Giulio Ferrero

Genome-wide activity of unliganded estrogen receptor-α in breast cancer cells

Significance Estrogen receptor-α (ERα) is a key protein in breast cancer and treatments targeting ERα are among the most widely used and effective in clinics. Although the role of estrogen-stimulated ERα in breast cancer has been exhaustively described, the functions of ERα in the absence of estrogen is hill-defined. In this work, we show that ERα binds extensively to the genome of breast cancer cells in the absence of estrogen, where it regulates the expression of hundreds of genes endowed with developmental functions. Our data suggest that ERα has a fundamental role in the homeostasis of luminal epithelial cells also when estrogen is ablated physiologically or pharmacologically. Estrogen receptor-α (ERα) has central role in hormone-dependent breast cancer and its ligand-induced functions have been extensively characterized. However, evidence exists that ERα has functions that are independent of ligands. In the present work, we investigated the binding of ERα to chromatin in the absence of ligands and its functions on gene regulation. We demonstrated that in MCF7 breast cancer cells unliganded ERα binds to more than 4,000 chromatin sites. Unexpectedly, although almost entirely comprised in the larger group of estrogen-induced binding sites, we found that unliganded-ERα binding is specifically linked to genes with developmental functions, compared with estrogen-induced binding. Moreover, we found that siRNA-mediated down-regulation of ERα in absence of estrogen is accompanied by changes in the expression levels of hundreds of coding and noncoding RNAs. Down-regulated mRNAs showed enrichment in genes related to epithelial cell growth and development. Stable ERα down-regulation using shRNA, which caused cell growth arrest, was accompanied by increased H3K27me3 at ERα binding sites. Finally, we found that FOXA1 and AP2γ binding to several sites is decreased upon ERα silencing, suggesting that unliganded ERα participates, together with other factors, in the maintenance of the luminal-specific cistrome in breast cancer cells.

Luminal long non-coding RNAs regulated by estrogen receptor alpha in a ligand-independent manner show functional roles in breast cancer

Estrogen Receptor alpha (ERα) activation by estrogenic hormones induces luminal breast cancer cell proliferation. However, ERα plays also important hormone-independent functions to maintain breast tumor cells epithelial phenotype. We reported previously by RNA-Seq that in MCF-7 cells in absence of hormones ERα down-regulation changes the expression of several genes linked to cellular development, representing a specific subset of estrogen-induced genes. Here, we report regulation of long non-coding RNAs from the same experimental settings. A list of 133 Apo-ERα-Regulated lncRNAs (AER-lncRNAs) was identified and extensively characterized using published data from cancer cell lines and tumor tissues, or experiments on MCF-7 cells. For several features, we ran validation using cell cultures or fresh tumor biopsies. AER-lncRNAs represent a specific subset, only marginally overlapping estrogen-induced transcripts, whose expression is largely restricted to luminal cells and which is able to perfectly classify breast tumor subtypes. The most abundant AER-lncRNA, DSCAM-AS1, is expressed in ERα+ breast carcinoma, but not in pre-neoplastic lesions, and correlates inversely with EMT markers. Down-regulation of DSCAM-AS1 recapitulated, in part, the effect of silencing ERα, i.e. growth arrest and induction of EMT markers. In conclusion, we report an ERα-dependent lncRNA set representing a novel luminal signature in breast cancer cells.

Differentially methylated microRNAs in prediagnostic samples of subjects who developed breast cancer in the European Prospective Investigation into Nutrition and Cancer (EPIC-Italy) cohort.

The crosstalk between microRNAs (miRNAs) and other epigenetic factors may lead to novel hypotheses about carcinogenesis identifying new targets for research. Because a single miRNA can regulate multiple downstream target genes, its altered expression may potentially be a sensitive biomarker to detect early malignant transformation and improve diagnosis and prognosis. In the current study, we tested the hypothesis that altered methylation of miRNA encoding genes, associated with deregulated mature miRNA expression, may be related to dietary and lifestyle factors and may contribute to cancer development. In a case-control study nested in a prospective cohort (EPIC-Italy), we analysed DNA methylation levels of miRNA encoding genes (2191 CpG probes related to 517 genes) that are present in the Infinium Human Methylation450 BeadChip array in prediagnostic peripheral white blood cells of subjects who developed colorectal cancer (CRC, n = 159) or breast cancer (BC, n = 166) and matched subjects who remained clinically healthy. In the whole cohort, several differentially methylated miRNA genes were observed in association with age, sex, smoking habits and physical activity. Interestingly, in the case-control study, eight differentially methylated miRNAs were identified in subjects who went on to develop BC (miR-328, miR-675, miR-1307, miR-1286, miR-1275, miR-1910, miR-24-1 and miR-548a-1; all Bonferroni-adjusted P < 0.05). No significant associations were found with CRC. Assuming that altered methylation of miRNAs detectable in blood may be present before diagnosis, it may represent a biomarker for early detection or risk of cancer and may help to understand the cascade of events preceding tumour onset.

