The biological revolution - towards a mechanistic understanding of the impact of diet on cancer risk

There is strong epidemiological evidence to show that differences in diet explain a significant proportion of the variation in cancer incidence worldwide. However, because of the complex nature of eating behaviour and the chemical heterogeneity of foods, it remains very difficult to ascertain which aspects of diet, in what quantities and over what time-frames are responsible for modifying risk. In addition, there are few dietary intervention studies demonstrating reduction in cancer risk. Much faster progress has been made in understanding the biological basis of cancer. It is now clear that damage to the genome resulting in aberrant expression of genes (principally suppression of tumour suppressor genes (TSGs) and inappropriate expression of oncogenes) is fundamental to tumorigenesis. It is also becoming clear that much of the inter-individual variation in cancer experience is due to differences in the amount of damage experienced and/or the capacity to repair that damage. Both of these processes are influenced strongly by dietary factors and by genetic predisposition (polymorphisms in the requisite genes). It is possible that understanding diet:gene interactions in DNA damage and in repair will not only explain much of the inter-individual variation in risk but also offer opportunities to design better dietary intervention studies aimed at chemoprevention. The Human Genome maps and the SNPs databases, together with the rapid development of tools suitable for investigating genetic and epigenetic changes in small tissue biopsies provide the means to begin to test hypotheses about the mechanisms by which diet influences cancer risk directly in human subjects. This is likely to form a significant component of the emerging science of nutrigenomics.     

Molecular epidemiology in cancer research

The use of molecular biomarkers in epidemiological investigations brings clear advantages of economy, speed and precision. Epidemiology--the study of the factors that control the patterns of incidence of disease--normally requires large numbers of subjects and/or long periods of time, because what is measured (the occurrence of disease) is a rare event. Biomarkers are measurable biological parameters that reflect, in some way, an individual's risk of disease-because they indicate exposure to a causative (or protective) agent, or because they represent an early stage in development of the disease, or because they allow an assessment of individual susceptibility. Biomarkers must be usable on one of the few materials available for biomonitoring of humans, i.e. blood, urine, exfoliated epithelial cells and, with some difficulty, biopsies. The approach of molecular epidemiology has a great potential is several areas of cancer research: investigating the aetiology of the disease; monitoring cancer risk in people exposed to occupational or environmental carcinogens; studying factors that protect from cancer; and assessing intrinsic factors that might predispose to cancer. The biomarkers most commonly employed in cancer epidemiology include: measurements of DNA damage--DNA breaks, altered bases, bulky adducts--in lymphocytes; the surrogate marker of chemical modifications to blood proteins, caused by agents that also damage DNA; the presence of metabolites of DNA-damaging agents (or the products of DNA damage themselves) in urine; chromosome alterations, including translocations, micronuclei and sister chromatid exchange, resulting from DNA damage; mutations in marker genes; DNA repair; and the differential expression of a variety of enzymes, involved in both activation and detoxification of carcinogens, that help to determine individual susceptibility. The molecular approach has been enthusiastically employed in several studies of occupational/environmental exposure to carcinogens. While the estimation of biological markers of exposure has certainly shown the expected effects in terms of DNA damage and adducts, the detection of the biological effects of exposure (e.g. at the level of chromosome alterations) has not been so clear-cut. This is true also when smokers are examined as a group compared with non-smokers. Several markers (especially of chromosome damage and mutation) show a strong correlation with age-indicating either an increasing susceptibility to damage with age, or an accumulation of long-lived changes. DNA repair--a crucial player in the removal of damage before it can cause mutation--may vary between individuals, and may be modulated by intrinsic or extrinsic factors, but limited data are available because of the lack of a reliable assay. Information on other enzymes determining individual susceptibility does exist, and some significant effects of phenotypic or genotypic polymorphisms have emerged, although the interactions between various enzymes make the situation very complex. The important question of whether oxidative DNA damage in normal cells is decreased by dietary antioxidants (vitamin C, carotenoids etc., from fruit and vegetables) has been tackled in antioxidant supplementation experiments. The use of poorly validated assays for base oxidation has not helped us to reach a definitive answer; it seems that, in any case, the level of oxidative damage has been greatly exaggerated. DNA-damaging agents lead to characteristic kinds of base changes (transitions, transversions, deletions). The investigation of the spectrum of mutations in cancer-related genes studied in tumour tissue should lead to a better understanding of the agents ultimately responsible for inducing the tumour. Similarly, studying mutations in a neutral marker gene (not involved in tumorigenesis) can tell us about the origins of the 'background' level of mutations. So far, interpretation of the growing databases is largely speculative. (ABSTRACT     

