The extracellular matrix protein fibulin-3/EFEMP1 promotes pleural mesothelioma growth by activation of PI3K/Akt signaling.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited therapeutic options. The extracellular matrix protein fibulin-3/EFEMP1 accumulates in the pleural effusions of MPM patients and has been proposed as a prognostic biomarker of these tumors. However, it is entirely unknown whether fibulin-3 plays a functional role on MPM growth and progression. Here, we demonstrate that fibulin-3 is upregulated in MPM tissue, promotes the malignant behavior of MPM cells, and can be targeted to reduce tumor progression. Overexpression of fibulin-3 increased the viability, clonogenic capacity and invasion of mesothelial cells, whereas fibulin-3 knockdown decreased these phenotypic traits as well as chemoresistance in MPM cells. At the molecular level, fibulin-3 activated PI3K/Akt signaling and increased the expression of a PI3K-dependent gene signature associated with cell adhesion, motility, and invasion. These pro-tumoral effects of fibulin-3 on MPM cells were disrupted by PI3K inhibition as well as by a novel, function-blocking, anti-fibulin-3 chimeric antibody. Anti-fibulin-3 antibody therapy tested in two orthotopic models of MPM inhibited fibulin-3 signaling, resulting in decreased tumor cell proliferation, reduced tumor growth, and extended animal survival. Taken together, these results demonstrate for the first time that fibulin-3 is not only a prognostic factor of MPM but also a relevant molecular target in these tumors. Further development of anti-fibulin-3 approaches are proposed to increase early detection and therapeutic impact against MPM.

The scaffolding protein DLG5 promotes glioblastoma growth by controlling Sonic Hedgehog signaling in tumor stem cells.

BACKGROUND: Tumor invasion, a hallmark of malignant gliomas, involves reorganization of cell polarity and changes in the expression and distribution of scaffolding proteins associated with polarity complexes. The scaffolding proteins of the DLG family are usually downregulated in invasive tumors and regarded as tumor suppressors. Despite their important role in regulating neurodevelopmental signaling, the expression and functions of DLG proteins have remained almost entirely unexplored in malignant gliomas. METHODS: Western blot, immunohistochemistry, and analysis of gene expression were used to quantify DLG members in glioma specimens and cancer datasets. Over-expression and knockdown of DLG5, the highest-expressed DLG member in glioblastoma, were used to investigate its effects on tumor stem cells and tumor growth. qRT-PCR, Western blotting, and co-precipitation assays were used to investigate DLG5 signaling mechanisms. RESULTS: DLG5 was upregulated in malignant gliomas compared to other solid tumors, being the predominant DLG member in all glioblastoma molecular subtypes. DLG5 promoted glioblastoma stem cell invasion, viability, and self-renewal. Knockdown of this protein in vivo disrupted tumor formation and extended survival. At the molecular level, DLG5 regulated Sonic Hedgehog (Shh) signaling, making DLG5-deficient cells insensitive to Shh ligand. Loss of DLG5 increased the proteasomal degradation of Gli1, underlying the loss of Shh signaling and tumor stem cell sensitization. CONCLUSIONS: The high expression and pro-tumoral functions of DLG5 in glioblastoma, including its dominant regulation of Shh signaling in tumor stem cells, reveal a novel role for this protein that is strikingly different from its proposed tumor-suppressor role in other solid tumors.

Ko143 Reverses MDR in Glioblastoma via Deactivating P-Glycoprotein, Sensitizing a Resistant Phenotype to TMZ Treatment.

BACKGROUND/AIM: Over-expression of both P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) has been associated with multidrug-resistance in glioblastoma (GBM). Though previously studied broad-spectrum inhibitors of drug efflux pumps have failed to progress in clinical studies due to in vivo toxicity, research into clinically viable targeted inhibitors is needed. This study evaluated the effects of Ko143, a non-toxic analog of fumitremorgin C, on temozolomide (TMZ) efficacy in resistant glioblastoma stem cells. MATERIALS AND METHODS: We used ATP-Glo assay to determine cell viabilities and flow cytometry to perform cell cycle analysis. Comparative gene expression was analysed through RT-qPCR. RESULTS: TMZ IC50 decreased 41.07% (p<0.01) in the resistant phenotype when delivered in combination with Ko143. Additionally, the TMZ-resistant phenotype (GBM146) displayed 44-fold greater P-gp expression than the TMZ-sensitive phenotype (GBM9) (p<0.01), yet a 0.6-fold lower BCRP expression. Ko143 potentiates TMZ efficacy and likely inhibits P-glycoprotein more potently than previously indicated. CONCLUSION: Further development of non-toxic, targeted inhibitors of drug efflux pumps for use in combinatorial chemotherapy may improve glioblastoma patient prognosis.

Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme.

The human genome encodes a variety of poorly understood RNA species that remain challenging to identify using existing genomic tools. We developed chromatin run-on and sequencing (ChRO-seq) to map the location of RNA polymerase for almost any input sample, including samples with degraded RNA that are intractable to RNA sequencing. We used ChRO-seq to map nascent transcription in primary human glioblastoma (GBM) brain tumors. Enhancers identified in primary GBMs resemble open chromatin in the normal human brain. Rare enhancers that are activated in malignant tissue drive regulatory programs similar to the developing nervous system. We identified enhancers that regulate groups of genes that are characteristic of each known GBM subtype and transcription factors that drive them. Finally we discovered a core group of transcription factors that control the expression of genes associated with clinical outcomes. This study characterizes the transcriptional landscape of GBM and introduces ChRO-seq as a method to map regulatory programs that contribute to complex diseases.

Brain repair by hematopoietic growth factors in the subacute phase of traumatic brain injury.

OBJECTIVETraumatic brain injury (TBI) is a major cause of long-term disability and death in young adults. The lack of pharmaceutical therapy for post-acute TBI recovery remains a crucial medical challenge. Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF), which are 2 key hematopoietic growth factors, have shown neuroprotective and neurorestorative effects in experimental stroke. The objective of this study was to determine the therapeutic efficacy of combined treatment (SCF + G-CSF) in subacute TBI.METHODSYoung-adult male C57BL mice were subject to TBI in the cortex of the right hemisphere. After TBI induction, mice were randomly divided into 2 groups: a vehicle control group and an SCF + G-CSF treatment group. Mice without TBI served as sham operative controls. Treatment was initiated 2 weeks after TBI induction. SCF (200 µg/kg) and G-CSF (50 µg/kg) or an equal volume of vehicle solution was subcutaneously injected daily for 7 days. A battery of neurobehavioral tests for evaluation of memory and cognitive function (water maze and novel object recognition tests), anxiety (elevated plus maze test), and motor function (Rota-Rod test) was performed during the period of 2-9 weeks after treatment. Neurodegeneration and dendritic density in both hemispheres were determined through histochemistry and immunohistochemistry at 11 weeks posttreatment.RESULTSWater maze testing showed that TBI-impaired spatial learning and memory was restored by SCF + G-CSF treatment. The findings from the elevated plus maze tests revealed that SCF + G-CSF treatment recovered TBI-caused anxiety and risk-taking behavior. There were no significant differences between the treated and nontreated TBI mice in both the Rota-Rod test and novel object recognition test. In the brain sections, the authors observed that widespread degenerating neurons were significantly increased in both hemispheres in the TBI-vehicle control mice. TBI-induced increases in neurodegeneration were significantly reduced by SCF + G-CSF treatment in the contralateral hemisphere, making it no different from that of the sham controls. Dendritic density in the frontal cortex of the contralateral hemisphere was significantly reduced in the TBI-vehicle control mice, whereas SCF + G-CSF-treated TBI mice showed significant increases of the dendritic density in the same brain region. SCF + G-CSF-treated TBI mice also showed a trend toward increasing dendritic density in the contralateral hippocampus.CONCLUSIONSSCF + G-CSF treatment in the subacute phase of TBI restored TBI-impaired spatial learning and memory, prevented posttraumatic anxiety and risk-taking behavior, inhibited TBI-induced neurodegeneration, and enhanced neural network remodeling. These findings suggest the therapeutic potential of hematopoietic growth factors for brain repair in the subacute phase of TBI.

Development of a Function-Blocking Antibody Against Fibulin-3 as a Targeted Reagent for Glioblastoma.

