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The role of the tumour-suppressor miR-34 family in breast physiology and in mammary stem cells (MaSCs) is largely unknown. Here, we revealed that miR-34 family, and miR-34a in particular, is implicated in mammary epithelium homoeostasis. Expression of miR-34a occurs upon luminal commitment and differentiation and serves to inhibit the expansion of the pool of MaSCs and early progenitor cells, likely in a p53-independent fashion. Mutant mice (miR34-KO) and loss-of-function approaches revealed two separate functions of miR-34a, controlling both proliferation and fate commitment in mammary progenitors by modulating several pathways involved in epithelial cell plasticity and luminal-to-basal conversion. In particular, miR-34a acts as endogenous inhibitor of the Wnt/beta-catenin signalling pathway, targeting up to nine upstream regulators at the same time, thus modulating the expansion of the MaSCs/early progenitor pool. These multiple roles of miR-34a are maintained in a model of human breast cancer, in which chronic expression of miR-34a in triple-negative mesenchymal-like cells (enriched in cancer stem cells-CSCs) could promote a luminal-like differentiation programme, restrict the CSC pool, and inhibit tumour propagation. Hence, activation of miR-34a-dependent programmes could provide a therapeutic opportunity for the subset of breast cancers, which are rich in CSCs and respond poorly to conventional therapies.

This work is concerned with the geometrical characterisation of bistable composite plates in order to investigate their curvatures interdependence. The main non linear feature of bistable asymmetric laminates concerns large deflection with small energy input. This is an aspect of particular interest for several applications. Accordingly, there is an increasing importance placed on accuracy/sensitivity of predicting techniques to model bistable deformation shapes. This paper presents a novel forecast model to map the surface profiles of bistable laminates. By Timoshenko and Ashwell approaches, an analytical model is developed to provide an interpretation of the bistable shapes, in terms of principal and anticlastic curvatures. The experimental data (via laser acquisitions), with analytical and Finite Element models, help to understand the relation between material properties, plate dimensions, stacking sequence, curing features of the composite laminates and the bistability phenomenon.

Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Here we report on the characterization of an intergenic lncRNA expressed in epithelial tissues which we termed EPR (Epithelial cell Program Regulator). EPR is rapidly downregulated by TGF-β and its sustained expression largely reshapes the transcriptome, favors the acquisition of epithelial traits, and reduces cell proliferation in cultured mammary gland cells as well as in an animal model of orthotopic transplantation. EPR generates a small peptide that localizes at epithelial cell junctions but the RNA molecule per se accounts for the vast majority of EPR-induced gene expression changes. Mechanistically, EPR interacts with chromatin and regulates Cdkn1a gene expression by affecting both its transcription and mRNA decay through its association with SMAD3 and the mRNA decay-promoting factor KHSRP, respectively. We propose that EPR enables epithelial cells to control proliferation by modulating waves of gene expression in response to TGF-β., Several lncRNAs are regulated by TGF-β. Here the authors report that an intergenic lncRNA —EPR— is a component of the TGF-β signaling pathway and controls epithelial cell proliferation by altering transcription and mRNA decay of Cdkn1a. EPR overexpression restrains tumor growth of orthotopically transplanted mice.

An innovative method based on nonlinear Lamb waves behavior and Local Defect Resonance (LDR) is proposed for locating ad evaluating disbonds in Single-Lap Joints (SLJ) typically used in aerospace industry. The presence of damages/defects such as disbonds leads to the presence of sub-and super-harmonics components in the frequency response. Particular wave excitation frequencies promote nonlinear response of the structure causing LDR. The LDR frequency was experimentally evaluated through the appearance of a single subharmonic component in the frequency spectrum of signals received by piezoelectric transducers (PZTs) bonded on the structure. The nonlinear properties of Lamb waves were exploited to make defects generate subharmonic wave packets at LDR frequency. An algorithm was developed for damage/defect localization that was accurately performed by knowing (i) PZTs positions, (ii) Time of Flight (ToF) and (iii) propagation properties of subharmonics packet. Several disbonds with different dimensions were artificially reproduced on an aluminum SLJ: experimental results showed good accordance both in usual (single damage) and critical (multi-damage) scenario. The paper proposes a baseline-free method for the disbonds detection and localization in SLJs that uses the PZT signals without affecting adhesive interface, thus allowing an active health monitoring.

Structural Health Monitoring (SHM) represents a fast-growing field of great interest specially for aerospace, civil and mechanical engineering. This work proposes an in-flight SHM method for impact detection by ultrasounds, that is based on the dynamic behavior of the inspected structure and is useful to establish critical and dangerous operational conditions. The proposed method can be used to detect impact events both in metallic or composite structures, it is specifically designed to be used on typical fuselage and wing panels and is based on the propagation of Lamb waves in the structure on which PZT sensors are bonded for receiving signals. Algorithms are implemented in order to evaluate the impact location by post-processing the acquired signals. Several test cases are numerically studied before being tested in laboratory and reproduced in-flight. A good agreement between the numerical, laboratory and in-flight results is achieved.

This study is focused on the development of a finite difference model to simulate Lamb wave propagation through Single Lap Joints (SLJs). The main advantage of this model is the mathematical ability to easily reproduce the presence of a damage (debonding) as a discontinuity in velocity values. This makes our model suitable for continuous and embedded Structural Health Monitoring (SHM) of a complex structure. Numerical simulations and experimental campaigns are presented in order to validate the proposed model.

MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Large Drosha Complex (LDC). Through high resolution mass spectrometry (MS) analysis we discovered that the LDC is extensively methylated, with 82 distinct methylated sites associated to 16 out of 23 subunits of the LDC. The majority of these modifications occurs on arginine (R)- residues (61), leading to 86 methylation events, while 29 lysine (K)-methylation events occurs on 21 sites of the complex. Interestingly, both depletion and pharmacological inhibition of PRMT1 lead to a widespread alteration of the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the specific impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the ILF3 subunit of the complex is linked to its diminished binding to the target pri-miRNAs. Overall, our study uncovers a previously uncharacterized role of R-methylation in the regulation of the LDC activity in mammalian cells, thus affecting global miRNA levels.

Structural Health Monitoring (SHM) gives a diagnosis of a structure assessing the structural integrity and predicting the residual life through appropriate data processing and interpretation. A structure must remain in the design domain, although it can be subjected to normal aging due to usage, action of the environment, and accidental events. SHM involves the integration of electronic devices in the inspected structure that sometimes are Piezoelectric Transducers (PZT). These are lightweight and small and can be produced in different geometries. They are used both in guided wave-based and electromechanical impedance-based methods. The PZT bonding requires essential steps such as preparation of the surfaces, application of the adhesive, and assembly that make the bonding process not so easy to be realised. Furthermore, adhesives are susceptible to environmental degradation. Transducer debonding or non-uniform distributed glue underneath the sensor causes the reduction of the performance and can affect the reliability of the SHM system. In this paper, a sensor diagnostic method for the monitoring of the PZT operational status is proposed in order to detect bonding defect/damage between a PZT patch and a host structure. The authors propose a method based on the nonlinear behaviour of the contact PZT/structure that allows the identification of the damaged PZT and the geometrical characterization of the debonding. The feasibility of the diagnostic procedure is demonstrated by numerical studies and experiments, where disbonds were created by inhibiting the adhesive action on a part of the interface through Teflon film. The proposed method can be used to evaluate the sensor functionality after an extreme loading event or over a long period of service time.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

MicroRNAs (miRNAs) are an evolutionary conserved class of short, single-stranded noncoding RNAs (18-22 nt in length) that act in posttranscriptional regulation of gene expression in higher eukaryotes. The abundance of a miRNA is a key feature in control of its activity and, therefore, a number of mechanisms finely regulate miRNA levels, acting at both transcriptional and posttranscriptional level. Recent evidences, including our research, highlighted the role of miRNA decay as a mechanism controlling the miRNA pool. We describe in this chapter an optimized methodology to determine miRNA degradation rates in mammalian cells. Our approach is based on metabolic pulse labeling with 4-thiouridine (4sU), a uridine analog that is incorporated in nascent RNA and allows thiol-specific biotinylation and selective pull-down of labeled RNA. In particular, given the long average half-life and the complex biogenetic process of miRNAs, we developed a "pulse-chase" protocol where 4sU is removed from the medium after a long labeling period (2-3 h pulse), and labeled RNA is purified at different time points to measure the decay of labeled molecules. By combining the 4sU-based "pulse-chase" approach with high-throughput small RNA sequencing (sRNAseq), it is possible to quantify at genome-wide level miRNA degradation rates.

