Chiocca Ea

MicroRNAs and glioblastoma; the stem cell connection

Recent data draw close parallels between cancer, including glial brain tumors, and the biology of stem and progenitor cells. At the same time, it has become clear that one of the major roles that microRNAs play is in the regulation of stem cell biology, differentiation, and cell ‘identity’. For example, microRNAs have been increasingly implicated in the regulation of neural differentiation. Interestingly, initial studies in the incurable brain tumor glioblastoma multiforme strongly suggest that microRNAs involved in neural development play a role in this disease. This encourages the idea that certain miRs allow continued tumor growth through the suppression of differentiation and the maintenance of the stem cell-like properties of tumor cells. These concepts will be explored in this article.

Cytomegalovirus Contributes to Glioblastoma in the Context of Tumor Suppressor Mutations

To study the controversial role of cytomegalovirus (CMV) in glioblastoma, we assessed the effects of murine CMV (MCMV) perinatal infection in a GFAP-cre; Nf1(loxP/+); Trp53(-/+) genetic mouse model of glioma (Mut3 mice). Early on after infection, MCMV antigen was predominantly localized in CD45+ lymphocytes in the brain with active viral replication and local areas of inflammation, but, by 7 weeks, there was a generalized loss of MCMV in brain, confirmed by bioluminescent imaging. MCMV-infected Mut3 mice exhibited a shorter survival time from their gliomas than control Mut3 mice perinatally infected with mock or with a different neurotropic virus. Animal survival was also significantly shortened when orthotopic gliomas were implanted in mice perinatally infected with MCMV versus controls. MCMV infection increased phosphorylated STAT3 (p-STAT3) levels in neural stem cells (NSC) harvested from Mut3 mice subventricular zone, and, in vivo, there was increased p-STAT3 in NSCs in MCMV-infected compared with control mice. Of relevance, human CMV (HCMV) also increased p-STAT3 and proliferation of patient-derived glioblastoma neurospheres, whereas a STAT3 inhibitor reversed this effect in vitro and in vivo. These findings thus associate CMV infection to a STAT3-dependent modulatory role in glioma formation/progression in the context of tumor suppressor mutations in mice and possibly in humans.

Interferon-stimulated Gene 15 (ISG15) and ISG15-linked Proteins Can Associate with Members of the Selective Autophagic Process, Histone Deacetylase 6 (HDAC6) and SQSTM1/p62

Background: ISG15 is a Ub-like protein that conjugates cellular and pathogenic proteins during the innate immune response. Results: ISG15 associates with the selective autophagic factors HDAC6 and p62, leading to degradation of ISG15 conjugates. Conclusion: The IFN response leads to ISG15 expression allowing for its association with HDAC and p62. This may mark proteins for autophagy. Significance: This finding provides evidence of an interferon-stimulated pathway linked to autophagy. The ubiquitin-like interferon (IFN)-stimulated gene 15 (ISG15) and its specific E1, E2, and E3 enzymes are transcriptionally induced by type I IFNs. ISG15 conjugates newly synthesized proteins. ISG15 linkage to proteins appears to be an important downstream IFN signaling event that discriminates cellular and pathogenic proteins synthesized during IFN stimulation from existing proteins. This eliminates potentially pathogenic proteins as the cell attempts to return to normal homeostasis after IFN “stressed” conditions. However, the molecular events that occur in this process are not well known. Here, we show that the C-terminal LRLRGG of ISG15 interacts with the binder of ubiquitin zinc finger (BUZ) domain of histone deacetylase 6 (HDAC6). Because HDAC6 is involved in the autophagic clearance of ubiquitinated aggregates during which SQSTM1/p62 plays a major role as a cargo adapter, we also were able to confirm that p62 binds to ISG15 protein and its conjugated proteins upon forced expression. Both HDAC6 and p62 co-localized with ISG15 in an insoluble fraction of the cytosol, and this co-localization was magnified by the proteasome inhibitor MG132. In addition, ISG15 was degraded via the lysosome. Overexpression of ISG15, which leads to an increased conjugation level of the cellular proteome, enhanced autophagic degradation independently of IFN signaling transduction. These results thus indicate that ISG15 conjugation marks proteins for interaction with HDAC6 and p62 upon forced stressful conditions likely as a step toward autophagic clearance.

Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity

A lack of experimental models of tumor heterogeneity limits our knowledge of the complex subpopulation dynamics within the tumor ecosystem. In high-grade gliomas (HGG), distinct hierarchical cell populations arise from different glioma stem-like cell (GSC) subpopulations. Extracellular vesicles (EV) shed by cells may serve as conduits of genetic and signaling communications; however, little is known about how HGG heterogeneity may impact EV content and activity. In this study, we performed a proteomic analysis of EVs isolated from patient-derived GSC of either proneural or mesenchymal subtypes. EV signatures were heterogeneous, but reflected the molecular make-up of the GSC and consistently clustered into the two subtypes. EV-borne protein cargos transferred between proneural and mesenchymal GSC increased protumorigenic behaviors in vitro and in vivo Clinically, analyses of HGG patient data from the The Cancer Genome Atlas database revealed that proneural tumors with mesenchymal EV signatures or mesenchymal tumors with proneural EV signatures were both associated with worse outcomes, suggesting influences by the proportion of tumor cells of varying subtypes in tumors. Collectively, our findings illuminate the heterogeneity among tumor EVs and the complexity of HGG heterogeneity, which these EVs help to maintain. Cancer Res; 76(10); 2876-81. ©2016 AACR.

Extracellular Vesicles and MicroRNAs: Their Role in Tumorigenicity and Therapy for Brain Tumors.

MicroRNAs are small non-coding RNAs which mediate post-transcriptional gene regulation. Recently, microRNAs have also been found to be localized to the extracellular space, often encapsulated in secreted extracellular vesicles (EVs). This tandem of EVs and tissue-specific expressed/secreted microRNAs that can be taken up by neighboring or distant recipient cells, leading to changes in gene expression—suggests a cell-specialized role in physiological and pathological conditions. The complexity of solid tumors and their distinct pathophysiology relies on interactive communications between the various cell types in the neoplasm (tumor, endothelial, or macrophages, for instance). Understanding how such EV/microRNA-mediated communication occurs may actually lead to avenues for therapeutic exploitation and/or intervention, particularly for the most formidable cancers, such as those in the brain. In this review, the role of microRNAs/EVs in brain tumors will be discussed with emphasis on how these molecules could be utilized for tumor therapy.

Comparative effectiveness of antinociceptive gene therapies in animal models of diabetic neuropathic pain

Peripheral neuropathic pain is one of the most common and debilitating complications of diabetes. Several genes have been shown to be effective in reducing neuropathic pain in animal models of diabetes after transfer to the dorsal root ganglion using replication-defective herpes simplex virus (HSV)1-based vectors, yet there has never been a comparative analysis of their efficacy. We compared four different HSV1-based vectors engineered to produce one of two opioid receptor agonists (enkephalin or endomorphin), or one of two isoforms of glutamic acid decarboxylase (GAD65 or GAD67), alone and in combination, in the streptozotocin-induced diabetic rat and mouse models. Our results indicate that a single subcutaneous hindpaw inoculation of vectors expressing GAD65 or GAD67 reduced diabetes-induced mechanical allodynia to a degree that was greater than daily injections of gabapentin in rats. Diabetic mice that developed thermal hyperalgesia also responded to GAD65 or endomorphin gene delivery. The results suggest that either GAD65 or GAD67 vectors are the most effective in the treatment of diabetic pain. The vector combinations, GAD67+endomorphin, GAD67+enkephalin or endomorphin+enkephalin also produced a significant antinociceptive effect but the combination did not appear to be superior to single gene treatment. These findings provide further justification for the clinical development of antinociceptive gene therapies for the treatment of diabetic peripheral neuropathies.

Combining HDAC inhibitors with oncolytic virotherapy for cancer therapy

Histone deacetylase (HDAC) enzymes play a critical role in the epigenetic regulation of cellular functions and signaling pathways in many cancers. HDAC inhibitors (HDACi) have been validated for single use or in combination with other drugs in oncologic therapeutics. An even more novel combination therapy with HDACi is to use them with an oncolytic virus. HDACi may lead to an amplification of tumor-specific lytic effects by facilitating increased cycles of viral replication, but there may also be direct anticancer effects of the drug by itself. Here, we review the molecular mechanisms of anti-cancer effects of the combination of oncolytic viruses with HDACi.

In vitro screening of clinical drugs identifies sensitizers of oncolytic viral therapy in glioblastoma stem-like cells.

Oncolytic viruses (OV) have broad potential as an adjuvant for the treatment of solid tumors. The present study addresses the feasibility of clinically applicable drugs to enhance the oncolytic potential of the OV Delta24-RGD in glioblastoma. In total, 446 drugs were screened for their viral sensitizing properties in glioblastoma stem-like cells (GSCs) in vitro. Validation was done for 10 drugs to determine synergy based on the Chou Talalay assay. Mechanistic studies were undertaken to assess viability, replication efficacy, viral infection enhancement and cell death pathway induction in a selected panel of drugs. Four viral sensitizers (fluphenazine, indirubin, lofepramine and ranolazine) were demonstrated to reproducibly synergize with Delta24-RGD in multiple assays. After validation, we underscored general applicability by testing candidate drugs in a broader context of a panel of different GSCs, various solid tumor models and multiple OVs. Overall, this study identified four viral sensitizers, which synergize with Delta24-RGD and two other strains of OVs. The viral sensitizers interact with infection, replication and cell death pathways to enhance efficacy of the OV.