Klinik und Genetik der Neurofibromatose Typ 1

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by mutations in the NF1 gene located on chromosome 17q11.2. NF1 is fully penetrant, meaning that every individual carrying the mutation exhibits symptoms of the disease, although with some considerably variable expressivity. NF1 is characterised by the eponymous neurofibromas, which are benign Schwann cell tumours. Among the other main characteristic features of NF1 are pigmentary anomalies such as café-au-lait spots, axillary or inguinal freckling, and Lisch nodules. NF1 is a member of the class of hereditary cancer syndromes, and patients with NF1 are at increased risk of developing specific NF1-associated tumours. These tumours are caused by the biallelic inactivation of the NF1 tumour suppressor gene, resulting in aberrant Ras regulation. Over the last few years, significant progress has been made in identifying and managing the clinical symptoms of NF1 as well as in developing novel therapeutic approaches.     

Systematic evaluation of genetic variation at the androgen receptor locus and risk of prostate cancer in a multiethnic cohort study

Repeat length of the CAG microsatellite polymorphism in exon 1 of the androgen receptor (AR) gene has been associated with risk of prostate cancer in humans. This association has been the focus of >20 primary epidemiological publications and multiple review articles, but a consistent and reproducible association has yet to be confirmed. We systematically addressed possible causes of false-negative and false-positive association in >4,000 individuals from a multiethnic, prospective cohort study of prostate cancer, comprehensively studying genetic variation by microsatellite genotyping, direct resequencing of exons in advanced cancer cases, and haplotype analysis across the 180-kb AR genomic locus. These data failed to confirm that common genetic variation in the AR gene locus influences risk of prostate cancer. A systematic approach that assesses both coding and noncoding genetic variation in large and diverse patient samples can help clarify hypotheses about association between genetic variants and disease.     

EGCG synergizes the therapeutic effect of irinotecan through enhanced DNA damage in human colorectal cancer cells

Irinotecan is a kind of alkaloid with antitumour activity, but its low solubility and high toxicity limit its application. Epigallocatechin-3-gallate (EGCG) is one of the main bioactive components in tea. The epidemiological investigation and animal and cell experiments show that EGCG has a preventive and therapeutic effect on many kinds of tumours. Here, colorectal cancer cells RKO and HCT116 were employed, and the CCK8 proliferation test was used to screen the appropriate concentration of EGCG and irinotecan, and the effects of single and/or combined drugs on migration, invasion, DNA damage, cell cycle and autophagy of tumour cells were investigated. The results showed that EGCG combined with irinotecan (0.5 μmol L⁻) not only had a stronger inhibitory effect on tumour cells than EGCG or irinotecan alone but also prevented tumour cell migration and invasion. EGCG alone did not cause DNA damage in colorectal cancer cells, but its combination with irinotecan could induce S or G2 phase arrest by inhibiting topoisomerase I to cause more extensive DNA damage. EGCG also induced apoptosis by promoting autophagy with irinotecan synergistically. These results indicated that EGCG in combination with irinotecan could be a promising strategy for colorectal cancer.     

An EMT‐related gene signature for the prognosis of human bladder cancer

The transition from non–muscle‐invasive bladder cancer (NMIBC) to muscle‐invasive bladder cancer (MIBC) is detrimental to bladder cancer (BLCA) patients. Here, we aimed to study the underlying mechanism of the subtype transition. Gene set variation analysis (GSVA) revealed the epithelial‐mesenchymal transition (EMT) signalling pathway with the most positive correlation in this transition. Then, we built a LASSO Cox regression model of an EMT‐related gene signature in BLCA. The patients with high risk scores had significantly worse overall survival (OS) and disease‐free survival (DFS) than those with low risk scores. The EMT‐related gene signature also performed favourably in the accuracy of prognosis and in the subtype survival analysis. Univariate and multivariate Cox regression analyses demonstrated that the EMT‐related gene signature, pathological N stage and age were independent prognostic factors for predicting survival in BLCA patients. Furthermore, the predictive nomogram model was able to effectively predict the outcome of BLCA patients by appropriately stratifying the risk score. In conclusion, we developed a novel EMT‐related gene signature that has tumour‐promoting effects, acts as a negative independent prognostic factor and might facilitate personalized counselling and treatment in BLCA.     