Summary: Physical exercise stimulates adult hippocampal neurogenesis and is considered a relevant strategy for preventing age-related cognitive decline in humans. The underlying mechanisms remains controversial. Here, we show that exercise increases proliferation of neural precursor cells (NPCs) of the mouse dentate gyrus (DG) via downregulation of microRNA 135a-5p (miR-135a). MiR-135a inhibition stimulates NPC proliferation leading to increased neurogenesis, but not astrogliogenesis, in DG of resting mice, and intriguingly it re-activates NPC proliferation in aged mice. We identify 17 proteins (11 putative targets) modulated by miR-135 in NPCs. Of note, inositol 1,4,5-trisphosphate (IP3) receptor 1 and inositol polyphosphate-4-phosphatase type I are among the modulated proteins, suggesting that IP3 signaling may act downstream miR-135. miR-135 is the first noncoding RNA essential modulator of the brain's response to physical exercise. Prospectively, the miR-135-IP3 axis might represent a novel target of therapeutic intervention to prevent pathological brain aging. : Pons-Espinal, Gasperini, and colleagues report that running induces NPC proliferation and neurogenesis via downregulation of miR-135a-5p in the mouse hippocampus. Remarkably, downregulation of miR-135a stimulates proliferation in the hippocampus of aged mice. ITPR1 and INPP4A, involved in IP3 signaling, are modulated by miR-135 in NPCs. Prospectively, therapeutic exploitation of the miR-135-IP3 axis might represent a novel intervention strategy for successful aging. Keywords: adult neurogenesis, running, aging, miR-135a, inositol 1,4,5-trisphosphate (IP3) pathway, ITPR1, INPP4A

Morphing structures for lightweight and energy-efficient aircraft mobile surfaces have been investigated for several years. This paper presents a novel lightweight, passive and low-energy morphing surface concept based on the “lever effect” of a bistable composite plate that can be integrated in aircraft moving surfaces. By using appropriate boundary conditions, it is demonstrated that the magnitude of the activation force on the bistable composite can be tailored to match the differential pressure on the aircraft's airfoil. As a consequence, the bistable laminate can be used as a passive morphing surface. Both numerical simulations and experimental testing are used to prove this concept on a NACA 2412 airfoil structure. The results show that, by choosing proper configuration of constraints, lay-up and aspect ratio of the bistable composite, it is possible to tailor and activate the snap-through mechanism in a passive manner. The proposed concept would save significant weight when compared to an active morphing concept.

This work aims at presenting techniques for the damage identification in single lap joints (SLJs). The two proposed experimental approaches, exploiting particular interactions of the structure with vibrational waves produced by piezoelectric sensors, allow to perform a Structural Health Monitoring (SHM) without a baseline. The first technique involves the excitation of the structure by means of stationary sinusoidal waves: the presence of a subharmonic in the frequency response spectrum at a receiver point indicates the presence of damage in the joint. In addition, through a simplified analytical model it is possible to relate the frequency of this subharmonic to the size of the damage. The second technique is based on the use of a tone burst: the exciting sensor sends this transient signal that travels through the bonded area and is subsequently read by the receiving sensor; the information received is the result of an interaction between the sent wave and the reflection of the boundaries, sensitive to possible damages. The attenuation of the burst, studied through the wave equations, gives indications on the size of the damage. Both experimental campaigns were carried out on aluminum SLJs bonded with acrylic adhesive, using piezoelectric sensors (one exciting and one receiving). Simplified analytical models were used to validate the experimental results. The good analytical-experimental correlation confirms the validity of the proposed approaches.

Asymmetric cell divisions balance stem cell proliferation and differentiation to sustain tissue morphogenesis and homeostasis. During asymmetric divisions, fate determinants and niche contacts segregate unequally between daughters, but little is known on how this is achieved mechanistically. In Drosophila neuroblasts and murine mammary stem cells, the association of the spindle orientation protein LGN with the stem cell adaptor Inscuteable has been connected to asymmetry. Here we report the crystal structure of Drosophila LGN in complex with the asymmetric domain of Inscuteable, which reveals a tetrameric arrangement of intertwined molecules. We show that Insc:LGN tetramers constitute stable cores of Par3–Insc-LGN-GαiGDP complexes, which cannot be dissociated by NuMA. In mammary stem cells, the asymmetric domain of Insc bound to LGN:GαiGDP suffices to drive asymmetric fate, and reverts aberrant symmetric divisions induced by p53 loss. We suggest a novel role for the Insc-bound pool of LGN acting independently of microtubule motors to promote asymmetric fate specification., During asymmetric divisions fate determinants and niche contacts segregate unequally between daughter cells, but the mechanism is unclear. Here the authors show that Insc:LGN tetramers promote assembly of Par3-Insc-LGN-GαiGDP complexes and asymmetric fate specification independently of microtubule motors.

We examined the influence of microenvironment stimuli on molecular events relevant to the biological functions of 1833-bone metastatic clone and the parental MDA-MB231 cells. (i) In both the cell lines, hepatocyte growth factor (HGF) and the osteoblasts’ biological products down regulated nuclear Ets-1-protein level in concomitance with endogenous miR-125b accumulation. In contrast, under hypoxia nuclear Ets-1 was unchanged, notwithstanding the miR-125b increase. (ii) Also, the 1833-cell invasiveness and the expression of Endothelin-1, the target gene of Ets-1/HIF-1, showed opposite patterns under HGF and hypoxia. We clarified the molecular mechanism(s) reproducing the high miR-125b levels with the mimic in 1833 cells. Under hypoxia, the miR-125b mimic maintained a basal level and functional Ets-1 protein, as testified by the elevated cell invasiveness. However, under HGF ectopic miR-125b downregulated Ets-1 protein and cell motility, likely involving an Ets-1-dominant negative form sensible to serum conditions; Ets-1-activity inhibition by HGF implicated HIF-1α accumulation, which drugged Ets-1 in the complex bound to the Endothelin-1 promoter. Altogether, 1833-cell exposure to HGF would decrease Endothelin-1 transactivation and protein expression, with the possible impairment of Endothelin-1-dependent induction of E-cadherin, and the reversion towards an invasive phenotype: this was favoured by Ets-1 overexpression, which inhibited HIF-1α expression and HIF-1 activity. (iii) In MDA-MB231 cells, HGF strongly and rapidly decreased Ets-1, hampering invasiveness and reducing Ets-1-binding to Endothelin-1 promoter; HIF-1α did not form a complex with Ets-1 and Endothelin-1-luciferase activity was unchanged. Overall, depending on the microenvironment conditions and endogenous miR-125b levels, bone-metastatic cells might switch from Ets-1-dependent motility towards colonization/growth, regulated by the balance between Ets-1 and HIF-1.

