Combinatorial targeting of menin and the histone methyltransferase DOT1L as a novel therapeutic strategy for treatment of chemotherapy-resistant ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is characterized by a low response rate and high frequency of resistance development to currently available treatments. The therapeutic potential of histone methyltransferase DOT1L inhibitor in OC cells has been demonstrated, but optimal efficacy and safety of this targeted therapy approach still require improvement. We set forth to evaluate if this problem can be overcome by combinatorial targeting of this epigenetic modifier and menin, one of its functional partners in chromatin. METHODS: siRNA-mediated gene knock-down and pharmacological inhibition of menin, a key component of the MLL/SET1 complex and a fitness gene in OC cells, coupled to cell proliferation assays on a panel of high grade serous OC cell lines, including chemotherapy-sensitive and -resistant clones, were applied in order to evaluate how depletion or blockade of this enzyme influences growth and viability of OC cells. RNA sequencing was applied to identify menin target genes and pathways, and the effects of combined inhibition of menin and DOT1L on growth and transcriptome of these OC models were evaluated. RESULTS: Silencing and pharmacological inhibition of menin exert antiproliferative effects in all OC cells tested and, in PEO1 and PEO4 cells, a profound impact on transcriptome via down-regulation of cell cycle regulatory pathways, aryl hydrocarbon receptor, MYC and KRAS signalling. We demonstrated association of menin and DOT1L in OC cells and identified a subset of genes co-regulated by the two factors. Interestingly, co-treatment with DOT1L and menin pharmacological inhibitors exerts an additive effect on growth inhibition on chemotherapy-sensitive and -refractory OC cells mediated by transcriptome changes controlled by menin and DOT1L activities. CONCLUSION: These results indicate that menin functionally cooperates with DOT1L in OC cells modulating transcription of genes involved in key cellular functions including, among others, cell proliferation and survival, that are strongly affected by combined inhibition of these two epigenetic regulators, suggesting that this may represent a novel therapeutic strategy for chemotherapy-resistant OCs. TRIAL REGISTRATION: NA; The manuscript does not contain clinical trials.

Targeted molecular profiling of epithelial ovarian cancer from Italian BRCA wild-type patients with a BRCA and PARP pathways gene panel.

Ovarian cancer (OC) is the fifth most common type of cancer in women and the fourth most common cause of cancer death in women. Identification of pathogenic variants in OC tissues has an important clinical significance for therapeutic and prevention purposes. This study aims to evaluate the mutational profile of a patient cohort, negative for BRCA1/2 germinal variants and Mismatch Repair defects, using next-generation sequencing (NGS) approach on DNA from formalin-fixed paraffin-embedded samples. We used a custom NGS panel, targeting 34 cancer-related genes, mainly of the BRCA and PARP pathways, and analyzed NGS data to identify somatic and germline variants in Italian patients affected by primary epithelial ovarian cancer. We analyzed 75 epithelial ovarian cancer tissues and identified 54 pathogenic variants and 56 variants of unknown significance. TP53 was characterized by the highest mutational rate, occurring in 55% of tested epithelial ovarian cancers (EOCs). Interestingly, a subset of 8 EOCs showed pathogenic variants of homologous recombination pathway, which could be sensitive to PARP-inhibitor therapies. Germline analysis of actionable genes revealed 4 patients carrier of pathogenic germline variants respectively of RAD51C (2 patients), RAD51D, and PALB2. Molecular profiling of EOCs using our custom NGS panel has enabled the detection of both somatic and germline variants, allowing the selection of patients suitable for targeted therapies, and the identification of high-risk OC families that can benefit from genetic counseling and testing.

HOME-BIO (sHOtgun MEtagenomic analysis of BIOlogical entities): a specific and comprehensive pipeline for metagenomic shotgun sequencing data analysis.

BACKGROUND: Next-Generation-Sequencing (NGS) enables detection of microorganisms present in biological and other matrices of various origin and nature, allowing not only the identification of known phyla and strains but also the discovery of novel ones. The large amount of metagenomic shotgun data produced by NGS require comprehensive and user-friendly pipelines for data analysis, that speed up the bioinformatics steps, relieving the users from the need to manually perform complex and time-consuming tasks. RESULTS: We describe here HOME-BIO (sHOtgun MEtagenomic analysis of BIOlogical entities), an exhaustive pipeline for metagenomics data analysis, comprising three independent analytical modules designed for an inclusive analysis of large NGS datasets. CONCLUSIONS: HOME-BIO is a powerful and easy-to-use tool that can be run also by users with limited computational expertise. It allows in-depth analyses by removing low-complexity/ problematic reads, integrating the analytical steps that lead to a comprehensive taxonomy profile of each sample by querying different source databases, and it is customizable according to specific users' needs.

