Jay W. Tichelaar

Principles for the post-GWAS functional characterization of cancer risk loci

Genome wide association studies (GWAS) have identified more than 200 mostly new common low-penetrance susceptibility loci for cancers. The predicted risk associated with each locus is generally modest (with a per-allele odds ratio typically less than 2) and so, presumably, are the functional effects of individual genetic variants conferring disease susceptibility. Perhaps the greatest challenge in the ‘post-GWAS’ era is to understand the functional consequences of these loci. Biological insights can then be translated to clinical benefits, including reliable biomarkers and effective strategies for screening and disease prevention. The purpose of this article is to propose principles for the initial functional characterization of cancer risk loci, with a focus on non-coding variants, and to define ‘post-GWAS’ functional characterization. By December 2010, there were 1,212 published GWAS studies1 reporting significant (P < 5 × 10−8) associations for 210 traits (Table 1), and the Catalog of Published GWAS states that by March 2011, 812 publications reported 3,977 SNP associations1. This is likely a small fraction of the common susceptibility loci of low penetrance that will eventually be identified. Despite these successes in identifying risk loci, the causal variant and/or the molecular basis of risk etiology has been determined for only a small fraction of these associations2–4. Plausible candidate genes can be based on proximity to risk loci, but few have so far been defined in a more systematic manner (Supplementary Table 1). Table 1 The genomic context in which a variant is found can be used as preliminary functional analysis Increased investment in post-GWAS functional characterization of risk loci5 has now been advocated across diseases and for cardiovascular disease and diabetes6. For cancer biology, the complex interplay between genetics and the environment in many cancers poses a particularly exciting challenge for post-GWAS research. Here we suggest a systematic strategy for understanding how cancer-associated variants exert their effects. We mostly refer to SNPs throughout the paper, but we recognize that other types of common genetic (for example, copy number variants) or epigenetic variation may influence risk. Our understanding of the way in which a risk variant initiates disease pathogenesis progresses from statistical association between genetic variation and trait or disease variation to functionality and causality. The functional consequences of variants in protein-coding regions causing most monogenic disorders are more readily interpreted because we know the genetic code. For non-Mendelian or multifactorial traits, most of the common DNA variants have so far mapped to non-protein–coding regions2, where our understanding of functional consequences and causality is more rudimentary. Our hypothesis is that the trait-associated alleles exert their effects by influencing transcriptional output (such as transcript levels and splicing) through multiple mechanisms. We emphasize appropriate assays and models to test the functional effects of both SNPs and genes mapping to cancer predisposition loci. Although much of what is written is applicable to alleles discovered for any trait, the section on modeling gene effects will emphasize measuring cancer-related phenotypes. At some loci, multiple, independently associated risk alleles rather than single risk alleles may be functionally responsible for the occurrence of disease. Genotyping susceptibility loci (and their correlated variants) in multiple populations with different linkage disequilibrium (LD) structures may prove effective in substantially reducing the number of potentially causative variants (that is, the same causal variant may segregate in multiple populations), as shown for the FGFR2 locus in breast cancer7, but for most loci there will remain a set of potentially causative variants that cannot be separated at the statistical level from case-control genotype data. A susceptibility locus should be re-sequenced to ascertain all genetic variation, identifying candidate functional or causal variants and identifying candidate causal genes. Ideally, the identification of a causal SNP would be the next step to reveal the molecular mechanisms of risk modification. Practically, however, it is unclear what the criteria for causality should be, particularly in non-protein–coding regions. Thus, although we propose a framework set of analyses (Box 1), we acknowledge that the techniques and methods will continue to evolve with the field. Box 1 Strategies to progress from tag SNP to mechanism Target resequencing efforts using linkage disequilibrium (LD) structure. Use other populations to refine LD regions (for example African ancestry with shorter LD and more heterogeneity). Determine expression levels of nearby genes as a function of genotype at each locus (eQTL). Characterize gene regulatory regions by multiple empirical techniques bearing in mind that these are tissue and context specific. Combine regulatory regions with risk loci using coordinates from multiple reference genomes to capture all variation within the shorter regulatory regions that correlates with the tag SNP at each locus. Multiple experimental manipulations in model systems are needed to progressively implicate transcription units (genes) in mechanisms relevant to the associated loci: Knockouts of regulatory regions in animal (difficult and may be limited by functional redundancy, but new targeting methods in rat are promising) models followed by genome-wide expression analysis. Use chromatin association methods (3C, CHIA-PET) of regulatory regions to determine the identity of target genes (compare with eQTL data). Targeted gene perturbations in somatic cell models. Explore fully genome-wide eQTL and miRNA quantitative variation correlation in relevant tissues and cells. Explore epigenetic mechanisms in the context of genome-wide genetic polymorphism. Employ cell models and tissue reconstructions to evaluate mechanisms using gene perturbations and polymorphic variants. The human cancer cell xenograft has re-emerged as a minimal in vivo validation of these models. Above all, resist the temptation to equate any partial functional evidence as sufficient. Published claims of functional relevance should be fully evaluated using the steps detailed above.

