Over the past two decades, it was discovered that introducing synthetic small interfering RNAs (siRNAs) into the cytoplasm facilitates effective gene-targeted silencing. This compromises gene ...expression and regulation by repressing transcription or stimulating sequence-specific RNA degradation. Substantial investments in developing RNA therapeutics for disease prevention and treatment have been made. We discuss the application to proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds to and degrades the low-density lipoprotein cholesterol (LDL-C) receptor, interrupting the process of LDL-C uptake into hepatocytes. PCSK9 loss-of-function modifications show significant clinical importance by causing dominant hypocholesterolemia and lessening the risk of cardiovascular disease (CVD). Monoclonal antibodies and small interfering RNA (siRNA) drugs targeting PCSK9 are a significant new option for managing lipid disorders and improving CVD outcomes. In general, monoclonal antibodies are restricted to binding with cell surface receptors or circulating proteins. Similarly, overcoming the intracellular and extracellular defenses that prevent exogenous RNA from entering cells must be achieved for the clinical application of siRNAs.
-acetylgalactosamine (GalNAc) conjugates are a simple solution to the siRNA delivery problem that is especially suitable for treating a broad spectrum of diseases involving liver-expressed genes. Inclisiran is a GalNAc-conjugated siRNA molecule that inhibits the translation of PCSK9. The administration is only required every 3 to 6 months, which is a significant improvement over monoclonal antibodies for PCSK9. This review provides an overview of siRNA therapeutics with a focus on detailed profiles of inclisiran, mainly its delivery strategies. We discuss the mechanisms of action, its status in clinical trials, and its prospects.
Ionizing radiation (IR) is effectively used in cancer therapy. However, in subsets of patients, a few radioresistant cancer cells survive and cause disease relapse with metastatic progression. The ...MET oncogene encodes the hepatocyte growth factor (HGF) receptor and is known to drive "invasive growth", a regenerative and prosurvival program unduly activated in metastasis.
Human tumor cell lines (MDA-MB-231, MDA-MB-435S, U251) were subjected to therapeutic doses of IR. MET mRNA, and protein expression and signal transduction were compared in treated and untreated cells, and the involvement of the DNA-damage sensor ataxia telangiectasia mutated (ATM) and the transcription factor nuclear factor kappa B (NF-κB) in activating MET transcription were analyzed by immunoblotting, chromatin immunoprecipitation, and use of NF-κB silencing RNA (siRNA). Cell invasiveness was measured in wound healing and transwell assays, and cell survival was measured in viability and clonogenic assays. MET was inhibited by siRNA or small-molecule kinase inhibitors (PHA665752 or JNJ-38877605). Combinations of MET-targeted therapy and radiotherapy were assessed in MDA-MB-231 and U251 xenografts (n = 5-6 mice per group). All P values were from two-sided tests.
After irradiation, MET expression in cell lines was increased up to fivefold via activation of ATM and NF-κB. MET overexpression increased ligand-independent MET phosphorylation and signal transduction, and rendered cells more sensitive to HGF. Irradiated cells became more invasive via a MET-dependent mechanism that was further enhanced in the presence of HGF. MET silencing by siRNA or inhibition of its kinase activity by treatment with PHA665752 or JNJ-38877605 counteracted radiation-induced invasiveness, promoted apoptosis, and prevented cells from resuming proliferation after irradiation in vitro. Treatment with MET inhibitors enhanced the efficacy of IR to stop the growth of or to induce the regression of xenografts (eg, at day 13, U251 xenografts, mean volume increase relative to mean tumor volume at day 0: vehicle = 438%, 5 Gy IR = 151%, 5 Gy IR + JNJ-38877605 = 76%; difference, IR vs JNJ-38877604 + IR = 75%, 95% CI = 59% to 91%, P = .01).
IR induces overexpression and activity of the MET oncogene through the ATM-NF-κB signaling pathway; MET, in turn, promotes cell invasion and protects cells from apoptosis, thus supporting radioresistance. Drugs targeting MET increase tumor cell radiosensitivity and prevent radiation-induced invasiveness.