Carcinogen: DNA adducts in tobacco smoke-associated cancer of the upper respiratory tract.

Mortality connected with tobacco smoke-associated laryngeal cancer in Poland markedly exceeds the relevant epidemiological data from other European countries. The main groups of genotoxic agents considered as potential carcinogens present in tobacco smoke are polycyclic aromatic hydrocarbons, aromatic amines, N-nitrosoamines and reactive oxygen species. Aromatic DNA adducts, N7-alkylated guanosines and oxidative DNA damage derived from tobacco smoke exposure were detected in laryngeal and oral (tumour and non-tumour) biopsies, and white blood cells of cancer subjects. Further, DNA lesions were analysed to estimate the significance of such confounders as intensity of smoking, subject's sex, age, topography of larynx, cancer staging and genetic factor. The number of cigarettes smoked per day was found to be the main determinant of an individual's DNA adduct level. The occurrence of DNA lesions was established as a reliable marker of former exposure to tobacco smoke genotoxicants. On the other hand, a comparison of DNA lesion levels in various regions of larynx indicates limited usefulness of DNA adduct analysis as an estimate of cancer risk. For a better risk estimation one has to take into account DNA lesions in proto-oncogenes and tumour suppressor genes and the efficacy of DNA repair. Altogether, DNA adducts formation and removal has to be considered as a single stage in the multistep carcinogenesis.     

Novel gene signatures for prognosis prediction in ovarian cancer

Ovarian cancer (OV) is one of the leading causes of cancer deaths in women worldwide. Late diagnosis and heterogeneous treatment result to poor survival outcomes for patients with OV. Therefore, we aimed to develop novel biomarkers for prognosis prediction from the potential molecular mechanism of tumorigenesis. Eight eligible data sets related to OV in GEO database were integrated to identify differential expression genes (DEGs) between tumour tissues and normal. Enrichment analyses discovered DEGs were most significantly enriched in G2/M checkpoint signalling pathway. Subsequently, we constructed a multi‐gene signature based on the LASSO Cox regression model in the TCGA database and time‐dependent ROC curves showed good predictive accuracy for 1‐, 3‐ and 5‐year overall survival. Utility in various types of OV was validated through subgroup survival analysis. Risk scores formulated by the multi‐gene signature stratified patients into high‐risk and low‐risk, and the former inclined worse overall survival than the latter. By incorporating this signature with age and pathological tumour stage, a visual predictive nomogram was established, which was useful for clinicians to predict survival outcome of patients. Furthermore, SNRPD1 and EFNA5 were selected from the multi‐gene signature as simplified prognostic indicators. Higher EFNA5 expression or lower SNRPD1 indicated poorer outcome. The correlation between signature gene expression and clinical characteristics was observed through WGCNA. Drug‐gene interaction was used to identify 16 potentially targeted drugs for OV treatment. In conclusion, we established novel gene signatures as independent prognostic factors to stratify the risk of OV patients and facilitate the implementation of personalized therapies.     

From gene mutations to tumours--stem cells in gastrointestinal carcinogenesis

Stem cells share many properties with malignant cells, such as the ability to self-renew and proliferate. Cancer is believed to be a disease of stem cells. The gastrointestinal tract has high cancer prevalence partly because of rapid epithelial cell turnover and exposure to dietary toxins. The molecular pathways of carcinogenesis differ according to the tissue. Work on hereditary cancer syndromes including familial adenomatous polyposis (FAP) has led to advances in our understanding of the events that occur in tumour development from a gastrointestinal stem cell. The initial mutation involved in the adenoma-carcinoma sequence is in the 'gatekeeper' tumour-suppressor gene adenomatous polyposis coli (APC). Somatic hits in this gene are non-random in FAP, with the type of mutation selected for by the position of the germline mutation. In the stomach, a metaplasia-dysplasia sequence occurs and is often related to Helicobacter pylori infection. Clonal expansion of mutated cells occurs by niche succession. Further expansion of the aberrant clone then occurs by the longitudinal division of crypts into two daughter units--crypt fission. Two theories seek to explain the early development of adenomas--the 'top down' and 'bottom up' hypotheses. Initial studies suggested that colorectal tumours were monoclonal; however, later work on chimeric mice and a sex chromosome mixoploid patient with FAP suggested that up to 76% of early adenomas were polyclonal. Introduction of a homozygous resistance allele has reduced tumour multiplicity in the mouse and has been used to rule out random collision of polyps as the cause of these observations. It is likely that short-range interaction between adjacent initiated crypts is responsible for polyclonality.     