Black Raspberry-Derived Anthocyanins Demethylate Tumor Suppressor Genes Through the Inhibition of DNMT1 and DNMT3B in Colon Cancer Cells

We previously reported that oral administration of black raspberry powder decreased promoter methylation of tumor suppressor genes in tumors from patients with colorectal cancer. The anthocyanins (ACs) in black raspberries are responsible, at least in part, for their cancer-inhibitory effects. In the present study, we asked if ACs are responsible for the demethylation effects observed in colorectal cancers. Three days of treatment of ACs at 0.5, 5, and 25 μg/ml suppressed activity and protein expression of DNMT1 and DNMT3B in HCT116, Caco2 and SW480 cells. Promoters of CDKN2A, and SFRP2, SFRP5, and WIF1, upstream of Wnt pathway, were demethylated by ACs. mRNA expression of some of these genes was increased. mRNA expression of β-catenin and c-Myc, downstream of Wnt pathway, and cell proliferation were decreased; apoptosis was increased. ACs were taken up into HCT116 cells and were differentially localized with DNMT1 and DNMT3B in the same cells visualized using confocal laser scanning microscopy. Although it was reported that DNMT3B is regulated by c-Myc in mouse lymphoma, DNMT3B did not bind with c-Myc in HCT116 cells. In conclusion, our results suggest that ACs are responsible, at least in part, for the demethylation effects of whole black raspberries in colorectal cancers.

Sustained CTL activation by murine pulmonary epithelial cells promotes the development of COPD-like disease

Chronic obstructive pulmonary disease (COPD) is a lethal progressive lung disease culminating in permanent airway obstruction and alveolar enlargement. Previous studies suggest CTL involvement in COPD progression; however, their precise role remains unknown. Here, we investigated whether the CTL activation receptor NK cell group 2D (NKG2D) contributes to the development of COPD. Using primary murine lung epithelium isolated from mice chronically exposed to cigarette smoke and cultured epithelial cells exposed to cigarette smoke extract in vitro, we demonstrated induced expression of the NKG2D ligand retinoic acid early transcript 1 (RAET1) as well as NKG2D-mediated cytotoxicity. Furthermore, a genetic model of inducible RAET1 expression on mouse pulmonary epithelial cells yielded a severe emphysematous phenotype characterized by epithelial apoptosis and increased CTL activation, which was reversed by blocking NKG2D activation. We also assessed whether NKG2D ligand expression corresponded with pulmonary disease in human patients by staining airway and peripheral lung tissues from never smokers, smokers with normal lung function, and current and former smokers with COPD. NKG2D ligand expression was independent of NKG2D receptor expression in COPD patients, demonstrating that ligand expression is the limiting factor in CTL activation. These results demonstrate that aberrant, persistent NKG2D ligand expression in the pulmonary epithelium contributes to the development of COPD pathologies.

Increased staining for phospho-Akt, p65/RELA and cIAP-2 in pre-neoplastic human bronchial biopsies

BackgroundThe development of non-small cell lung carcinoma proceeds through a series of well-defined pathological steps before the appearance of invasive lung carcinoma. The molecular changes that correspond with pathology changes are not well defined and identification of the molecular events may provide clues on the progression of intraepithelial neoplasia in the lung, as well as suggest potential targets for chemoprevention. The acquisition of anti-apoptotic signals is critical for the survival of cancer cells but the pathways involved are incompletely characterized in developing intra-epithelial neoplasia (IEN).MethodsWe used immunohistochemistry to determine the presence, relative levels, and localization of proteins that mediate anti-apoptotic pathways in developing human bronchial neoplasia.ResultsBronchial epithelial protein levels of the phosphorylated (active) form of AKT kinase and the caspase inhibitor cIAP-2 were increased in more advanced grades of bronchial IEN lesions than in normal bronchial epithelium. Additionally, the percentage of biopsies with nuclear localization of p65/RELA in epithelial cells increased with advancing pathology grade, suggesting that NF-κB transcriptional activity was induced more frequently in advanced IEN lesions.ConclusionOur results indicate that anti-apoptotic pathways are elevated in bronchial IEN lesions prior to the onset of invasive carcinoma and that targeting these pathways therapeutically may offer promise in prevention of non-small cell lung carcinoma.

