Breast Carcinoma-amplified Sequence 2 Regulates Adult Neurogenesis via β-catenin

Background Breast carcinoma-amplified sequence 2 (BCAS2) regulates β-catenin gene splicing. The conditional knockout of BCAS2 expression in the forebrain (BCAS2 cKO) of mice confers impaired learning and memory along with decreased β-catenin expression. Because β-catenin reportedly regulates adult neurogenesis, we wondered whether BCAS2 could regulate adult neurogenesis via β-catenin. Methods BCAS2 regulating neurogenesis was investigated by characterizing BCAS2 cKO mice. Also, lentivirus-shBCAS2 was intracranially injected into the hippocampus of wild-type mice to knock down BCAS2 expression. We evaluated the rescue effects of BCAS2 cKO by intracranial injection of adeno-associated virus encoding BCAS2 (AAV-DJ8-BCAS2) and AAV-β-catenin gene therapy. Results To show that BCAS2-regulating adult neurogenesis via β-catenin, first, BCAS2 cKO mice showed low SRY-box 2–positive (Sox2+) neural stem cell proliferation and doublecortin-positive (DCX+) immature neurons. Second, stereotaxic intracranial injection of lentivirus-shBCAS2 knocked down BCAS2 in the hippocampus of wild-type mice, and we confirmed the BCAS2 regulation of adult neurogenesis via β-catenin. Third, AAV-DJ8-BCAS2 gene therapy in BCAS2 cKO mice reversed the low proliferation of Sox2+ neural stem cells and the decreased number of DCX+ immature neurons with increased β-catenin expression. Moreover, AAV-β-catenin gene therapy restored neuron stem cell proliferation and immature neuron differentiation, which further supports BCAS2 regulating adult neurogenesis via β-catenin. In addition, cells targeted by AAV-DJ8 injection into the hippocampus included Sox2 and DCX immature neurons, interneurons, and astrocytes. BCAS2 may regulate adult neurogenesis by targeting Sox2+ and DCX+ immature neurons for autocrine effects and interneurons or astrocytes for paracrine effects. Conclusions BCAS2 can regulate adult neurogenesis in mice via β-catenin.

Exogenous Transforming Growth Factor-β in Brain-Induced Symptoms of Central Fatigue and Suppressed Dopamine Production in Mice

Myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS) is one of the most refractory diseases in humans and is characterized by severe central fatigue accompanied with various symptoms that affect daily life, such as impaired memory, depression, and somatic pain. However, the etiology and pathophysiological mechanisms of CFS remain unknown. To investigate the pathophysiological role of transforming growth factor (TGF)-β1, we injected a cytokine into the lateral ventricle of a C57BL/6 mouse. The intracranial injection of TGF-β1 increased the immobility duration in a forced swimming test (FST) and time spent at the closed arm in elevated plus maze (EPM) analysis. The mice injected with TGF-β1 into their brain showed increased sensitivity to pain in a von Frey test, and had a decreased retention time on rotarod and latency time in a bright box in a passive avoidance test. In addition, the serum levels of muscle fatigue biomarkers, lactate dehydrogenase (LDH) and creatine kinase (CK), were significantly increased after administration of TGF-β1. Intracranial injection of TGF-β1 significantly reduced the production of tyrosine hydroxylase (TH) in the ventral tegmental area, accompanied by a decreased level of dopamine in the striatum. The suppression of TH expression by TGF-β1 was confirmed in the human neuroblastoma cell line, SH-SY5Y. These results, which show that TGF-β1 induced fatigue-like behaviors by suppressing dopamine production, suggest that TGF-β1 plays a critical role in the development of central fatigue and is, therefore, a potential therapeutic target of the disease.

Overexpression of the Aryl Hydrocarbon Receptor (Ahr) Mediates an Oxidative Stress Response following Injection of Fine Particulate Matter in the Temporal Cortex

Studies have shown that particulate matter (PM) induces the expression of the aryl hydrocarbon receptor (Ahr) leading to the activation of the oxidative stress response. This study is aimed at characterizing the specific impact of fine PM on the expression profile of the Ahr and oxidative stress response in the primary auditory cortex. PM2.5 (<1.8 μm)-loaded filters were suspended in sterile saline to 102.6–111.82 μg/ml. Next, 10 μl of PM2.5 or an equal volume of saline was administered intracranially into the temporal cortex of two groups of rats (PM2.5 and control; n = 14 per group), respectively. One week after intracranial injection, the temporal cortex was harvested. Transmission electron microscopy was performed to evaluate the distribution of PM2.5 within the temporal cortex. Additionally, the mRNA and protein expression levels of cytochrome P450 1A1 (CYP1A1), CYP1B1, inducible nitric oxide synthase (iNOS), Ahr, and brevican mRNA and protein were measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) or western blotting, respectively. Finally, the protein expression levels of the receptor for advanced glycation end products (RAGE) were estimated using enzyme-linked immunosorbent assay (ELISA). PM2.5 was observed in intracellular vesicles within the temporal cortex following intracranial injection. Levels of oxidative stress molecules (i.e., CYP1A1, CYP1B1, and iNOS), Ahr, Brevican, and RAGE were higher in the PM2.5 group compared with the control group. Intracranial administration of PM2.5 led to increased levels of Ahr and markers of an oxidative stress response in the temporal cortex. The oxidative stress response-mediated increases in the levels of brevican and RAGE.