Purpose: We sought a novel approach against glioblastomas (GBM) focused on targeting signaling molecules localized in the tumor extracellular matrix (ECM). We investigated fibulin-3, a glycoprotein that forms the ECM scaffold of GBMs and promotes tumor progression by driving Notch and NFκB signaling.Experimental Design: We used deletion constructs to identify a key signaling motif of fibulin-3. An mAb (mAb428.2) was generated against this epitope and extensively validated for specific detection of human fibulin-3. mAb428.2 was tested in cultures to measure its inhibitory effect on fibulin-3 signaling. Nude mice carrying subcutaneous and intracranial GBM xenografts were treated with the maximum achievable dose of mAb428.2 to measure target engagement and antitumor efficacy.Results: We identified a critical 23-amino acid sequence of fibulin-3 that activates its signaling mechanisms. mAb428.2 binds to that epitope with nanomolar affinity and blocks the ability of fibulin-3 to activate ADAM17, Notch, and NFκB signaling in GBM cells. mAb428.2 treatment of subcutaneous GBM xenografts inhibited fibulin-3, increased tumor cell apoptosis, and enhanced the infiltration of inflammatory macrophages. The antibody reduced tumor growth and extended survival of mice carrying GBMs as well as other fibulin-3-expressing tumors. Locally infused mAb428.2 showed efficacy against intracranial GBMs, increasing tumor apoptosis and reducing tumor invasion and vascularization, which are enhanced by fibulin-3.Conclusions: To our knowledge, this is the first rationally developed, function-blocking antibody against an ECM target in GBM. Our results offer a proof of principle for using "anti-ECM" strategies toward more efficient targeted therapies for malignant glioma. Clin Cancer Res; 24(4); 821-33. ©2017 AACR.

Bay846, a new irreversible small molecule inhibitor of EGFR and Her2, is highly effective against malignant brain tumor models.

The epidermal growth factor receptor (EGFR) pathway is aberrantly activated in tumors and plays a key role in promoting tumor growth. Small molecule inhibitors which bind reversibly to EGFR have demonstrated limited clinical activity. Thus, there is a continued need to develop novel EGFR inhibitors with improved anti-tumor activity. Bay846 is a newly developed small molecule inhibitor that binds irreversibly to the tyrosine kinase domains of EGFR and Her2. The in vitro and in vivo efficacy of Bay846 was tested using a panel of nine human malignant brain tumor (glioma) models. Lapatinib, a reversible inhibitor of EGFR and Her2, was included for comparison. Six glioma cell lines were sensitive to Bay846 treatment. Bay846 strongly suppressed tumor cell growth in vitro by inducing cell lysis/death rather than cell cycle arrest. Consistent with this, Bay846 had potent anti-tumor activity which led to regressions in tumor size. The active, phosphorylated form of EGFR was reduced by Bay846 treatment in vitro and in tumors. Importantly, the efficacy of Bay846 was significantly greater than lapatinib in all assays. Bay846-sensitivity was associated with expression of a wild-type PTEN in conjunction with high levels of an oncogenic EGFR variant (A289V or EGFRvIII). These studies demonstrate that targeting the EGFR pathway with the irreversible inhibitor Bay846 has great potential to increase the efficacy of this cancer therapy.

Anti-epidermal growth factor receptor monoclonal antibody cetuximab augments radiation effects in glioblastoma multiforme in vitro and in vivo.

OBJECTIVE: Previously, we demonstrated that the anti-epidermal growth factor receptor (EGFR) antibody cetuximab alone was effective against EGFR-amplified glioblastoma multiforme (GBM) cells in vivo and in vitro. The purpose of the present work was to study further the effectiveness of cetuximab as a monotherapy as well as combining it with radiation therapy or chemotherapy. METHODS: EGFR-amplified GBM cells were implanted either in the flanks of nude mice to determine the effectiveness of cetuximab on larger tumor burden or intracranially to assess the ability of cetuximab to cross the blood-brain barrier. Cells were also exposed to cetuximab in combination with radiation in vivo or chemotherapeutic agents in vitro. RESULTS: Increasing tumor burden in the flanks of mice decreased the amount of tumor growth inhibition. For the first two intracranial models using cetuximab for 5 weeks, the treated mice had a significant increase in median survival compared with controls. When cetuximab was given indefinitely, the results were encouraging, with an increase in median survival for the treated group not yet reached but at least 900%. Mice with flank GBM exposed to cetuximab and radiation had a larger increase in median survival than those with either treatment alone. Preliminary in vitro experiments using cetuximab and chemotherapeutic agents showed increased cytotoxicity. CONCLUSION: These results were encouraging, demonstrating the effectiveness of cetuximab against EGFR-amplified GBM. Surprisingly, cetuximab was effective when administered systemically in an intracranial model. Radiation augmented the effect of cetuximab on GBM in vitro and in vivo. In vitro analysis demonstrated additive effects for chemotherapeutic agents as well. These results confirm EGFR blockade with cetuximab as a potential treatment against human GBM.

