in tumours, expression of WT1 was assessed and found to be significantly underexpressed in the majority of NSCLC tumours compared to a normal lung reference. Collection of more samples and further integrative analysis is currently underway. Conclusion Differences in methylation between these two groups may explain why some former smokers develop cancer while others remain cancer free despite similar lifestyle changes. As methylation is a reversible DNA modification, this knowledge would prompt the development and application of DNA demethylation chemopreventative agents and unique therapeutic strategies. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A27.