BRCA Methylation Testing Identifies a Subset of Ovarian Carcinomas without Germline Variants That Can Benefit from PARP Inhibitor.

Homologous Recombination Deficiency (HRD) is a frequent feature of high-grade epithelial ovarian carcinoma (EOC), associated with sensitivity to PARP-inhibitors (PARPi). The best characterized causes of HRD in EOCs are germline or somatic mutations in BRCA1 and BRCA2 genes. Although promoter methylation is a well-known mechanism of gene transcriptional repression, few data have been published about BRCA gene methylation in EOCs. In this retrospective study, we quantitatively analyzed by pyrosequencing a selected series of 90 formalin-fixed (FFPE) primary EOCs without BRCA germline mutations. We identified 20/88 (22.7%) EOCs showing BRCA promoter methylation, including 17/88 (19.3%) in BRCA1 and 4/86 (4.6%) in BRCA2 promoters, one of which showing concomitant BRCA1 methylation. Mean methylation levels were 49.6% and 45.8% for BRCA1 and BRCA2, respectively, with methylation levels ≥50% in 10/20 methylated EOCs. Constitutive BRCA methylation was excluded by testing blood-derived DNA. In conclusion, pyrosequencing methylation analysis of BRCA genes is a robust, quantitative and sensitive assay applicable to FFPE samples. Remarkably, a considerable subset of germline BRCA-negative EOCs showed somatic methylation and, likely, HRD. A subpopulation of women with BRCA methylation, even without BRCA mutations, could potentially benefit from PARP-inhibitors; further clinical studies are needed to clarify the predictive role of somatic BRCA methylation of PARP-therapy response.

Uncoupling effects of estrogen receptor α on LKB1/AMPK interaction upon adiponectin exposure in breast cancer.

Adipose tissue is a metabolic and endocrine organ that secretes bioactive molecules called adipocytokines. Among these, adiponectin has a crucial role in obesity-associated breast cancer. The key molecule of adiponectin signaling is AMPK, which is mainly activated by liver kinase B1 (LKB1). Here, we demonstrated that estrogen receptor-α (ERα)/LKB1 interaction may negatively interfere with the LKB1 capability to phosphorylate AMPK and inhibit its downstream signaling TSC2/mTOR/p70S6k. In adiponectin-treated MCF-7 cells, AMPK signaling was not working, resulting in its downstream target acetyl-CoA carboxylase (ACC) being still active. In contrast, in MDA-MB-231 cells, AMPK and ACC phosphorylation was enhanced by adiponectin, inhibiting lipogenesis and cell growth. Upon adiponectin, ERα signaling switched the energy balance of breast cancer cells toward a lipogenic phenotype. Therefore, adiponectin played an inhibitory role on ERα-negative cell growth and progression in vitro and in vivo. In contrast, low adiponectin levels, similar to those circulating in obese patients, acted on ERα-positive cells as a growth factor, stimulating proliferation. The latter effect was blunted in vivo by high adiponectin concentration. All this may have translational relevance, addressing how the handling of adiponectin, as a therapeutic tool in breast cancer treatment, needs to be carefully considered in ERα-positive obese patients, where circulating levels of this adipocytokine are relatively low. In other words, in ERα-positive breast cancer obese patients, higher adiponectin doses should be administered with respect to ERα-negative breast cancer, also opportunely combined with antiestrogen therapy. -Mauro, L., Naimo, G. D., Gelsomino, L., Malivindi, R., Bruno, L., Pellegrino, M., Tarallo, R., Memoli, D., Weisz, A., Panno, M. L., Andò, S. Uncoupling effects of estrogen receptor α on LKB1/AMPK interaction upon adiponectin exposure in breast cancer.

iSmaRT: a toolkit for a comprehensive analysis of small RNA-Seq data.

