Extracellular vesicle microRNAs contribute to Notch signaling pathway in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive T-cell malignancy characterized by genotypically-defined and phenotypically divergent cell populations, governed by adaptive landscapes. Clonal expansions are associated to genetic and epigenetic events, and modulation of external stimuli that affect the hierarchical structure of subclones and support the dynamics of leukemic subsets. Recently, small extracellular vesicles (sEV) such as exosomes were also shown to play a role in leukemia. Here, by coupling miRNome, bulk and single cell transcriptome profiling, we found that T-ALL-secreted sEV contain NOTCH1-dependent microRNAs (EV-miRs), which control oncogenic pathways acting as autocrine stimuli and ultimately promoting the expansion/survival of highly proliferative cell subsets of human T-cell leukemias. Of interest, we found that NOTCH1-dependent EV-miRs mostly comprised members of miR-17-92a cluster and paralogues, which rescued in vitro the proliferation of T-ALL cells blocked by γ-secretase inhibitors (GSI) an regulated a network of genes characterizing patients with relapsed/refractory early T-cell progenitor (ETP) ALLs. All these findings suggest that NOTCH1 dependent EV-miRs may sustain the growth/survival of immunophenotypically defined cell populations, altering the cell heterogeneity and the dynamics of T-cell leukemias in response to conventional therapies

Deciphering the Molecular Profile of Lung Cancer: New Strategies for the Early Detection and Prognostic Stratification

Recent advances in radiological imaging and genomic analysis are profoundly changing the way to manage lung cancer patients. Screening programs which couple lung cancer risk prediction models and low-dose computed tomography (LDCT) recently showed their effectiveness in the early diagnosis of lung tumors. In addition, the emerging field of radiomics is revolutionizing the approach to handle medical images, i.e., from a “simple” visual inspection to a high-throughput analysis of hundreds of quantitative features of images which can predict prognosis and therapy response. Yet, with the advent of next-generation sequencing (NGS) and the establishment of large genomic consortia, the whole mutational and transcriptomic profile of lung cancer has been unveiled and made publicly available via web services interfaces. This has tremendously accelerated the discovery of actionable mutations, as well as the identification of cancer biomarkers, which are pivotal for development of personalized targeted therapies. In this review, we will describe recent advances in cancer biomarkers discovery for early diagnosis, prognosis, and prediction of chemotherapy response

Role of the Circadian Gas-Responsive Hemeprotein NPAS2 in Physiology and Pathology

Simple Summary NPAS2, short for Neuronal PAS Domain Protein 2, is a transcription factor involved in regulating the circadian rhythms and sleep-wake cycles in mammals, including humans. It is a key component of the molecular clockwork that governs daily biological processes. NPAS2 binds heme as a prosthetic group and CO at micromolar concentrations, with ensuing changes in DNA affinity. In this way, gaseous signaling plus heme-based sensing and redox balance modify NPAS2 transcriptional activity and the expression of target genes. NPAS2 plays a crucial role in metabolism regulation and in maintaining the body's internal clock synchronized with the day-night cycle. Dysregulation of NPAS2 can lead to disruptions in circadian rhythms and may contribute to sleep disturbances, psychiatric disorders and other health issues, such as neoplastic, cardiovascular and cerebrovascular diseases. Alternatively, NPAS2 could represent a valuable predictive biomarker for prevention/stratification strategies and a promising druggable target for innovative therapeutic approaches.Abstract Neuronal PAS domain protein 2 (NPAS2) is a hemeprotein comprising a basic helix-loop-helix domain (bHLH) and two heme-binding sites, the PAS-A and PAS-B domains. This protein acts as a pyridine nucleotide-dependent and gas-responsive CO-dependent transcription factor and is encoded by a gene whose expression fluctuates with circadian rhythmicity. NPAS2 is a core cog of the molecular clockwork and plays a regulatory role on metabolic pathways, is important for the function of the central nervous system in mammals, and is involved in carcinogenesis as well as in normal biological functions and processes, such as cardiovascular function and wound healing. We reviewed the scientific literature addressing the various facets of NPAS2 and framing this gene/protein in several and very different research and clinical fields

Innovative Renewable Technology Integration for Nearly Zero-Energy Buildings within the Re-COGNITION Project

