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Simple Summary The HPV oncoproteins E6 and E7 can modulate the expression and activity of the maintenance DNA methyltransferase 1, suggesting that HPV carcinogenic mechanisms may include aberrant DNA methylation. Some studies previously proposed both gene-associated DNA methylation signatures and a global hypermethylation profile in HPV-positive head and neck cancer, but the validation of such signatures and a more detailed analysis of the methylation profile of transposable elements (TEs) in oropharyngeal squamous cell carcinoma (OPSCC) are still missing. TEs account for approximately 50% of the human genome and their hypomethylation and reactivation have been consistently reported in cancer, usually being associated with worse prognosis. Based on this, this study aimed at validating a previously established 5-CpG methylation signature in FFPE OPSCC from a middle-income population, in which the frequency of HPV infection is only 6.1%, and dissecting the methylation profile of TEs, focusing on their impact on gene expression and overall survival. Abstract HPV oncoproteins can modulate DNMT1 expression and activity, and previous studies have reported both gene-specific and global DNA methylation alterations according to HPV status in head and neck cancer. However, validation of these findings and a more detailed analysis of the transposable elements (TEs) are still missing. Here we performed pyrosequencing to evaluate a 5-CpG methylation signature and Line1 methylation in an oropharyngeal squamous cell carcinoma (OPSCC) cohort. We further evaluated the methylation levels of the TEs, their correlation with gene expression and their impact on overall survival (OS) using the TCGA cohort. In our dataset, the 5-CpG signature distinguished HPV-positive and HPV-negative OPSCC with 66.67% sensitivity and 84.33% specificity. Line1 methylation levels were higher in HPV-positive cases. In the TCGA cohort, Line1, Alu and long terminal repeats (LTRs) showed hypermethylation in a frequency of 60.5%, 58.9% and 92.3%, respectively. ZNF541 and CCNL1 higher expression was observed in HPV-positive OPSCC, correlated with lower methylation levels of promoter-associated Alu and LTR, respectively, and independently associated with better OS. Based on our findings, we may conclude that a 5-CpG methylation signature can discriminate OPSCC according to HPV status with high accuracy and TEs are differentially methylated and may regulate gene expression in HPV-positive OPSCC.

International audience; Omics approaches are continuously providing new clues on the mechanisms of action of contaminants in species of environmental relevance, contributing to the emergence of molecular ecotoxicology. Co-expression network approaches represent a suitable methodological framework for studying the rich content of omics datasets. This study aimed to find evidence of key pathways and proteins related to the testicular toxicity in the sentinel crustacean species Gammarus fossarum exposed to endocrine disruptors using a weighted protein co-expression network analysis. From a shotgun proteomics dataset of male gonads of G. fossarum organisms exposed to cadmium (Cd), pyriproxyfen (Pyr) and methoxyfenozide (Met) in laboratory conditions, four distinct modules were identified as significantly correlated to contaminants' exposure. Protein set enrichment analysis identified modules involved in cytoskeleton organization and oxidative stress response associated with the Cd exposure. The module associated with Pyr exposure was associated with endoplasmic reticulum stress (ER) response, and the module correlated with Met exposure was characterized by a significant proportion of amphipod-restricted proteins whose functions are still not characterized. Our results show that co-expression networks are efficient and adapted tools to identify new potential mode of actions from environmental sentinel species, such as G. fossarum, using a proteogenomic approach, even without an annotated genome.

Ambient particulate matter is the environmental factor with the highest contribution to global disease burden and mortality. Open questions remain regarding causality at low doses and the effects of specific pollutants. Establishing causality in regards to air pollution is methodologically challenging, affecting the establishment of regulatory policies. In an effort to address this problem, we suggest combining the concept of the exposome with the Meet-in-the-Middle approach (MITM). This approach consists of measuring molecular fingerprints and relating them retrospectively to measurements of external exposure and prospectively to a health outcome. Markers robustly associated with both ends of the exposure-to-disease continuum, validate a causal hypothesis5. In the context of carcinogenesis, this approach allows establishing the relationship between the middle-to-outcome nature of the hallmarks of cancer with the bottom-to-middle approaches of the key characteristics of carcinogens. We recently demonstrated proof of principle of this approach, by investigating the temporal sequence of hallmarks of cancer from the point of view of pathological specimens of cancer (branched mutational spectra), and then by considering the key characteristics of the carcinogen, benzo(a)pyrene. The main cancer pathways affected follow a generally common sequence: resisting cell death, insensitivity to anti-growth signals, sustained proliferation (almost simultaneous), deregulated energetics, replicative immortality, and activation of invasion and metastasis. Angiogenesis and avoiding immune destruction display a varying position in the above sequence. At the same time, “key characteristics” of BaP were found associated with most hallmarks of cancer, supporting its carcinogenicity. A MITM approach, using exposomic evidence, is a promising approach that can successfully address causality concerns in regards to air-pollution toxicology and the need for regulatory policies. Key messages Investigation of tumour mutational spectra and of the mechanisms of action of a carcinogen, reveals an overlap between the hallmarks of cancer and the key characteristics of the carcinogen. This investigation provides proof of principle that the exposome/meet-in-the-middle approach can address concerns in air-pollution toxicology and provide evidence to support regulatory policies.

The Human Leucocyte Antigen (HLA) locus and other DNA sequence variants identified in Genome-Wide Association (GWA) studies explain around 50% of the heritability of celiac disease (CD). However, the pathogenesis of CD could be driven by other layers of genomic information independent from sequence variation, such as DNA methylation, and it is possible that allele-specific methylation explains part of the SNP associations. Since the DNA methylation landscape is expected to be different among cell types, we analyzed the methylome of the epithelial and immune cell populations of duodenal biopsies in CD patients and controls separately. We found a cell type-specific methylation signature that includes genes mapping to the HLA region, namely TAP1 and HLA-B. We also performed Immunochip SNP genotyping of the same samples and interrogated the expression of some of the affected genes. Our analysis revealed that the epithelial methylome is characterized by the loss of CpG island (CGI) boundaries, often associated to altered gene expression, and by the increased variability of the methylation across the samples. The overlap between differentially methylated positions (DMPs) and CD-associated SNPs or variants contributing to methylation quantitative trait loci (mQTLs) is minimal. In contrast, there is a notable enrichment of mQTLs among the most significant CD-associated SNPs. Our results support the notion that DNA methylation alterations constitute a genotype-independent event and confirm its role in the HLA region (apart from the well-known, DQ allele-specific effect). Finally, we find that a fraction of the CD-associated variants could exert its phenotypic effect through DNA methylation. The authors thank the technical and human support provided by SGIker of the UPV/EHU and the Genomics Platform at the CIC bioGUNE. We also thank Dr Florence Le Calvez-Kelm and Mr Geoffroy Durand from the Genetic Cancer Susceptibility group (IARC, Lyon) for their help with the HM450 beadchip assays. The work was funded by ISCIII Research Project Grants PI13/01201 and PI16/00258, cofunded by the European Union ERDF/ESF "A way to make Europe" co-financed by the Spanish Ministry of Economy and Competitiveness -http://www.mineco.gob.es/-and by the European Union ERDF/ESF "A way to make Europe" and project 2011/111034 from the Basque Department of Health -http://www.euskadi.eus/gobierno-vasco/departamento-salud/inicio/-to J.R.B. N.F.J. was supported by an IARC Postodctoral Fellowship (FP7 Marie Curie Actions-People-COFUND) and a Postdoctoral Fellowship from the Basque Department of Education -http://training.iarc.fr/en/fellowships/postdoc.php-.I.R.G.and A.J.M. are supported by Predoctoral Fellowship grants from the UPV/EHU and the Basque Department of Education -http://www.euskadi.eus/gobierno-vasco/departamento-educacion/-, respectively. S.E. was supported by the abovementioned IARC Postdoctoral Fellowship program. ACR is an Ikerbasque Research Fellow -http://www.ikerbasque.net/-.The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data nor in writing the manuscript.

