Activity In Glioblastoma Research Articles

Obtusaquinone: A Cysteine-Modifying Compound That Targets Keap1 for Degradation.

We have previously identified the natural product obtusaquinone (OBT) as a potent antineoplastic agent with promising in vivo activity in glioblastoma and breast cancer through the activation of oxidative stress; however, the molecular properties of this compound remained elusive. We used a multidisciplinary approach comprising medicinal chemistry, quantitative mass spectrometry-based proteomics, functional studies in cancer cells, and pharmacokinetic analysis, as well as mouse xenograft models to develop and validate novel OBT analogs and characterize the molecular mechanism of action of OBT. We show here that OBT binds to cysteine residues with a particular affinity to cysteine-rich Keap1, a member of the CUL3 ubiquitin ligase complex. This binding promotes an overall stress response and results in ubiquitination and proteasomal degradation of Keap1 and downstream activation of the Nrf2 pathway. Using positron emission tomography (PET) imaging with the PET-tracer 2-[18F]fluoro-2-deoxy-d-glucose (FDG), we confirm that OBT is able to penetrate the brain and functionally target brain tumors. Finally, we show that an OBT analog with improved pharmacological properties, including enhanced potency, stability, and solubility, retains the antineoplastic properties in a xenograft mouse model.

Spider venom administration impairs glioblastoma growth and modulates immune response in a non-clinical model

Molecules from animal venoms are promising candidates for the development of new drugs. Previous in vitro studies have shown that the venom of the spider Phoneutria nigriventer (PnV) is a potential source of antineoplastic components with activity in glioblastoma (GB) cell lines. In the present work, the effects of PnV on tumor development were established in vivo using a xenogeneic model. Human GB (NG97, the most responsive line in the previous study) cells were inoculated (s.c.) on the back of RAG−/− mice. PnV (100 µg/Kg) was administrated every 48 h (i.p.) for 14 days and several endpoints were evaluated: tumor growth and metabolism (by microPET/CT, using 18F-FDG), tumor weight and volume, histopathology, blood analysis, percentage and profile of macrophages, neutrophils and NK cells isolated from the spleen (by flow cytometry) and the presence of macrophages (Iba-1 positive) within/surrounding the tumor. The effect of venom was also evaluated on macrophages in vitro. Tumors from PnV-treated animals were smaller and did not uptake detectable amounts of 18F-FDG, compared to control (untreated). PnV-tumor was necrotic, lacking the histopathological characteristics typical of GB. Since in classic chemotherapies it is observed a decrease in immune response, methotrexate (MTX) was used only to compare the PnV effects on innate immune cells with a highly immunosuppressive antineoplastic drug. The venom increased monocytes, neutrophils and NK cells, and this effect was the opposite of that observed in the animals treated with MTX. PnV increased the number of macrophages in the tumor, while did not increase in the spleen, suggesting that PnV-activated macrophages were led preferentially to the tumor. Macrophages were activated in vitro by the venom, becoming more phagocytic; these results confirm that this cell is a target of PnV components. Spleen and in vitro PnV-activated macrophages were different of M1, since they did not produce pro- and anti-inflammatory cytokines. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. The identification, optimization and synthesis of antineoplastic drugs from PnV molecules may lead to a new multitarget chemotherapy. Glioblastoma is associated with high morbidity and mortality; therefore, research to develop new treatments has great social relevance. Natural products and their derivatives represent over one-third of all new molecular entities approved by FDA. However, arthropod venoms are underexploited, although they are a rich source of new molecules. A recent in vitro screening of the Phoneutria nigriventer spider venom (PnV) antitumor effects by our group has shown that the venom significantly affected glioblastoma cell lines. Therefore, it would be relevant to establish the effects of PnV on tumor development in vivo, considering the complex neoplastic microenvironment. The venom was effective at impairing tumor development in murine xenogeneic model, activating the innate immune response and increasing tumor infiltrating macrophages. In addition, PnV activated macrophages in vitro for a different profile of M1. These activated PnV-macrophages have potential to fight the tumor without promoting tumorigenesis. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. We aim to synthesize and carry out a formulation with these antineoplastic molecules for clinical trials. Spider venom biomolecules induced smaller and necrotic xenogeneic GB; spider venom activated the innate immune system; venom increased blood monocytes and the migration of macrophages to the tumor; activated PnV-macrophages have a profile different of M1 and have a potential to fight the tumor without promote tumorigenesis.

