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Hans Skovgaard Poulsen,1,2,* Thomas Urup,1,2,* Signe Regner Michaelsen,1,2 Mikkel Staberg,1,2 Mette Villingshøj,1,2 Ulrik Lassen1–3 1Department of Radiation Biology, 2Department of Oncology, 3Phase I Unit, The Finsencenter, Copenhagen University Hospital, Copenhagen, Denmark *These authors contributed equally to this work Abstract: Glioblastoma multiforme (GBM) remains one of the most devastating tumors, and patients have a median survival of 15 months despite aggressive local and systemic therapy, including maximal surgical resection, radiation therapy, and concomitant and adjuvant temozolomide. The purpose of antineoplastic treatment is therefore to prolong life, with a maintenance or improvement of quality of life. GBM is a highly vascular tumor and overexpresses the vascular endothelial growth factor A, which promotes angiogenesis. Preclinical data have suggested that anti-angiogenic treatment efficiently inhibits tumor growth. Bevacizumab is a humanized monoclonal antibody against vascular endothelial growth factor A, and treatment has shown impressive response rates in recurrent GBM. In addition, it has been shown that response is correlated to prolonged survival and improved quality of life. Several investigations in newly diagnosed GBM patients have been performed during recent years to test the hypothesis that newly diagnosed GBM patients should be treated with standard multimodality treatment, in combination with bevacizumab, in order to prolong life and maintain or improve quality of life. The results of these studies along with relevant preclinical data will be described, and pitfalls in clinical and paraclinical endpoints will be discussed. Keywords: primary treatment, VEGF, quality of life, monoclonal antibody, patient survival, vascular tumor

Background: Glioblastoma ranks among the most lethal cancers, with current therapies offering only palliation. Paracrine vascular endothelial growth factor (VEGF) signaling has been targeted using anti-angiogenic agents, whereas autocrine VEGF/VEGF receptor 2 (VEGFR2) signaling is poorly understood. Bevacizumab resistance of VEGFR2-expressing glioblastoma cells prompted interrogation of autocrine VEGF-C/VEGFR2 signaling in glioblastoma., Methods: Autocrine VEGF-C/VEGFR2 signaling was functionally investigated using RNA interference and exogenous ligands in patient-derived xenograft lines and primary glioblastoma cell cultures in vitro and in vivo. VEGF-C expression and interaction with VEGFR2 in a matched pre- and post-bevacizumab treatment cohort were analyzed by immunohistochemistry and proximity ligation assay., Results: VEGF-C was expressed by patient-derived xenograft glioblastoma lines, primary cells, and matched surgical specimens before and after bevacizumab treatment. VEGF-C activated autocrine VEGFR2 signaling to promote cell survival, whereas targeting VEGF-C expression reprogrammed cellular transcription to attenuate survival and cell cycle progression. Supporting potential translational significance, targeting VEGF-C impaired tumor growth in vivo, with superiority to bevacizumab treatment., Conclusions: Our results demonstrate VEGF-C serves as both a paracrine and an autocrine pro-survival cytokine in glioblastoma, promoting tumor cell survival and tumorigenesis. VEGF-C permits sustained VEGFR2 activation and tumor growth, where its inhibition appears superior to bevacizumab therapy in improving tumor control.

Glioblastoma (GBM) ranks among the most lethal cancers, with current therapies offering only palliation. Inter- and intrapatient heterogeneity is a hallmark of GBM, with epigenetically distinct cancer stem-like cells (CSCs) at the apex. Targeting GSCs remains a challenging task because of their unique biology, resemblance to normal neural stem/progenitor cells, and resistance to standard cytotoxic therapy. Here, we find that the chromatin regulator, JmjC domain histone H3K36me2/me1 demethylase KDM2B, is highly expressed in glioblastoma surgical specimens compared to normal brain. Targeting KDM2B function genetically or pharmacologically impaired the survival of patient-derived primary glioblastoma cells through the induction of DNA damage and apoptosis, sensitizing them to chemotherapy. KDM2B loss decreased the GSC pool, which was potentiated by coadministration of chemotherapy. Collectively, our results demonstrate KDM2B is crucial for glioblastoma maintenance, with inhibition causing loss of GSC survival, genomic stability, and chemoresistance., (© 2018 Danish Cancer Society. Published by FEBS Press and John Wiley & Sons Ltd.)