Summary: The interest in investigating the biological roles of small non-coding RNAs (sncRNAs) is increasing, due to the pleiotropic effects of these molecules exert in many biological contexts. While several methods and tools are available to study microRNAs (miRNAs), only few focus on novel classes of sncRNAs, in particular PIWI-interacting RNAs (piRNAs). To overcome these limitations, we implemented iSmaRT ( i ntegrative Sm all R NA T ool-kit), an automated pipeline to analyze smallRNA-Seq data. Availability and Implementation: iSmaRT is a collection of bioinformatics tools and own algorithms, interconnected through a Graphical User Interface (GUI). In addition to performing comprehensive analyses on miRNAs, it implements specific computational modules to analyze piRNAs, predicting novel ones and identifying their RNA targets. A smallRNA-Seq dataset generated from brain samples of Huntington's Disease patients was used here to illustrate iSmaRT performances, demonstrating how the pipeline can provide, in a rapid and user friendly way, a comprehensive analysis of different classes of sncRNAs. iSmaRT is freely available on the web at ftp://labmedmolge-1.unisa.it (User: iSmart - Password: password). Contact: aweisz@unisa.it or ggiurato@unisa.it. Supplementary information: Supplementary data are available at Bioinformatics online.

Design and expression of peptides with antimicrobial activity against Salmonella typhimurium.

We showed previously that insertion of Synechocystis Δ12 -desaturase in salmonella's membrane alters membrane physical state (MPS), followed by the expression of stress genes causing inability to survive within murine macrophages (MΦ). Recently, we showed that expression of one membrane lipid domain (MLD) of Δ12 -desaturase (ORF200) interferes with salmonella MPS, causing loss of virulence in mice and immunoprotection. Here, we postulate that an α-antimicrobial peptide (α-AMP) intercalates within membrane lipids, and depending on its amino acid sequence, it does so within specific key sensors of MLD. In this study, we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg2+ concentration. We synthesised a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in salmonella typhimurium. We showed that the pattern of gene expression controlled by PhoP/PhoQ highlights dysregulation of pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens. RNA-Seq of strain expressing ORF200 showed that the pattern of those genes is also altered here. Accumulation of NUF conferred temporary immunoprotection. This represents a powerful procedure to address synthetic α-AMPs to a specific MLD generating live non-virulent bacterial strains.

miR-196a Is Able to Restore the Aggressive Phenotype of Annexin A1 Knock-Out in Pancreatic Cancer Cells by CRISPR/Cas9 Genome Editing.

Annexin A1 (ANXA1) is a Ca2+-binding protein that is involved in pancreatic cancer (PC) progression. It is able to mediate cytoskeletal organization maintaining a malignant phenotype. Our previous studies showed that ANXA1 Knock-Out (KO) MIA PaCa-2 cells partially lost their migratory and invasive capabilities and also the metastatization process appeared affected in vivo. Here, we investigated the microRNA (miRNA) profile in ANXA1 KO cells finding that the modification in miRNA expression suggests the significant involvement of ANXA1 in PC development. In this study, we focused on miR-196a which appeared down modulated in absence of ANXA1. This miRNA is a well known oncogenic factor in several tumour models and it is able to trigger the agents of the epithelial to mesenchymal transition (EMT), like ANXA1. Our results show that the reintroduction in ANXA1 KO cells of miR-196a through the mimic sequence restored the early aggressive phenotype of MIA PaCa-2. Then, ANXA1 seems to support the expression of miR-196a and its role. On the other hand, this miRNA is able to mediate cytoskeletal dynamics and other protein functions promoting PC cell migration and invasion. This work describes the correlation between ANXA1 and specific miRNA sequences, particularly miR-196a. These results could lead to further information on ANXA1 intracellular role in PC, explaining other aspects that are apart from its tumorigenic behaviour.

Estrogen receptor beta impacts hormone-induced alternative mRNA splicing in breast cancer cells.