This work is concerned with the mechanical characterization of bistable composite plates in order to investigate their nonlinear behavior dependence on mechanical factors, e.g. strain, stress trends and potential energy. The bistable laminates have two stable shapes that are actuated by a variety of mechanisms (piezoelectric ceramic based actuators, shape memory alloys or thermal actuation) to induce “snap-through” between states. These composite structures are receiving interest in several aeronautic applications such as shape changing applications without the need of servoactivated control systems. Scope of the work is to describe the “0” strain-stress status of the asymmetric bistable laminates, immediately after the manufacturing process. An experimental testing is carried out with the purpose of collecting enough data for the numerical and analytical analyses. Numerical simulations based on Finite Element Models (FEM) are used to study strain and stress fields of the laminates and successively to validate semi-analytical results. By the Classic Plate Lamination Theory (CLPT), an analytical model is developed to provide an interpretation of the bistability phenomenon. The experimental results, with FE and CLPT models, help to understand the relation between the mechanical features of the composite laminate and the bistability phenomenon. This paper reports on detailed nonlinear characterization of bistable plates using numerical, analytical and experimental data in order to provide a starting point for future works characterizing bistable strain-stress evolution over the time.

MicroRNAs (miRNAs) are an evolutionary conserved class of short, single-stranded noncoding RNAs (

This study is concerned with the activation energy threshold of bistable composite plates in order to tailor a bistable system for specific aeronautical applications. The aim is to explore potential configurations of the bistable plates and their dynamic behavior for designing novel morphing structure suitable for aerodynamic surfaces and, as a possible further application, for power harvesters. Bistable laminates have two stable mechanical shapes that can withstand aerodynamic loads without additional constraint forces or locking mechanisms. This kind of structures, when properly loaded, snap-through from one stable configuration to another, causing large strains that can also be used for power harvesting scopes. The transition between the stable states of the composite laminate can be triggered, in principle, simply by aerodynamic loads (pilot, disturbance or passive inputs) without the need of servo-activated control systems. Both numerical simulations based on Finite Element models and experimental testing based on different activating forcing spectra are used to validate this concept. The results show that dynamic activation of bistable plates depend on different parameters that need to be carefully managed for their use as aircraft passive wing flaps.

Adhesion is attracting increasing interest in the aerospace field since composite materials have become, together with aluminium alloys, the main structural materials for aircraft primary structures. Nano-graphite was demonstrated to improve the mechanical performance of several polymers used as composite matrices. In this work Single Lap Joints (SLJs) of unidirectional composite laminates were manufactured, tested and simulated: two families of specimens were investigated and compared, one joined using conventional epoxy resin, the other joined with an adhesive obtained mixing the same epoxy resin with nano-graphite particles. The dispersion of expanded and sonicated graphene stacks (EGS, 3% wt) in the epoxy matrix was obtained by the swelling method, dispersing first the filler in acetone and then mixing it with the epoxy oligomers. Finally the solvent was evaporated and the filler-epoxy mixture was degassed under vacuum before adding an amine curing agent in a stoichiometric quantity. The research demonstrates the superior mechanical properties of the adhesive with the addition of nano-graphite through experimental characterization of its behaviour in terms of strength and energy absorption. Finite element numerical simulations have been carried out using the Cohesive Zone Model (CZM) element, obtaining as parameters the maximum shear stress and the critical fracture energy for the two adhesives. A good correlation between numerical and experimental results has been achieved and the criteria for developing reliable and accurate non-linear models of the adhesive failure have been established.

Summary Adult neurogenesis requires the precise control of neuronal versus astrocyte lineage determination in neural stem cells. While microRNAs (miRNAs) are critically involved in this step during development, their actions in adult hippocampal neural stem cells (aNSCs) has been unclear. As entry point to address that question we chose DICER, an endoribonuclease essential for miRNA biogenesis and other RNAi-related processes. By specific ablation of Dicer in aNSCs in vivo and in vitro, we demonstrate that miRNAs are required for the generation of new neurons, but not astrocytes, in the adult murine hippocampus. Moreover, we identify 11 miRNAs, of which 9 have not been previously characterized in neurogenesis, that determine neurogenic lineage fate choice of aNSCs at the expense of astrogliogenesis. Finally, we propose that the 11 miRNAs sustain adult hippocampal neurogenesis through synergistic modulation of 26 putative targets from different pathways., Graphical Abstract, Highlights • Dicer depletion in aNSCs impairs neurogenesis and stimulates astrogliogenesis • Synergy of 11 miRNAs sustains neuronal fate of aNSCs • miRNA converge on multiple targets in different pathways to induce neurogenesis, In this article, the authors demonstrate that Dicer-dependent miRNAs are required for the generation of new neurons, but not astrocytes, in the adult hippocampus in vivo and in vitro. The authors identify a new set of 11 miRNAs that synergistically converge on multiple targets in different pathways to sustain neurogenic lineage fate commitment in aNSCs.

In this study the amplitude and the phase of the structural response of samples of Single Lap Joint (SLJ) subjected to ultrasonic harmonic excitation was evaluated to identify and characterize the defects within the bonded region. Different parameters such as frequency, shape, and amplitude of the response signal coming back from the adhesive joint are key criteria for understanding the quality of the adhesion. Different metallic samples with the same geometry were experimentally tested: the defects were artificially introduced bonding partially two plates and changing the extension of the debonded region: two piezoelectric sensors (one exciting, one receiver) were attached on each of the two bonded plates. In this way, different experimental tests were carried out in order to study the influence of debonded regions on SLJ structural behavior. The structural dynamic response of the debonded samples was investigated and compared with the predictions of numerical models, for each SLJ, introducing viscoelastic properties for the adhesive layer, and applying the harmonic excitation. Moreover the numerical modal analysis was used to understand the experimental results by a proper description of viscoelastic tape behavior. The numerical simulations were used to find correlation between the content of the acquired signals and the defects of adhesion.

A reduced-order short-period model is derived, which allows for representing the effects of structural flexibility on aircraft response to pilot and disturbance inputs on the basis of a minimum set of relevant information on aerodynamic and structural configuration. The model is derived from first principles by means of a Lagrangian approach, featuring vertical (heave) and pitch degrees of freedom, together with deformation variables for flexural deformation of wing and after portion of the fuselage. As a byproduct, an explicit formulation for stability derivatives with respect to deformation variables is derived. Numerical results are reported for a configuration representative of a modern commercial jet aircraft.

Summary Loss of p53 function is invariably associated with cancer. Its role in tumor growth was recently linked to its effects on cancer stem cells (CSCs), although the underlying molecular mechanisms remain unknown. Here, we show that c-myc is a transcriptional target of p53 in mammary stem cells (MaSCs) and is activated in breast tumors as a consequence of p53 loss. Constitutive Myc expression in normal mammary cells leads to increased frequency of MaSC symmetric divisions, extended MaSC replicative-potential, and MaSC-reprogramming of progenitors, whereas Myc activation in breast cancer is necessary and sufficient to maintain the expanding pool of CSCs. Concomitant p53 loss and Myc activation trigger the expression of 189 mitotic genes, which identify patients at high risk of mortality and relapse, independently of other risk factors. Altogether, deregulation of the p53:Myc axis in mammary tumors increases CSC content and plasticity and is a critical determinant of tumor growth and clinical aggressiveness., Graphical Abstract, Highlights • Myc is overexpressed and deregulated in breast tumors because of p53 signaling attenuation • Myc activation favors SC symmetric divisions and SC reprogramming of progenitors • Myc activation is necessary and sufficient to sustain the cancer SC phenotype • Expression of 189 mitotic p53:Myc targets identifies high-risk breast cancer patients, Santoro et al. demonstrate the existence of a regulatory axis instructed by the tumor-suppressor p53, executed by the Myc oncogene, and involving 189 p53:Myc targets. The authors show that activation of this axis maintains the expanding pool of cancer stem cells (SCs), thus promoting tumor growth, and predicts adverse prognosis.