Molecular epidemiology studies of carcinogenic environmental pollutants. Effects of polycyclic aromatic hydrocarbons (PAHs) in environmental pollution on exogenous and oxidative DNA damage

Exposure to high levels of environmental air pollution is known to be associated with an increased carcinogenic risk. The individual contribution to this risk derived from specific carcinogenic chemicals within the complex mixture of air pollution is less certain, but may be explored by the use of molecular epidemiological techniques. Measurements of biomarkers of exposure, of effect and of susceptibility provide information of potential benefit for epidemiological and cancer risk assessment. The application of such techniques has been mostly concerned in the past with the carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) that are associated with particulate matter in air pollution, and has showed clear evidence of genotoxic effects, such as DNA adducts, chromosome aberrations (CA) and ras oncogene overexpression, in environmentally exposed Czech and Polish populations. We are currently extending these studies by an investigation of populations exposed to environmental pollution in three European countries, Czech Republic, Slovak Republic and Bulgaria. This pays particular attention to PAHs, but also investigates the extent of radically induced (oxidative) DNA damage in the exposed populations. Policemen, bus drivers and controls, who carried personal monitors to determine their exposures to PAHs have been studied, and blood and urine were collected. Antioxidant and dietary status were assessed in these populations. Stationary monitors were also used for ambient air monitoring. Amongst the parameters studied in the biological samples were: (a) exposure biomarkers, such as PAH adducts with DNA, p53 and p21(WAF1) protein levels, (b) oxidative DNA damage, (c) the biological effect of the exposure by measurement of chromosome damage by fluorescence in situ hybridisation (FISH) or conventional methods, and (d) polymorphisms in carcinogen metabolising and DNA repair enzymes. Repair ability was also measured by the Comet assay. In vitro systems are being evaluated to characterise the genotoxicity of the organic compounds adsorbed to air particles.     

MTHFR and MTRR genotype and haplotype analysis and colorectal cancer susceptibility in a case-control study from the Czech Republic

Polymorphic variants in genes involved in one-carbon metabolism, in particular of dietary folate, may modulate the risk for colorectal cancer through aberrant DNA-methylation and altered nucleotide synthesis and repair. In the present study, we have assessed the association of six polymorphisms and relative haplotypes in the MTHFR gene (rs1801133 and rs1801131) and in the MTRR gene (rs1801394, rs1532268, rs162036, and rs10380) with the risk for colorectal cancer in 666 patients and 1377 controls from the Czech Republic. We found that the 677 C>T polymorphism in the MTHFR gene significantly decreased the risk for colorectal cancer in homozygous carriers of the variant allele (OR, 0.58; 95% CI, 0.39-0.87). Also, we noted a significantly different distribution of genotypes between cases and controls for the 66A>G polymorphism in the MTRR gene. In particular, homozygous carriers of the G-containing allele of this polymorphism were at an increased risk for colorectal cancer (OR, 1.39; 95% CI, 1.04-1.85). Haplotype analysis of the two MTHFR polymorphisms showed a moderate difference in the distribution of the TA haplotype between cases and controls. In comparison to the most common haplotype (CA), the TA haplotype was associated with a decreased risk for colorectal cancer (OR, 0.84; 95% CI, 0.71-0.99). No difference in the distribution between cases and controls was observed for the haplotypes based on the four polymorphisms in the MTRR gene. The present study suggests that the 677TT genotype and the TA haplotype in the MTHFR gene may also have a role in colorectal cancer risk in the Czech population, indicating the importance of genes involved in folate metabolism with respect to cancer risk. For MTRR, additional studies on larger populations are needed to clarify the possible role of variation in this gene in colorectal carcinogenesis.     