E2 regulates epigenetic signature on neuroglobin enhancer-promoter in neuronal cells

Estrogens are neuroprotective factors in several neurological diseases. Neuroglobin (NGB) is one of the estrogen target genes involved in neuroprotection, but little is known about its transcriptional regulation. Estrogen genomic pathway in gene expression regulation is mediated by estrogen receptors (ERα and ERβ) that bind to specific regulatory genomic regions. We focused our attention on 17β-estradiol (E2)-induced NGB expression in human differentiated neuronal cell lines (SK-N-BE and NT-2). Previously, using bioinformatics analysis we identified a putative enhancer in the first intron of NGB locus. Therefore, we observed that E2 increased the enrichment of the H3K4me3 epigenetic marks at the promoter and of the H3K4me1 and H3K27Ac at the intron enhancer. In these NGB regulatory regions, we found estrogen receptor alpha (ERα) binding suggesting that ERα may mediate chromatin remodeling to induce NGB expression upon E2 treatment. Altogether our data show that NGB expression is regulated by ERα binding on genomic regulatory regions supporting hormone therapy applications for the neuroprotection against neurodegenerative diseases.

Small non-coding RNA profiling in human biofluids and surrogate tissues from healthy individuals: description of the diverse and most represented species

The role of non-coding RNAs in different biological processes and diseases is continuously expanding. Next-generation sequencing together with the parallel improvement of bioinformatics analyses allows the accurate detection and quantification of an increasing number of RNA species. With the aim of exploring new potential biomarkers for disease classification, a clear overview of the expression levels of common/unique small RNA species among different biospecimens is necessary. However, except for miRNAs in plasma, there are no substantial indications about the pattern of expression of various small RNAs in multiple specimens among healthy humans. By analysing small RNA-sequencing data from 243 samples, we have identified and compared the most abundantly and uniformly expressed miRNAs and non-miRNA species of comparable size with the library preparation in four different specimens (plasma exosomes, stool, urine, and cervical scrapes). Eleven miRNAs were commonly detected among all different specimens while 231 miRNAs were globally unique across them. Classification analysis using these miRNAs provided an accuracy of 99.6% to recognize the sample types. piRNAs and tRNAs were the most represented non-miRNA small RNAs detected in all specimen types that were analysed, particularly in urine samples. With the present data, the most uniformly expressed small RNAs in each sample type were also identified. A signature of small RNAs for each specimen could represent a reference gene set in validation studies by RT-qPCR. Overall, the data reported hereby provide an insight of the constitution of the human miRNome and of other small non-coding RNAs in various specimens of healthy individuals.

Sequence alignment tools: one parallel pattern to rule them all?

In this paper, we advocate high-level programming methodology for next generation sequencers (NGS) alignment tools for both productivity and absolute performance. We analyse the problem of parallel alignment and review the parallelisation strategies of the most popular alignment tools, which can all be abstracted to a single parallel paradigm. We compare these tools to their porting onto the FastFlow pattern-based programming framework, which provides programmers with high-level parallel patterns. By using a high-level approach, programmers are liberated from all complex aspects of parallel programming, such as synchronisation protocols, and task scheduling, gaining more possibility for seamless performance tuning. In this work, we show some use cases in which, by using a high-level approach for parallelising NGS tools, it is possible to obtain comparable or even better absolute performance for all used datasets.

Biologic Data of Cynomolgus Monkeys Maintained under Laboratory Conditions

The cynomolgus monkey (Macaca fascicularis) is a well-known non-human primate species commonly used in non-clinical research. It is important to know basal clinical pathology parameters in order to have a reference for evaluating any potential treatment-induced effects, maintaining health status among animals and, if needed, evaluating correct substantiative therapies. In this study, data from 238 untreated cynomolgus monkeys (119 males and 119 females of juvenile age, 2.5 to 3.5 years) kept under laboratory conditions were used to build up a reference database of clinical pathology parameters. Twenty-two hematology markers, 24 clinical chemistry markers and two blood coagulation parameters were analyzed. Gender-related differences were evaluated using statistical analyses. To assess the possible effects of stress induced by housing or handling involved in treatment procedures, 78 animals (35 males and 35 females out of 238 juvenile monkeys and four adult males and four adult females) were used to evaluate cortisol, corticosterone and behavioral assessment over time. Data were analyzed using a non-parametric statistical test and machine learning approaches. Reference clinical pathology data obtained from untreated animals may be extremely useful for investigators employing cynomolgus monkeys as a test system for non-clinical safety studies.