Dual metabolic reprogramming by ONC201/TIC10 and 2-Deoxyglucose induces energy depletion and synergistic anti-cancer activity in glioblastoma

BackgroundDysregulation of the metabolome is a hallmark of primary brain malignancies. In this work we examined whether metabolic reprogramming through a multi-targeting approach causes enhanced anti-cancer activity in glioblastoma.MethodsPreclinical testing of a combined treatment with ONC201/TIC10 and 2-Deoxyglucose was performed in established and primary-cultured glioblastoma cells. Extracellular flux analysis was used to determine real-time effects on OXPHOS and glycolysis. Respiratory chain complexes were analysed by western blotting. Biological effects on tumour formation were tested on the chorioallantoic membrane (CAM).ResultsONC201/TIC10 impairs mitochondrial respiration accompanied by an increase of glycolysis. When combined with 2-Deoxyglucose, ONC201/TIC10 induces a state of energy depletion as outlined by a significant decrease in ATP levels and a hypo-phosphorylative state. As a result, synergistic anti-proliferative and anti-migratory effects were observed among a broad panel of different glioblastoma cells. In addition, this combinatorial approach significantly impaired tumour formation on the CAM.ConclusionTreatment with ONC201/TIC10 and 2-Deoxyglucose results in a dual metabolic reprogramming of glioblastoma cells resulting in a synergistic anti-neoplastic activity. Given, that both agents penetrate the blood–brain barrier and have been used in clinical trials with a good safety profile warrants further clinical evaluation of this therapeutic strategy.

ASPM promotes glioblastoma growth by regulating G1 restriction point progression and Wnt-β-catenin signaling.

Increasing evidence has indicated that the disorganized expression of certain genes promotes tumour progression. In this study, we elucidate the potential key differentially expressed genes (DEGs) between glioblastoma (GBM) and normal brain tissue by analysing three different mRNA expression profiles downloaded from the Gene Expression Omnibus (GEO) database. DEGs were sorted, and key candidate genes and signalling pathway enrichments were analysed. In our analysis, the highest fold change DEG was found to be abnormal spindle-like microcephaly associated (ASPM). The ASPM expression pattern from the database showed that it is highly expressed in GBM tissue, and patients with high expression of ASPM have a poor prognosis. Moreover, ASPM showed aberrantly high expression in GBM cell lines. Loss-of-function assay indicated that ASPM enhances tumorigenesis in GBM cells in vitro. Xenograft growth verified the oncogenic activity of ASPM in vivo. Furthermore, downregulation of ASPM could arrest the cell cycle of GBM cells at the G0/G1 phase and attenuate the Wnt/β-catenin signalling activity in GBM. These data suggest that ASPM may serve as a new target for the therapeutic treatment of GBM.

Abstract CN08-02: BMP signaling in glioblastoma

Abstract Glioma-initiating cells (GICs) play central roles in the initiation, progression, recurrence, tumor heterogeneity and drug resistance of glioblastoma. Bone morphogenetic proteins (BMPs) regulate normal embryonic neural development and exert anti-tumor activities in glioblastoma. However, its mechanisms of action remain to be elucidated. Through RNA-seq analyses, we have identified several BMP-target genes in GICs, including SMAD6, Noggin (NOG), Distal-less homeobox 2 (DLX2), and Paired-related homeobox 1 (PRRX1). DLX2 promotes apoptosis and neural differentiation of GICs. We also show that the expression of PRRX1 is induced by BMP stimulation, and that PRRX1 induces decrease in the CD133-positive GIC population and inhibits in vivotumorigenic activity of GICs. Of the two spliced isoforms of PRRX1, the longer form of PRRX1, but not the shorter form, is involved in the induction of differentiation of GICs through epigenetic regulation of the CD133/PROM1gene. In addition, we show that BMP-4 represses the expression of a transmembrane protein, which is preferentially expressed in mesenchymal subtype of glioblastoma. Potential use of these proteins for diagnosis and treatment of glioblastoma will be discussed. Citation Format: Kohei Miyazono, Ryo Tanabe. BMP signaling in glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN08-02. doi:10.1158/1535-7163.TARG-19-CN08-02