The large geographic variations in the incidence of gastric cancer (GC) are likely due to differential environmental exposures, in particular to Helicobacter pylori (H. pylori) infection. We aimed to investigate the impact of H. pylori on the epigenome in normal gastric mucosa and methylation changes associated with cancer risk independent of H. pylori. A discovery set of normal gastric mucosa from GC cases (n = 42) and controls (n = 42), nested in a large case-control study and stratified by H. pylori status, were subjected to genome-wide methylation profiling. Single-nucleotide polymorphism arrays from peripheral blood leukocytes were used to conduct methylation quantitative trait loci (mQTL) analysis. A validation set of gastric mucosa samples (n = 180) was used in the replication phase. We found 1,924 differentially methylated positions (DMPs) and 438 differentially methylated regions (DMRs) associated with H. pylori infection, most of which were hypermethylated. Significant methylation alterations identified in the initial set were successfully replicated. Furthermore, the H. pylori-associated DMP/Rs showed marked stability ('epigenetic memory') after H. pylori clearance. Interestingly, we found 152 DMRs associated with cancer risk independent of the H. pylori status in normal gastric mucosa. The methylation score derived from three biomarkers was a strong predictor of GC. Finally, the mQTL analysis indicated that the H. pylori- and cancer-specific methylation signatures were minimally affected by genetic variation. The comprehensively characterized methylome changes associated with H. pylori infection and GC risk in our study might serve as potential biomarkers for early cancer progression in tumour-free gastric mucosa.

Purpose: Breast cancer is among the leading causes of cancer-related death; discovery of novel prognostic markers is needed to improve outcomes. Combining systems biology and epidemiology, we investigated miRNA-associated genes and breast cancer survival in a well-characterized population-based study. Experimental Design: A recently developed algorithm, ActMiR, was used to identify key miRNA “activities” corresponding to target gene degradation, which were predictive of breast cancer mortality in published databases. We profiled miRNA-associated genes in tumors from our well-characterized population-based cohort of 606 women with first primary breast cancer. Cox proportional hazards models were used to estimate HRs and 95% confidence intervals (CI), after 15+ years of follow-up with 119 breast cancer–specific deaths. Results: miR-500a activity was identified as a key miRNA for estrogen receptor–positive breast cancer mortality using public databases. From a panel of 161 miR-500a–associated genes profiled, 73 were significantly associated with breast cancer–specific mortality (FDR < 0.05) in our population, among which two clusters were observed to have opposing directions of association. For example, high level of SUSD3 was associated with reduced breast cancer–specific mortality (HR = 0.3; 95% CI, 0.2–0.4), whereas the opposite was observed for TPX2 (HR = 2.7; 95% CI, 1.8–3.9). Most importantly, we identified set of genes for which associations with breast cancer–specific mortality were independent of known prognostic factors, including hormone receptor status and PAM50–derived risk-of-recurrence scores. These results are validated in independent datasets. Conclusions: We identified novel markers that may improve prognostic efficiency while shedding light on molecular mechanisms of breast cancer progression. Clin Cancer Res; 24(3); 581–91. ©2017 AACR.

Simple Summary Squamous cell carcinomas of the upper aerodigestive tract are highly incident, lethal, and share the same epithelial lining of origin, risk factors and genetic alterations. However, their biological and clinical behaviors differ, having an impact on patient survival. This study aimed at identifying the main DNA methylation differences between these tumors, giving an overview of the main genomic regions affected, whether DNA methylation gains or losses are more common, the impact on gene expression and the signaling pathways affected. This knowledge will help identifying potential site-specific biomarkers as well as shedding light on whether epigenetic mechanisms explain, at least in part, the diverse behavior of upper aerodigestive tract tumors. Abstract Upper aerodigestive tract (UADT) tumors present different biological behavior and prognosis, suggesting specific molecular mechanisms underlying their development. However, they are rarely considered as single entities (particularly head and neck subsites) and share the most common genetic alterations. Therefore, there is a need for a better understanding of the global DNA methylation differences among UADT tumors. We performed a genome-wide DNA methylation analysis of esophageal (ESCC), laryngeal (LSCC), oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinomas, and their non-tumor counterparts. The unsupervised analysis showed that non-tumor tissues present markedly distinct DNA methylation profiles, while tumors are highly heterogeneous. Hypomethylation was more frequent in LSCC and OPSCC, while ESCC and OSCC presented mostly hypermethylation, with the latter showing a CpG island overrepresentation. Differentially methylated regions affected genes in 127 signaling pathways, with only 3.1% of these being common among different tumor subsites, but with different genes affected. The WNT signaling pathway, known to be dysregulated in different epithelial tumors, is a frequent hit for DNA methylation and gene expression alterations in ESCC and OPSCC, but mostly for genetic alterations in LSCC and OSCC. UADT tumor subsites present differences in genome-wide methylation regarding their profile, intensity, genomic regions and signaling pathways affected.

Next generation sequencing and mass spectrometry technologies have recently expanded the availability of whole transcriptomes and proteomes beyond classical model organisms in molecular biology, even in absence of an annotated genome. However, the fragmented nature of transcriptomic and proteomic data reduces the ability to interpret the data, notably in non-model organisms. Network-based approaches may help extracting important biological information from -omics datasets. The reproductive cycle of the freshwater crustacean Gammarus fossarum.provides an excellent case study to test the relevance of a network analysis in non-model organisms. Here, we illustrated how the use of a co-expression network analysis (based on Weighted Gene Co-expression Network Analysis algorithm, WGCNA) allowed identifying protein modules whose expression profiles described germ cell maturation and embryonic development in the freshwater crustacean Gammarus fossarum. Proteome datasets included testes, ovaries or embryos samples at different maturation or developmental stages, respectively. We identified an embryonic module correlated with mid-developmental stages corresponding to the organogenesis and it was characterized by enrichment in proteins involved in RNA editing and splicing. An ovarian module was enriched in vitellogenin-like proteins and clottable proteins, confirming the diversity of proteins belonging to the large lipid transfer family involved in oocytes maturations in this freshwater amphipod. Moreover, our results found evidence of a fine-tuned regulation between energy production by glycolysis and actin-myosin-dependent events in G. fossarum spermatogenesis. This study illustrates the importance of applying systems biology approaches to emergent animal models to improve the understanding of the molecular mechanisms regulating important physiological events with ecological relevance.