P09.10 Local immunotherapy of brain cancer harnessing high-retention Fc-fusion constructs

BackgroundGlioblastoma is a highly aggressive cancer type and despite aggressive therapy, patients’ survival remains poor. Immunotherapy of brain cancer is particularly difficult because of its location behind the blood-brain-barrier and the immunosuppressive tumour microenvironment. In order to (re-)activate the immune system, and reverse the local immunosuppression, we employ the pro-inflammatory cytokine interleukin 12 (IL-12). This highly potent immune-stimulatory agent is known for its anti-cancer effect. Unfortunately, IL-12 was found to induce severe toxicity when applied intravenously, impeding its way into clinics. Thus, currently the only valid therapeutic option is local application into the tumour site.Materials and MethodsEngineered proteins were expressed in HEK293T cells and purified by affinity chromatography. In vivo experiments were performed in glioma-bearing mice using intracranial injection of bioluminescent GL-261 cell line. Treatments were performed on day 21 and 28 post tumour injection through intracranial injection using a step-catheter modelling convection enhanced delivery in mice. Blood or tissue was analysed using immunohistochemistry, flow cytometry and ELISA.ResultsBased on an IL-12-IgG fusion protein, we engineered a molecule for exclusively local therapy of brain cancer. We showed anti-cancer efficacy and increased tissue retention of the fusion molecule in glioma in mice. However, molecular analysis of treated tissue confirmed an upregulation of the immunosuppressive molecule PD-L1 in the tumour microenvironment. This means that, despite its efficacy, IL-12 induces an adaptive resistance mechanism, counteracting the therapeutic effect. We thus hypothesised that local IL-12 therapy combined with local blockade of the PD-1/PD-L1-axis would further improve therapeutic efficacy, while exclusively local administration would avoid increased side effects, which usually accompany combination immunotherapy. We showed significantly enhanced long-term survival of glioma-bearing mice treated with IL-12 therapy in combination with PD-L1 blockade compared to single or control treatments. In a next step, we engineered a novel, bifunctional molecule. Optimized for local application and minimized leakage into the systemic circulation, it combines immune-stimulation and checkpoint blockade in one entity. We showed anti-cancer efficacy and increased tissue retention in glioma in mice.ConclusionsThe potent anti-cancer effect of the cytokine IL-12 can be used in therapy when applied locally into the brain tumour. Besides fusion to IgG, we introduced several specific modifications on the molecule, which are crucial to prevent systemic exposure and associated toxic side effects. To overcome the dampening of the immune reaction through induced PD-L1 expression, we introduced a combination therapy of IL-12 with a PD-L1-blocking antibody in a single molecule. We showed this combination superior to single treatments in the context of exclusively local brain tumour therapy.Disclosure InformationL. Schellhammer: None. M. Beffinger: None. S. Pantelyushin: None. T. Buch: None. J. vom Berg: None.

ΔNp73/ETS2 complex drives glioblastoma pathogenesis— targeting downstream mediators by rebastinib prolongs survival in preclinical models of glioblastoma

Background Glioblastoma (GBM) remains one of the least successfully treated cancers. It is essential to understand the basic biology of this lethal disease and investigate novel pharmacological targets to treat GBM. The aims of this study were to determine the biological consequences of elevated expression of ΔNp73, an N-terminal truncated isoform of TP73, and to evaluate targeting of its downstream mediators, the angiopoietin 1 (ANGPT1)/tunica interna endothelial cell kinase 2 (Tie2) axis, by using a highly potent, orally available small-molecule inhibitor (rebastinib) in GBM. Methods ΔNp73 expression was assessed in glioma sphere cultures, xenograft glioblastoma tumors, and glioblastoma patients by western blot, quantitative reverse transcription PCR, and immunohistochemistry. Immunoprecipitation, chromatin immunoprecipitation (ChiP) and sequential ChIP were performed to determine the interaction between ΔNp73 and E26 transformation-specific (ETS) proto-oncogene 2 (ETS2) proteins. The oncogenic consequences of ΔNp73 expression in glioblastomas were examined by in vitro and in vivo experiments, including orthotopic zebrafish and mouse intracranial-injection models. Effects of rebastinib on growth of established tumors and survival were examined in an intracranial-injection mouse model. Results ΔNp73 upregulates both ANGPT1 and Tie2 transcriptionally through ETS conserved binding sites on the promoters by interacting with ETS2. Elevated expression of ΔNp73 promotes tumor progression by mediating angiogenesis and survival. Therapeutic targeting of downstream ΔNp73 signaling pathways by rebastinib inhibits growth of established tumors and extends survival in preclinical models of glioblastoma. Conclusion Aberrant expression of ΔNp73 in GBM promotes tumor progression through autocrine and paracrine signaling dependent on Tie2 activation by ANGPT1. Disruption of this signaling by rebastinib improves tumor response to treatment in glioblastoma.