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Identifying master epigenetic factors controlling proliferation and survival of cancer cells allows to discover new molecular targets exploitable to overcome resistance to current pharmacological regimens. In breast cancer (BC), resistance to endocrine therapy (ET) arises from aberrant Estrogen Receptor alpha (ERα) signaling caused by genetic and epigenetic events still mainly unknown. Targeting key upstream components of the ERα pathway provides a way to interfere with estrogen signaling in cancer cells independently from any other downstream event. By combining computational analysis of genome-wide 'drop-out' screenings with siRNA-mediated gene knock-down (kd), we identified a set of essential genes in luminal-like, ERα + BC that includes BRPF1, encoding a bromodomain-containing protein belonging to a family of epigenetic readers that act as chromatin remodelers to control gene transcription. To gather mechanistic insights into the role of BRPF1 in BC and ERα signaling, we applied chromatin and transcriptome profiling, gene ablation and targeted pharmacological inhibition coupled to cellular and functional assays. Results indicate that BRPF1 associates with ERα onto BC cell chromatin and its blockade inhibits cell cycle progression, reduces cell proliferation and mediates transcriptome changes through the modulation of chromatin accessibility. This effect is elicited by a widespread inhibition of estrogen signaling, consequent to ERα gene silencing, in antiestrogen (AE) -sensitive and -resistant BC cells and pre-clinical patient-derived models (PDOs). Characterization of the functional interplay of BRPF1 with ERα reveals a new regulator of estrogen-responsive BC cell survival and suggests that this epigenetic factor is a potential new target for treatment of these tumors.
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Neoplasias de la Mama , Proliferación Celular , Resistencia a Antineoplásicos , Receptor alfa de Estrógeno , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/genética , Femenino , Receptor alfa de Estrógeno/metabolismo , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Línea Celular Tumoral , Genes Esenciales , Antineoplásicos Hormonales/farmacología , Antineoplásicos Hormonales/uso terapéutico , Células MCF-7 , Cromatina/metabolismo , Cromatina/genética , Epigénesis Genética , Transducción de Señal/efectos de los fármacos , Perfilación de la Expresión GénicaRESUMEN
The ongoing COVID-19 pandemic caused by SARS-CoV-2 has affected millions of people worldwide and has significant implications for public health. Host transcriptomics profiling provides comprehensive understanding of how the virus interacts with host cells and how the host responds to the virus. COVID-19 disease alters the host transcriptome, affecting cellular pathways and key molecular functions. To contribute to the global effort to understand the virus's effect on host cell transcriptome, we have generated a dataset from nasopharyngeal swabs of 35 individuals infected with SARS-CoV-2 from the Campania region in Italy during the three outbreaks, with different clinical conditions. This dataset will help to elucidate the complex interactions among genes and can be useful in the development of effective therapeutic pathways.
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COVID-19 , Transcriptoma , Humanos , Italia , Pandemias , SARS-CoV-2RESUMEN
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.
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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.
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Neoplasias Ováricas , Humanos , Femenino , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/patología , Neoplasias Ováricas/patología , Proteína BRCA2/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteína BRCA1/genética , Formaldehído , Mutación de Línea Germinal/genéticaRESUMEN
In December 2019, several patients were hospitalized and diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which subsequently led to a global pandemic. To date, there are no studies evaluating the relationship between the respiratory phageome and the SARS-CoV-2 infection. The current study investigated the phageome profiles in the nasopharyngeal swabs collected from 55 patients during the three different waves of coronavirus disease 2019 (COVID-19) in the Campania Region (Southern Italy). Data obtained from the taxonomic profiling show that phage families belonging to the order Caudovirales have a high abundance in the patient samples. Moreover, the severity of the COVID-19 infection seems to be correlated with the phage abundance.
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COVID-19 , Humanos , Pandemias , SARS-CoV-2 , Índice de Severidad de la Enfermedad , ViromaRESUMEN
The histone lysine methyltransferase DOT1L (DOT1-like histone lysine methyltransferase) is responsible for the epigenetic regulation of gene expression through specific methylation of lysine79 residue of histone H3 (H3K79) in actively transcribed genes. Its normal activity is crucial for embryonic development and adult tissues functions, whereas its aberrant functioning is known to contribute to leukemogenesis. DOT1L is the only lysine methyltransferase that does not contain a SET domain, which is a feature that allowed the development of selective DOT1L inhibitors that are currently investigated in Phase I clinical trials for cancer treatment. Recently, abnormal expression of this enzyme has been associated with poor survival and increased aggressiveness of several solid tumors. In this review evidences of aberrant DOT1L expression and activity in breast, ovarian, prostate, colon, and other solid tumors, and its relationships with biological and clinical behavior of the disease and response to therapies, are summarized. Current knowledge of the structural basis of DOT1L ability to regulate cell proliferation, invasion, plasticity and stemness, cell cycle progression, cell-to-cell signaling, epithelial-to-mesenchymal transition, and chemoresistance, through cooperation with several molecular partners including noncoding RNAs, is also reviewed. Finally, available options for the treatment of therapeutically challenging solid tumors by targeting DOT1L are discussed.