Functional characterization of long non-coding RNAs (lncRNAs) and their pathological relevance is still a challenging task. Abnormal expression of a few long non-coding RNAs have been found associated with hepatocellular carcinoma, with potential implications to both improve our understanding of molecular mechanism of liver carcinogenesis and to discover biomarkers for early diagnosis or therapy. However, the understanding of the global role of lncRNAs during HCC development is still in its infancy. In this study, we produced RNA-Seq data from 23 liver tissues (controls, cirrhotic and HCCs) and applied statistical and gene network analysis approaches to identify and characterize expressed lncRNAs. We detected 5,525 lncRNAs across different tissue types and identified 57 differentially expressed lncRNAs in HCC compared with adjacent non-tumour tissues using stringent criteria (FDR2). Using weighted gene co-expression network analysis (WGCNA), we found that differentially expressed lncRNAs are co-expressed with genes involved in cell cycle regulation, TGF-β signalling and liver metabolism. Furthermore, we found that more than 20% of differentially expressed lncRNAs are associated to actively transcribed enhancers and that the co-expression patterns with their closest genes change dramatically during HCC development. Our study provides the most comprehensive compendium of lncRNAs expressed in HCC, as well as in control or cirrhotic livers. Our results identified both known oncogenic lncRNAs (such as H19 and CRNDE) and novel lncRNAs involved in cell cycle deregulation and liver metabolism deficits occurring during HCC development.

Systems biology has been driven by technology (the development of omics) and by statistical modelling and bioinformatics. We aim to bring biological thinking back. We suggest that three traditions of thought need to be considered: (a) causality in epidemiology, for example the “sufficient-component-cause framework”, and causality in other sciences, for example the Salmon and Dowe approach; (b) new acquisitions about disease pathogenesis, for example the “branched evolution model” in cancer, and the role of biomarkers in this process; (c) the burgeoning of omic research, with a large number of “signals” that need to be interpreted. To address the new challenges of epidemiology, the concept of the “exposome” has been proposed. We show examples from recent projects in the field, namely, new omic approaches applied to epidemiological studies; and in particular, the identification of hallmarks of cancer as intermediate steps between exposure to carcinogens and the cancer phenotype, according to the “meet-in-the-middle” concept. We use examples derived from the study of mutational spectra in tumours and benzo(a)pyrene and bisphenol A as model carcinogens. We suggest conceptualising the detection and tracing of signals in terms of information transmission.

BackgroundCancer is believed to arise through the perturbation of pathways and the order of pathway perturbation events can enhance understanding and evaluation of carcinogenicity. This order has not been examined so far, and this study aimed to fill this gap by attempting to gather evidence on the potential temporal sequence of events in carcinogenesis.DesignThe methodology followed was to discuss first the temporal sequence of hallmarks of cancer from the point of view of pathological specimens of cancer (essentially branched mutations) and then to consider the hallmarks of cancer that one well‐known carcinogen, benzo(a)pyrene, can modify.ResultsEven though the sequential order of driving genetic alterations can vary between and within tumours, the main cancer pathways affected are almost ubiquitous and follow a generally common sequence: resisting cell death, insensitivity to antigrowth signals, sustained proliferation, deregulated energetics, replicative immortality and activation of invasion and metastasis. The first 3 hallmarks can be regarded as almost simultaneous while angiogenesis and avoiding immune destruction are perhaps the only hallmarks with a varying position in the above sequence.ConclusionsOur review of hallmarks of cancer and their temporal sequence, based on mutational spectra in biopsies from different cancer sites, allowed us to propose a hypothetical temporal sequence of the hallmarks. This sequence can add molecular support to the evaluation of an agent as a carcinogen as it can be used as a conceptual framework for organising and evaluating the strength of existing evidence.

Previous studies indicate that the herb black cohosh (Actaea racemosa L.) and the triterpene glycoside actein inhibit the growth of human breast cancer cells and activate stress-associated responses. This study assessed the transcriptomic effects of black cohosh and actein on rat liver tissue, using Ingenuity and ToxFX analyses. Sprague-Dawley rats were treated with an extract of black cohosh enriched in triterpene glycosides (27%) for 24 h or actein for 6 and 24 h, at 35.7 mg/kg, and liver tissue collected for gene expression analysis. Ingenuity analysis indicates the top canonical pathways are, for black cohosh, RAR Activation, and, for actein, Superpathway of Cholesterol Biosynthesis, at 24 h. Actein alters the expression of cholesterol biosynthetic genes, but does not inhibit HMG-CoA reductase activity. Black cohosh and actein inhibited the growth of human breast and colon cancer cells and synergized with the statin simvastatin. Combinations of black cohosh with certain classes of statins could enhance their activity, as well as toxic, such as inflammatory liver, side effects. Transcriptomic analysis indicates black cohosh and actein warrant further study to prevent and treat cancer and lipid disorders. This study lays the basis for an approach to characterize the mode of action and toxicity of herbal medicines.

Introduction and aim. Epigenetic alterations pl Introduction and aim. ay an essential role in cancer onset and progression, thus studies of drugs targeting the epigenetic machinery are a principal concern for cancer treatment. Here, we evaluated the potential of the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5aza-dC) and the pan-deacetylase inhibitor Trichostatin A (TSA), at low cytotoxic concentrations, to modulate the canonical Wnt/E-catenin pathway in liver cancer cells. Material and Material and methods. methods. Pyrosequencing was used for DNA methylation analyses of LINE-1 sequences and the Wnt/E-catenin pathway antagonist DKK3, SFRP1, WIF1 and CDH1. qRT-PCR was employed to verify the expression of the antagonist. Pathway regulation were evaluated looking at the expression of E-catenin and E-cadherin by confocal microscopy and the antitumoral effects of the drugs was studied by wound healing and clonogenic assays. Results. Our resul Results. t suggest that 5aza-dC and TSA treatments were enough to induce a significant expression of the pathway antagonists, decrease of E-catenin protein levels, re-localization of the protein to the plasma membrane, and pathway transcriptional activity reduction. These important effects exerted an antitumoral outcome shown by the reduction of the migration and clonogenic capabilities of the cells. Conclusion. We were able to demonstrate Wnt/ Conclusion. E-catenin pathway modulation through E-cadherin up-regulation induced by 5aza-dC and TSA treatments, under an activation-pathway background, like CTNNB1 and TP53 mutations. These findings provide evidences of the potential effect of epigenetic modifier drugs for liver cancer treatment. However, further research needs to be conducted, to determine the in vivo potential of this treatment regimen for the management of liver cancer. COL0006769

The interaction between the (epi)genetic makeup of an individual and his/her environmental exposure record (exposome) is accepted as a determinant factor for a significant proportion of human malignancies. Recent evidence has highlighted the key role of epigenetic mechanisms in mediating gene-environment interactions and translating exposures into tumorigenesis. There is also growing evidence that epigenetic changes may be risk factor-specific ("fingerprints") that should prove instrumental in the discovery of new biomarkers in cancer. Here, we review the state of the science of epigenetics associated with environmental stimuli and cancer risk, highlighting key developments in the field. Critical knowledge gaps and research needs are discussed and advances in epigenomics that may help in understanding the functional relevance of epigenetic alterations. Key elements required for causality inferences linking epigenetic changes to exposure and cancer are discussed and how these alterations can be incorporated in carcinogen evaluation and in understanding mechanisms underlying epigenome deregulation by the environment.