GM-CSF modulates pulmonary resistance to influenza A infection

Alveolar type II epithelial or other pulmonary cells secrete GM-CSF that regulates surfactant catabolism and mucosal host defense through its capacity to modulate the maturation and activation of alveolar macrophages. GM-CSF enhances expression of scavenger receptors MARCO and SR-A. The alveolar macrophage SP-R210 receptor binds the surfactant collectin SP-A mediating clearance of respiratory pathogens. The current study determined the effects of epithelial-derived GM-CSF in host resistance to influenza A pneumonia. The results demonstrate that GM-CSF enhanced resistance to infection with 1.9×10(4) ffc of the mouse-adapted influenza A/Puerto Rico/8/34 (PR8) H1N1 strain, as indicated by significant differences in mortality and mean survival of GM-CSF-deficient (GM(-/-)) mice compared to GM(-/-) mice in which GM-CSF is expressed at increased levels. Protective effects of GM-CSF were observed both in mice with constitutive and inducible GM-CSF expression under the control of the pulmonary-specific SFTPC or SCGB1A1 promoters, respectively. Mice that continuously secrete high levels of GM-CSF developed desquamative interstitial pneumonia that impaired long-term recovery from influenza. Conditional expression of optimal GM-CSF levels at the time of infection, however, resulted in alveolar macrophage proliferation and focal lymphocytic inflammation of distal airways. GM-CSF enhanced alveolar macrophage activity as indicated by increased expression of SP-R210 and CD11c. Infection of mice lacking the GM-CSF-regulated SR-A and MARCO receptors revealed that MARCO decreases resistance to influenza in association with increased levels of SP-R210 in MARCO(-/-) alveolar macrophages. In conclusion, GM-CSF enhances early host resistance to influenza. Targeting of MARCO may reinforce GM-CSF-mediated host defense against pathogenic influenza.

Effect of dietary green tea extract and aerosolized difluoromethylornithine during lung tumor progression in A/J strain mice

Chemoprevention strategies to prevent the development of lung cancer in at-risk individuals are a key component in disease management. In addition to being highly effective, an ideal chemopreventive agent will require low toxicity as patients are likely to require treatment for several years before their risk of cancer is lowered to background levels. In principle, a combination of safe agents that work through distinct mechanisms will improve efficacy while simultaneously maintaining a favorable safety profile. Here, we describe the use of the decaffeinated green tea extract Polyphenon E (Poly E) (1% in diet) and aerosolized difluoromethylornithine (DFMO) (20 mg/kg/day, 5 days/week) in a mouse lung cancer chemoprevention study using a progression protocol. Female A/J mice were injected with benzo[a]pyrene (B[a]P) at 8 weeks of age and precancerous lesions allowed to form over a period of 21 weeks before chemoprevention treatment for an additional 25 weeks. Poly E treatment did not significantly inhibit average tumor multiplicity but reduced per animal tumor load. Analysis of tumor pathology revealed a specific inhibition of carcinomas, with the largest carcinomas significantly decreased in Poly E-treated animals. Aerosolized DFMO did not have a significant effect on lung tumor progression. Magnetic resonance imaging of B[a]P-induced lung tumors confirmed the presence of a subset of large, rapidly growing tumors in untreated mice. Our results suggest a potential role for green tea extracts in preventing the progression of large, aggressive lung adenocarcinomas.

Neutrophils Are Required for 3-Methylcholanthrene-Initiated, Butylated Hydroxytoluene-Promoted Lung Carcinogenesis

Multiple studies have shown a link between chronic inflammation and lung tumorigenesis. Inbred mouse strains vary in their susceptibility to methylcholanthrene (MCA)‐initiated butylated hydroxytoluene (BHT)‐promoted lung carcinogenesis. In the present study we investigated whether neutrophils play a role in strain dependent differences in susceptibility to lung tumor promotion. We observed a significant elevation in homeostatic levels of neutrophils in the lungs of tumor‐susceptible BALB/cByJ (BALB) mice compared to tumor‐resistant C57BL/6J (B6) mice. Additionally, BHT treatment further elevated neutrophil numbers as well as neutrophil chemoattractant keratinocyte‐derived cytokine (KC)/chemokine (C‐X‐C motif) ligand 1 (Cxcl1) levels in BALB lung airways. Lung CD11c+ cells were a major source of KC expression and depletion of neutrophils in BALB mice resulted in a 71% decrease in tumor multiplicity. However, tumor multiplicity did not depend on the presence of T cells, despite the accumulation of T cells following BHT treatment. These data demonstrate that neutrophils are essential to promote tumor growth in the MCA/BHT two‐step lung carcinogenesis model.

