Francesco Nicassio

miR-Test: A Blood Test for Lung Cancer Early Detection

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.

Degradation dynamics of microRNAs revealed by a novel pulse-chase approach

The regulation of miRNAs is critical to the definition of cell identity and behavior in normal physiology and disease. To date, the dynamics of miRNA degradation and the mechanisms involved in remain largely obscure, in particular, in higher organisms. Here, we developed a pulse-chase approach based on metabolic RNA labeling to calculate miRNA decay rates at genome-wide scale in mammalian cells. Our analysis revealed heterogeneous miRNA half-lives, with many species behaving as stable molecules (T1/2> 24 h), while others, including passenger miRNAs and a number (25/129) of guide miRNAs, are quickly turned over (T1/2= 4-14 h). Decay rates were coupled with other features, including genomic organization, transcription rates, structural heterogeneity (isomiRs), and target abundance, measured through quantitative experimental approaches. This comprehensive analysis highlighted functional mechanisms that mediate miRNA degradation, as well as the importance of decay dynamics in the regulation of the miRNA pool under both steady-state conditions and during cell transitions.

IsomiRage: From Functional Classification to Differential Expression of miRNA Isoforms.

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, trimmingu2009…) 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.

DEPDC1B coordinates de-adhesion events and cell-cycle progression at mitosis.

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.

The role of non-coding RNAs in the regulation of stem cells and progenitors in the normal mammary gland and in breast tumors

The outlook on stem cell (SC) biology is shifting from a rigid hierarchical to a more flexible model in which the identity and the behavior of adult SCs, far from being fixed, are determined by the dynamic integration of cell autonomous and non-autonomous mechanisms. Within this framework, the recent discovery of thousands of non-coding RNAs (ncRNAs) with regulatory function is redefining the landscape of transcriptome regulation, highlighting the interplay of epigenetic, transcriptional, and post-transcriptional mechanisms in the specification of cell fate and in the regulation of developmental processes. Furthermore, the expression of ncRNAs is often tissue- or even cell type-specific, emphasizing their involvement in defining space, time and developmental stages in gene regulation. Such a role of ncRNAs has been investigated in embryonic and induced pluripotent SCs, and in numerous types of adult SCs and progenitors, including those of the breast, which will be the topic of this review. We will focus on ncRNAs with an important role in breast cancer, in particular in mammary cancer SCs and progenitors, and highlight the ncRNA-based circuitries whose subversion alters a number of the epigenetic, transcriptional, and post-transcriptional events that control “stemness” in the physiological setting.

Parent-of-origin genetic background affects the transcriptional levels of circadian and neuronal plasticity genes following sleep loss

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.

Synergic Functions of miRNAs Determine Neuronal Fate of Adult Neural Stem Cells

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.

MicroRNAs in Cancer Management: Big Challenges for Small Molecules

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. n nAccording to the last release of miRNA database (miRBaseu2009u2009http://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. n nBesides 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.). n nThe unique pattern of altered miRNA expression provides a fingerprint that may serve for cancer diagnosis and prediction of patients 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. n nThe 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. n nDue 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. n nOverall 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. n n nPaolou2009u2009Gandellini n nElisau2009u2009Giovannetti n nFrancescou2009u2009Nicassio

Microenvironment Stimuli HGF and Hypoxia Differently Affected miR-125b and Ets-1 Function with Opposite Effects on the Invasiveness of Bone Metastatic Cells: A Comparison with Breast Carcinoma Cells

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.

Endogenous transcripts control miRNA levels and activity in mammalian cells by target-directed miRNA degradation

Little is known about miRNA decay. A target-directed miRNA degradation mechanism (TDMD) has been suggested, but further investigation on endogenous targets is necessary. Here, we identify hundreds of targets eligible for TDMD and show that an endogenous RNA (Serpine1) controls the degradation of two miRNAs (miR-30b-5p and miR-30c-5p) in mouse fibroblasts. In our study, TDMD occurs when the target is expressed at relatively low levels, similar in range to those of its miRNAs (100–200 copies per cell), and becomes more effective at high target:miRNA ratios (>10:1). We employ CRISPR/Cas9 to delete the miR-30 responsive element within Serpine1 3UTR and interfere with TDMD. TDMD suppression increases miR-30b/c levels and boosts their activity towards other targets, modulating gene expression and cellular phenotypes (i.e., cell cycle re-entry and apoptosis). In conclusion, a sophisticated regulatory layer of miRNA and gene expression mediated by specific endogenous targets exists in mammalian cells.Via the target-directed miRNA degradation process, RNAs can induce degradation of miRNAs by binding with extensive complementarity. Here, the authors show Serpine1 mRNA as one such RNA that can control the levels of the endogenous miRNA miR-30b/c-5p by modulating miRNA degradation.