Background Oxidative stress is increased in hyperuricemic patients with acute myocardial infarction (AMI). In these patients, use of sulfhydrylACE-inhibitors (ACEIs), such as zofenopril or captopril, and xanthine oxidase inhibitors (XOIs), may potentially result in an enhanced antioxidant effect and improved survival. However, the benefit of such combination in post-myocardial infarction has never been verified. Objectives To test the usefulness of the combination therapy Zofenopril + XOI in improving survival free from MACE in post-AMI patients Methods We re-analyzed the data of the four SMILE (Survival of Myocardial Infarction Long-term Evaluation) studies by grouping patients according to the type of ACEIs and the use of XOIs. 165 (31.4%) of the 525 patients were treated with XOIs (79 under zofenopril and 86 under placebo, lisinopril or ramipril), whereas 360 were not (192 zofenopril and 168 placebo or other ACEIs). In these four groups, we separately estimated the 1-year combined risk of major cardiovascular events (MACE, death or hospitalization for cardiovascular causes). Results MACE occurred in 10.1% of patients receiving zofenopril + XOIs, in 18.6% receiving placebo or other ACEIs + XOIs, in 13.5% receiving zofenopril without XOIs and in 22.0% receiving placebo or other ACEIs, but no XOIs (p=0.034 across groups). Rate of survival free from MACE was significantly larger in patients treated with zofenopril and XOIs than with other ACEIs with no XOIs [hazard ratio: 2.29 (1.06, 4.91), p=0.034]. A non-significant trend for superiority of zofenopril + XOIs combination was observed vs. zofenopril alone [1.19 (0.54, 2.64), p=0.669] or vs. placebo or other ACEIs combined with XOIs [1.82 (0.78, 4.26), p=0.169]. Conclusions Our retrospective analysis suggests an improved survival free from MACE in post-AMI patients treated with a combination of an ACEI and urate lowering drug with antioxidant activity. Disclosure of Interest None declared

MicroRNAs (miRNAs) are small regulatory non-coding RNAs with a diversity of cellular functions, and are frequently dysregulated in cancer. Using a novel computational method (ActMir) that we recently developed, the “activity” of miRNA hsa-miR-500a was implicated in estrogen receptor (ER) positive breast cancer; however its targets and functional impact remain poorly understood. Here, we performed an extensive gene expression analysis in ER+ breast cancer cell lines, to reveal the targets of miR-500a-5p after experimental modulation of its levels. We found that among mRNAs targeted by miR-500a-5p there was enrichment in oxidative stress response genes. Moreover, in vitro exposure to oxidative stress using H2O2 induces miR-500a-5p overexpression and downregulation of the oxidative stress targets TXNRD1 and NFE2L2. Finally, expression of several of the identified miR-500a-5p targets related to oxidative stress, including TXNRD1, was associated with ER+ breast cancer survival in multiple datasets. Overall, we identify miR-500a-5p as an oxidative stress response miRNA whose activity may define breast cancer progression and survival.

Background Head and neck squamous cell carcinomas (HNSCCs) represent a heterogeneous group of cancers for which human papilloma virus (HPV) infection is an emerging risk factor. Previous studies showed promoter hypermethylation in HPV(+) oropharyngeal cancers, but only few consistent target genes have been so far described, and the evidence of a functional impact on gene expression is still limited. Methods We performed global and stratified pooled analyses of epigenome-wide data in HNSCCs based on the Illumina HumanMethylation450 bead-array data in order to identify tissue-specific components and common viral epigenetic targets in HPV-associated tumours. Results We identified novel differentially methylated CpGs and regions associated with viral infection that are independent of the anatomic site. In particular, most hypomethylated regions were characterized by a marked loss of CpG island boundaries, which showed significant correlations with expression of neighbouring genes. Moreover, a subset of only five CpGs in a few hypomethylated regions predicted HPV status with a high level of specificity in different cohorts. Finally, this signature was a better predictor of survival compared with HPV status determined by viral gene expression by RNA sequencing in The Cancer Genome Atlas cohort. Conclusions We identified a novel epigenetic signature of HPV infection in HNSCCs which is independent of the anatomic site, is functionally correlated with gene expression and may be leveraged for improved stratification of prognosis in HNSCCs. Electronic supplementary material The online version of this article (doi:10.1186/s13073-017-0419-z) contains supplementary material, which is available to authorized users.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver pathology characterized by fat accumulation in a context of metabolic syndrome or insulin resistance. It can be associated with obesity, diabetes, hyperinsulinemia, dyslipidemia as well as hypertension. NAFLD consists of a large spectrum of hepatic lesions including benign steatosis, non-alcoholic steatohepatitis (NASH), cirrhosis or hepatocellular carcinoma. Upon chronic stress, NASH would occur via at least "two-hits" process involving modulation of a high number of genes and proteins. Firstly, the accumulation of fat, either due to the increased inflow of free fatty acids or de novo lipogenesis, leads to steatosis. Secondly, when adaptive mechanisms for stress tolerance are overwhelmed, lipotoxicity and chronic inflammation trigger major hepatic damages, mainly via oxidative and inflammatory stress, lipid peroxidation and cell death. As a consequence, all these processes concur to favor steatohepatitis, fibrosis and cancer. Recently, the elucidation of physiopathological signaling cascades controlling NAFLD and NASH benefited from large-scale studies, namely the omics, such as transcriptomics, genomics, proteomics, and lipidomics. The advent of lipidomics would allow shedding light upon the respective roles of triglyceride and fatty acid metabolites in the lipotoxic liver injury hypothesis for the pathogenesis of NASH. In this review, the contribution of the omics to the understanding of the molecular basis of NASH is discussed that could offer perspectives for novel biomarkers discovery.

Background S tudies indicate that extracts and purified components from black cohosh inhibit the growth of human breast cancer cells, but the molecular targets and signaling pathways have not yet been defined. Purpose This study examines the pharmacological mechanisms and toxicological effects in the short term of the herb black cohosh on female Sprague–Dawley rats. Materials and methods To assess effects on gene activity and lipid content, we treated female Sprague–Dawley rats with an extract of black cohosh enriched in triterpene glycosides (27%) at 35.7 or 0 mg/kg. Four animals for each group were sacrificed at 1, 6 and 24 h after treatment; liver tissue and serum samples were obtained for gene expression and lipid analysis. Results Microarray analysis of rat liver tissue indicated that black cohosh markedly downregulated mitochondrial oxidative phosphorylation genes. Phospholipid biosynthesis and remodeling, PI3-Kinase and sphingosine signaling were upregulated, driven largely by an upregulation of several isoforms of phospholipase C. Hierarchical clustering indicated that black cohosh clustered with antiproliferative compounds, specifically tubulin binding vinca alkaloids and DNA alkylators. In support of this, black cohosh repressed the expression of cyclin D1 and ID3, and inhibited the proliferation of HepG2, p53 positive, liver cancer cells. Black cohosh reduced the level of free fatty acids at 6 and 24 h and triglycerides at 6 h in the serum, but increased the free fatty acid and triglyceride content of the treated livers at 24 h. Conclusion Our results suggest that black cohosh warrants further study for breast cancer prevention and therapy.