When talking about regulatory RNAs, and microRNAs in particular, attention should be paid to what types of molecules are present in the cell and in what numbers, rather than to their sizes. The distinction between small and long RNAs is based on RNA isolation protocols and is not a reflection of specific biogenesis pathways, regulatory mechanisms or biological roles. Conversely, sequence and quantity of expressed miRNAs are of paramount importance.

Summary Cancer genetic heterogeneity offers a wide repertoire of molecular determinants to be screened as therapeutic targets. Here, we identify potential anticancer targets by exploiting negative genetic interactions between genes with driver loss-of-function mutations (recessive cancer genes) and their functionally redundant paralogs. We identify recessive genes with additional copies and experimentally test our predictions on three paralogous pairs. We confirm digenic negative interactions between two cancer genes ( SMARCA4 and CDH1 ) and their corresponding paralogs ( SMARCA2 and CDH3 ). Furthermore, we identify a trigenic negative interaction between the cancer gene DNMT3A , its functional paralog DNMT3B , and a third gene, DNMT1 , which encodes the only other human DNA-methylase domain. Although our study does not exclude other causes of synthetic lethality, it suggests that functionally redundant paralogs of cancer genes could be targets in anticancer therapy.

Recent evidence indicates that the miRNA biogenesis factors DROSHA, DGCR8, and DICER exert non-overlapping functions, and have also roles in miRNA-independent regulatory mechanisms. However, it is currently unknown whether miRNA-independent functions of DGCR8 play any role in the maintenance of neuronal progenitors and during corticogenesis. Here, by phenotypic comparison of cortices from conditional Dgcr8 and Dicer knockout mice, we show that Dgcr8 deletion, in contrast to Dicer depletion, leads to premature differentiation of neural progenitor cells and overproduction of TBR1-positive neurons. Remarkably, depletion of miRNAs upon DCGR8 loss is reduced compared to DICER loss, indicating that these phenotypic differences are mediated by miRNA-independent functions of DGCR8. We show that Dgcr8 mutations induce an earlier and stronger phenotype in the developing nervous system compared to Dicer mutants and that miRNA-independent functions of DGCR8 are critical for corticogenesis. Finally, our data also suggest that the Microprocessor complex, with DROSHA and DGCR8 as core components, directly regulates the Tbr1 transcript, containing evolutionarily conserved hairpins that resemble miRNA precursors, independently of miRNAs.

BACKGROUND The identification of circulating microRNAs (miRNAs) in the blood has been recently exploited for the development of minimally invasive tests for the early detection of cancer. Nevertheless, the clinical transferability of such tests is uncertain due to still-insufficient standardization and optimization of methods to detect circulating miRNAs in the clinical setting. METHODS We performed a series of tests to optimize the quantification of serum miRNAs that compose the miR-Test, a signature for lung cancer early detection, and systematically analyzed variables that could affect the performance of the test. We took advantage of a large-scale (>1000 samples) validation study of the miR-Test that we recently published, to evaluate, in clinical samples, the effects of analytical and preanalytical variables on the quantification of circulating miRNAs and the clinical output of the signature (risk score). RESULTS We developed a streamlined and standardized pipeline for the processing of clinical serum samples that allows the isolation and analysis of circulating miRNAs by quantitative reverse-transcription PCR, with a throughput compatible with screening trials. The major source of analytical variation came from RNA isolation from serum, which could be corrected by use of external (spike-in) or endogenous miRNAs as a reference for normalization. We also introduced standard operating procedures and QC steps to check for unspecific fluctuations that arise from the lack of standardized criteria in the collection or handling of the samples (preanalytical factors). CONCLUSIONS We propose our methodology as a reference for the development of clinical-grade blood tests on the basis of miRNA detection.

http://hdl.handle.net/20.500.11768/96454 Lung cancer is the first cause of cancer mortality worldwide, and its early detection is currently the main available strategy to improve disease prognosis. While early diagnosis can be successfully achieved through tomography-based population screenings in high-risk individuals, simple methodologies are needed for effective cancer prevention programs. We developed a test, based on the detection of 34 microRNAs (miRNAs) from serum, that could identify patients with early stage non-small cell lung carcinomas (NSCLCs) in a population of asymptomatic high-risk individuals with 80% accuracy. The signature could assign disease probability accurately either in asymptomatic or symptomatic patients, is able to distinguish between benign and malignant lesions, and to capture the onset of the malignant disease in individual patients over time. Thus, our test displays a number of features of clinical relevance that project its utility in programs for the early detection of NSCLC.

Background: Cancer stem cells (CSCs) have inherent or developed drug resistance for which chemotherapy fails to eliminate all tumor cells, a property that is critically contributing to recurrence and metastasis. Here, we combine miR-34a, a well-known tumor suppressor miRNA that controls proliferation, apoptosis and self-renewal, with Paclitaxel (PTX), a commonly used first-line chemotherapy drug. Based on our preliminary observations (Bonetti et al., manuscript under revision), we hypothesized a combinatorial approach, coupling miRNA- and chemo- treatment, aimed at reducing CSCs in vitro and recurrence in vivo using triple-negative breast cancer (TNBC) as a model. Methodology: As a model, we exploited SUM159pt cells (TNBC mesenchymal-like, with CSC features) to mimic aggressive breast cancer, which harbor a vector for pLUC, which is induced by luciferin in vivo, to monitor tumor growth in real time. Furthermore, we used i) pSlik34a, with tunable expression of miR-34a by doxycycline; ii) treatment with PTX to select for resistant cells (RC); and combined the two, by treating RC with doxycycline. CSC content was evaluated by ALDH (Aldehyde Dehydrogenase) activity, sphere-forming efficiency (SFE) and qRT-PCR of known markers. A similar approach was followed for in vivo studies, with NSG female injected through tail vein injection with 500,000 SUM159/pLUC/pSlik34a cells, and separated into three groups: not treated (NT), PTX alone, and PTX+34a. Results: As observed for aggressive breast cancer, treatment of SUM159 with PTX generated resistant cells (RC) with increased CSC content (ALDH activity and spheres forming ability). When miR-34a was induced in the RC, cells changed morphology, slowed proliferation rate, and decreased CSC content (ALDH activity and spheres). Moreover, a second PTX treatment almost completely abolished the ALDH activity and SFE. The combined effect was evaluated in vivo, following by luminescence the growth of metastases after lung colonization, with PTX+34a mice reducing lung colonization than the PTX treatment or miR-34a treatment alone. Conclusions: Overall, these results indicate that miR-34a sensitize PTX-resistant cells reducing cancer stem-cell capacities, and in this preclinical setting limited the aggressive phenotype of breast cancer, including proliferation, drug resistance, relapse and metastasis causing in delay of lung colonization in vivo. Citation Format: Montserrat Climent, Paola Bonetti, Francesco Nicassio. miRNA-34a sensitizes triple-negative breast cancer cells to paclitaxel reducing cancer stem cell population and lung colonization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4410.