Activity of anti-epidermal growth factor receptor monoclonal antibody C225 against glioblastoma multiforme.

OBJECTIVE: Overexpression of epidermal growth factor receptor (EGFR) in glioblastoma multiforme (GBM) secondary to EGFR gene amplification is associated with a more aggressive tumor phenotype and a worse clinical outcome. The purpose of this study was to analyze whether blocking this receptor with the anti-EGFR chimeric monoclonal antibody C225 would decrease proliferation and increase apoptosis in GBM cells. METHODS: EGFR expression and amplification were analyzed for seven human GBM cell lines. These lines were then exposed to different concentrations of C225 for 48 hours, 72 hours, and 7 days, after which time cytotoxicity, apoptosis, and vascular endothelial growth factor expression were assessed in vitro. Two EGFR-amplified human GBM were implanted in the flanks of nude mice, and the animals received C225 twice per week intraperitoneally for 5 weeks. Tumor volumes and survival times were compared with those of sham-treated mice. RESULTS: EGFR gene amplification was demonstrated in three of the primary GBM lines. C225 treatment produced significant cytotoxicity in all three EGFR-amplified GBM lines, but not in unamplified lines. Flow cytometry demonstrated increased apoptosis in C225-treated, EGFR-amplified GBM lines, but not in unamplified lines. There was a decrease in vascular endothelial growth factor expression in all GBM lines with exposure to C225. Tumor-bearing mice treated with C225 experienced significant inhibition of tumor growth as well as a 200% increase in median survival. CONCLUSION: Blocking EGFR in GBM cells that overexpress this receptor significantly changes tumor cell biology by promoting apoptosis while decreasing proliferation and vascular endothelial growth factor expression. This approach holds great promise for the treatment of patients with GBMs.

Apoptosis and survival in high-grade astrocytomas as related to tumor Fas (APO-1/CD95) expression.

Although a majority of high-grade gliomas express the apoptosis-inducing receptor Fas, little is known about the extent of apoptosis or prognostic significance of Fas expression in these tumors. In situ labeling of apoptotic cells and Ki-67 immunohistochemistry were performed on 51 high-grade human astrocytomas previously characterized for Fas expression. Survival data was compiled from patient records and correlated with tumor grade, apoptotic index (AI) and Fas expression. A significant correlation was found between tumor grade and the AI and Ki-67 labeling index (LI); however, only the AI increased significantly with Fas expression. The AI increased from 0.39 +/- 0.12% to 0.82 +/- 0.10% in grade III vs. IV astrocytomas (P = 0.003). The Ki-67-LI increased from 3.64 +/- 1.5% to 11.35 +/- 2.1% in grade III vs. IV astrocytomas (P = 0.004). Additionally, tumors expressing higher Fas levels had a greater AI than those expressing lower levels (0.81 +/- 0.11% vs. 0.43 +/- 0.11%) (P = 0.017). Despite longer median survivals for patients with tumors exhibiting high Fas expression, statistical significance was not achieved. Patients with grade III astrocytomas demonstrated a median survival of 20 vs. 18 months for tumors with high vs. low Fas expression (P = 0.51). Patients with grade IV astrocytomas demonstrated a median survival of 9 vs. 7.4 months for tumors with high vs. low Fas expression, respectively (P = 0.77). Although the degree of Fas expression in high-grade astrocytomas appears to correlate with the apoptotic rate, no overall differences in survival could be demonstrated between tumors expressing high vs. low Fas levels.