A multi targeting conditionally replicating adenovirus displays enhanced oncolysis while maintaining expression of immunotherapeutic agents

Studies have demonstrated that oncolytic adenoviruses based on a 24 base pair deletion in the viral E1A gene (D24) may be promising therapeutics for treating a number of cancer types. In order to increase the therapeutic potential of these oncolytic viruses, a novel conditionally replicating adenovirus targeting multiple receptors upregulated on tumors was generated by incorporating an Ad5/3 fiber with a carboxyl terminus RGD ligand. The virus displayed full cytopathic effect in all tumor lines assayed at low titers with improved cytotoxicity over Ad5-RGD D24, Ad5/3 D24 and an HSV oncolytic virus. The virus was then engineered to deliver immunotherapeutic agents such as GM-CSF while maintaining enhanced heterogenic oncolysis

Kinomic exploration of temozolomide and radiation resistance in Glioblastoma multiforme xenolines

Glioblastoma multiforme (GBM) represents the most common and deadly primary brain malignancy, particularly due to temozolomide (TMZ) and radiation (RT) resistance. To better understand resistance mechanisms, we examined global kinase activity (kinomic profiling) in both treatment sensitive and resistant human GBM patient-derived xenografts (PDX or “xenolines”)

MARCKS regulates growth and radiation sensitivity and is a novel prognostic factor for glioma

Purpose This study assessed whether Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) can regulate glioblastoma (GBM) growth, radiation sensitivity and clinical outcome. Experimental Design MARCKS protein levels were analyzed in five GBM explant cell lines and eight patient-derived xenograft tumors by immunoblot, and these levels were correlated to proliferation rates and intracranial growth rates, respectively. Manipulation of MARCKS protein levels was assessed by lentiviral-mediated shRNA knockdown in the U251 cell line and MARCKS over-expression in the U87 cell line. The effect of manipulation of MARCKS on proliferation, radiation sensitivity and senescence was assessed. MARCKS gene expression was correlated with survival outcomes in the Repository of Molecular Brain Neoplasia Data (REMBRANDT) Database and The Cancer Genome Atlas (TCGA). Results MARCKS protein expression was inversely correlated with GBM proliferation and intracranial xenograft growth rates. Genetic silencing of MARCKS promoted GBM proliferation and radiation resistance, while MARCKS overexpression greatly reduced GBM growth potential and induced senescence. We found MARCKS gene expression to be directly correlated with survival in both the REMBRANDT and TCGA databases. Specifically, patients with high MARCKS expressing tumors of the Proneural molecular subtype had significantly increased survival rates. This effect was most pronounced in tumors with unmethylated O6-methylguanine DNA methyltransferase (MGMT) promoters, a traditionally poor prognostic factor. Conclusions MARCKS levels impact GBM growth and radiation sensitivity. High MARCKS expressing GBM tumors are associated with improved survival, particularly with unmethylated MGMT promoters. These findings suggest the use of MARCKS as a novel target and biomarker for prognosis in the Proneural subtype of GBM

Expression Signature of IFN/STAT1 Signaling Genes Predicts Poor Survival Outcome in Glioblastoma Multiforme in a Subtype-Specific Manner

Previous reports have implicated an induction of genes in IFN/STAT1 (Interferon/STAT1) signaling in radiation resistant and prosurvival tumor phenotypes in a number of cancer cell lines, and we have hypothesized that upregulation of these genes may be predictive of poor survival outcome and/or treatment response in Glioblastoma Multiforme (GBM) patients. We have developed a list of 8 genes related to IFN/STAT1 that we hypothesize to be predictive of poor survival in GBM patients. Our working hypothesis that over-expression of this gene signature predicts poor survival outcome in GBM patients was confirmed, and in addition, it was demonstrated that the survival model was highly subtype-dependent, with strong dependence in the Proneural subtype and no detected dependence in the Classical and Mesenchymal subtypes. We developed a specific multi-gene survival model for the Proneural subtype in the TCGA (the Cancer Genome Atlas) discovery set which we have validated in the TCGA validation set. In addition, we have performed network analysis in the form of Bayesian Network discovery and Ingenuity Pathway Analysis to further dissect the underlying biology of this gene signature in the etiology of GBM. We theorize that the strong predictive value of the IFN/STAT1 gene signature in the Proneural subtype may be due to chemotherapy and/or radiation resistance induced through prolonged constitutive signaling of these genes during the course of the illness. The results of this study have implications both for better prediction models for survival outcome in GBM and for improved understanding of the underlying subtype-specific molecular mechanisms for GBM tumor progression and treatment response

CD133 Is a Marker of Bioenergetic Stress in Human Glioma

Mitochondria dysfunction and hypoxic microenvironment are hallmarks of cancer cell biology. Recently, many studies have focused on isolation of brain cancer stem cells using CD133 expression. In this study, we investigated whether CD133 expression is regulated by bioenergetic stresses affecting mitochondrial functions in human glioma cells. First, we determined that hypoxia induced a reversible up-regulation of CD133 expression. Second, mitochondrial dysfunction through pharmacological inhibition of the Electron Transport Chain (ETC) produced an up-regulation of CD133 expression that was inversely correlated with changes in mitochondrial membrane potential. Third, generation of stable glioma cells depleted of mitochondrial DNA showed significant and stable increases in CD133 expression. These glioma cells, termed rho0 or ρ0, are characterized by an exaggerated, uncoupled glycolytic phenotype and by constitutive and stable up-regulation of CD133 through many cell passages. Moreover, these ρ0 cells display the ability to form “tumor spheroids” in serumless medium and are positive for CD133 and the neural progenitor cell marker, nestin. Under differentiating conditions, ρ0 cells expressed multi-lineage properties. Reversibility of CD133 expression was demonstrated by transfering parental mitochondria to ρ0 cells resulting in stable trans-mitochondrial “cybrid” clones. This study provides a novel mechanistic insight about the regulation of CD133 by environmental conditions (hypoxia) and mitochondrial dysfunction (genetic and chemical). Considering these new findings, the concept that CD133 is a marker of brain tumor stem cells may need to be revised