Glioblastoma (GBM) is known as an intractable, highly heterogeneous tumor encompassing multiple subclones, each supported by a distinct glioblastoma stem cell (GSC). The contribution of GSC genetic ...and transcriptional heterogeneity to tumor subclonal properties is debated. In this study, we describe the systematic derivation, propagation, and characterization of multiple distinct GSCs from single, treatment-naive GBMs (GSC families). The tumorigenic potential of each GSC better correlates with its transcriptional profile than its genetic make-up, with classical GSCs being inherently more aggressive and mesenchymal more dependent on exogenous growth factors across multiple GBMs. These GSCs can segregate and recapitulate different histopathological aspects of the same GBM, as shown in a paradigmatic tumor with two histopathologically distinct components, including a conventional GBM and a more aggressive primitive neuronal component. This study provides a resource for investigating how GSCs with distinct genetic and/or phenotypic features contribute to individual GBM heterogeneity and malignant escalation.
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•Distinct GSCs can be derived from single GBMs by different growth factor cocktails•GSCs segregate different RTK expression and gene amplifications•GSCs segregate distinct transcriptional profiles, each related to different malignancy•GSCs with a neural progenitor profile recapitulate the GBM primitive neuronal component
De Bacco et al. devise a methodology to propagate distinct glioblastoma stem-like cells (GSCs) from individual tumors, stably segregating different features coexisting in the original tumor. The GSCs' transcriptional profile is a superior indicator of their tumorigenicity compared with their genetic make-up, uncovering diverse phenotypes that drive intratumoral heterogeneity.
Glioblastoma (GBM) contains stem‐like cells (GSCs) known to be resistant to ionizing radiation and thus responsible for therapeutic failure and rapidly lethal tumor recurrence. It is known that GSC ...radioresistance relies on efficient activation of the DNA damage response, but the mechanisms linking this response with the stem status are still unclear. Here, we show that the MET receptor kinase, a functional marker of GSCs, is specifically expressed in a subset of radioresistant GSCs and overexpressed in human GBM recurring after radiotherapy. We elucidate that MET promotes GSC radioresistance through a novel mechanism, relying on AKT activity and leading to (i) sustained activation of Aurora kinase A, ATM kinase, and the downstream effectors of DNA repair, and (ii) phosphorylation and cytoplasmic retention of p21, which is associated with anti‐apoptotic functions. We show that MET pharmacological inhibition causes DNA damage accumulation in irradiated GSCs and their depletion in vitro and in GBMs generated by GSC xenotransplantation. Preclinical evidence is thus provided that MET inhibitors can radiosensitize tumors and convert GSC‐positive selection, induced by radiotherapy, into GSC eradication.
Synopsis
GBM radioresistance relies on the inherent properties of its stem‐like cell population (GSCs). MET, which encodes for the HGF receptor, is shown to be a functional marker of GSC radioresistance and a therapeutic target for GSC radiosensitization.
MET expression identifies radioresistant GSCs derived from a subset of patients and propagated as neurospheres.
In GBMs recurring after radiotherapy, MET expression is more frequent, as compared with matched primary tumors (85% versus 45%).
MET drives positive selection of irradiated GSCs, by promoting the DNA damage response via AKT, which sustains ATM activation, and p21 phosphorylation and cytoplasmic retention.
When combined with radiotherapy, MET inhibition impairs the GSC DNA damage response and converts GSC selection from positive to negative.
GBM radioresistance relies on the inherent properties of its stem‐like cell population (GSCs). MET, which encodes for the HGF receptor, is shown to be a functional marker of GSC radioresistance and a therapeutic target for GSC radiosensitization.
Current glioma diagnostic guidelines call for molecular profiling to stratify patients into prognostic and treatment subgroups. In case the tumor tissue is inaccessible, cerebrospinal fluid (CSF) has ...been proposed as a reliable tumor DNA source for liquid biopsy. We prospectively investigated the use of CSF for molecular characterization of newly diagnosed gliomas.