CBMT-35. METABOLIC REWIRING BY ONC201/TIC10 AND 2-DEOXYGLUCOSE HAS SYNERGISTIC ANTI-GLIOBLASTOMA ACTIVITY

Abstract BACKGROUND Metabolic dysregulation is a common feature of cancers such as primary brain malignancies. In this work we examined whether a rewiring of the metabolome by a multi-targeting approach would induce enhanced anti-neoplastic activity in glioblastoma. METHODS Preclinical testing of a combined treatment with ONC201/TIC10 and 2-Deoxyglucose was performed in established and primary cultured glioblastoma cells. Extracellular flux analysis was used to determine real-time effects on OXPHOS (OCR) and glycolysis (ECAR). Expression of respiratory chain complexes was analysed by Western blotting. Biological effects on tumor formation were tested in patient-derived model systems on the chorion allantoic membrane (CAM). Protein array analyses were performed to determine effects on phospho protein kinase expression. RESULTS Treatment with ONC201/TIC10 leads to impaired mitochondrial respiration and a dose-dependent increase of glycolysis. ONC201/TIC10 combined with 2-Deoxyglucose, induces a state of energy deprivation characterized by a significant decrease in ATP levels. On the molecular level, pAMPK α1 was significantly up-regulated while a hypo-phosphorylation signature was noted including mTOR signaling, src family kinases and receptor tyrosine kinases such as EGFR and PDGFR-β. As a result, synergistic anti-proliferative and anti-migratory effects were observed among established and primary cultured glioblastoma cells. In addition, tumor formation on the CAM was significantly impaired following the combination treatment. CONCLUSIONS Simultaneous treatment with ONC201/TIC10 and 2-Deoxyglucose causes a reprogramming of the metabolic circuitry and results in a synergistic anti-glioblastoma activity. Since both agents were tested in clinical trials with good tolerability, and they both penetrate the blood-brain barrier, further clinical evaluation of this therapeutic strategy seems promising.

Role of myeloid cells in the immunosuppressive microenvironment in gliomas.

Glioma is the most common primary brain cancer, and half of patients present a diagnosis of glioblastoma (GBM), its most aggressive and lethal form. Conventional therapies, including surgery, radiotherapy, and chemotherapy, have not resulted in major ameliorations in GBM survival outcome, which remains extremely poor. Recent immunotherapy improvements for other tumors, coupled with growing knowledge of the complex interactions between malignant glioma cells and the immune system, led to an exponential increase in glioma immunotherapy research. However, immunotherapeutic strategies in GBM have not yet reached their full potential, mainly due to the limited understanding of the strong immunosuppressive microenvironment (TME) characterizing this tumor. Glioma-associated macrophages and microglia (GAMs) are key drivers of the local immunosuppression promoting tumor progression and its resistance to immunomodulating therapeutic strategies. Together with other myeloid cells, such as dendritic cells and neutrophils, GAMs actively shape glioma TME, modulate anti-tumoral immune response and support angiogenesis, tumor cell invasion and proliferation. In this review, we discuss the role of myeloid cells in the complex TME of glioma and the available clinical data on therapeutic strategies focusing on approaches that affect myeloid cells activity in GBM.