With the 2010/31/EU directive, all new buildings shall be nearly zero-energy buildings (nZEB) from 2020 onward, with the aim of strongly reducing the energy consumption related to the building sector. To achieve this goal, it is not sufficient to focus on the design of the building envelope; smart and efficient energy management is necessary. Moreover, to ensure the adoption of RES systems in the built environment, innovative technologies need to be further developed in order to increase their cost-effectiveness, energy efficiency and integration capability. This paper proposes a synthesis, design and operation optimization of an integrated multi-energy system composed of traditional and innovative renewable technologies, developed within the European project Re-COGNITION. A biogas-based micro cogeneration unit, lightweight glass-free photovoltaic modules, a passive variable geometry small wind turbine optimized for an urban environment and latent heat thermal storage based on phase change materials are some of the technologies developed within the Re-COGNITION project. The optimization problem is solved to contemporarily evaluate (a) the optimal design and (b) the optimal operations of the set of technologies considering both investment and operating costs, using mixed integer non-linear programming. The optimization is applied to the four pilots that are developed during the project, in various European cities (Turin (Italy), Corby (United Kingdom), Thessaloniki (Greece), Cluj-Napoca (Romania). Simulation results show that the development and optimal exploitation of new technologies through optimization strategies provide significant benefits in terms of cost (between 11% and 42%) and emissions (between 10% and 25%), managing building import/export energy and charge/discharge storage cycles

Abstract 2582: Investigating the origin and function of circulating miRNA in lung cancer

MicroRNAs (miRNAs) are small, highly conserved noncoding RNA molecules involved in the regulation of gene expression. MicroRNAs are resistant to harsh conditions and stably exist in body fluids (e.g. saliva, urine, breast milk, blood). Circulating cell-free miRNAs (cf-miRNA) were shown to be effective biomarkers for the early diagnosis of cancer. Furthermore, cf-miRNA were also found in exosomes that are nano-sized extracellular vesicles which exchange molecular information among cells. However, the understanding of origin of cf-miRNAs and of biological function still remains elusive. We recently identified a signature of cf-miRNAs diagnostic for lung cancer which we now hypothesize being released by lung cancer cells (EP-cf-miRNA) and by tumor microenvironment (TME-cf-miRNA). In particular, two cf-miRNAs, miR-29a and 223-3p, were selected as prototypes of EP- and TME-cf-miRNAs for further investigations.We found that the expression of miR-223-3p was higher in TME than in cancer cells of a cohort of 19 lung adenocarcinoma. In contrast, miR-29a-3p was frequently overexpressed (≥2 fold) in cancer cells. Interestingly, the serum concentration of cf-miR-29a was lower than miR-223-3p and this also in exosomes.We next investigated the expression profile of miR-29a/223-3p in a panel of lung normal and adenocarcinoma cell lines (N=14). miR-29a/223-3p were both expressed intracellularly and in exosomes. Of note, the miR-29a expression level resembles that observed in lung cancer cells from FFPE samples. Conversely, miR-223-3p expression was lower in the adenocarcinoma cell lines which further confirm a more TME origin. We are now expanding our analysis by screening the whole set of cf-miRNAs by high-throughput qPCR profile (OpenArray technology) and by applying laser capture microdissection to investigate single cancer epithelial cells and TME. Our work provides proof of principle demonstration of an effective approach to investigate cf-miRNA origin to unravel the role of cf-miRNA in lung adenocarcinoma

Proteomic characterization of peroxisome proliferator‐activated receptor‐γ (PPARγ) overexpressing or silenced colorectal cancer cells unveils a novel protein network associated with an aggressive phenotype