Lung cancer is the leading cause of cancer death worldwide. Low-dose computed tomography screening (LDCT) was recently shown to anticipate the time of diagnosis, thus reducing lung cancer mortality. However, concerns persist about the feasibility and costs of large-scale LDCT programs. Such concerns may be addressed by clearly defining the target "high-risk" population that needs to be screened by LDCT. We recently identified a serum microRNA signature (the miR-Test) that could identify the optimal target population. Here, we performed a large-scale validation study of the miR-Test in high-risk individuals (n = 1115) enrolled in the Continuous Observation of Smoking Subjects (COSMOS) lung cancer screening program. The overall accuracy, sensitivity, and specificity of the miR-Test are 74.9% (95% confidence interval [CI] = 72.2% to 77.6%), 77.8% (95% CI = 64.2% to 91.4%), and 74.8% (95% CI = 72.1% to 77.5%), respectively; the area under the curve is 0.85 (95% CI = 0.78 to 0.92). These results argue that the miR-Test might represent a useful tool for lung cancer screening in high-risk individuals.

Np95 is an important determinant in cell cycle progression. Its expression is tightly regulated and becomes detectable shortly before the entry of cells into S phase. Accordingly, Np95 is absolutely required for the G1/S transition. Its continued expression throughout the S/G2/M phases further suggests additional roles. Indeed, Np95 has been implicated in DNA damage response. Here, we show that Np95 is tightly bound to chromatin in vivo and that it binds to histones in vivo and in vitro. The binding to histones is direct and shows a remarkable preference for histone H3 and its N-terminal tail. A novel protein domain, the SRA-YDG domain, contained in Np95 is indispensable both for the interaction with histones and for chromatin binding in vivo. Np95 contains a RING finger. We show that this domain confers E3 ubiquitin ligase activity on Np95, which is specific for core histones, in vitro. Finally, Np95 shows specific E3 activity for histone H3 when the endogenous core octamer, coimmunoprecipitating with Np95, is used as a substrate. Histone ubiquitination is an important determinant in the regulation of chromatin structure and gene transcription. Thus, the demonstration that Np95 is a chromatin-associated ubiquitin ligase suggests possible molecular mechanisms for its action as a cell cycle regulator.

As more small RNA sequencing libraries are becoming available, it clearly emerges that microRNAs (miRNAs) are highly heterogeneous both in length and sequence. In comparison to canonical miRNAs, miRNA isoforms (termed as “isomiRs”) might exhibit different biological properties, such as a different target repertoire, or enhanced/reduced stability. Nonetheless, this layer of information has remained largely unexplored due to the scarcity of small RNA NGS-datasets and the absence of proper analytical tools. Here, we present a workflow for the characterization and analysis of miRNAs and their variants in next-generation sequencing datasets. IsomiRs can originate from an alternative dicing event (“templated” forms) or from the addition of nucleotides through an enzymatic activity or target-dependent mechanisms (“non-templated” forms). Our pipeline allows distinguishing canonical miRNAs from templated and non-templated isomiRs by alignment to a custom database, which comprises all possible 3′-, 5′-, and trimmed variants. Functionally equivalent isomiRs can be grouped together according to the type of modification (e.g., uridylation, adenylation, trimming …) to assess which miRNAs are more intensively modified in a given biological context. When applied to the analysis of primary epithelial breast cancer cells, our methodology provided a 40% increase in the number of detected miRNA species and allowed to easily identify and classify more than 1000 variants. Most modifications were compatible with templated IsomiRs, as a consequence of imprecise Drosha or Dicer cleavage. However, some non-templated variants were consistently found either in the normal or in the cancer cells, with the 3′-end adenylation and uridylation as the most frequent events, suggesting that miRNA post-transcriptional modification frequently occurs. In conclusion, our analytical tool permits the deconvolution of miRNA heterogeneity and could be used to explore the functional role of miRNA isoforms.

Summary Cells entering mitosis become rounded, lose attachment to the substrate, and increase their cortical rigidity. Pivotal to these events is the dismantling of focal adhesions (FAs). How mitotic reshaping is linked to commitment to divide is unclear. Here, we show that DEPDC1B, a protein that accumulates in G2, coordinates de-adhesion events and cell-cycle progression at mitosis. DEPDC1B functions as an inhibitor of a RhoA-based signaling complex, which assembles on the FA-associated protein tyrosine phosphatase, receptor type, F (PTPRF) and mediates the integrity of FAs. By competing with RhoA for the interaction with PTPRF, DEPDC1B promotes the dismantling of FAs, which is necessary for the morphological changes preceding mitosis. The circuitry is relevant in whole organisms, as shown by the control exerted by the DEPDC1B/RhoA/PTPRF axis on mitotic dynamics during zebrafish development. Our results uncover an adhesion-dependent signaling mechanism that coordinates adhesion events with the control of cell-cycle progression., Graphical Abstract, Highlights • DEPDC1B is a cell-cycle gene involved in the transition from G2 phase to mitosis • Persistent adhesion at G2 phase delays CycB/CDK1 activation and G2/M transition • DEPDC1B controls RhoA/ROCK-dependent adhesion dynamics at G2 phase • DEPDC1B inhibits RhoA activation by displacing it from the PTPRF/GEF-H1 complex, During mitosis, cells become rounded and lose attachment to the substrate. Marchesi et al. show that DEPDC1B, a cell-cycle-regulated protein, binds to the focal-adhesion-associated receptor PTPRF, thus inhibiting RhoA activation and leading to dismantling of focal adhesions upon mitotic entry. DEPDC1B thus links mitotic progression to de-adhesion.

Sleep homoeostasis refers to a process in which the propensity to sleep increases as wakefulness progresses and decreases as sleep progresses. Sleep is tightly organized around the circadian clock and is regulated by genetic and epigenetic mechanisms. The homoeostatic response of sleep, which is classically triggered by sleep deprivation, is generally measured as a rebound effect of electrophysiological measures, for example delta sleep. However, more recently, gene expression changes following sleep loss have been investigated as biomarkers of sleep homoeostasis. The genetic background of an individual may affect this sleep-dependent gene expression phenotype. In this study, we investigated whether parental genetic background differentially modulates the expression of genes following sleep loss. We tested the progeny of reciprocal crosses of AKR/J and DBA/2J mouse strains and we show a parent-of-origin effect on the expression of circadian, sleep and neuronal plasticity genes following sleep deprivation. Thus, we further explored, by in silico , specific functions or upstream mechanisms of regulation and we observed that several upstream mechanisms involving signalling pathways (i.e. DICER1, PKA), growth factors (CSF3 and BDNF) and transcriptional regulators (EGR2 and ELK4) may be differentially modulated by parental effects. This is the first report showing that a behavioural manipulation (e.g. sleep deprivation) in adult animals triggers specific gene expression responses according to parent-of-origin genomic mechanisms. Our study suggests that the same mechanism may be extended to other behavioural domains and that the investigation of gene expression following experimental manipulations should take seriously into account parent-of-origin effects.

