Sprouty2 enhances the tumorigenic potential of glioblastoma cells

Background: Sprouty2 (SPRY2), a feedback regulator of receptor tyrosine kinase (RTK) signaling, has been shown to be associated with drug resistance and cell proliferation in glioblastoma (GBM), but the underlying mechanisms are still poorly defined. Methods: SPRY2 expression and survival patterns of patients with gliomas were analyzed using publicly available databases. Effects of RNA interference targeting SPRY2 on cellular proliferation in established GBM or patient-derived GBM stemlike cells were examined. Loss- or gain-of-function of SPRY2 to regulate the tumorigenic capacity was assessed in both intracranial and subcutaneous xenografts. Results: SPRY2 was found to be upregulated in GBM, which correlated with reduced survival in GBM patients. SPRY2 knockdown significantly impaired proliferation of GBM cells but not of normal astrocytes. Silencing of SPRY2 increased epidermal growth factor-induced extracellular signal-regulated kinase (ERK) and Akt activation causing premature onset of DNA replication, increased DNA damage, and impaired proliferation, suggesting that SPRY2 suppresses DNA replication stress. Abrogating SPRY2 function strongly inhibited intracranial tumor growth and led to significantly prolonged survival of U87 xenograft-bearing mice. In contrast, SPRY2 overexpression promoted tumor propagation of low-tumorigenic U251 cells. Conclusions: The present study highlights an antitumoral effect of SPRY2 inhibition that is based on excessive activation of ERK signaling and DNA damage response, resulting in reduced cell proliferation and increased cytotoxicity, proposing SPRY2 as a promising pharmacological target in GBM patients

SSTR2 in Nasopharyngeal Carcinoma:Relationship with Latent EBV Infection and Potential as a Therapeutic Target

SIMPLE SUMMARY: Nasopharyngeal cancer (NPC) is a malignant epithelial tumor endemic to parts of Asia and associated with infection by the Epstein–Barr virus (EBV) in these regions. The cancer is often detected at a late stage which is associated with poor outcomes (63% 5-year survival). Advances for the management of this disease have remained largely stagnant and treatment relies primarily on radiotherapy and chemotherapy, as well as surgery when indicated. Nevertheless, our understanding of its underlying biology has grown rapidly in the past two decades, laying the foundation for the development of improved therapeutics which have the potential to improve outcomes. This review offers a comprehensive, up-to-date summary of this disease, with a focus on the role of somatostatin receptor 2 (SSTR2) in NPC and how this increased knowledge may lead to improved diagnosis and management of this disease. ABSTRACT: Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor, most commonly located in the pharyngeal recess and endemic to parts of Asia. It is often detected at a late stage which is associated with poor prognosis (5-year survival rate of 63%). Treatment for this malignancy relies predominantly on radiotherapy and/or systemic chemotherapy, which can be associated with significant morbidity and impaired quality of life. In endemic regions NPC is associated with infection by Epstein–Barr virus (EBV) which was shown to upregulate the somatostatin receptor 2 (SSTR2) cell surface receptor. With recent advances in molecular techniques allowing for an improved understanding of the molecular aetiology of this disease and its relation to SSTR2 expression, we provide a comprehensive and up-to-date overview of this disease and highlight the emergence of SSTR2 as a key tumor biomarker and promising target for imaging and therapy