A C19MC-LIN28A-MYCN Oncogenic Circuit Driven by Hijacked Super-enhancers Is a Distinct Therapeutic Vulnerability in ETMRs: A Lethal Brain Tumor

© 2019 Elsevier Inc. Embryonal tumors with multilayered rosettes (ETMRs) are highly lethal infant brain cancers with characteristic amplification of Chr19q13.41 miRNA cluster (C19MC) and enrichment of pluripotency factor LIN28A. Here we investigated C19MC oncogenic mechanisms and discovered a C19MC-LIN28A-MYCN circuit fueled by multiple complex regulatory loops including an MYCN core transcriptional network and super-enhancers resulting from long-range MYCN DNA interactions and C19MC gene fusions. Our data show that this powerful oncogenic circuit, which entraps an early neural lineage network, is potently abrogated by bromodomain inhibitor JQ1, leading to ETMR cell death. Sin-Chan et al. uncover a C19MC-LIN28A-MYCN super-enhancer-dependent oncogenic circuit in embryonal tumors with multilayered rosettes (ETMRs). The circuit entraps an early neural lineage network to sustain embryonic epigenetic programming and is vulnerable to bromodomain inhibition, which promotes ETMR cell death

Acquisition of Chemoresistance in Gliomas Is Associated with Increased Mitochondrial Coupling and Decreased ROS Production

Temozolomide (TMZ) is an alkylating agent used for treating gliomas. Chemoresistance is a severe limitation to TMZ therapy; there is a critical need to understand the underlying mechanisms that determine tumor response to TMZ. We recently reported that chemoresistance to TMZ is related to a remodeling of the entire electron transport chain, with significant increases in the activity of complexes II/III and cytochrome c oxidase (CcO). Moreover, pharmacologic and genetic manipulation of CcO reverses chemoresistance. Therefore, to test the hypothesis that TMZ-resistance arises from tighter mitochondrial coupling and decreased production of reactive oxygen species (ROS), we have assessed mitochondrial function in TMZ-sensitive and -resistant glioma cells, and in TMZ-resistant glioblastoma multiform (GBM) xenograft lines (xenolines). Maximum ADP-stimulated (state 3) rates of mitochondrial oxygen consumption were greater in TMZ-resistant cells and xenolines, and basal respiration (state 2), proton leak (state 4), and mitochondrial ROS production were significantly lower in TMZ-resistant cells. Furthermore, TMZ-resistant cells consumed less glucose and produced less lactic acid. Chemoresistant cells were insensitive to the oxidative stress induced by TMZ and hydrogen peroxide challenges, but treatment with the oxidant L-buthionine-S,R-sulfoximine increased TMZ-dependent ROS generation and reversed chemoresistance. Importantly, treatment with the antioxidant N-acetyl-cysteine inhibited TMZ-dependent ROS generation in chemosensitive cells, preventing TMZ toxicity. Finally, we found that mitochondrial DNA-depleted cells (ρ°) were resistant to TMZ and had lower intracellular ROS levels after TMZ exposure compared with parental cells. Repopulation of ρ° cells with mitochondria restored ROS production and sensitivity to TMZ. Taken together, our results indicate that chemoresistance to TMZ is linked to tighter mitochondrial coupling and low ROS production, and suggest a novel mitochondrial ROS-dependent mechanism underlying TMZ-chemoresistance in glioma. Thus, perturbation of mitochondrial functions and changes in redox status might constitute a novel strategy for sensitizing glioma cells to therapeutic approaches

Referral from primary care to a physical activity programme : establishing long-term adherence? A randomized controlled trial. Rationale and study design

Background: Declining physical activity is associated with a rising burden of global disease. There is little evidence about effective ways to increase adherence to physical activity. Therefore, interventions are needed that produce sustained increases in adherence to physical activity and are cost-effective. The purpose is to assess the effectiveness of a primary care physical activity intervention in increasing adherence to physical activity in the general population seen in primary care. Method and design: Randomized controlled trial with systematic random sampling. A total of 424 subjects of both sexes will participate; all will be over the age of 18 with a low level of physical activity (according to the International Physical Activity Questionnaire, IPAQ), self-employed and from 9 Primary Healthcare Centres (PHC). They will volunteer to participate in a physical activity programme during 3 months (24 sessions; 2 sessions a week, 60 minutes per session). Participants from each PHC will be randomly allocated to an intervention (IG) and control group (CG). The following parameters will be assessed pre and post intervention in both groups: (1) health-related quality of life (SF-12), (2) physical activity stage of change (Prochaska's stages of change), (3) level of physical activity (IPAQ-short version), (4) change in perception of health (vignettes from the Cooperative World Organization of National Colleges, Academies, and Academic Associations of Family Physicians, COOP/WONCA), (5) level of social support for the physical activity practice (Social Support for Physical Activity Scale, SSPAS), and (6) control based on analysis (HDL, LDL and glycated haemoglobin). Participants' frequency of visits to the PHC will be registered over the six months before and after the programme. There will be a follow up in a face to face interview three, six and twelve months after the programme, with the reduced version of IPAQ, SF-12, SSPAS, and Prochaska's stages. Discussion: The pilot study showed the effectiveness of an enhanced low-cost, evidence-based intervention in increased physical activity and improved social support. If successful in demonstrating long-term improvements, this randomised controlled trial will be the first sustainable physical activity intervention based in primary care in our country to demonstrate long-term adherence to physical activity