Open-Label Phase II Evaluation of Imatinib in Primary Inoperable or Incompletely Resected and Recurrent Glioblastoma

Purpose:Preclinical studies indicated that imatinib may have single-agent activity in glioblastoma through inhibition of tyrosine kinase activity and also that it might enhance the efficacy of radiotherapy. We therefore sought to investigate clinical efficacy in patients with newly diagnosed and recurrent glioblastoma in combination with radiotherapy. Methods: We conducted a nonrandomized, 2-arm, open-label phase II trial including patients aged 18 years or older with an ECOG performance status of 0–2 that were either newly diagnosed (arm A) with a measurable tumor (i.e., after incomplete resection or biopsy) or that were diagnosed with progression of a glioblastoma after initial therapy (arm B). Patients in arm A received 600 mg/day imatinib in combination with hypofractionated radiotherapy (2.5 Gy per fraction, 22 fractions). Patients in arm B received 600 mg/day imatinib alone or in combination with re-irradiation at various doses. In case tumor progression occurred, CCNU was added (2 cycles, 100 mg/m²) to imatinib. The primary end point was progression-free survival (PFS). The secondary end point was safety, defined as per Common Terminology Criteria for Adverse Events (version 2.0). Overall survival (OS) was analyzed as an exploratory end point. Results: Fifty-one patients were enrolled, of which 19 were included in arm A and 32 in arm B. The median follow-up was 4 (0.5–30) months in arm A and 6.5 (0.3–51.5) months in arm B. The median PFS was 2.8 months (95% CI 0–8.7) in arm A and 2.1 months (95% CI 0–11.8) in arm B. The median OS was 5.0 (0.8–30) months (95% CI 0–24.1) in arm A and 6.5 (0.3–51.5) months (95% CI 0–32.5) in arm B. The major grade 3 events were seizure (present in 17 patients), pneumonia (11 patients), and vigilance decrease (7 patients). Conclusions: Imatinib showed no measurable activity in patients with newly diagnosed or recurrent glioblastoma.

MicroRNA-1 suppresses glioblastoma in preclinical models by targeting fibronectin.

Glioblastoma (GBM) is a deadly and incurable brain tumor. Although microRNAs (miRNAs) play critical roles in regulating the cancer cell phenotype, the underlying mechanisms of how they regulate tumorigenesis are incompletely understood. We found that miR-1 is expressed at relatively low levels in brain cancer patients, especially GBM. Ectopic miR-1 expression in GBM cells inhibited proliferation and migration, increased sensitivity to apoptosis induced by the DNA alkylating agent temozolomide in vitro, and inhibited GBM tumorigenesis in vivo. Expression of miR-1 in GBM cell lines directly targets fibronectin. High fibronectin expression in GBM correlates with poor patient survival and fibronectin expression is inversely correlated with miR-1 expression. Knockout of fibronectin expression in GBM cell lines inhibited proliferation and migration, increased sensitivity to apoptosis induced by temozolomide in vitro, and markedly suppressed GBM tumor growth and promoted animal survival. In contrast, restoring fibronectin levels in GBM cells ectopically expressing miR-1 increased tumorigenicity and decreased animal survival. Therefore, these results confirm that miR-1 has tumor suppressive activity in GBM by targeting fibronectin, and that the miR-1/fibronectin pathway may be a potential drug target in this devastating cancer.

Abstract 1787: Selective activation of a PI3K catalytic isoform by G protein-coupled receptors in glioblastoma

Abstract Objective: To determine how PI3K catalytic isoforms become dysregulated in glioblastoma. Background: Recurrence in glioblastoma (GBM) is common and even more resistant to treatment than initial GBM. The Sheng Lab has previously identified that PIK3CB expression levels, but not other PI3K subtypes, are higher in recurrent GBM and are associated with GBM recurrence in newly diagnosed individuals. PI3KCB encodes for PI3K catalytic subunit p110β, and PI3K/AKT pathway activation is a well-known promoter of cell survival. Understanding mechanisms of PIK3CB activation in GBM may identify new therapeutic targets and improve GBM survival. Current knowledge of PI3K isoform activity in GBM is insufficient and contradictory. Given previous studies, it was hypothesized that G-Coupled Protein Receptor (GPCR) promoters preferentially activate PI3K/AKT in PIK3CBhigh GBM cells. Further, that knockdown of PIK3CB, but not other PI3K isoforms, inhibits PI3K/AKT reactivation in PIK3CBhigh cell lines. Design/Methods: U87MG cells, an established p110βhigh GBM cell line, were starved overnight in serum free media, and then treated with growth factors, promoters or inhibitors of several previously hypothesized pathways. Cell lysates were collected and PI3K/AKT pathway activity was measured with immunoblotting. The ratio of pAKT/AKT/ACTB quantified PI3K/AKT pathway activity. Results: Overnight serum starvation depleted pAKT, and incubation with 1% FBS restored pAKT signaling in a time dependent manner. Neither EGF nor FGF-2 activated PI3K/AKT signaling. Gefitinib, an EGFR inhibitor, failed to inhibit reactivation when cells were co-treated with 1% FBS. However, GPCR stimulation by Lysophosphatidic acid (LPA) increased pAKT/AKT ratios in a dose dependent manner. More significantly, Gallein, a GPCR inhibitor, suppressed PI3K/AKT signaling when cells were co-treated with 1% FBS. Selective PI3K inhibitors showed little PI3K/AKT reactivation at multiple doses. Conclusions: These results suggest that GPCR signaling is responsible for PI3K/AKT pathway activation in p110βhigh cells. Citation Format: Abigail R. Winn, Zhi Sheng. Selective activation of a PI3K catalytic isoform by G protein-coupled receptors in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1787.