BACKGROUND: Estrogens play an important role in breast cancer (BC) development and progression; when the two isoforms of the estrogen receptor (ERα and ERß) are co-expressed each of them mediate specific effects of these hormones in BC cells. ERß has been suggested to exert an antagonist role toward the oncogenic activities of ERα, and for this reason it is considered an oncosuppressor. As clinical evidence regarding a prognostic role for this receptor subtype in hormone-responsive BC is still limited and conflicting, more knowledge is required on the biological functions of ERß in cancer cells. We have previously described the ERß and ERα interactomes from BC cells, identifying specific and distinct patterns of protein interactions for the two receptors. In particular, we identified factors involved in mRNA splicing and maturation as important components of both ERα and ERß pathways. Guided by these findings, here we performed RNA sequencing to investigate in depth the differences in the early transcriptional events and RNA splicing patterns induced by estradiol in cells expressing ERα alone or ERα and ERß. RESULTS: Exon skipping was the most abundant splicing event in the post-transcriptional regulation by estradiol. We identified several splicing events induced by ERα alone and by ERα+ERß, demonstrating for the first time that ERß significantly affects estrogen-induced splicing in BC cells, as revealed by modification of a subset of ERα-dependent splicing by ERß, as well as by the presence of splicing isoforms only in ERß+cells. In particular, we observed that ERß+BC cell lines exhibited around 2-fold more splicing events than the ERß- cells. Interestingly, we identified putative direct targets of ERß-mediated alternative splicing by correlating the genomic locations of ERß and ERα binding sites with estradiol-induced differential splicing in the corresponding genes. CONCLUSIONS: Taken together, these results demonstrate that ERß significantly affects estrogen-induced early transcription and mRNA splicing in hormone-responsive BC cells, providing novel information on the biological role of ERß in these tumors.

Ultrasound-guided fine needle aspiration cytology of a primary lymph node leiomyoma: a flexible procedure for a complex case.

BACKGROUND: A primary lymph node leiomyoma diagnosed by fine needle aspiration cytology (FNAC) is reported. CASE: A 22-year-old male complained of right groin swelling; ultrasound examination (US) showed a lymph node containing a 20-mm hypoechoic nodule. The residual lymph node was oval, with a well-characterized cortex and hilum. US-FNAC of the nodule showed oval spindle cells embedded in fibrillar matrix. Nuclei were naked and oval with dispersed chromatin but without nucleoli. Immunocytochemistry showed positivity for vimentin and actin, and negativity for cytokeratin, S100, CD23 and CD31. A smear of the residual lymph node showed a reactive lymphoid cell population. FNAC diagnosis was mesenchymal cell proliferation with smooth muscle phenotype; a lymph node is part of the lesion. A CT scan did not detect any inguinal or abdominal mass. The surgical sample was a lymph node containing a spindle cell tumor, which was actin and desmin positive, and S100, CD21, HMB45, CD23 and CD31 negative; MIB1 was positive in <5% of the cells. The residual lymph node was normal. CONCLUSION: The final diagnosis was primary benign leiomyoma in a lymph node. US-FNAC may frame complex lymph node lesions and provide treatment options.

Small non-coding RNA transcriptomic profiling in adult and fetal human brain.

Small non-coding RNAs (sncRNAs) make up ~1% of the transcriptome; nevertheless, they play significant roles in regulating cellular processes. Given the complexity of the central nervous system, sncRNAs likely hold particular importance in the human brain. In this study, we provide sncRNA transcriptomic profiles in a range of adult and prenatal brain regions, with a focus on piRNAs, due to their underexplored expression in somatic cells and tissue-specific nature. Using the WIND workflow, which combines two detection methods, we found 1333 (731 miRNAs, 249 piRNAs, 285 snoRNAs, and 68 other sncRNAs) and 1445 unique sncRNAs (770 miRNAs, 307 piRNAs, 289 snoRNAs, and 79 other sncRNAs) in developing and adult brains, respectively. Significant variations were found upon comparison of fetal and adult brain groups, with 82 miRNAs, 17 piRNAs, and 70 snoRNAs enriched in fetal brains and 22 miRNAs, 11 piRNAs in adult brains. This dataset represents a valuable resource for exploring the sncRNA roles in brain function, their involvement in neurological diseases, and the molecular mechanisms behind brain region interactions.

Expression of targets of the RNA-binding protein AUF-1 in human airway epithelium indicates its role in cellular senescence and inflammation.