PITX1 is a regulator of TERT expression in prostate cancer with prognostic power

Simple Summary Most prostate cancer is of an indolent form and is curable. However, some prostate cancer belongs to rather aggressive subtypes leading to metastasis and death, and immediate therapy is mandatory. However, for these, the therapeutic options are highly invasive, such as radical prostatectomy, radiation or brachytherapy. Hence, a precise diagnosis of these tumor subtypes is needed, and the thus far applied diagnostic means are insufficient for this. Besides this, for their endless cell divisions, prostate cancer cells need the enzyme telomerase to elongate their telomeres (chromatin endings). In this study, we developed a gene regulatory model based on large data from transcription profiles from prostate cancer and chromatin-immuno-precipitation studies. We identified the developmental regulator PITX1 regulating telomerase. Besides observing experimental evidence of PITX1′s functional role in telomerase regulation, we also found PITX1 serving as a prognostic marker, as concluded from an analysis of more than 15,000 prostate cancer samples. Abstract The current risk stratification in prostate cancer (PCa) is frequently insufficient to adequately predict disease development and outcome. One hallmark of cancer is telomere maintenance. For telomere maintenance, PCa cells exclusively employ telomerase, making it essential for this cancer entity. However, TERT, the catalytic protein component of the reverse transcriptase telomerase, itself does not suit as a prognostic marker for prostate cancer as it is rather low expressed. We investigated if, instead of TERT , transcription factors regulating TERT may suit as prognostic markers. To identify transcription factors regulating TERT , we developed and applied a new gene regulatory modeling strategy to a comprehensive transcriptome dataset of 445 primary PCa. Six transcription factors were predicted as TERT regulators, and most prominently, the developmental morphogenic factor PITX1. PITX1 expression positively correlated with telomere staining intensity in PCa tumor samples. Functional assays and chromatin immune-precipitation showed that PITX1 activates TERT expression in PCa cells. Clinically, we observed that PITX1 is an excellent prognostic marker, as concluded from an analysis of more than 15,000 PCa samples. PITX1 expression in tumor samples associated with (i) increased Ki67 expression indicating increased tumor growth, (ii) a worse prognosis, and (iii) correlated with telomere length

Electrophysiological study of ion channels of retinal pigment epithelium cells of transgenic mice with Norrie's disease gene defect