Targeting of CD317 by the immunotoxin HM1.24-ETA’ to allow immunotherapy in glioblastoma patients.

e13560 Background: Glioblastoma is the most common primary malignant brain tumor with a poor prognosis. CD317 (HM1.24) is a transmembrane protein and may exist in differently spliced variants. It is highly expressed on plasma cells in multiple myeloma, as well as in certain solid tumor types. While several antibody drug conjugates are already in clinical practice, small immunotoxins with a different intracellular mode of action are only established in hairy cell leukemia. The immunotoxin HM1.24-ETA’ protein is a CD317 single chain Fv (scFv) antibody fused to a truncated version of Pseudomonas aeruginosa exotoxin A (ETA’). Methods: In vivo CD317 mRNA expression in human glioma of different grades and survival probabilities of glioblastoma patients based on CD317 mRNA expression were analyzed using the database of the Cancer Genome Atlas network (TCGA). CD317 protein expression was analyzed by immunohistochemistry in a human tissue microarray (TMA). In vitro CD317 mRNA expression was assessed by RT-PCR and CD317 protein levels by flow cytometry in several human glioblastoma cell lines. A cytotoxicity assay after treatment with HM1.24-ETA’ immunotoxin was performed in human glioblastoma cell lines. Results: Data on mRNA expression from the TCGA database demonstrated, that CD317 was upregulated in human glioblastomas compared to lower grade gliomas. In the group of glioblastoma patients increased CD317 mRNA expression was associated with decreased probability of survival ( p< 0.001). CD317 protein levels correlated directly with the tumor grade of astrocytic gliomas in the TMA. CD317 was expressed heterogeneously on mRNA and protein levels in the tested cell-lines in vitro. HM1.24-ETA’ induced cytotoxicity in CD317-positive glioblastoma cells in a concentration-dependent manner. Animal experiments currently performed suggest activity in glioblastoma xenografted mice. Conclusions: These data highlight CD317 as an interesting target antigen and HM1.24-ETA’ immunotoxin as a strategy for immunotherapy of glioblastoma patients.

A Role for NF-κB in Organ Specific Cancer and Cancer Stem Cells.

Cancer stem cells (CSCs) account for tumor initiation, invasiveness, metastasis, and recurrence in a broad range of human cancers. Although being a key player in cancer development and progression by stimulating proliferation and metastasis and preventing apoptosis, the role of the transcription factor NF-κB in cancer stem cells is still underestimated. In the present review, we will evaluate the role of NF-κB in CSCs of glioblastoma multiforme, ovarian cancer, multiple myeloma, lung cancer, colon cancer, prostate cancer, as well as cancer of the bone. Next to summarizing current knowledge regarding the presence and contribution of CSCs to the respective types of cancer, we will emphasize NF-κB-mediated signaling pathways directly involved in maintaining characteristics of cancer stem cells associated to tumor progression. Here, we will also focus on the status of NF-κB-activity predominantly in CSC populations and the tumor mass. Genetic alterations leading to NF-κB activity in glioblastoma, ependymoma, and multiple myeloma will be discussed.