Purpose: Glioblastoma (GBM) ranks among the deadliest solid cancers worldwide and its prognosis has remained dismal, despite the use of aggressive chemo-irradiation treatment regimens. Limited drug delivery into the brain parenchyma and frequent resistance to currently available therapies are problems that call for a prompt development of novel therapeutic strategies. While only displaying modest efficacies as mono-therapy in pre-clinical settings, histone deacetylase inhibitors (HDACi) have shown promising sensitizing effects to a number of cytotoxic agents. Here, we sought to investigate the sensitizing effect of the HDACi trichostatin A (TSA) to the alkylating agent lomustine (CCNU), which is used in the clinic for the treatment of GBM., Methods: Twelve primary GBM cell cultures grown as neurospheres were used in this study, as well as one established GBM-derived cell line (U87 MG). Histone deacetylase (HDAC) expression levels were determined using quantitative real-time PCR and Western blotting. The efficacy of either CCNU alone or its combination with TSA was assessed using various assays, i.e., cell viability assays (MTT), cell cycle assays (flow cytometry, FACS), double-strand DNA break (DSB) quantification assays (microscopy/immunofluorescence) and expression profiling assays of proteins involved in apoptosis and cell stress (Western blotting and protein array)., Results: We found that the HDAC1, 3 and 6 expression levels were significantly increased in GBM samples compared to non-neoplastic brain control samples. Additionally, we found that pre-treatment of GBM cells with TSA resulted in an enhancement of their sensitivity to CCNU, possibly via the accumulation of DSBs, decreased cell proliferation and viability rates, and an increased apoptotic rate., Conclusion: From our data we conclude that the combined administration of TSA and CCNU eradicates GBM cells with a higher efficacy than either drug alone, thereby opening a novel avenue for the treatment of GBM.

Background: For Glioblastoma (GBM) patients, a number of anti-neoplastic strategies using specifically targeting drugs have been tested; however, the effects on survival have been limited. One explanation could be treatment resistance due to redundant signaling pathways, which substantiates the need for combination therapies. In GBM, both the epidermal growth factor receptor (EGFR) and the notch signaling pathways are often deregulated and linked to cellular growth, invasion and angiogenesis. Several studies have confirmed cross-talk and co-dependence of these pathways. Therefore, this study aimed at testing a combination treatment strategy using inhibitors targeting the notch and EGFR pathways., Methods: For evaluation of cell viability a standard MTT assay was used. Western blotting (WB) and Q-RT-PCR were employed in order to assess the protein- and mRNA expression levels, respectively. In order to determine angiogenic processes, we used an endothelial spheroid sprouting assay. For assessment of secreted VEGF from GBM cells we performed a VEGF-quantikine ELISA., Results: GBM cells were confirmed to express EGFR and Notch and to have the capacity to induce endothelial cell sprouting. Inhibition of EGFR and Notch signaling was achieved using either Iressa (gefitinib) or the gamma-secretase inhibitor DAPT. Our data showed that DAPT combined with Iressa treatment displayed increased inhibitory effect on cell viability and abrogated expression and activation of major pro-survival pathways. Similarly, the combinational treatment significantly increased abrogation of GBM-induced endothelial cell sprouting suggesting reduced GBM angiogenesis., Conclusion: This study finds that simultaneous targeting of notch and EGFR signaling leads to enhanced inhibitory effects on GBM-induced angiogenesis and cell viability, thereby stressing the importance of further evaluation of this targeting approach in a clinical setting.