The Fc Domain of Immunoglobulin Is Sufficient to Bridge NK Cells with Virally Infected Cells

Summary Clearance of pathogens or tumor cells by antibodies traditionally requires both Fab and Fc domains of IgG. Here, we show the Fc domain of IgG alone mediates recognition and clearance of herpes simplex virus (HSV1)‐infected cells. The human natural killer (NK) cell surface is naturally coated with IgG bound by its Fc domain to the Fc&ggr; receptor CD16a. NK cells utilize the Fc domain of bound IgG to recognize gE, an HSV1‐encoded glycoprotein that also binds the Fc domain of IgG but at a site distinct from CD16a. The bridge formed by the Fc domain between the HSV1‐infected cell and the NK cell results in NK cell activation and lysis of the HSV1‐infected cell in the absence of HSV1‐specific antibody in vitro and prevents fatal HSV1 infection in vivo. This mechanism also explains how bacterial IgG‐binding proteins regulate NK cell function and may be broadly applicable to Fc&ggr;‐receptor‐bearing cells. HighlightsDC‐MEGE identified HSV1 viral proteins regulating NK cell cytotoxicityHSV1 gE activates NK cells through an IgGFc bridge with CD16a on NK cellsIgGFc alone protects mice from lethal HSV1 infection; NK cells are requiredBacterial IgG‐binding proteins activate NK cells through the IgGFc bridge &NA; IgG conventionally utilizes its Fab and Fc domains to engage antigens and immune effector cells, respectively. Dai et al. show that the Fc domain alone allows CD16+ NK cells to recognize and lyse virus‐infected cells that express IgG‐binding proteins.

Abstract 2169: SapC-DOPS induces lethal mitophagy in glioblastoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The goal of this study is to evaluate SapC-DOPS, a novel cancer nanotherapeutic, for glioblastoma multiforme (GBM). SapC-DOPS delivered intravenously (i.v) was found to specifically target intracranial tumors in mice bearing spontaneous brain tumor, as well in nude mice intracranially implanted with human GBM cells. Treatment of tumor bearing mice with SapC-DOPS (i.v.) significantly increased survival: 25% and 75% long-term survivors in U87ΔEGFR-Luc and X12v2 implanted mice, respectively (P 0.05). In contrast, SapC-DOPS treatment increased levels of an autophagic marker LC3-II via western blot. Autophagosome formation was also confirmed through transmission electron microscopy. Utilizing a stable GBM cell line expressing a GFP-LC3 fusion protein, we observed punctuated GFP expression following treatment, indicative of autophagosome formation. Quantification of GFP punctated cells showed a significant increase in SapC-DOPS treated cells compared to control (P<.001). Analysis of red/green fluorescence following acridine orange staining showed an induction of acidic vesicular organelles indicative of autophagolysosomes. In addition, inhibition of autophagosome formation using 3-methyladeneine or inhibition of auotphagic vacuole maturation with bafilomycin A1 resulted in a significant rescue of SapC-DOPS-induced killing (P<.001). Knockdown of ATG5 using siRNA also resulted in a rescue of SapC-DOPS-induced cell death and autophagy induction (P<.001). Interestingly, we did not observe a decrease in the activation of mTOR as determined by the phosphorylation of 4EBP1 and p70S6K which are typically induced during autophagy. This led us to test the use of rapamycin, a known inhibitor of mTOR and inducer of autophagy, in combination with SapC-DOPS. By using the Chou Talalay analysis, we observed strong synergy for multiple drug combinations in primary GBM neurospheres (combination index < .4). To investigate whether SapC-DOPS induced autophagy could be preferentially targeting mitochondria (mitophagy), we utilized MitoTracker Green (mitochondria) and LysoTracker Red (autophagolysosomes). By using confocal microscopy analysis, we were able to observe a decrease in mitochondrial mass in cells treated with SapC-DOPS as well as co-localization of mitochondria with autophagolysosomes. In addition to this, we observed a significant decrease in ATP levels following SapC-DOPS treatment (P<.01). These findings suggest therapeutic implications for treating GBM by using SapC-DOPS alone and in combination with an AKT/mTOR inhibitor. Citation Format: Jeffrey Wojton, Naduparambil K. Jacob, Nicholas Denton, Nina Dmitrieva, Hiroshi Nakashima, Chang-Hyuk Kwon, Lionel Chow, Ennio A. Chiocca, Arnab Chakravarti, Balveen Kaur, Xiaoyang Qi. SapC-DOPS induces lethal mitophagy in glioblastoma. [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 2169. doi:10.1158/1538-7445.AM2013-2169