Assessment of Glioblastoma Response in the Era of Bevacizumab: Longstanding and Emergent Challenges in the Imaging Evaluation of Pseudoresponse.

Glioblastoma is the deadliest primary malignant brain neoplasm, and despite the availability of many treatment options, its prognosis remains somber. Enhancement detected by magnetic resonance imaging (MRI) was considered the best imaging marker of tumor activity in glioblastoma for decades. However, its role as a surrogate marker of tumor viability has changed with the appearance of new treatment regimens and imaging modalities. The antiangiogenic therapy created an inflection point in the imaging assessment of glioblastoma response in clinical trials and clinical practice. Although BEV led to the improvement of enhancement, it did not necessarily mean tumor response. The decrease in the enhancement intensity represents a change in the permeability properties of the blood brain barrier, and presumably, the switch of the tumor growth pattern to an infiltrative non-enhancing phenotype. New imaging techniques for the assessment of cellularity, blood flow hemodynamics, and biochemistry have emerged to overcome this hurdle; nevertheless, designing tools to assess tumor response more accurately, and in so doing, improve the assessment of response to standard of care (SOC) therapies and to novel therapies, remains challenging.

NMDA Receptor-Mediated Signaling Pathways Enhance Radiation Resistance, Survival and Migration in Glioblastoma Cells-A Potential Target for Adjuvant Radiotherapy.

Glioblastoma is one of the most aggressive malignant brain tumors, with a survival time less than 15 months and characterized by a high radioresistance and the property of infiltrating the brain. Recent data indicate that the malignancy of glioblastomas depends on glutamatergic signaling via ionotropic glutamate receptors. In this study we revealed functional expression of Ca2+-permeable NMDARs in three glioblastoma cell lines. Therefore, we investigated the impact of this receptor on cell survival, migration and DNA double-strand break (DSB) repair in the presence of both, glutamate and NMDAR antagonists, and after clinically relevant doses of ionizing radiation. Our results indicate that treatment with NMDAR antagonists slowed the growth and migration of glutamate-releasing LN229 cells, suggesting that activation of NMDARs facilitate tumor expansion. Furthermore, we found that DSB-repair upon radiation was more effective in the presence of glutamate. In contrast, antagonizing the NMDAR or the Ca2+-dependent transcription factor CREB impaired DSB-repair similarly and resulted in a radiosensitizing effect in LN229 and U-87MG cells, indicating a common link between NMDAR signaling and CREB activity in glioblastoma. Since the FDA-approved NMDAR antagonists memantine and ifenprodil showed differential radiosensitizing effects, these compounds may constitute novel optimizations for therapeutic interventions in glioblastoma.

Chromatin landscapes reveal developmentally encoded transcriptional states that define human glioblastoma.

Glioblastoma is an incurable brain cancer characterized by high genetic and pathological heterogeneity. Here, we mapped active chromatin landscapes with gene expression, whole exomes, copy number profiles, and DNA methylomes across 44 patient-derived glioblastoma stem cells (GSCs), 50 primary tumors, and 10 neural stem cells (NSCs) to identify essential super-enhancer (SE)-associated genes and the core transcription factors that establish SEs and maintain GSC identity. GSCs segregate into two groups dominated by distinct enhancer profiles and unique developmental core transcription factor regulatory programs. Group-specific transcription factors enforce GSC identity; they exhibit higher activity in glioblastomas versus NSCs, are associated with poor clinical outcomes, and are required for glioblastoma growth in vivo. Although transcription factors are commonly considered undruggable, group-specific enhancer regulation of the MAPK/ERK pathway predicts sensitivity to MEK inhibition. These data demonstrate that transcriptional identity can be leveraged to identify novel dependencies and therapeutic approaches.

HPCAL1 promotes glioblastoma proliferation via activation of Wnt/β-catenin signalling pathway.