In the last decade, the scientific community has been shaken by what we call the “noncoding revolution.” We have witnessed the discovery of an increasing amount of RNA molecules, which play a critical role in normal physiology as well as disease, without providing any protein product. The smallest regulatory RNA species, known as the ~22 nucleotide-long microRNAs (miRNAs), were at the forefront of such a revolution. According to the last release of miRNA database (miRBase http://www.mirbase.org—release 21) more than 35,000 miRNA species have been identified, of which a total of more than 2,500 mature miRNAs exist only in humans [1]. Since their first discovery in C. elegans in 1993, it has become clear that these tiny molecules have an enormous regulatory potential, being able to exert negative posttranscriptional regulation on hundreds of protein coding genes, even simultaneously, and ultimately act as master regulators of entire biological processes. Besides their role in development, the involvement of miRNAs in human disease, and cancer in particular, has attracted major attention. In fact, deregulated miRNA expression could lead to aberrant expression of targeted oncogenes or tumor suppressors, thus resulting in tumor development and progression. Accordingly, altered expression of miRNAs has been observed in almost every tumor type, including haematological malignancies, carcinomas, sarcomas, and central nervous system neoplasms. Relevant examples of the role of miRNAs on specific cancer types are reported in this issue, with a focus on childhood acute lymphoblastic leukemia (original contribution by M. Duyu et al.), ovarian and cervical cancer (reviewed by Y. Kinose et al. and S. M. Diaz-Gonzalez et al.), soft tissue sarcomas (reviewed by T. Fujiwara et al.), or medulloblastoma (reviewed by S. Lopez-Ochoa et al.). The unique pattern of altered miRNA expression provides a fingerprint that may serve for cancer diagnosis and prediction of patient's prognosis or response to treatment. In addition, a number of miRNAs have been shown to directly participate in tumorigenesis by acting as “oncoMirs” or “tumor suppressive miRNAs,” thus becoming potential key targets or tools for anticancer therapy. The review by A. Saumet and colleagues suitably introduces the current approaches and applications of miRNAs in cancer and human diseases, from expression analyses aimed at identifying miRNAs potentially useful for tumor diagnosis or prognosis to the potential use of them as novel therapeutic agents. The opportunities and challenges of miRNAs as cancer biomarkers have been addressed in the issue by H. Lan and colleagues. Specific focuses have been also provided on the clinicopathological significance of miR-155 in breast cancer (H. Zeng et al.), diagnostic and prognostic miRNAs in sarcomas (T. Fujiwara et al.), and miRNAs exploitable for prostate cancer risk assessment (A. Cannistraci et al.). Based on their relative stability in body fluids, a great effort has been devoted in the last years to the study of circulating miRNAs as noninvasive biomarkers for tumor diagnosis or disease monitoring. Though attracting, miRNA quantification in liquid samples is far from being trivial. Challenges encountered in the field have been extensively addressed in the issue by P. Tiberio and colleagues. Due to their nature as physiological molecules able to control multiple genes at the same time, miRNAs are also considered as very promising therapeutic targets or tools. As tumors typically evade cancer treatment by acquiring secondary mutations on targeted proteins, it appears more difficult for a tumor to escape from miRNA effects, which are directed on multiple proteins at the same time. Controlling the delivery and activity of miRNA-modulating agents into specific tissues or organs still appears as the “the big challenge,” albeit a number of promising approaches are under validation. This contention is extensively explored in this issue in different contexts. The review by O. Fortunato et al. focuses on lung cancer, one of the big cancer killers, and discusses the most likely miRNA targets as well as the methodological approaches for in vivo delivery of miRNAs. The use of miRNA modulators for a direct antitumor effect, when administered alone or in combination with chemo- or radiotherapy, has been described in the prostate cancer context by A. Cannistraci and colleagues. Furthermore, the use of miRNAs for indirect anticancer effects has been reviewed by S. Gallach et al., who discussed on the possibility to modulate miR-126 or miR-92a to target tumor angiogenesis, thus limiting tumor growth and metastatic spread, or act on hypoxia. Finally the role of miRNAs in determining and regulating chemoresistance of pancreatic cancer has been discussed by I. Garajova and colleagues. Overall the issue is intended to provide evidence of the potential usefulness of miRNAs in cancer management as well as focus on the still unsolved technical and conceptual challenges in miRNA research. Paolo Gandellini Elisa Giovannetti Francesco Nicassio

Bistable energy harvesting has become a major field of research due to some unique features for converting mechanical energy into electrical power. When properly loaded, bistable structures snap-through from one stable configuration to another, causing large strains and consequently power generation. Moreover, bistable structures can harvest energy across a broad-frequency bandwidth due to their nonlinear characteristics. Despite the fact that snap-through may be triggered regardless of the form or frequency of exciting vibration, the external force must reach a specific snap-through activation threshold value to trigger the transition from one stable state to another. This aspect is a limiting factor for realistic vibration energy harvesting application with bistable devices. This paper presents a novel power harvesting concept for bistable composites based on a 'lever effect' aimed at minimising the activation force to cause the snap through by choosing properly the bistable structures' constraints. The concept was demonstrated with the help of numerical simulation and experimental testing. The results showed that the actuation force is one order of magnitude smaller (3%-6%) than the activation force of conventionally constrained bistable devices. In addition, it was shown that the output voltage was higher than the conventional configuration, leading to a significant increase in power generation. This novel concept could lead to a new generation of more efficient bistable energy harvesters for realistic vibration environments. © 2016 IOP Publishing Ltd.

Transcriptional regulation by Rb–E2F and posttranscriptional regulation by microRNAs control the expression of cell cycle and DNA replication genes and restrict cellular proliferation., The cancer-associated loss of microRNA (miRNA) expression leads to a proliferative advantage and aggressive behavior through largely unknown mechanisms. Here, we exploit a model system that recapitulates physiological terminal differentiation and its reversal upon oncogene expression to analyze coordinated mRNA/miRNA responses. The cell cycle reentry of myotubes, forced by the E1A oncogene, was associated with a pattern of mRNA/miRNA modulation that was largely reciprocal to that induced during the differentiation of myoblasts into myotubes. The E1A-induced mRNA response was preponderantly Retinoblastoma protein (Rb)-dependent. Conversely, the miRNA response was mostly Rb-independent and exerted through tissue-specific factors and Myc. A subset of these miRNAs (miR-1, miR-34, miR-22, miR-365, miR-29, miR-145, and Let-7) was shown to coordinately target Rb-dependent cell cycle and DNA replication mRNAs. Thus, a dual level of regulation—transcriptional regulation via Rb–E2F and posttranscriptional regulation via miRNAs—confers robustness to cell cycle control and provides a molecular basis to understand the role of miRNA subversion in cancer.

Antisense transcription has long been recognized as a mechanism involved in the regulation of gene expression. Therefore, several human diseases associated with abnormal patterns of gene expression might display antisense RNA-mediated pathogenetic mechanisms. Such issue could be particularly relevant for cancer pathogenesis, since deregulated gene expression has long been established as a hallmark of cancer cells. Herein, we report on a bioinformatic search for antisense transcription in two cancer-associated regions of human chromosome 6 (6q21 and 6q27). Natural antisense transcripts (NATs) for several genes in both genomic regions were predicted in silico and subsequently validated by strand-specific RT-PCR. Detailed experimental validation by quantitative real-time RT-PCR of five putative cancer related sense-antisense transcript pairs revealed a single candidate tumor suppressor gene (RPS6KA2) whose expression levels display marked cancer-related changes that are likely mediated by its antisense RNA in a breast cancer cell line model.

Expression profiling analysis of human cancers is a promising approach to obtain precise molecular classification of cancers, to develop stratification tools for therapeutic regimens, and to predict the biological behavior of neoplasia. Direct profiling of human cancers (herein defined as "the unbiased approach") presents, however, intrinsic problems connected with the high genetic noise embedded in the system. This, in turn, leads to fitting of the noise in the data (the so-called "overtraining") with consequent instability of the identified signatures, when applied on different cohorts of patients. To circumvent these problems, "biased approaches" - which exploit the molecular knowledge of cancer obtained in model systems - are being developed. Biased approaches, however, are not problem-free, in that they provide information limited to single oncogenic events, thereby failing, at least in principle, to capture the complex repertoire of alterations of human cancers. In this review, we compare the two approaches and provide a test case, from our studies, of how "integrated" strategies, which combine biased and unbiased approaches, might lead to the identification of stable and reliable predictive signatures in cancer.