We recruited two cohorts of newly diagnosed patients with glioma, one (n = 45) providing CSF collected in proximity of the tumor, the other (n = 39) CSF collected by lumbar puncture (LP). Both cohorts provided tumor tissues by surgery concomitant with CSF sampling. DNA samples retrieved from CSF and matched tumors were systematically characterized and compared by comprehensive (NGS, next-generation sequencing) or targeted (ddPCR, droplet digital PCR) methodologies. Conventional and molecular diagnosis outcomes were compared.
We report that tumor DNA is abundant in CSF close to the tumor, but scanty and mostly below NGS sensitivity threshold in CSF from LP. Indeed, tumor DNA is mostly released by cells invading liquoral spaces, generating a gradient that attenuates by departing from the tumor. Nevertheless, in >60% of LP CSF samples, tumor DNA is sufficient to assess a selected panel of genetic alterations (IDH and TERT promoter mutations, EGFR amplification, CDKN2A/B deletion: ITEC protocol) and MGMT methylation that, combined with imaging, enable tissue-agnostic identification of main glioma molecular subtypes.
This study shows potentialities and limitations of CSF liquid biopsy in achieving molecular characterization of gliomas at first clinical presentation and proposes a protocol to maximize diagnostic information retrievable from CSF DNA.
Semaphorins are a large family of molecular cues implicated in neural development and in a variety of functions outside the nervous system. Semaphorin 5A (Sema5A) is a transmembrane semaphorin, ...containing seven thrombospondin type‐1 repeats, which was recently found to control axon guidance. Here we show that plexin‐B3 is a high‐affinity receptor specific for Sema5A. We further demonstrate that plexin‐B3 activation by Sema5A mediates functional responses in plexin‐B3‐expressing cells (either fibroblasts, epithelial and primary endothelial cells). In addition, Sema5A can trigger the intracellular signalling of the hepatocyte growth factor/scatter factor receptor, Met, associated in a complex with plexin‐B3. We thus conclude that Sema5A is able to elicit multiple functional responses through its receptor plexin‐B3.
Treatment of aortic valve stenosis is evolving, indications for transcatheter approach (TAVI) have increased but also surgical valve replacement has changed with the use of minimally invasive ...approaches. Comparisons between TAVI and surgery have rarely been done with minimally invasive techniques (mini-SAVR) in the surgical arm. Aim of the present study is to compare mini-SAVR and TAVI in a multicenter recent cohort.
Evaluated were 2904 patients undergone mini-SAVR (2407) or TAVI (497) in 10 different centers in the period 2011–2016. The Heart Team approved treatment for complex cases. The primary outcome is the incidence of 30-day mortality following mini-SAVR and TAVI. Secondary outcomes are the occurrence of major complications following both procedures. Propensity matched comparisons was performed based on multivariable logistic regression model.
In the overall population TAVI patients had increased surgical risk (median EuroSCORE II 3.3% vs. 1.7%, p ≤ 0.001) and 30-day mortality was higher (1.5% and 2.8% in mini-SAVR and TAVI respectively, p = 0.048). Propensity score identified 386 patients per group with similar baseline profile (median EuroSCORE II ~3.0%). There was no difference in 30-day mortality (3.4% in mini-SAVR and 2.3% in TAVI; p = 0.396) and stroke, surgical patients had more blood transfusion, kidney dysfunction and required longer ICU and hospital length of stay while TAVI patients had more permanent pace maker insertion.
Mini-SAVR and TAVI are both safe and effective to treat aortic stenosis in elderly patients with comorbidities. A joint evaluation by the heart-team is essential to direct patients to the proper approach.
•A comparison between TAVI and minimally invasive techniques has rarely been done•In a propensity-matched comparison from 10 different hospitals, TAVI and mini-SAVR provided optimal short-term outcome.•30-day mortality was 3.4% in mini-SAVR and 2.3% in TAVI (p = 0.396)•Mini-SAVR and TAVI are both safe and effective to treat aortic stenosis in elderly patients with comorbidities.