A dominant-negative c-jun mutant inhibits lung carcinogenesis in mice.

Lung cancer is the leading cause of cancer mortality in the United States and worldwide. The identification of key regulatory and molecular mechanisms involved in lung tumorigenesis is therefore critical to increase our understanding of this disease and could ultimately lead to targeted therapies to improve prevention and treatment. Induction of members of the activator protein-1 (AP-1) transcription factor family has been described in human non–small cell lung carcinoma. Activation of AP-1 can either stimulate or repress transcription of multiple gene targets, ultimately leading to increased cell proliferation and inhibition of apoptosis. In the present study, we show induction of AP-1 in carcinogen-induced mouse lung tumors compared with surrounding normal lung tissue. We then used a transgenic mouse model directing conditional expression of the dominant-negative c-jun mutant TAM67 in lung epithelial cells to determine the effect of AP-1 inhibition on mouse lung tumorigenesis. Consistent with low AP-1 activity in normal lung tissue, TAM67 expression had no observed effects in adult mouse lung. TAM67 decreased tumor number and overall lung tumor burden in chemically induced mouse lung tumor models. The most significant inhibitory effect was observed on carcinoma burden compared with lower-grade lesions. Our results support the concept that AP-1 is a key regulator of mouse lung tumorigenesis, and identify AP-1–dependent transcription as a potential target to prevent lung tumor progression. Cancer Prev Res; 3(9); 1148–56. ©2010 AACR.

Nonredundant Functions of αβ and γδ T Cells in Acrolein-Induced Pulmonary Pathology

Acrolein exposure represents a significant human health hazard. Repeated acrolein exposure causes the accumulation of monocytes/macrophages and lymphocytes, mucous cell metaplasia, and epithelial injury. Currently, the mechanisms that control these events are unclear, and the relative contribution of T-cell subsets to pulmonary pathologies following repeated exposures to irritants is unknown. To examine whether lymphocyte subpopulations regulate inflammation and epithelial cell pathology, we utilized a mouse model of pulmonary pathology induced by repeated acrolein exposures. The role of lymphocyte subsets was examined by utilizing transgenic mice genetically deficient in either alphabeta T cells or gammadelta T cells, and changes in cellular, molecular, and pathologic outcomes associated with repeated inhalation exposure to 2.0 and 0.5 ppm acrolein were measured. To examine the potential functions of lymphocyte subsets, we purified these cells from the lungs of mice repeatedly exposed to 2.0 ppm acrolein, isolated and amplified messenger RNA, and performed microarray analysis. Our data demonstrate that alphabeta T cells are required for macrophage accumulation, whereas gammadelta T cells are critical regulators of epithelial cell homeostasis, as identified by epithelial cell injury and apoptosis, following repeated acrolein exposure. This is supported by microarray analyses that indicated the T-cell subsets are unique in their gene expression profiles following acrolein exposures. Microarray analyses identified several genes that may contribute to phenotypes mediated by T-cell subpopulations including those involved in cytokine receptor signaling, chemotaxis, growth factor production, lymphocyte activation, and apoptosis. These data provide strong evidence that T-cell subpopulations in the lung are major determinants of pulmonary pathology and highlight the advantages of dissecting their effector functions in response to toxicant exposures.

DURATION-DEPENDENT CYTOPROTECTIVE VERSUS INFLAMMATORY EFFECTS OF LUNG EPITHELIAL FIBROBLAST GROWTH FACTOR-7 EXPRESSION

Fibroblast growth factor-7 (FGF7) is a lung epithelial cell mitogen that is cytoprotective during injury. Transgenic mice that conditionally expressed FGF7 were used to dissect the mechanisms of FGF7 protection during lung injury. FGF7 improved survival when induced 3 days prior to acute lung injury. In contrast, FGF7 caused pulmonary inflammation and lung injury after 7 days or longer. Gene expression analysis of mouse lung mRNA identified mRNAs that contribute to the protective effects of FGF7. FGF7 improved survival during acute lung injury in adult mouse lung after short-term expression, but paradoxically induced inflammation and injury after persistent expression.