Adenocarcinoma is the predominant histological subtype of lung cancer, the leading cause of cancer deaths in the world. At stage I, the tumor is cured by surgery alone in about 60% of cases. Markers are needed to stratify patients by prognostic outcomes and may help in devising more effective therapies for poor prognosis patients. To achieve this goal, we used an integrated strategy combining meta-analysis of published lung cancer microarray data with expression profiling from an experimental model. The resulting 80-gene model was tested on an independent cohort of patients using RT-PCR, resulting in a 10-gene predictive model that exhibited a prognostic accuracy of approximately 75% in stage I lung adenocarcinoma when tested on 2 additional independent cohorts. Thus, we have identified a predictive signature of limited size that can be analyzed by RT-PCR, a technology that is easy to implement in clinical laboratories.

In recent years, enormous effort has been dedicated to transcriptomic profiling of various physiological and pathological conditions, in particular in cancer biology field. Microarray gene expression (mRNA) datasets of thousands of human tumors are now publicly available and can be used to get insights of cancer processes by systems-based analysis and to identify biomarkers for improvement of cancer therapy. However, the high complexity of human transcriptome may be difficult to handle. In this regard, shifting the attention to the miRNome could be an advantage, since its complexity is at least 20-fold lower than that of a reference transcriptome (∼1000 miRNAs vs. ∼20,000 genes). Importantly, patterns of distinct miRNA expression profiles were observed in tumors and there is a growing interest in miRNAs, behaving as potential cancer determinants and biomarkers. Knowing that almost 50% of human miRNA genes are located within introns of coding genes and that they usually share transcriptional regulation, those publicly available datasets are likely to contain “latent” information on intronic-miRNA expression. In other words, it could be possible to predict the regulation of intronic-miRNA expression by simply analyzing their host genes profile (miR-HG). The aim of our work is to take advantage of cancer datasets, focusing mainly on breast cancer datasets, to provide proof of principle evidences that meta-analysis of miR-HG expression profiles can pinpoint intronic-miRNAs with a role in breast cancer cell biology, and a potential utility as cancer biomarkers. Using this approach, we successfully discovered a diagnostic microRNA signature enabling the identification of breast cancer molecular subtypes. Importantly, qRT-PCR analysis of just three intronic-miRNAs, using FFPE samples, was sufficient to identify more aggressive breast tumor subtypes (i.e. basal, HER2 and luminal B subtypes), with a ∼80% of accuracy, in patients with moderately differentiated breast cancer. Unexpectedly, in a number of cases, the regulation of expression of intronic-miRNAs was more relevant to cancer phenotypes than the expression of their host genes. In line with these encouraging results, we propose our data mining strategy as a valid tool for cancer research and other biomedical fields. Citation Format: Simona Monterisi, Giovanni D'Ario, Elisa Dama, Nicole Rotmensz, Stefano Confalonieri, Chiara Tordonato, Flavia Troglio, Giovanni Bertalot, Patrick Maisonneuve, Giuseppe Viale, Francesco Nicassio, Pier Paolo Di Fiore, Fabrizio Bianchi. Mining cancer gene expression databases for latent information on intronic microRNAs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 233. doi:10.1158/1538-7445.AM2015-233

Background: Lung cancer is the leading cause of cancer death worldwide. As lung cancer is asymptomatic in its early stages, the majority of patients are diagnosed with advanced disease, when the tumor is unresectable. Consequently, the survival rate is very low: 15% at 5 years. It is vital, therefore, that screening programs and novel diagnostic tools are developed, which will increase the detection of lung cancer in its early stages (stage I-II), when the tumor is still curable, to reduce lung cancer mortality. Recently, we described a serum microRNA signature diagnostic for asymptomatic, early stage, lung cancer. The availability of reliable biomarkers to identify high-risk individuals might help to reduce the size of the target population for LDCT-based programs, thereby reducing costs and probably increasing compliance Methods: We performed a large-scale validation study of a miRNA blood test based on our signature (the miR-Test) in a population of high-risk individuals (N = 1115) enrolled in the lung cancer screening program COSMOS (Continuous Observation of SMOking Subjects), and other 74 lung cancer patients diagnosed outside of screening. Results: The miR-Test showed overall accuracy, specificity and sensitivity of 75%, 78%, and 75%, respectively, with an AUC of 0.85. The test appears to have a dual origin: the first from epithelial cells (the epithelial-like component); the second from cells of hematopoietic origin (the inflammatory-like component). Of note, we found that both components are needed to maintain a good performance of the miR-Test. Conclusions: The relatively high sensitivity of the miR-Test in detecting asymptomatic lung cancer and its high negative predictive value (NPV > 99%), confirm the effectiveness of the test, both interms of its ability to identify asymptomatic lung cancer patients and to reduce significantly unnecessary CTs on healthy individuals. Citation Format: Francesca Montani, Matteo Jacopo Marzi, Fabio Dezi, Elisa Dama, Rose Mary Carletti, Giuseppina Bonizzi, Raffaella Bertolotti, Massimo Bellomi, Cristiano Rampinelli, Patrick Maisonneuve, Lorenzo Spaggiari, Giulia Veronesi, Francesco Nicassio, Pier Paolo Di Fiore, Fabrizio Bianchi. miR-Test: a blood test for lung cancer early detection. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1573. doi:10.1158/1538-7445.AM2015-1573

Lung cancer is leading cause of cancer death worldwide. Being lung cancer asymptomatic in its early stages, the majority of patients are diagnosed with advanced disease. Therefore, it is vital that screening programs and novel diagnostic tools are developed to increase lung cancer early detection. The development of a minimally invasive blood-based diagnostic tool would be ideal as a first-line screening procedure. An increasing number of studies are demonstrating that fluctuations of circulating miRNAs are associated to lung cancer. Recently, we described a serum circulating miRNA signature (miR-Test) diagnostic for asymptomatic, early stage, lung cancer, that was validated in a large cohort of individuals (N = 1115) enrolled in the lung cancer screening program COSMOS (Continuous Observation of SMOking Subjects). However, the transfer to the clinic of a blood test based on circ-miRNAs requires the establishment of standardized operating procedures (SOPs), working instructions and guidelines for all pre-analytical and analytical procedures. We identified possible sources of variability affecting circulating miRNAs, analyzed their impact on the miR-Test performance, and defined a standardized protocol to optimize miR-Test application. Analysis of all possible technical and biological variation affecting circ-miRNAs level, revealed two main sources of variability: one related to analytical procedures for miRNAs extraction and quantification, and the other due to pre-analytical conditions, on how samples are prepared. The extraction causes the main source of analytical imprecision. In conclusion, we identified an optimal protocol for the application of miR-Test for lung cancer early diagnosis. Citation Format: Francesca Montani, Matteo Marzi, Fabio Dezi, Elisa Dama, Rose Mary Carletti, Giulia Veronesi, Francesco Nicassio, Pier Paolo Di Fiore, Fabrizio Bianchi. Serum circulating miR–Test application: Standardized protocol for miR–Test clinical application. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1575. doi:10.1158/1538-7445.AM2015-1575

The molecular anatomy of cancer cells is being explored through unbiased approaches aimed at the identification of cancer-specific transcriptional signatures. An alternative biased approach is exploitation of molecular tools capable of inducing cellular transformation. Transcriptional signatures thus identified can be readily validated in real cancers and more easily reverse-engineered into signaling pathways, given preexisting molecular knowledge. We exploited the ability of the adenovirus early region 1 A protein (E1A) oncogene to force the reentry into the cell cycle of terminally differentiated cells in order to identify and characterize genes whose expression is upregulated in this process. A subset of these genes was activated through a retinoblastoma protein/E2 viral promoter required factor–independent (pRb/E2F-independent) mechanism and was overexpressed in a fraction of human cancers. Furthermore, this overexpression correlated with tumor progression in colon cancer, and 2 of these genes predicted unfavorable prognosis in breast cancer. A proof of principle biological validation was performed on one of the genes of the signature, skeletal muscle cell reentry-induced (SKIN) gene, a previously undescribed gene. SKIN was found overexpressed in some primary tumors and tumor cell lines and was amplified in a fraction of colon adenocarcinomas. Furthermore, knockdown of SKIN caused selective growth suppression in overexpressing tumor cell lines but not in tumor lines expressing physiological levels of the transcript. Thus, SKIN is a candidate oncogene in human cancer.