Introduction: The RNA-binding protein AU-rich-element factor-1 (AUF-1) participates to posttranscriptional regulation of genes involved in inflammation and cellular senescence, two pathogenic mechanisms of chronic obstructive pulmonary disease (COPD). Decreased AUF-1 expression was described in bronchiolar epithelium of COPD patients versus controls and in vitro cytokine- and cigarette smoke-challenged human airway epithelial cells, prompting the identification of epithelial AUF-1-targeted transcripts and function, and investigation on the mechanism of its loss. Results: RNA immunoprecipitation-sequencing (RIP-Seq) identified, in the human airway epithelial cell line BEAS-2B, 494 AUF-1-bound mRNAs enriched in their 3'-untranslated regions for a Guanine-Cytosine (GC)-rich binding motif. AUF-1 association with selected transcripts and with a synthetic GC-rich motif were validated by biotin pulldown. AUF-1-targets' steady-state levels were equally affected by partial or near-total AUF-1 loss induced by cytomix (TNFα/IL1ß/IFNγ/10 nM each) and siRNA, respectively, with differential transcript decay rates. Cytomix-mediated decrease in AUF-1 levels in BEAS-2B and primary human small-airways epithelium (HSAEC) was replicated by treatment with the senescence- inducer compound etoposide and associated with readouts of cell-cycle arrest, increase in lysosomal damage and senescence-associated secretory phenotype (SASP) factors, and with AUF-1 transfer in extracellular vesicles, detected by transmission electron microscopy and immunoblotting. Extensive in-silico and genome ontology analysis found, consistent with AUF-1 functions, enriched RIP-Seq-derived AUF-1-targets in COPD-related pathways involved in inflammation, senescence, gene regulation and also in the public SASP proteome atlas; AUF-1 target signature was also significantly represented in multiple transcriptomic COPD databases generated from primary HSAEC, from lung tissue and from single-cell RNA-sequencing, displaying a predominant downregulation of expression. Discussion: Loss of intracellular AUF-1 may alter posttranscriptional regulation of targets particularly relevant for protection of genomic integrity and gene regulation, thus concurring to airway epithelial inflammatory responses related to oxidative stress and accelerated aging. Exosomal-associated AUF-1 may in turn preserve bound RNA targets and sustain their function, participating to spreading of inflammation and senescence to neighbouring cells.

Rapid and sensitive detection of SARS-CoV-2 variants in nasopharyngeal swabs and wastewaters.

The SARS-CoV-2 virus is continuously evolving, with appearance of new variants characterized by multiple genomic mutations, some of which can affect functional properties, including infectivity, interactions with host immunity, and disease severity. The rapid spread of new SARS-CoV-2 variants has highlighted the urgency to trace the virus evolution, to help limit its diffusion, and to assess effectiveness of containment strategies. We propose here a PCR-based rapid, sensitive and low-cost allelic discrimination assay panel for the identification of SARS-CoV-2 genotypes, useful for detection in different sample types, such as nasopharyngeal swabs and wastewater. The tests carried out demonstrate that this in-house assay, whose results were confirmed by SARS-CoV-2 whole-genome sequencing, can detect variations in up to 10 viral genome positions at once and is specific and highly sensitive for identification of all tested SARS-CoV-2 clades, even in the case of samples very diluted and of poor quality, particularly difficult to analyze.

ABCA1, TCF7, NFATC1, PRKCZ, and PDGFA DNA methylation as potential epigenetic-sensitive targets in acute coronary syndrome via network analysis.

Acute coronary syndrome (ACS) is the most severe clinical manifestation of coronary heart disease.We performed an epigenome-wide analysis of circulating CD4+ and CD8+ T cells isolated from ACS patients and healthy subjects (HS), enrolled in the DIANA clinical trial, by reduced-representation bisulphite sequencing (RRBS). In CD4+ T cells, we identified 61 differentially methylated regions (DMRs) associated with 57 annotated genes (53% hyper- and 47% hypo-methylated) by comparing ACS patients vs HS. In CD8+ T cells, we identified 613 DMRs associated with 569 annotated genes (28% hyper- and 72% hypo-methylated) in ACS patients as compared to HS. In CD4+vs CD8+ T cells of ACS patients we identified 175 statistically significant DMRs associated with 157 annotated genes (41% hyper- and 59% hypo-methylated). From pathway analyses, we selected six differentially methylated hub genes (NFATC1, TCF7, PDGFA, PRKCB, PRKCZ, ABCA1) and assessed their expression levels by q-RT-PCR. We found an up-regulation of selected genes in ACS patients vs HS (P < 0.001). ABCA1, TCF7, PDGFA, and PRKCZ gene expression was positively associated with CK-MB serum concentrations (r = 0.75, P = 0.03; r = 0.760, P = 0.029; r = 0.72, P = 0.044; r = 0.74, P = 0.035, respectively).This pilot study is the first single-base resolution map of DNA methylome by RRBS in CD4+ and CD8+ T cells and provides specific methylation signatures to clarify the role of aberrant methylation in ACS pathogenesis, thus supporting future research for novel epigenetic-sensitive biomarkers in the prevention and early diagnosis of this pathology.