Einleitung: Das Norrie-Syndrom ist eine X-chromosomal vererbte Erkrankung mit angeborener Blindheit, oftmals assoziierter Taubheit und geistiger Retardierung. Aus Elektroretinogramm (ERG) Ableitungen lässt sich eine Beteiligung des retinalen Pigmentepithels (RPE) an der Pathogenese nicht ausschließen. In dieser Arbeit wurden mit Hilfe der Patch-Clamp-Technik Kalzium- und Kaliumkanäle an kultivierten RPE Zellen von Wildtyp- und transgenen Mäusen mit Norrie-Gendefekt gemessen und verglichen. Ziel war, eine potentielle Dysfunktion des RPE bei Norrie-Mäusen zu erfassen und damit eine mögliche ätiologische Beteiligung an der retinalen Degeneration zu beschreiben. Methoden: Kulturen von RPE-Zellen von Wildtyp-Mäusen und transgenen Mäusen mit Norrie- Gendefekt wurden angelegt 12-14 Tage nach Geburt. Zellen wurden elektrophysiologisch analysiert mittels Patch-Clamp- Technik in der Whole-Cell-Konfiguration. Immunozytochemie wurde zum weiteren Nachweis der Ionenkanalidentität angewandt. Ergebnisse: Die durchschnittliche Membrankapazität aller gemessenen Zellen betrug 120.9 ± 6.5 pF (n=103). Ca2+ Ströme wurden gemessen und konnten anhand ihrer Spannungs-abhängigkeit, Stromkinetik, Nifedipine-Sensitivität und immunozytochemischem Nachweis der α1D-Untereinheit als L-type Ca2+ Kanäle identifiziert werden. Der L-type Ca2+ Kanal konnte durch den Tyrosinkinase-Hemmer Genistein sowohl bei Wildtyp- als auch bei Norrie-Mäusen gehemmt werden. Bei Untersuchungen zu Kaliumkanälen zeigten die meisten Zellen einen auswärtsrektifizierenden Strom mit typischer Charakteristik von Delayed-Rectifier K+ Kanälen, deren Identität mittels immunozytochemischen Nachweis der Kv1.3-Untereinheit bestätigt werden konnte. Das Ruhemembranpotential am RPE von Norrie-Mäusen betrug - 40.0 ± 6.9 mV (n=7), was ohne signifikanten Unterschied zu den Werten von Kontrollmäusen war (- 37.6 ± 3.5 mV (n=7)). Der Delayed-Rectifier artige K+ Kanal ließ sich an RPE-Zellen sowohl von Norrie- als auch von Wildtyp-Mäusen durch Genistein reversibel hemmen, wodurch seine Regulation durch Tyrosinkinasen nachgewiesen werden konnte. Schlussfolgerung: Im Vergleich der elektrophysiologischen Charakteristik der L-type Ca2+ Kanäle und der Delayed-Rectifier artigen K+ Kanäle zeigten sich keine statistisch signifikanten Unterschiede zwischen RPE- Zellen von Norrie-Mäusen und Wildtyp-Mäusen. Jedoch repräsentieren die Ergebnisse eine Erstbeschreibung von elektrophysiologischen Untersuchungen zur Ionenkanalcharakterisierung von RPE-Zellen von Mäusen. In Anbetracht der Möglichkeiten der Transgenese bei Mäusen und der generell weit verbreiteten Nutzung als Tiermodell kommt dieser Charakterisierung von Maus RPE-Zellen eine besondere Bedeutung zu. Die hier charakterisierten Ionenkanäle sind mit denen in der Literatur an RPE-Zellen von Ratte, Rind und Mensch beschriebenen vergleichbar.Introduction: Norrie disease is an X-linked recessive disorder characterized by congenital blindness often coupled with deafness and mental retardation. Electroretinogram (ERG) studies indicate that the retinal pigment epithelium (RPE) might be involved in ocular pathogenesis. We compared calcium and potassium channels of cultured RPE cells of wild-type and transgenic mice with Norrie disease to find out whether altered channel characteristics are involved in RPE-dysfunction and the etiology of retinal degeneration. Methods: RPE cells from wild-type mice and Norrie mutant mice were analyzed using the whole-cell configuration of the patch-clamp technique. We compared basic properties of calcium and potassium conductances. In addition, ion channels were characterized by subunit-directed immunocytochemistry. Results: Membrane capacitance of investigated RPE cells was 120.9 ± 6.5 pF (n=103). Ca2+ currents could be identified as currents through L-type Ca2+ channels by means of voltage-dependence, current kinetics, nifedipine sensitivity and detection of the α1D-subunit by immunocytochemistry. Application of the tyrosine kinase inhibitor genistein caused a decrease in L-type current amplitudes in both cell populations. Examining K+ channels, most cells showed outwardly rectifying whole-cell currents with characteristics typical for delayed rectifier K+ channels, which was verified by Kv1.3-subunit-directed immunostaining. Resting membrane potential was - 40.0 ± 6.9 mV (n=7) in RPE from Norrie mice and did not differ significantly from control cells with - 37.6 ± 3.5 mV (n=7). The delayed-rectifier current amplitude could be reversibly reduced by genistein in Norrie cells as well as in wild type controls. Conclusions: Comparing general features of L-type Ca2+ channels and delayed rectifier K+ channels, no significant difference could be observed between RPE cells from Norrie mice and wild type mice. However, this study presents for the first time the culture of murine RPE cells and presents electrophysiological data on these cells. Using mice as a new animal model for investigating RPE function can break new ground for future experiments, especially by means of transgenic or knock out mice. In addition, this study revealed that the electrophysiological characteristics for L-type Ca2+ channels and delayed rectifier K+ channels are comparable with data from human RPE cells and cells from established animal models like rat and cow

Combining Oncolytic Viruses and Small Molecule Therapeutics: Mutual Benefits

The focus of treating cancer with oncolytic viruses (OVs) has increasingly shifted towards achieving efficacy through the induction and augmentation of an antitumor immune response. However, innate antiviral responses can limit the activity of many OVs within the tumor and several immunosuppressive factors can hamper any subsequent antitumor immune responses. In recent decades, numerous small molecule compounds that either inhibit the immunosuppressive features of tumor cells or antagonize antiviral immunity have been developed and tested for. Here we comprehensively review small molecule compounds that can achieve therapeutic synergy with OVs. We also elaborate on the mechanisms by which these treatments elicit anti-tumor effects as monotherapies and how these complement OV treatment

Targeting Human Glioblastoma Cells: Comparison of Nine Viruses with Oncolytic Potential