Peroxisome proliferator‐activated receptor‐γ (PPARγ) is a transcription factor of the nuclear hormone receptor superfamily implicated in a wide range of processes, including tumorigenesis. Its role in colorectal cancer (CRC) is still debated; most reports support that PPARγ reduced expression is associated with poor prognosis. We employed 2‐Dimensional Differential InGel Electrophoresis (2‐D DIGE) followed by Liquid Chromatography (LC)‐tandem Mass Spectrometry (MS/MS) to identify differentially expressed proteins and the molecular pathways underlying PPARγ expression in CRC progression. We identified several differentially expressed proteins in HT29 and HCT116 CRC cells and derived clones either silenced or overexpressing PPARγ, respectively. In Ingenuity Pathway Analysis (IPA) they showed reciprocal relation with PPARγ and a strong relationship with networks linked to cell death, growth and survival. Interestingly, five of the identified proteins, ezrin (EZR), isoform C of prelamin‐A/C (LMNA), alpha‐enolase (ENOA), prohibitin (PHB) and RuvB‐like 2 (RUVBL2) were shared by the two cell models with opposite expression levels, suggesting a possible regulation by PPARγ. mRNA and western blot analysis were undertaken to obtain a technical validation and confirm the expression trend observed by 2‐D DIGE data. We associated EZR upregulation with increased cell surface localization in PPARγ‐overexpressing cells by flow cytometry and immunofluorescence staining. We also correlated EZR and PPARγ expression in our series of CRC specimens and the expression profiling of all five proteins levels in the publicly available colon cancer genomic data from Oncomine and Cancer Genome Atlas (TCGA) colon adenocarcinoma (COAD) datasets. In summary, we identified a panel of proteins correlated with PPARγ expression that could be associated with CRC unveiling new pathways to be investigated for the selection of novel potential prognostic/predictive biomarkers and/or therapeutic targets

Analysis of extracellular-vesicle miRNAs in a new aggressive molecular subtype of lung cancer

Non-Small Cell Lung cancer is the most frequently diagnosed lung cancer type (80-90%) and lung adenocarcinoma (LUAD) is the major subtype. Early diagnosis by low-dose computed tomography was shown to be effective in reducing mortality (~20% at 5 years). However, the management of lung cancer patients can be difficult due to pathological and genetic heterogeneity of the disease. We discovered a new molecular subtype namely C1-LUAD using a 10-genes signature, which correlates with the worst prognosis and has peculiar features of mesenchymal and stem-like cells. Of note, analyzing intracellular differentially expressed miRNAs in C1 patients, we found a targeted network of genes as hubs of mechanisms for lung cancer progression. Moreover, we discovered 5 miRNAs up-regulated and 6 miRNAs down-regulated in C1 exosomes extracted from serum, which pave the way to make an early diagnosis for this aggressive subtype of LUAD. Finally, we highlighted that C1-like cells are more prone to internalized labeled-exosomes in a time and dose dependent manner, suggesting a role of these extracellular-vesicles in the maintenance of the metastatic features

Analysis of extracellular-vesicle miRNAs in a new aggressive molecular subtype of lung cancer

Non-Small Cell Lung cancer is the most frequently diagnosed lung cancer type (80-90% of all cases) and lung adenocarcinoma (LUAD) is the major subtype. Early diagnosis by low-dose computed tomography was recently shown to be effective in reducing lung cancer mortality (~20% at 5 years). However, the management of lung cancer patients can be difficult due to pathological and genetic heterogeneity of the disease. We, recently, discovered a new molecular subtype of lung adenocarcinoma using a 10-genes signature, namely C1- LUAD, which correlates with the worst patients prognosis and has peculiar features of mesenchymal and stem-like cells. Yet, we also identified an intracellular signature of 7 microRNAs as a surrogate of the 10-genes to distinguish the C1-LUAD subtype. Of note, analyzing intracellular differentially expressed miRNAs in C1 patients, we found a targeted network of genes as hubs of mechanisms for lung cancer progression. Importantly, miRNAs can be secreted in body fluids (e.g., Urine, breast milk, blood) via extracellular vesicles (i.e. exosomes). In the last years, tumor exosomes have also drawn increasing attention due to their stability and accessibility as diagnostic and prognostic biomarkers. For these reasons, we selected 22-miRNAs which target genes involved in cancer progression and we analyzed their expression in isolated exosomes derived from C1 and nonC1 serum samples. Performing a differential expression analysis we found 5 miRNAs up-regulated and 6 miRNAs down-regulated in C1 compared with nonC1 patients. Next, using differential centrifugation protocol, we purified and characterized exosomes from a panel of LUAD cell lines classified by gene expression and phenotypic behaviors in C1-like and nonC1-like. Notably, we evaluated the heterogenic exosomes kinetic up-take in C1-like and nonC1-like cells, highlighting that C1-like cells are more prone to internalized labeled-exosomes, in a time and dose dependent manner. Overall, we identified for the first time a set of exosomal-miRNAs linked to C1-LUAD subtype, which pave the way to make an early diagnosis for this new aggressive subtype of lung cancer. Moreover, we found that C1-like cells internalize more efficiently exosomes, suggesting a role of these extracellular vesicles in the maintenance of the metastatic features characteristic of the C1 tumors