A high resistance and heterogeneous response to conventional anti-cancer chemotherapies characterize malignant cutaneous melanoma, the most aggressive and deadly form of skin cancer. Withaferin A (WFA), a withanolide derived from the medicinal plant Withania somnifera, has been reported for its anti-tumorigenic activity against various cancer cells. For the first time, we examined the death-inducing potential of WFA against a panel of four different human melanoma cells and investigated the cellular mechanisms involved. WFA induces apoptotic cell death with various IC50 ranging from 1.8 to 6.1 μM. The susceptibility of cells toward WFA-induced apoptosis correlated with low Bcl-2/Bax and Bcl-2/Bim ratios. In all cell lines, the apoptotic process triggered by WFA involves the mitochondrial pathway and was associated with Bcl-2 down regulation, Bax mitochondrial translocation, cytochrome c release into the cytosol, transmembrane potential (ΔΨm) dissipation, caspase 9 and caspase 3 activation and DNA fragmentation. WFA cytotoxicity requires early reactive oxygen species (ROS) production and glutathione depletion, the inhibition of ROS increase by the antioxidant N-acetylcysteine resulting in complete suppression of mitochondrial and nuclear events. Altogether, these results support the therapeutic potential of WFA against human melanoma.

The purpose of this study was to assess in rats the pharmacological parameters and effects on gene expression in the liver of the triterpene glycoside actein. Actein, an active component from the herb black cohosh, has been shown to inhibit the proliferation of human breast cancer cells. To conduct our assessment, we determined the molecular effects of actein on livers from Sprague-Dawley rats treated with actein at 35.7 mg/kg for 6 and 24 h. Chemogenomic analyses indicated that actein elicited stress and statin-associated responses in the liver; actein altered expression of cholesterol and fatty acid biosynthetic genes, p53 pathway genes, CCND1 and ID3. Real-time RT-PCR validated that actein induced three time-dependent patterns of gene expression in the liver: (i) a decrease followed by a significant increase of HMGCS1, HMGCR, HSD17B7, NQO1, S100A9; (ii) a progressive increase of BZRP and CYP7A1 and (iii) a significant increase followed by a decrease of CCND1 and ID3. Consistent with actein's statin- and stress-associated responses, actein reduced free fatty acid and cholesterol content in the liver by 0.6-fold at 24 h and inhibited the growth of human HepG2 liver cancer cells. To determine the bioavailability of actein, we collected serum samples for pharmacokinetic analysis at various times up to 24 h. The serum level of actein peaked at 2.4 microg/mL at 6 h. Actein's ability to alter pathways involved in lipid disorders and carcinogenesis may make it a new agent for preventing and treating these major disorders.

The increased incidence of cancer over the last 50-60 years may be largely attributed to two factors: the ageing of the population and the diffusion of agents and situations presenting carcinogenic risks. Today, we have entered into a new era in which populations are ever-increasingly exposed to diffuse carcinogenic risks, present not only in the occupational, but also in the general environment. We must now also consider an additional factor in the carcinogenic process, that is, the age in which exposure to carcinogenic risks begins. Apart from the paradigmatic cases of diethylstilboestrol and ionizing radiation, the available epidemiological data concerning the adult consequences of developmental exposure to carcinogens is very limited. However, important data have been provided by long-term experimental carcinogenicity bioassays conducted using rodents. This paper reports a selection of studies conducted in the laboratories of the Cesare Maltoni Cancer Research Center of the European Ramazzini Foundation in which exposure to the chemical agents vinyl acetate monomer, ethyl alcohol and aspartame was started during developmental life and continued into adulthood. The results of these studies provide supporting evidence that lifespan exposure to carcinogenic agents beginning during developmental life produces an overall increase in the carcinogenic effects observed. Moreover, when comparing prenatal and postnatal exposure, the data demonstrate that the development of cancers may appear earlier in life.

Aspartame (APM) is one of the most widely used artificial sweeteners in the world. First approved by the U.S. Food and Drug Administration (FDA) for limited use in solid food in 1981, its authorization was extended to soft drinks in 1983 and then approved as a general sweetener in 1996 (FDA 1981, 1983, 1996). Likewise, the sweetener was approved for general use in the European Union in 1994 (EC Directive 1994). APM is now present in > 6,000 consumer packaged goods and in nearly 500 pharmaceutical products, including children’s medicines (Aspartame Information Center 2005). In the United States, > 70% of aspartame sales are attributed to soft drinks (American Dietetic Association 2004). The acceptable daily intake (ADI) of aspartame is currently 50 mg/kg body weight (bw) in the United States and 40 mg/kg bw in the European Union for both children and adults. Daily consumption of artificial sweeteners by women of childbearing age and by children has been estimated at 2.5–5.0 mg/kg bw (Butchko et al. 2002). In a study of Swedish diabetics, the general APM intake was lower than the ADI, but the worst-case calculation of intake in the children’s group was 114% of the ADI (Ilback et al. 2003). APM is metabolized in the gastric tract of rodents, nonhuman primates, and humans to its three constituents: aspartic acid, phenylalanine, and methanol. When absorbed, aspartic acid is transformed into alanine plus oxaloacetate (Stegink 1984); phenylalanine is transformed mainly into tyrosine and, to a lesser extent, phenylethylamine and phenyl-pyruvate (Harper 1984); and methanol is transformed into formaldehyde and then to formic acid (Opperman 1984). In vitro and in vivo tests demonstrate that APM is not genotoxic. Likewise, long-term carcinogenicity studies conducted by the manufacturers of aspartame using rats and mice in the 1970s and 1980s did not demonstrate any carcinogenic effects. A detailed review of the genotoxicity and carcinogenicity studies available to date on APM has been published previously (Belpoggi et al. 2006; Soffritti et al. 2005, 2006). In our opinion, the small number of animals used per sex and per group and the termination of these experiments after 110 weeks of age, rather than observing animals over their life span, represent limiting factors when evaluating the carcinogenic risk or safety of artificial sweeteners such as aspartame. It was for this reason, together with the growing use of APM in industrialized countries, that we designed and performed a mega-experiment using seven groups of Sprague-Dawley rats (100–150 per sex per group) treated with APM in feed at various dose levels (including one very close to the ADI for humans), from 8 weeks of age until natural death (Belpoggi et al. 2006; Soffritti et al. 2005, 2006). The study demonstrated for the first time that APM is a multipotential carcinogenic agent, capable of inducing, in our experimental conditions a) a significant, dose-related increased incidence of malignant tumor–bearing animals in males (p ≤ 0.05) and in females (p ≤ 0.01), particularly in females treated at 50,000 ppm (p ≤ 0.01); b) a significant dose-related increase in lymphomas/leukemias in both males (p ≤ 0.05) and females (p ≤ 0.01), particularly in females treated at doses of 100,000 (p ≤ 0.01), 50,000 (p ≤ 0.01), 10,000 (p ≤ 0.05), 2,000 (p ≤ 0.05), or 400 ppm (p ≤ 0.01); c) a significant, dose-related increased incidence (p ≤ 0.01) of transitional cell carcinomas of the renal pelvis and ureter and their precursors (dysplasias) in females treated at 100,000 (p ≤ 0.01), 50,000 (p ≤ 0.01), 10,000 (p ≤ 0.01), 2,000 (p ≤ 0.05), or 400 ppm (p ≤ 0.05); d ) a significant, dose-related increased incidence of malignant schwannomas of peripheral nerves (p ≤ 0.05) in males (Belpoggi et al. 2006; Soffritti et al. 2005, 2006). Given the consolidated experience of the European Ramazzini Foundation (ERF) in the conduct of long-term bioassays and the large number of rodents used in the study, the results attracted the attention of the scientific community, consumer and industry associations, and the national and international agencies responsible for food safety, including the Italian Superior Council of Health, the European Food Safety Authority (EFSA), the U.S. FDA, Health Canada, and others. At their request, we provided each of these agencies with all available raw data related to the study. To our knowledge, only the EFSA has issued an official opinion on our study, releasing on 5 May 2006 a 40-page report in which they concluded that it is not necessary to revise their previous opinion on the absolute safety of APM (EFSA 2006). Subsequent to our findings of hematopoietic cancers in rats, and in light of persistent concerns among the scientific community of an association between APM and brain cancers, Lim et al. (2006) published the results of a study that assessed the correlation between the consumption of aspartame-containing beverages and the incidence of these types of cancers. The findings were based on data derived from a prospective study conducted by the U.S. National Institutes of Health and the American Association of Retired Persons, using a cohort of > 285,000 men and > 188,000 women between 50 and 71 years of age, who had satisfactorily responded to a self-administered food frequency questionnaire. The questionnaire included questions on the consumption of beverages (soft drinks, fruit drinks, sweetened iced tea) potentially containing APM during the previous year. The questionnaires were mailed from 1995 to 1996 and the follow-up lasted until 2000. The conclusions of the study (Lim et al. 2006) did not support the hypothesis that APM increases hematopoietic or brain cancer risks. Recently a group of Italian authors (Gallus et al. 2007) published the results of an integrated network of case–control studies conducted in Italy between 1991 and 2004 on the potential correlation between artificial sweeteners (including APM) and cancer. The authors interviewed patients with histologically confirmed cancers of the oral cavity and pharynx (598), esophagus (304), colon (1,225), rectum (728), larynx (460), breast (2,569), ovary (1,031), prostate (1,294), and kidney (renal cell carcinoma 767). Controls were 7,028 patients (3,301 men and 3,727 women) admitted to the same hospitals for acute, nonneoplastic disorders. Cases and controls were interviewed during their hospital stay, using a questionnaire on subjects’ usual diet in the 2 years before diagnosis. The results reported a lack of association between artificial sweeteners and the risk of the aforementioned cancers. As soon as we perceived the carcinogenic effects of APM during the elaboration of the data in our first mega-experiment (Belpoggi et al. 2006; Soffritti et al. 2005, 2006), we planned an integrated program of long-term bioassays, beginning treatment from prenatal life, on > 4,000 rats and mice in order to better quantify the carcinogenic risks of aspartame. In this report we present the results of a second study on APM in which male and female Sprague-Dawley rats were exposed to very low doses of APM in feed (100 or 20 mg/kg bw) from fetal life until natural death.