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Since its first appearance, the SARS-CoV-2 has spread rapidly in the human population, reaching the pandemic scale with >280 million confirmed infections and more than 5 million deaths to date (https://covid19.who.int/). These data justify the urgent need to enhance our understanding of SARS-CoV-2 effects in the respiratory system, including those linked to co-infections. The principal aim of our study is to investigate existing correlations in the nasopharynx between the bacterial community, potential pathogens, and SARS-CoV-2 infection. The main aim of this study was to provide evidence pointing to possible relationships between components of the bacterial community and SARS-CoV-2 in the nasopharynx. Meta-transcriptomic profiling of the nasopharyngeal microbial community was carried out in 89 SARS-Cov-2 positive subjects from the Campania Region in Italy. To this end, RNA extracted from nasopharyngeal swabs collected at different times during the initial phases of the pandemic was analyzed by Next-Generation Sequencing (NGS). Results show a consistently high presence of members of the Proteobacteria (41.85%), Firmicutes (28.54%), and Actinobacteria (16.10%) phyla, and an inverted correlation between the host microbiome, co-infectious bacteria, and super-potential pathogens such as Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Neisseria gonorrhoeae. In depth characterization of microbiota composition in the nasopharynx can provide clues to understand its potential contribution to the clinical phenotype of Covid-19, clarifying the interaction between SARS-Cov-2 and the bacterial flora of the host, and highlighting its dysbiosis and the presence of pathogens that could affect the patient's disease progression and outcome.
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COVID-19 , Coinfección , Microbiota , Bacterias/genética , Coinfección/epidemiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Italia/epidemiología , Microbiota/genética , Nasofaringe/microbiología , Pandemias , SARS-CoV-2/genéticaRESUMEN
From December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has spread rapidly, leading to a global pandemic. Little is known about possible relationships between SARS-CoV-2 and other viruses in the respiratory system affecting patient prognosis and outcomes. This study aims to characterize respiratory virome profiles in association with SARS-CoV-2 infection and disease severity, through the analysis in 89 nasopharyngeal swabs collected in a patient's cohort from the Campania region (Southern Italy). Results show coinfections with viral species belonging to Coronaviridae, Retroviridae, Herpesviridae, Poxviridae, Pneumoviridae, Pandoraviridae, and Anelloviridae families and only 2% of the cases (2/89) identified respiratory viruses.
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COVID-19 , Nasofaringe , COVID-19/epidemiología , COVID-19/terapia , COVID-19/virología , Humanos , Italia/epidemiología , Nasofaringe/virología , Pandemias , SARS-CoV-2 , ViromaRESUMEN
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.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Humanos , Nasofaringe , SARS-CoV-2/genética , Aguas ResidualesRESUMEN
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.
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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.
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Colesterol/biosíntesis , Receptor beta de Estrógeno/metabolismo , MicroARNs/metabolismo , ARN Pequeño no Traducido/genética , Neoplasias de la Mama Triple Negativas/genética , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Receptor beta de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/metabolismo , Análisis de Supervivencia , Regulación hacia Arriba/genéticaRESUMEN
Triple-negative breast cancer (TNBC) is characterized by poor response to therapy and low overall patient survival. Recently, Estrogen Receptor beta (ERß) has been found to be expressed in a fraction of TNBCs where, because of its oncosuppressive actions on the genome, it represents a potential therapeutic target, provided a better understanding of its actions in these tumors becomes available. To this end, the cell lines Hs 578T, MDA-MB-468 and HCC1806, representing the claudin-low, basal-like 1 and 2 TNBC molecular subtypes respectively, were engineered to express ERß under the control of a Tetracycline-inducible promoter and used to investigate the effects of this transcription factor on gene activity. The antiproliferative effects of ERß in these cells were confirmed by multiple functional approaches, including transcriptome profiling and global mapping of receptor binding sites in the genome, that revealed direct negative regulation by ERß of genes, encoding for key components of cellular pathways associated to TNBC aggressiveness representing novel therapeutic targets such as angiogenesis, invasion, metastasis and cholesterol biosynthesis. Supporting these results, interaction proteomics by immunoprecipitation coupled to nano LC-MS/MS mass spectrometry revealed ERß association with several potential nuclear protein partners, including key components of regulatory complexes known to control chromatin remodeling, transcriptional and post-transcriptional gene regulation and RNA splicing. Among these, ERß association with the Polycomb Repressor Complexes 1 and 2 (PRC1/2), known for their central role in gene regulation in cancer cells, was confirmed in all three TNBC subtypes investigated, suggesting its occurrence independently from the cellular context. These results demonstrate a significant impact of ERß in TNBC genome activity mediated by its cooperation with regulatory multiprotein chromatin remodeling complexes, providing novel ground to devise new strategies for the treatment of these diseases based on ligands affecting the activity of this nuclear receptor or some of its protein partners.
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Colesterol/biosíntesis , Cromatina/metabolismo , Receptor beta de Estrógeno/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Perfilación de la Expresión Génica , Humanos , Proteómica , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
Estrogen Receptor alpha (ERα) is a ligand-inducible transcription factor that mediates estrogen signaling in hormone-responsive cells, where it controls key cellular functions by assembling in gene-regulatory multiprotein complexes. For this reason, interaction proteomics has been shown to represent a useful tool to investigate the molecular mechanisms underlying ERα action in target cells. RNAs have emerged as bridging molecules, involved in both assembly and activity of transcription regulatory protein complexes. By applying Tandem Affinity Purification (TAP) coupled to mass spectrometry (MS) before and after RNase digestion in vitro, we generated a dataset of nuclear ERα molecular partners whose association with the receptor involves RNAs. These data provide a useful resource to elucidate the combined role of nuclear RNAs and the proteins identified here in ERα signaling to the genome in breast cancer and other cell types.
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Neoplasias de la Mama/genética , Núcleo Celular/metabolismo , Receptor alfa de Estrógeno/metabolismo , ARN Neoplásico/metabolismo , Neoplasias de la Mama/metabolismo , Núcleo Celular/genética , Receptor alfa de Estrógeno/genética , Femenino , Humanos , Espectrometría de Masas , Proteómica , ARN Neoplásico/genéticaRESUMEN
BACKGROUND: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. METHODS: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. RESULTS: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. CONCLUSIONS: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.