Terminal differentiation exerts a remarkably tight control on cell proliferation. However, the oncogenic products of DNA tumor viruses, such as adenovirus E1A, can force postmitotic cells to proliferate, thus representing a powerful tool to study progression into S phase. In this study, we identified the gene encoding Np95, a murine nuclear phosphoprotein, as an early target of E1A-induced transcriptional events. In terminally differentiated (TD) cells, the activation of Np95 was specifically induced by E1A, but not by overexpression of E2F-1 or of the cyclin E (cycE)–cyclin-dependent kinase 2 (cdk2) complex. In addition, the concomitant expression of Np95 and of cycE–cdk2 was alone sufficient to induce S phase in TD cells. In NIH-3T3 cells, the expression of Np95 was tightly regulated during the cell cycle, and its functional ablation resulted in abrogation of DNA synthesis. Thus, expression of Np95 is essential for S phase entry. Previous evidence suggested that E1A, in addition to its well characterized effects on the pRb/E2F-1 pathway, activates a parallel and complementary pathway that is also required for the reentry in S phase of TD cells (Tiainen, M., D. Spitkousky, P. Jansen-Dürr, A. Sacchi, and M. Crescenzi. 1996. Mol. Cell. Biol. 16:5302–5312). From our results, Np95 appears to possess all the characteristics to represent the first molecular determinant identified in this pathway.

Background. The recent development of high-throughput sequencing technologies (Next Generation Sequencing - NGS) provided instruments to reveal the complexity of nucleic acids, highlighting the existence of numerous species of non-coding RNAs (ncRNAs) that play a significant regulatory role in complex organisms, impacting on both physiology and disease. In particular, microRNAs (miRNAs) have been proposed as “next-generation” markers for their ability to unambiguously distinguish cellular states, including SCs, progenitors and differentiated cells, as well as tumour types, even among closely related cancers or in different biological fluids. However, a comprehensive approach for the identification and characterization of small non-coding RNAs from pathological samples (blood, FFPE, primary cultures) has not been established nor compared with current available methodologies (i.e. QPCR). Aim: Our aim is to develop a simultaneous and comparative protocol for the analysis of small non-coding RNAs from pathological samples, which encompasses both high-throughput QPCR and sequencing analysis in order to reveal the complexity of cancer associated small ncRNAs. Results: An optimized protocol to analyse small ncRNAs from different pathological samples has been developed and compared with high-throughput QPCR. Both platforms resulted as highly efficient and quantitative, although NGS manages to score many molecules and RNA species that could not be analysed by current QPCR platforms, thus revealing a major complexity of cancer associated non-coding RNAs. Conclusion: We propose that the combined use of NGS and QPCR platforms would allow a wider and more detailed analysis of ncRNAs, expanding our ability to fish out robust and efficient molecular markers for diagnostic (early detection), prognostic or therapeutic use. Note: This abstract was not presented at the meeting. Citation Format: Francesca Montani, Matteo Marzi, Rose Mary Carletti, Pier Paolo Di Fiore, Francesco Nicassio. Revealing the complexity of cancer associated small non-coding RNAs by next generation sequencing (NGS) and low-density array. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 522. doi:10.1158/1538-7445.AM2014-522

Cancer stem cells (CSCs) are cells with aberrantly regulated stem-like characteristics, such as self-renewal and quiescence. In breast cancer, CSCs have been shown to drive the tumorigenic process and to contribute to the emergence of therapeutic resistance and disease relapse. Based on this background, we decided to focus on the mechanisms that sustain CSC growth, namely self-renewal, to reveal molecules and pathways that control SC biology under physiological and pathological conditions. Recent research suggests a key role for non-coding RNAs (microRNAs - miRNAs; and long non coding RNAs - lncRNAs) in the control of genetic program that specifies stem cell identity and properties. Nevertheless, we still do not know which non-coding RNAs are involved and how they influence self-renewal or differentiation properties of the normal as well the cancer SC pool in the breast tissue. Based on these premises we aimed at isolating those non-coding RNAs (either lncRNAs or miRNAs) that significantly associates with the self-renewal properties and the content of stem cells in the normal mammary gland and in human breast cancer to search for: i) “markers” that improve the stratification and prognosis of human breast cancer; and ii) “modifiers” (enhancing or suppressing) of self-renewal properties of breast normal and cancer stem cells. In the long-term, our findings could serve as a basis to develop alternative strategies for the treatment of breast cancer that could radically improve the clinical management of breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Paola Bonetti, Matteo J. Marzi, francesco nicassio. microRNAs as modifiers of stem cell properties in breast cancer: a whole genome approach. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 538. doi:10.1158/1538-7445.AM2014-538

Background. High incidence and mortality rate tumours present a more favourable prognosis if diagnosed in early stages (Jemal A. et al, 2011). Screening programs based on effective radiological techniques (e.g. mammography or low dose spiral CT) promise a mortality reduction, but their application on large scale suffers of disadvantages such as the use of ionizing radiation, overdiagnosis risk and high costs. Thus, the identification of molecular markers for cancer early diagnosis, especially if detectable through a simple non-invasive blood test, would provide powerful tools for the improvement of screening programs. Recently, a novel class of small non-coding RNAs, namely microRNAs, has been identified. MiRNA are extremely stable in biological fluids and their levels are different in malignant vs. control sera, making them a promising class of biological markers for early diagnosis. Indeed, studies from our and other labs led to the identification of “miRNA signatures” able to predict the presence of tumours even within a population of asymptomatic high-risk individuals (Bianchi F. et al., 2011; Boeri M. et al., 2011). However, methods used in these studies rely on QPCR approaches, which require previous knowledge on the molecules to be investigated. Indeed, the real complexity of circulating RNAs is still obscure, comprising other classes of molecules not yet investigated that could behave as novel biomarkers. The recent development of high-throughput sequencing technologies (Next Generation Sequencing - NGS) provided instruments to reveal the complexity of nucleic acids, but a complete protocol for the identification of circulating RNAs has not been established nor compared with current available approaches (i.e. QPCR). Aim: Our aim is to develop a simultaneous and comparative protocol for serum miRNA analysis, which encompasses both high-throughput QPCR and sequencing analysis in order to reveal the complexity of circulating small-RNAs. Methods: Total RNA has been isolated from the serum of healthy donors and analysed simultaneously by low-density QPCR (TaqMan Low Density Array, Life Technologies) and by NGS (Illumina). Results: A step-wise protocol to simultaneously analyze non-coding RNAs using NGS technology (Illumina) and high-throughput QPCR has been developed. The protocol has been optimized to allow both analyses using even limited amount of serum (up to 1mL). Both platforms resulted as highly efficient and quantitative, although NGS manages to score many molecules and RNA species that could not be analysed by current QPCR platforms, thus revealing a major complexity of circulating non-coding RNAs. Conclusion: We proposed that the combined use of NGS and QPCR platforms would allow a wider and more detailed analysis of circulating RNAs, expanding our ability to fish out robust and efficient molecular markers for early detection of cancer. Citation Format: Francesca Montani, Rose Mary Carletti, Matteo J. Marzi, Gabriele Bucci, Francesco Nicassio, Pier Paolo Di Fiore. Identification of serum circulating non-coding RNAs as diagnostic markers by Next Generation Sequencing (NGS) and low-density array. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5280. doi:10.1158/1538-7445.AM2013-5280