Background Hepatocellular carcinoma (HCC) is among the most frequent human malignancies and a major cause of cancer-related death worldwide. It is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be one of the molecular mechanisms leading to hepatocarcinogenesis. Previous studies from our group revealed that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression. Imprinted genes are mono-allelically expressed in a parent-of-origin-dependent manner and have been suggested to be more susceptible to deregulation in cancer. To test this notion, we performed a targeted analysis of DNA methylation in known imprinted genes, using HCC samples and in vitro models of carcinogenic exposure. Results Analysis of HCC DNA methylation in two independent datasets showed that differentially methylated loci are significantly enriched in imprinted genes. Most of the promoters of imprinted genes were found hypomethylated in HCC tumors compared to surrounding tissues, contrasting with the frequent promoter hypermethylation observed in tumors. We next investigated the status of methylation of the imprinting control region (ICR) of different imprinted clusters and found that the 15q11-13 ICR was significantly hypomethylated in tumors relative to their surrounding tissues. In addition, expression of imprinted genes within this cluster was frequently deregulated in a gene-specific manner, suggesting distinct mechanisms of regulation in this region. Finally, primary human hepatocytes and hepatocyte-like HepaRG cells displayed higher methylation variability in certain imprinted loci after natural hepatitis B virus (HBV) infection and after lipid accumulation, respectively. Conclusion The methylation status of a large panel of imprinted genes was found deregulated in HCC, suggesting a major role of this mechanism during hepatocarcinogenesis. In vitro models support the hypothesis of imprinted gene methylation as a potential marker of environmental exposures. Electronic supplementary material The online version of this article (doi:10.1186/s13148-015-0053-9) contains supplementary material, which is available to authorized users.

Background Ischemia-reperfusion (I/R) injury is the main cause of graft failure in liver transplantation (LT). Ischemic postconditioning (IPo) has shown to be beneficial against I/R injury. Our objective was to compare the results of LT with or without IPo. Methods One hundred patients undergoing LT alternatively received IPo or not. At the time of arterial reperfusion, IPo consisted of three 1-minute arterial occlusions, interspersed with 1-minute reperfusion pauses. The primary endpoint was postoperative aspartate aminotransferase (AST) peak value; early graft dysfunction and histological I/R injury were secondary endpoints. Results Median postoperative AST peak values was similar in both groups (426 vs 463 IU/L, P = 0.21); no difference was found in other postoperative liver function tests. In the IPo group, fewer grafts presented severe histological I/R injury (12% vs 28%; P = 0.029). Ischemic postconditioning did not induce changes in cellular apoptosis but triggered autophagy in periportal areas. Independent predictors of severe I/R injury were IPo (odds ratio, 0.20; P = 0.008) and arterial warm ischemia duration (odds ratio, 1.05; P = 0.008). Early graft dysfunction rate was similar in both groups (20% versus 26%, P = 0.47) and was associated with severe histological I/R injury and longer cold ischemia. Morbidity, mortality, and 1-year graft and patient survival were similar in both groups. Conclusions Ischemic postconditioning did not influence postoperative AST peak values or other liver function tests. However, our results showed a better tolerance to I/R injury on histological findings of grafts receiving IPo. Future studies are necessary to optimize the IPo protocol in LT, to clarify its clinical impact, and to deepen the molecular understanding.

To propose a test to evaluate endothelial function, based on VO(2) on-transition kinetics in sub-anaerobic threshold (AT) constant load exercise, we tested healthy subjects and patients with ischemic-hypertensive cardiopathy by two cardiopulmonary tests on a cycle ergometer endowed with an electric motor to overcome initial inertia: a pre-test and, after at least 24 h, one 6 min constant load exercise at 90 % AT. We measured net phase 3 VO(2)-on kinetics and, by phase 2 time constant (tau), valued endothelial dysfunction. We found shorter tau in repeated tests, shorter time between first and second test, by persisting endothelium-dependent arteriolar vasodilatation and/or several other mechanisms. Reducing load to 80 % and 90 % AT did not produce significant changes in tau of healthy volunteers, while in heart patients an AT load of 70 %, compared to 80 % AT, shortened tau (delta=4.38+/-1.65 s, p=0.013). In heart patients, no correlation was found between NYHA class, ejection fraction (EF), and the two variables derived from incremental cycle cardio-pulmonary exercise, as well as between EF and tau; while NYHA class groups were well correlated with tau duration (r=0.92, p=0.0001). Doxazosin and tadalafil also significantly reduced tau. In conclusion, the O(2) consumption kinetics during the on-transition of constant load exercise below the anaerobic threshold are highly sensitive to endothelial function in muscular microcirculation, and constitute a marker for the evaluation of endothelial dysfunction.