Glioblastoma (GBM) is the most prevalent primary malignancy of the central nervous system with obvious aggressiveness, and is associated with poor clinical outcome. Studies have indicated that calcium ion (Ca2+) can positively regulate the initiation of malignancy with regard to GBM by modulating quiescence, proliferation, migration and maintenance. Hippocalcin like‐1 protein (HPCAL1) serves as a sensor of Ca2+. However, the understanding of HPCAL1 activity in GBM is limited. The present study revealed that the gene HPCAL1 was up‐regulated by Ca2+ in the tissues and cells of GBM. Ectopic expression of HPCAL1 promoted proliferation of cells. Exhaustion of HPCAL1 inhibited cell growth not only in vivo, but also in vitro. In addition, HPCAL1 enhanced the Wnt pathway by stimulating β‐catenin accumulation and nuclear translocation in GBM cells, while β‐catenin silencing significantly inhibited the proliferation and growth of the GBM cells. Our results showed that Ser9 phosphorylation of GSK3β was significantly decreased after HPCAL1 knockdown in GBM cells, and knockdown of the gene GSK3β in GBM cells enhanced cell proliferation and promoted transcription of the genes CCND1 and c‐Myc. Furthermore, the phosphorylation of ERK was decreased in the cells with HPCAL1 knockdown, while it was promoted via overexpression of HPCAL1. The suppression or depletion of the gene ERK decreased proliferation triggered by overexpression of HPCAL1 and impaired transcription of the genes c‐Myc and CCND1. These studies elucidate the tumour‐promoting activity of HPCAL1. They also offer an innovative therapeutic strategy focusing on the HPCAL1‐Wnt/β‐catenin axis to regulate proliferation and development of GBM.

Statins affect human glioblastoma and other cancers through TGF-β inhibition.

The cholesterol-lowering statins have known anti-cancer effects, but the mechanisms and how to utilize statins in oncology have been unclear. We noted in the CellMiner database that statin activity against cancer lines correlated with higher expression of TGF-β target genes such as SERPINE1 and ZYX. This prompted us to assess whether statins affected TGF-β activity in glioblastoma (GBM), a cancer strongly influenced by TGF-β and in dire need of new therapeutic approaches. We noted that statins reduced TGF-β activity, cell viability and invasiveness, Rho/ROCK activity, phosphorylation and activity of the TGF-β mediator Smad3, and expression of TGF-β targets ZYX and SERPINE1 in GBM and GBM-initiating cell (GIC) lines. Statins were most potent against GBM, GIC, and other cancer cells with high TGF-β activity, and exogenous TGF-β further sensitized mesenchymal GICs to statins. Statin toxicity was rescued by addition of exogenous mevalonolactone or geranylgeranyl pyrophosphate, indicating that the observed effects reflected inhibition of HMG CoA-reductase by the statins. Simvastatin significantly inhibited the growth of subcutaneous GIC grafts and prolonged survival in GIC intracranially grafted mice. These results indicate where the statins might best be applied as adjunct therapies in oncology, against GBM and other cancers with high TGF-β activity, and have implications for other statin roles outside of oncology.

Encapsulation of echinomycin in cyclodextrin inclusion complexes into liposomes: in vitro anti-proliferative and anti-invasive activity in glioblastoma.

Echinomycin, a DNA bis-intercalator peptide, belongs to the family of quinoxaline antibiotics. Echinomycin exhibits potent antitumor and antimicrobial activity. However, it is highly water insoluble and suffers from low bioavailability and unwanted side effects. Therefore, developing new formulations and delivery systems that can enhance echinomycin solubility and therapeutic potency is needed for further clinical application. In this study, echinomycin has been complexed into the hydrophobic cavity of γ-cyclodextrin (γCD) then encapsulated into PEGylated liposomes. The anti-proliferative and anti-invasive effect has been evaluated against U-87 MG glioblastoma cells. Echinomycin-in-γCD inclusion complexes have been characterized by phase solubility assay, TLC, and 1H-NMR. The echinomycin-in-γCD inclusion complexes have been loaded into liposomes using a thin film hydration method to end up with echinomycin-in-γCD-in-liposomes. Drug-loaded liposomes were able to inhibit cell proliferation with IC50 of 1.0 nM. Moreover, echinomycin-in-γCD-in-liposomes were found to inhibit the invasion of U-87 MG cells using the spheroid gel invasion assay. In conclusion, the current work describes for the first time γCD-echinomycin complexes and their encapsulation into PEGylated liposomes.