Ki67 in Gleason Pattern 3 as a Marker of the Presence of Higher-Grade Prostate Cancer.

PURPOSE: Prostate biopsies may undergrade up to half of all prostate cancers (PCs), delaying definitive treatment by up to 3 years. One cause of undergrading is the partial sampling inherent in the technique. Because of this, a prostate biopsy that appears to be Gleason 3+3=6 may come either from a true 3+3=6 tumor or from a higher-grade tumor that has been sampled only partially. The main goal of the present study is to identify a way to distinguish these 2 kinds of "Gleason 3+3=6" biopsies.Mounting evidence hints at the possibility that Gleason pattern 3 associated with higher-grade PC (aG3) is biologically distinct from pure Gleason pattern 3 (pG3). MATERIALS AND METHODS: In this study, we used immunohistochemistry and computer-aided image analysis to compare the expression of Ki67, cyclin D1, MYC, and p53 between foci of aG3 and pG3, to search for a marker that could distinguish them. RESULTS: The expression of Ki67 differed significantly between pG3 and aG3. The average Ki67 labeling index was 1.63% for pG3 and 7.62% for aG3 (P<0.01); the average number of Ki67+ cells per high-power field was 17 for pG3 and 60 for aG3 (P<0.01). The other markers did not differ significantly between pG3 and aG3. CONCLUSIONS: When a biopsy only shows Gleason pattern 3 PC, Ki67 immunohistochemistry could be used to distinguish the nodules of true Gleason score 3+3=6 from those that only appear to be 3+3=6 because of a sampling error. This would dramatically improve the diagnostic performance of prostate biopsies and the management of early PC.

Posttranscriptional Gene Regulatory Networks in Chronic Airway Inflammatory Diseases: In silico Mapping of RNA-Binding Protein Expression in Airway Epithelium.

Background: Posttranscriptional gene regulation (PTGR) contributes to inflammation through alterations in messenger RNA (mRNA) turnover and translation rates. RNA-binding proteins (RBPs) coordinate these processes but their role in lung inflammatory diseases is ill-defined. We evaluated the expression of a curated list of mRNA-binding RBPs (mRBPs) in selected Gene Expression Omnibus (GEO) transcriptomic databases of airway epithelium isolated from chronic obstructive pulmonary disease (COPD), severe asthma (SA) and matched control subjects, hypothesizing that global changes in mRBPs expression could be used to infer their pathogenetic roles and identify novel disease-related regulatory networks. Methods: A published list of 692 mRBPs [Nat Rev Genet 2014] was searched in GEO datasets originated from bronchial brushings of stable COPD patients (C), smokers (S), non-smokers (NS) controls with normal lung function (n = 6/12/12) (GEO ID: GSE5058) and of (SA) and healthy control (HC) (n = 6/12) (GSE63142). Fluorescence intensity data were extracted and normalized on the medians for fold change (FC) comparisons. FCs were set at ≥ |1.5| with a false discovery rate (FDR) of ≤ 0.05. Pearson correlation maps and heatmaps were generated using tMEV tools v4_9_0.45. DNA sequence motifs were searched using PScan-ChIP. Gene Ontology (GO) was performed with Ingenuity Pathway Analysis (IPA) tool. Results: Significant mRBP expression changes were detected for S/NS, COPD/NS and COPD/S (n = 41, 391, 382, respectively). Of those, 32% of genes changed by FC ≥ |1.5| in S/NS but more than 60% in COPD/NS and COPD/S (n = 13, 267, 257, respectively). Genes were predominantly downregulated in COPD/NS (n = 194, 73%) and COPD/S (n = 202, 79%), less so in S/NS (n = 4, 31%). Unsupervised cluster analysis identified in 4 out of 12 S the same mRBP pattern seen in C, postulating subclinical COPD. Significant DNA motifs enrichment for transcriptional regulation was found for downregulated RBPs. Correlation analysis identified five clusters of co-expressed mRBPs. GO analysis revealed significant enrichments in canonical pathways both specific and shared among comparisons. Unexpectedly, no significant mRBPs modulation was found in SA compared to controls. Conclusions: Airway epithelial mRBPs profiling reveals a COPD-specific global downregulation of RBPs shared by a subset of control smokers, the potential of functional cooperation by coexpressed RBPs and significant impact on relevant pathogenetic pathways in COPD. Elucidation of PTGR in COPD could identify disease biomarkers or pathways for therapeutic targeting.