Arsenic (As) is a metal found in nature whose acute and chronic toxic effects have been known for decades. Hundreds of millions of people are at risk of exposure to As and its various chemical forms which can occur in the occupational and general environment in air, water, soil, food, and medicines. Several epidemiological studies have shown that prolonged exposure to As can induce various types of malignant tumors in humans, namely, skin, lung, liver, kidney, and bladder cancers. These effects have been observed particularly in geographic areas where people are exposed to well water with high concentrations of As. While the risks of As at high concentrations are well documented, there is still a great deal of uncertainty regarding the risk of exposure to As at very low levels. This uncertainty is due to the absence of adequate epidemiological data and the insufficiency of experimental data currently available. Given the limited evidence demonstrating the carcinogenic potential of As in animals, a long-term carcinogenicity bioassay on sodium arsenite (NaAsO(2)) was performed at the Cesare Maltoni Cancer Research Center (CMCRC) of the European Ramazzini Foundation (ERF). NaAsO(2) was administrated with drinking water at concentrations of 200, 100, 50, or 0 mg/L, for 104 weeks to Sprague-Dawley rats (50/sex/group), 8 weeks old at the start of the study. The animals were monitored until spontaneous death at which time each animal underwent complete necropsy. Histopathological evaluation of all pathological lesions and of all organs and tissues collected was routinely performed on each animal. The results demonstrate that in our experimental conditions NaAsO(2) induces sparse benign and malignant tumors among treated rats. The types of tumors observed are infrequent in the strain of Sprague-Dawley rats of the colony used in our laboratory, namely, lung adenomas and carcinomas, kidney adenomas/papillomas and carcinomas, and bladder carcinomas. Notably, an elevated incidence of these types of oncological lesions is also observed among people living in geographical areas where As is present at higher concentrations in drinking water.

Aspartame (APM) is one of the most widely used artificial sweeteners in the world. Its ever-growing use in more than 6000 products, such as soft drinks, chewing gum, candy, desserts, etc., has been accompanied by rising consumer concerns regarding its safety, in particular its potential long-term carcinogenic effects. In light of the inadequacy of the carcinogenicity bioassays performed in the 1970s and 1980s, a long-term mega-experiment on APM was undertaken at the Cesare Maltoni Cancer Research Center of the European Ramazzini Foundation on groups of male and female Sprague-Dawley rats (100-150/sex/group), 8 weeks old at the start of the experiment. APM was administered in feed at concentrations of 100,000, 50,000, 10,000, 2,000, 400, 80, or 0 ppm. Treatment lasted until spontaneous death of the animals. The results of the study demonstrate that APM causes: (a) an increased incidence of malignant tumor-bearing animals, with a positive significant trend in both sexes, and in particular in females treated at 50,000 ppm (P < or = 0.01) when compared to controls; (b) an increase in lymphomas-leukemias, with a positive significant trend in both sexes, and in particular in females treated at doses of 100,000 (P < or = 0.01), 50,000 (P < or = 0.01), 10,000 (P < or = 0.05), 2000 (P < or = 0.05), and 400 ppm (P < or = 0.01); (c) a statistically significant increased incidence, with a positive significant trend, of transitional cell carcinomas of the renal pelvis and ureter in females and particularly in those treated at 100,000 ppm (P < or = 0.05); and (d) an increased incidence of malignant schwannomas of the peripheral nerves, with a positive trend in males (P < or = 0.05). The results of this mega-experiment indicate that APM, in the tested experimental conditions, is a multipotential carcinogenic agent.

The Cesare Maltoni Cancer Research Center of the European Ramazzini Foundation has conducted a long-term bioassay on aspartame (APM), a widely used artificial sweetener. APM was administered with feed to 8-week-old Sprague-Dawley rats (100-150/sex/group), at concentrations of 100,000, 50,000, 10,000, 2,000, 400, 80, or 0 ppm. The treatment lasted until natural death, at which time all deceased animals underwent complete necropsy. Histopathologic evaluation of all pathologic lesions and of all organs and tissues collected was routinely performed on each animal of all experimental groups. The results of the study show for the first time that APM, in our experimental conditions, causes a) an increased incidence of malignant-tumor-bearing animals with a positive significant trend in males (p < or = 0.05) and in females (p < or = 0.01), in particular those females treated at 50,000 ppm (p < or = 0.01); b) an increase in lymphomas and leukemias with a positive significant trend in both males (p < or = 0.05) and females (p < or = 0.01), in particular in females treated at doses of 100,000 (p < or = 0.01), 50,000 (p < or = 0.01), 10,000 (p < or = 0.05), 2,000 (p < or = 0.05), or 400 ppm (p < or = 0.01); c) a statistically significant increased incidence, with a positive significant trend (p < or = 0.01), of transitional cell carcinomas of the renal pelvis and ureter and their precursors (dysplasias) in females treated at 100,000 (p < or = 0.01), 50,000 (p < or = 0.01), 10,000 (p < or = 0.01), 2,000 (p < or = 0.05), or 400 ppm (p < or = 0.05); and d) an increased incidence of malignant schwannomas of peripheral nerves with a positive trend (p < or = 0.05) in males. The results of this mega-experiment indicate that APM is a multipotential carcinogenic agent, even at a daily dose of 20 mg/kg body weight, much less than the current acceptable daily intake. On the basis of these results, a reevaluation of the present guidelines on the use and consumption of APM is urgent and cannot be delayed.

This 12-week randomized, parallel-group, multicenter study compared fixed combinations of delapril (D) 30 mg plus indapamide (I) 2.5 mg and fosinopril (F) 20 mg plus hydrochlorothiazide (H) 12.5 mg in 171 adult patients with mild to moderate essential hypertension. After a 2-week placebo run-in, sitting and standing systolic (SBP) and diastolic blood pressure (DBP) was measured by conventional sphygmomanometry. The primary efficacy endpoint was the percentage of normalized (sitting DBP < or =90 mm Hg) and responder (sitting DBP reduction of 10 mm Hg or DBP < or =90 mm Hg) patients. Treatment effects were analyzed in the intention-to-treat (ITT; n = 171) and the per-protocol (PP; n = 167) populations. The percentage of normalized and responder patients did not differ significantly between the D + I (87.4% and 92%) and the F + H (81% and 86.9%) ITT groups. Similar results were seen in the PP population. In ITT and PP patients, sitting and standing SBP and DBP values were comparable at baseline in the two groups and were significantly (P

Taxol transport in Taxus baccata L. cell suspension cultures was studied using 〚 14 C〛-taxol as a tracer. The time course of uptake showed a saturable absorption that reached a maximum within 20 min. The uptake depended on its exogenous concentration and its accumulation was highly stimulated in the presence of 10–15 μM exogenous taxol. The absorbed molecule was found to localise both in the cell walls (20 %) and in the cell protoplasts (80 %), suggesting an accumulation within the vacuoles. Taxol uptake was strongly inhibited by Na-orthovanadate and verapamil, while Ca 2+ was found to be one of the factors required for the active absorption of the molecule, since in the absence of this cation, the uptake was reduced by about 40 % and occurred mainly through a non-energy dependent mechanism. Taxol release into the culture medium was demonstrated not to depend on cell lysis, occurred through a mechanism that reached its maximum after 10–15 min and was strongly enhanced by treatment with Na-orthovanadate and verapamil, although the effect was found to be transient.