PLEXIN genes encode receptors for secreted and membrane-bound semaphorins. It was proposed that the extracellular domain of plexins
acts as an inhibitory moiety, preventing receptor activation. Here ...we show that plexin-B1 and plexin-B2 undergo proteolytic
processing in their extracellular portion, thereby converting single-chain precursors into non-disulfide-linked, heterodimeric
receptors. We demonstrate that plexin processing is mediated by subtilisin-like proprotein convertases, by inhibition with
α1-antitrypsin Portland, and by mutagenesis of the substrate-cleavage sites. We provide evidence indicating that proprotein
convertases cleave plexins in a post-Golgi compartment and, likely, at the cell surface. In addition, we find that both cell
surface targeting and proteolytic processing of plexin-B1 depend on protein-protein interaction motifs in the cytoplasmic
domain of the receptor. We then show that proteolytic conversion of plexin-B1 into a heterodimeric receptor greatly increases
the binding and the functional response to its specific ligand semaphorin 4D/CD100. Thus, we conclude that cleavage by proprotein
convertases is a novel regulatory step for semaphorin receptors localized at the cell surface.
Plexins are a large family of transmembrane receptors for the Semaphorins, known for their role in the assembly of neural circuitry. More recently, Plexins have been implicated in diverse biological ...functions, including vascular growth, epithelial tissue morphogenesis and tumour development. In particular, PlexinB1, the receptor for Sema4D, has been suggested to play a role in neural development and in tumour angiogenesis, based on in vitro studies. However, the tissue distribution of PlexinB1 has not been extensively studied and the functional relevance of this receptor in vivo still awaits experimental testing. In order to shed light on PlexinB1 function in vivo, we therefore undertook the genomic targeting of the mouse gene to obtain loss of function mutants.
This study shows that PlexinB1 receptor and its putative ligand, Sema4D, have a selective distribution in nervous and epithelial tissues during development and in the adult. PlexinB1 and Sema4D show largely complementary cell distribution in tissues, consistent with the idea that PlexinB1 acts as the receptor for Sema4D in vivo. Interestingly, PlexinB1 is also expressed in certain tissues in the absence of Sema4D, suggesting Sema4D independent activities. High expression of PlexinB1 was found in lung, kidney, liver and cerebellum. Mutant mice lacking expression of semaphorin receptor PlexinB1 are viable and fertile. Although the axon collapsing activity of Sema4D is impaired in PlexinB1 deficient neurons, we could not detect major defects in development, or in adult histology and basic functional parameters of tissues expressing PlexinB1. Moreover, in the absence of PlexinB1 the angiogenic response induced by orthotopically implanted tumours was not affected, suggesting that the expression of this semaphorin receptor in endothelial cells is redundant.
Our expression analysis suggests a multifaceted role of PlexinB1 during mouse development and tissue homeostasis in the adult. Nonetheless, the genetic deletion of PlexinB1 does not result in major developmental defects or clear functional abnormalities. We infer that PlexinB1 plays a redundant role in mouse development and it is not strictly required for tumour induced angiogenesis.
GO4SEM (Global Opportunities for Small/medium enterprises in Electro-Mobility) aims to support the European small and medium enterprises (SMEs) in assessing emerging global markets by creating a ...framework that informs and advices on the business opportunities and market requirements for selected countries in Asia and America.
The main goal of the EU coordination GO4SEM was two folded: on the one hand it aimed to support the European SMEs for their entry in global emerging e-mobility markets with a focus on Brazil, India and China. On the other hand it also assessed the business opportunities in more mature markets, with a known or renewed interest in e-mobility, such as Japan, South Korea and the USA. The objective was to spread awareness of global market trends and opportunities and to trigger the creation of dedicated professional networks in the field of electric mobility. Moreover, policy advice, e.g. on standardization or education, is given. The project identified specific product-, service- and technology-needs of emerging e-mobility supply chains. As a result a set of guidelines and recommendations were developed, targeting feasible business models for European SMEs aiming to enter the aforementioned value chains on a global platform.
This contribution presents first the innovation capabilities of the European SME community with respect to the development of e-mobility. Then based on that analysis and the global market needs, a European action plan is proposed for strengthening the competitive position of EU SMEs. And finally the recommendations towards the European Commission, which would facilitate the entering of European SMEs the global markets, are introduced.
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