Insights into the Role of Estrogen Receptor ß in Triple-Negative Breast Cancer.

Estrogen receptors (ERα and ERß) are ligand-activated transcription factors that play different roles in gene regulation and show both overlapping and specific tissue distribution patterns. ERß, contrary to the oncogenic ERα, has been shown to act as an oncosuppressor in several instances. However, while the tumor-promoting actions of ERα are well-known, the exact role of ERß in carcinogenesis and tumor progression is not yet fully understood. Indeed, to date, highly variable and even opposite effects have been ascribed to ERß in cancer, including for example both proliferative and growth-inhibitory actions. Recently ERß has been proposed as a potential target for cancer therapy, since it is expressed in a variety of breast cancers (BCs), including triple-negative ones (TNBCs). Because of the dependence of TNBCs on active cellular signaling, numerous studies have attempted to unravel the mechanism(s) behind ERß-regulated gene expression programs but the scenario has not been fully revealed. We comprehensively reviewed the current state of knowledge concerning ERß role in TNBC biology, focusing on the different signaling pathways and cellular processes regulated by this transcription factor, as they could be useful in identifying new diagnostic and therapeutic approaches for TNBC.

Small Non-Coding RNA Profiling Identifies miR-181a-5p as a Mediator of Estrogen Receptor Beta-Induced Inhibition of Cholesterol Biosynthesis in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly heterogeneous disease, representing the most aggressive breast cancer (BC) subtype with limited treatment options due to a lack of estrogen receptor alpha (ERα), progesterone receptor (PR), and Erb-B2 receptor tyrosine kinase 2 (HER2/neu) expression. Estrogen receptor beta (ERß) is present in a fraction of TNBC patients, where its expression correlates with improved patient outcomes, supported by the fact that it exerts oncosuppressive effects in TNBC cell models in vitro. ERß is involved in microRNA-mediated regulation of gene expression in hormone-responsive BC cells and could mediate its actions through small noncoding RNAs (sncRNAs) in TNBCs also. To verify this possibility, smallRNA sequencing was performed on three ERß-expressing cell lines from different TNBC molecular subtypes. Several sncRNAs resulted modulated by ERß, with a subset being regulated in a tumor subtype-independent manner. Interestingly, sncRNA profiling of 12 ERß+and 32 ERß- primary TNBC biopsies identified 7 microRNAs, 1 PIWI-interacting RNA (piRNA), and 1 transfer RNA (tRNA) differentially expressed in ERß+ compared to ERß- tumors and cell lines. Among them, miR-181a-5p was found to be overexpressed in ERß+ tumors and predicted target key components of the cholesterol biosynthesis pathway previously found to be inhibited by ERß in TNBC cells.

Inhibition of histone methyltransferase DOT1L silences ERα gene and blocks proliferation of antiestrogen-resistant breast cancer cells.

Breast cancer (BC) resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor α (ERα) signaling, and ways to block ERα pathway in these tumors are sought after. We identified the H3K79 methyltransferase DOT1L as a novel cofactor of ERα in BC cell chromatin, where the two proteins colocalize to regulate estrogen target gene transcription. DOT1L blockade reduces proliferation of hormone-responsive BC cells in vivo and in vitro, consequent to cell cycle arrest and apoptotic cell death, with widespread effects on ER-dependent gene transcription, including ERα and FOXA1 gene silencing. Antiestrogen-resistant BC cells respond to DOT1L inhibition also in mouse xenografts, with reduction in ERα levels, H3K79 methylation, and tumor growth. These results indicate that DOT1L is an exploitable epigenetic target for treatment of endocrine therapy-resistant ERα-positive BCs.