Recent development in molecular phenotyping of breast cancer to guide treatment strategy has improved breast cancer survival. While several prognostic panels, such as Oncotype DX (21 genes), MammaPrint (70 genes), ProSigna (50 genes), have been approved for clinical use with demonstrated utility, there is still room for improvement. Using a novel bioinformatics approach, we previously demonstrated that higher “activity” (instead of level of expression) of miR-500a-5p in tumor was associated with shorter survival of ER+ breast cancer patients. However, the activity of miR-500a cannot be directly measured and can only be inferred by the expression of its associated genes. Using NanoString platform, we profiled 162 miR-500a-related genes as well as PAM50 genes (22 genes overlapped between PAM50 set and miR-500a-related set) from tumors collected as part of the Long Island Breast Cancer Study Project, where a population-based sample of 1508 women, predominantly Caucasians aged 25-98 (median age 58), newly diagnosed with first primary breast cancer in 1996-1997, were followed up for 15+ years using the National Death Index. We were able to obtain sufficient RNA for profiling from paraffin-embedded tumors tissues for 609 cases, among which 119 breast cancer deaths were reported at the end of follow up. Survival analyses were carried out using Cox proportional hazards models adjusting for known prognostic factors. Consistent with our a priori hypothesis, most of the selected miR-500a related genes (81 out of 162) significantly associated with breast cancer-specific mortality. Co-expression analysis revealed two major clusters of genes with opposite associations with breast cancer-specific mortality. For example, expression of SUSD3 from the first cluster showed inverse associations with breast cancer-specific mortality [high vs low expression: Hazard Ratio (HR) 0.33, 95% CI: 0.23-0.47] while TPX2 from the second cluster showed the opposite [HR 2.64, CI: 1.84-3.80]. These associations were also confirmed in independent combined datasets (described in “KM-Plotter”, kmplot.com). Most importantly, these associations remained significant after adjusting for known prognostic factors including tumor hormonal receptor status and PAM50-derived risk of recurrence category, implicating they are independent markers for breast cancer survival. Our study identified novel predictors that may improve prognostic efficiency of current molecular phenotyping. In addition, these results shed light on molecular mechanism of breast cancer progression and may point to the targets for treatment of the disease. Citation Format: Vasily N. Aushev, Marilie D. Gammon, Humberto Parada, Davide Degli Esposti, Hector Hernandez-Vargas, Zdenko Herceg, Susan Teitelbaum, Jun Zhu, Eunjee Lee, Jia Chen. Novel prognostic gene profiles in tumors for breast cancer survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-253. doi:10.1158/1538-7445.AM2017-LB-253

Hepatocellular carcinoma (HCC) is the second most common cause of cancer death worldwide, but is still lacking sensitive and specific biomarkers for early diagnosis and prognosis. In this study, we applied targeted massively parallel semiconductor sequencing to assess methylation on a panel of genes (FBLN1, HINT2, LAMC1, LTBP1, LTBP2, PSMA2, PSMA7, PXDN, TGFB1, UBE2L3, VIM and YWHAZ) in plasma circulating cell-free DNA (cfDNA) and to evaluate the potential of these genes as HCC biomarkers in two different series, one from France (42 HCC cases and 42 controls) and one from Thailand (42 HCC cases, 26 chronic liver disease cases and 42 controls). We also analyzed a set of HCC and adjacent tissues and liver cell lines to further compare with ‘The Cancer Genome Atlas’ (TCGA) data. The methylation in cfDNA was detected for FBLN1, PSMA7, PXDN and VIM, with differences in methylation patterns between cases and controls for FBLN1 and VIM. The average methylation level across analyzed CpG-sites was associated with higher odds of HCC for VIM (1.48 [1.02, 2.16] for French cases and 2.18 [1.28, 3.72] for Thai cases), and lower odds of HCC for FBLN1 (0.89 [0.76, 1.03] for French cases and 0.75 [0.63, 0.88] for Thai cases). In conclusion, our study provides evidence that changes in VIM and FBLN1 methylation levels in cfDNA are associated with HCC and could represent useful plasma-based biomarkers. Also, the potential to investigate methylation patterns in cfDNA could bring new strategies for HCC detection and monitoring high-risk groups and response to treatment.

Intrinsic and acquired chemoresistance are frequent causes of cancer eradication failure. Thus, long-term cis-diaminedichloroplatine(II) (CDDP) or cisplatin treatment is known to promote tumor cell resistance to apoptosis induction via multiple mechanisms involving gene expression modulation of oncogenes, tumor suppressors and blockade of pro-apoptotic mitochondrial membrane permeabilization. Here, we demonstrate that CDDP-resistant non-small lung cancer cells undergo profound remodeling of their endoplasmic reticulum (ER) proteome (>80 proteins identified by proteomics) and exhibit a dramatic overexpression of two protein disulfide isomerases, PDIA4 and PDIA6, without any alteration in ER-cytosol Ca(2+) fluxes. Using pharmacological and genetic inhibition, we show that inactivation of both proteins directly stimulates CDDP-induced cell death by different cellular signaling pathways. PDIA4 inactivation restores a classical mitochondrial apoptosis pathway, while knockdown of PDIA6 favors a non-canonical cell death pathway sharing some necroptosis features. Overexpression of both proteins has also been found in lung adenocarcinoma patients, suggesting a clinical importance of these proteins in chemoresistance.

The effectiveness of ischemic preconditioning (IP) against hepatic ischemia/reperfusion injury during human liver surgery is linked to decreased apoptotic cell death as well as preservation of the ATP content in liver tissue. Overproduction of Bcl-2 is reported in preconditioned organs. In human liver biopsies exhibiting steatosis and/or vascular injuries (mainly peliosis) induced by chemotherapy, we find that the expression of Bcl-2 in centrolobular and peliotic areas colocalizes with the autophagy protein Beclin 1 in IP livers. Increased expression of phosphorylated Bcl-2 in preconditioned livers is associated with decreased immunoprecipitation of Beclin 1 and increased expression of LC3-II. The increased number of autophagic vacuoles seen by electron microscopy confirmed that IP could trigger autophagy in chemotherapy-injured livers, probably to reduce the pro-inflammatory necrotic cell death of hepatocytes or endothelial cells and to increase ATP levels. Indeed, necrosis is less frequent (p = 0.04) in IP livers than in the others although no change in apoptosis as assessed by TUNEL assay or caspase-3, -8 and -9 expressions is observed. In conclusion, Bcl-2 and Beclin 1 could be major targets in the regulation of cell death during ischemia/reperfusion injury modulating autophagy to switch on/off necrosis and/or apoptosis.