Luminal breast cancer-specific circular RNAs uncovered by a novel tool for data analysis

Circular RNAs are highly stable molecules present in all eukaryotes generated by distinct transcript processing. We have exploited poly(A-) RNA-Seq data generated in our lab in MCF-7 breast cancer cells to define a compilation of exonic circRNAs more comprehensive than previously existing lists. Development of a novel computational tool, named CircHunter, allowed us to more accurately characterize circRNAs and to quantitatively evaluate their expression in publicly available RNA-Seq data from breast cancer cell lines and tumor tissues. We observed and confirmed, by ChIP analysis, that exons involved in circularization events display significantly higher levels of the histone post-transcriptional modification H3K36me3 than non-circularizing exons. This result has potential impact on circRNA biogenesis since H3K36me3 has been involved in alternative splicing mechanisms. By analyzing an Ago-HITS-CLIP dataset we also found that circularizing exons overlapped with an unexpectedly higher number of Ago binding sites than non-circularizing exons. Finally, we observed that a subset of MCF-7 circRNAs are specific to tumor versus normal tissue, while others can distinguish Luminal from other tumor subtypes, thus suggesting that circRNAs can be exploited as novel biomarkers and drug targets for breast cancer.

Reproducible bioinformatics project: a community for reproducible bioinformatics analysis pipelines

BackgroundReproducibility of a research is a key element in the modern science and it is mandatory for any industrial application. It represents the ability of replicating an experiment independently by the location and the operator. Therefore, a study can be considered reproducible only if all used data are available and the exploited computational analysis workflow is clearly described. However, today for reproducing a complex bioinformatics analysis, the raw data and the list of tools used in the workflow could be not enough to guarantee the reproducibility of the results obtained. Indeed, different releases of the same tools and/or of the system libraries (exploited by such tools) might lead to sneaky reproducibility issues.ResultsTo address this challenge, we established the Reproducible Bioinformatics Project (RBP), which is a non-profit and open-source project, whose aim is to provide a schema and an infrastructure, based on docker images and R package, to provide reproducible results in Bioinformatics. One or more Docker images are then defined for a workflow (typically one for each task), while the workflow implementation is handled via R-functions embedded in a package available at github repository. Thus, a bioinformatician participating to the project has firstly to integrate her/his workflow modules into Docker image(s) exploiting an Ubuntu docker image developed ad hoc by RPB to make easier this task. Secondly, the workflow implementation must be realized in R according to an R-skeleton function made available by RPB to guarantee homogeneity and reusability among different RPB functions. Moreover she/he has to provide the R vignette explaining the package functionality together with an example dataset which can be used to improve the user confidence in the workflow utilization.ConclusionsReproducible Bioinformatics Project provides a general schema and an infrastructure to distribute robust and reproducible workflows. Thus, it guarantees to final users the ability to repeat consistently any analysis independently by the used UNIX-like architecture.

Dissecting the genomic activity of a transcriptional regulator by the integrative analysis of omics data

In the study of genomic regulation, strategies to integrate the data produced by Next Generation Sequencing (NGS)-based technologies in a meaningful ensemble are eagerly awaited and must continuously evolve. Here, we describe an integrative strategy for the analysis of data generated by chromatin immunoprecipitation followed by NGS which combines algorithms for data overlap, normalization and epigenetic state analysis. The performance of our strategy is illustrated by presenting the analysis of data relative to the transcriptional regulator Estrogen Receptor alpha (ERα) in MCF-7 breast cancer cells and of Glucocorticoid Receptor (GR) in A549 lung cancer cells. We went through the definition of reference cistromes for different experimental contexts, the integration of data relative to co-regulators and the overlay of chromatin states as defined by epigenetic marks in MCF-7 cells. With our strategy, we identified novel features of estrogen-independent ERα activity, including FoxM1 interaction, eRNAs transcription and a peculiar ontology of connected genes.