Tumour size can have an impact on the outcomes of epidemiological studies on second cancers after radiotherapy

Obtaining a correct dose–response relationship for radiation-induced cancer after radiotherapy presents a major challenge for epidemiological studies. The purpose of this paper is to gain a better understanding of the associated uncertainties. To accomplish this goal, some aspects of an epidemiological study on breast cancer following radiotherapy of Hodgkin’s disease were simulated with Monte Carlo methods. It is demonstrated that although the doses to the breast volume are calculated by one treatment plan, the locations and sizes of the induced secondary breast tumours can be simulated and, based on these simulated locations and sizes, the absorbed doses at the site of tumour incidence can also be simulated. For the simulations of point dose at tumour site, linear and non-linear mechanistic models which predict risk of cancer induction as a function of dose were applied randomly to the treatment plan. These simulations provided for each second tumour and each simulated tumour size the predicted dose. The predicted-dose–response-characteristic from the analysis of the simulated epidemiological study was analysed. If a linear dose–response relationship for cancer induction was applied to calculate the theoretical doses at the simulated tumour sites, all Monte-Carlo realizations of the epidemiological study yielded strong evidence for a resulting linear risk to predicted-dose–response. However, if a non-linear dose–response of cancer induction was applied to calculate the theoretical doses, the Monte Carlo simulated epidemiological study resulted in a non-linear risk to predicted-dose–response relationship only if the tumour size was small (<?1.5 cm). If the diagnosed breast tumours exceeded an average diameter of 1.5 cm, an applied non-linear theoretical-dose–response relationship for second cancer falsely resulted in strong evidence for a linear predicted-dose relationship from the epidemiological study realizations. For a typical distribution of breast cancer sizes, the model selection probability for a resulting predicted-dose linear model was 61% although a non-linear theoretical-dose–response relationship for cancer induction had been applied. The results of this study, therefore, provide evidence that the shapes of epidemiologically obtained dose–response relationships for cancer induction can be biased by the finite size of the diagnosed second tumour, even though the epidemiological study was done correctly.     

Identification of cancer genes using a statistical framework for multiexperiment analysis of nondiscretized array CGH data

Tumor formation is in part driven by DNA copy number alterations (CNAs), which can be measured using microarray-based Comparative Genomic Hybridization (aCGH). Multiexperiment analysis of aCGH data from tumors allows discovery of recurrent CNAs that are potentially causal to cancer development. Until now, multiexperiment aCGH data analysis has been dependent on discretization of measurement data to a gain, loss or no-change state. Valuable biological information is lost when a heterogeneous system such as a solid tumor is reduced to these states. We have developed a new approach which inputs nondiscretized aCGH data to identify regions that are significantly aberrant across an entire tumor set. Our method is based on kernel regression and accounts for the strength of a probe's signal, its local genomic environment and the signal distribution across multiple tumors. In an analysis of 89 human breast tumors, our method showed enrichment for known cancer genes in the detected regions and identified aberrations that are strongly associated with breast cancer subtypes and clinical parameters. Furthermore, we identified 18 recurrent aberrant regions in a new dataset of 19 p53-deficient mouse mammary tumors. These regions, combined with gene expression microarray data, point to known cancer genes and novel candidate cancer genes.     

The combination of genetic variations in the PRDX3 gene and dietary fat intake contribute to obesity risk

Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and the antioxidant capacity of the cell. This imbalance and an excess of ROS induce tissue/cellular damage, which are implicated in chronic inflammation disorders such as obesity, insulin resistance, and metabolic syndromes. Peroxiredoxins (Prxs) are the most abundant and ancient cellular antioxidant proteins that help to control intracellular peroxide levels and ROS-dependent signaling. Of the six mammalian isoforms, Prx III is specifically localized in mitochondria. In this study, we detected novel associations between genetic variations of the PRDX3 gene and BMI and obesity risk in the general Japanese population. In addition, these associations were observed only in the subjects with high dietary fat intake, but not in the subjects with low dietary fat intake. These findings indicate that the interaction between genetic variations in the PRDX3 gene and dietary fat intake is important for modulation of BMI and obesity risk.     

DNA methylation in endometrial cancer

Endometrial cancer is the most commonly diagnosed gynecological cancer, and it has been shown to be a complex disease driven by abnormal genetic, and epigenetic alterations, as well as environmental factors. Epigenetic changes resulting in aberrant gene expression are dynamic and modifiable features of many cancer types. A significant epigenetic change is aberrant DNA methylation. In this review, we review evidence on the role of aberrant DNA methylation, examining changes in relation to endometrial carcinogenesis, and report on recent advances in the understanding of the contribution of aberrant DNA methylation to endometrial cancer with the emphasis on the role of dietary/ lifestyle and environmental factors, as well as opportunities and challenges of DNA methylation in endometrial cancer management and prevention.