The Disruption of the β-Catenin/TCF-1/STAT3 Signaling Axis by 4-Acetylantroquinonol B Inhibits the Tumorigenesis and Cancer Stem-Cell-Like Properties of Glioblastoma Cells, In Vitro and In Vivo.

Background: Glioblastoma (GBM), a malignant form of glioma, is characterized by resistance to therapy and poor prognosis. Accumulating evidence shows that the initiation, propagation, and recurrence of GBM is attributable to the presence of GBM stem cells (GBM-CSCs). Experimental approach: Herein, we investigated the effect of 4-Acetylantroquinonol B (4-AAQB), a bioactive isolate of Antrodia cinnamomea, on GBM cell viability, oncogenic, and CSCs-like activities. Results: We observed that aberrant expression of catenin is characteristic of GBM, compared to other glioma types (p = 0.0001, log-rank test = 475.2), and correlates with poor prognosis of GBM patients. Lower grade glioma and glioblastoma patients (n = 1152) with low catenin expression had 25% and 21.5% better overall survival than those with high catenin expression at the 5 and 10-year time-points, respectively (p = 3.57e-11, log-rank test = 43.8). Immunohistochemistry demonstrated that compared with adjacent non-tumor brain tissue, primary and recurrent GBM exhibited enhanced catenin expression (~10-fold, p < 0.001). Western blot analysis showed that 4-AAQB significantly downregulated β-catenin and dysregulated the catenin/LEF1/Stat3 signaling axis in U87MG and DBTRG-05MG cells, dose-dependently. 4-AAQB–induced downregulation of catenin positively correlated with reduced Sox2 and Oct4 nuclear expression in the cells. Furthermore, 4-AAQB markedly reduced the viability of U87MG and DBTRG-05MG cells with 48 h IC50 of 9.2 M and 12.5 M, respectively, effectively inhibited the nuclear catenin, limited the migration and invasion of GBM cells, with concurrent downregulation of catenin, vimentin, and slug; similarly, colony and tumorsphere formation was significantly attenuated with reduced expression of c-Myc and KLF4 proteins. Conclusions: Summarily, we show for the first time that 4-AAQB suppresses the tumor-promoting catenin/LEF1/Stat3 signaling, and inhibited CSCs-induced oncogenic activities in GBM in vitro, with in vivo validation; thus projecting 4-AAQB as a potent therapeutic agent for anti-GBM target therapy.

Icaritin inhibits glioblastoma cell viability and glycolysis by blocking the IL-6/Stat3 pathway.

Glioblastoma (GBM) is a common and aggressive brain tumor that is associated with significant increase in glycolysis for energy production. Icaritin is a natural compound and exhibits anticancer activity in GBM. However, the effect of icaritin on glycolysis in GBM cells remains unclear. The aim of the current study was to investigate the effect of icaritin on glycolysis in GBM cells. The human GBM cell lines U87 and T98G were treated with icaritin or the inhibitor of Stat3 (S3I-201) in the presence or absence of recombinant human interleukin (IL)-6. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. The glycolysis was analyzed by detecting the glucose consumption and lactate production. The Western blot analysis was conducted to detect the expressions of hexokinase 2 (HK2), signal transducer and activator of transcription 3 (Stat3), p-Stat3, and B lymphoma Mo-MLV insertion region 1 (Bmi-1). Results showed that icaritin inhibited the viability of U87 and T98G cells in a dose-dependent manner. The decreased glucose consumption and lactate production, accompanied by reduced expressions of HK2, were found in both U87 and T98G cells. Icaritin inhibited the IL-6/Stat3 pathway, which is evidenced by the decreased expressions of p-Stat3 and Bmi-1. IL-6 treatment induced the phosphorylation of Stat3 and Bmi-1 expression, increased cell viability, as well as elevated glucose consumption, lactate production, and HK2 expression; however, the effects of IL-6 were attenuated by icaritin or S3I-201 treatment. In conclusion, icaritin exerted inhibitory effects on cell viability and glycolysis in GBM cells, which was mediated by the IL-6/Stat3 pathway.