Brain tumors classified as glioblastomas have proven refractory to treatment and generally result in death within a year of diagnosis. We used seven in vitro tests and one in vivo trial to compare the efficacy of nine different viruses for targeting human glioblastoma. Green fluorescent protein (GFP)-expressing vesicular stomatitis (VSV), Sindbis virus, pseudorabies virus (PRV), adeno-associated virus (AAV), and minute virus of mice i-strain (MVMi) and MVMp all infected glioblastoma cells. Mouse and human cytomegalovirus, and simian virus 40 showed only low levels of infection or GFP expression. VSV and Sindbis virus showed strong cytolytic actions and high rates of replication and spread, leading to an elimination of glioblastoma. PRV and both MVM strains generated more modest lytic effects and replication capacity. VSV showed a similar oncolytic profile on U-87 MG and M059J glioblastoma. In contrast, Sindbis virus showed strong preference for U-87 MG, whereas MVMi and MVMp preferred M059J. Sindbis virus and both MVM strains showed highly tumor-selective actions in glioblastoma plus fibroblast coculture. VSV and Sindbis virus were serially passaged on glioblastoma cells; we isolated a variant, VSV-rp30, that had increased selectivity and lytic capacity in glioblastoma cells. VSV and Sindbis virus were very effective at replicating, spreading within, and selectively killing human glioblastoma in an in vivo mouse model, whereas PRV and AAV remained at the injection site with minimal spread. Together, these data suggest that four (VSV, Sindbis virus, MVMi, and MVMp) of the nine viruses studied merit further analysis for potential therapeutic actions on glioblastoma

FailSim: A Numerical Toolbox for the Study of Fast Failures and Their Impact on Machine Protection at the CERN Large Hadron Collider

The High Luminosity LHC (HL-LHC) foresees to reach a nominal, levelled luminosity of 5·10³⁴ cm⁻² s⁻¹ through a higher beam brightness and by using new equipment, such as larger aperture final focusing quadrupole magnets. The HL-LHC upgrade has critical impacts on the machine protection strategy, as the stored beam energy reaches 700 MJ for each of the two beams. Some failure modes of the novel active superconducting magnet protection system of the inner triplet magnets, namely the Coupling-Loss Induced Quench (CLIQ) systems, have been identified as critical. This paper reports on FailSim, a Python-language framework developed to study the machine protection impact of failure cases and their proposed mitigation. It provides seamless integration of the successive phases required by the simulation studies, i.e., verifying the optics, preparing and running a MAD-X instance for multiple particle tracking, processing and analysing the simulation results and summarising them with the relevant plots to provide a solid estimate of the beam losses, their location and time evolution. The paper also presents and discusses the result of its application on the spurious discharge of a CLIQ unit

Peripheral Immunization Blocks Lethal Actions of Vesicular Stomatitis Virus within the Brain▿

Vesicular stomatitis virus (VSV) is the prototype virus for 75 or more negative-strand RNA viruses in the rhabdovirus family. Some of these viruses, including VSV, can cause neurological impairment or death upon brain infection. VSV has shown promise in the prevention and treatment of disease as a vaccine vector and an oncolytic virus, but infection of the brain remains a concern. Three VSV variants, the wild-type-related VSV-G/GFP and two attenuated viruses, VSV-CT1 and VSV-CT9-M51, were compared for neuroinvasiveness and neuromorbidity. In nonimmunized mice, direct VSV-G/GFP injection into the brain invariably resulted in lethal encephalitis; in contrast, partial survival was seen after direct injection of the attenuated VSV strains. In addition, both attenuated VSV strains showed significantly reduced neuroinvasiveness after intranasal inoculation of young postnatal day 16 mice. Of the three tested variants, VSV-CT9-M51 generated the lowest degree of neuropathology. Despite its attenuated state, peripheral inoculations of VSV-CT9-M51 targeted and killed human glioblastoma implanted into the mouse brain. Importantly, we show here that intranasal or intramuscular immunization prevents the lethal effects of subsequent VSV-G/GFP, VSV-CT1, and VSV-CT9-M51 injections into the brain. These results indicate that attenuated recombinant viruses show reduced neurovirulence and that peripheral immunization blocks the lethal actions of all VSVs tested