Background: Brain cancer stem-like cells (bCSC) are cancer cells with neural stem cell (NSC)-like properties found in the devastating brain tumor glioblastoma multiforme (GBM). bCSC are proposed a central role in tumor initiation, progression, treatment resistance and relapse and as such present a promising target in GBM research. The Notch signaling pathway is often deregulated in GBM and we have previously characterized GBM-derived bCSC cultures based on their expression of the Notch-1 receptor and found that it could be used as predictive marker for the effect of Notch inhibition. The aim of the present project was therefore to further elucidate the significance of Notch pathway activity for the tumorigenic properties of GBM-derived bCSC., Methods: Human-derived GBM xenograft cells previously established as NSC-like neurosphere cultures were used. Notch inhibition was accomplished by exposing the cells to the gamma-secretase inhibitor DAPT prior to gene expression analysis and intracranial injection into immunocompromised mice., Results: By analyzing the expression of several Notch pathway components, we found that the cultures indeed displayed different Notch pathway signatures. However, when DAPT-treated neurosphere cells were injected into the brain of immunocompromised mice, no increase in survival was obtained regardless of Notch pathway signature and Notch inhibition. We did however observe a decrease in the expression of the stem cell marker Nestin, an increase in the proliferative marker Ki-67 and an increased number of abnormal vessels in tumors formed from DAPT-treated, high Notch-1 expressing cultures, when compared with the control., Conclusion: Based on the presented results we propose that Notch inhibition partly induces differentiation of bCSC, and selects for a cell type that more strongly induces angiogenesis if the treatment is not sustained. However, this more differentiated cell type might prove to be more sensitive to conventional therapies.

Background: Brain cancer stem-like cells (bCSC) are cancer cells with neural stem cell (NSC)-like properties found in glioblastoma multiforme (GBM) and they are assigned a central role in tumor initiation, progression and relapse. The Notch pathway is important for maintenance and cell fate decisions in the normal NSC population. Notch signaling is often deregulated in GBM and recent results suggest that this pathway plays a significant role in bCSC as well. We therefore wished to further elucidate the role of Notch activation in GBM-derived bCSC., Methods: Human-derived GBM xenograft cells were cultured as NSC-like neurosphere cultures. Notch modulation was accomplished either by blocking the pathway using the γ-secretase inhibitor DAPT or by activating it by transfecting the cells with the constitutive active Notch-1 receptor., Results: GBM neurosphere cultures with high endogenous Notch activation displayed sensitivity toward Notch inhibition with regard to tumorigenic features as demonstrated by increased G0/G1 population and reduced colony formation capacity. Of the NSC-like characteristics, only the primary sphere forming potential was affected, while no effect was observed on self-renewal or differentiation. In contrast, when Notch signaling was activated a decrease in the G0/G1 population and an enhanced capability of colony formation was observed, along with increased self-renewal and de-differentiation., Conclusion: Based on the presented results we propose that active Notch signaling plays a role for cell growth and stem cell-like features in GBM neurosphere cultures and that Notch-targeted anti-bCSC treatment could be feasible for GBM patients with high endogenous Notch pathway activation.

Glioblastoma multiforme (GBM) is the most common, and most aggressive primary brain tumor among adults. A vast majority of the tumors express high levels of the epidermal growth factor receptor (EGFR) as a consequence of gene amplification. Furthermore, gene amplification is often associated with mutation of EGFR, and the constitutive activated deletion variant EGFRvIII is the most common EGFR mutation found in GBM. Activated EGFR signaling, through overexpression and/or mutation, is involved in increased tumorigenic potential. As such, EGFR is an attractive target for GBM therapy. However, clinical studies with EGFR inhibitors have shown inconsistent results, and as such, further knowledge regarding the role of EGFR and EGFRvIII in GBM is needed. For this, an appropriate in vivo/in vitro tumor model is required. Here, we report the establishment of an experimental GBM model in which the expressions of EGFR and EGFRvIII are maintained both in xenograft tumors growing subcutaneously on mice and in cell cultures established in stem cell conditions. With this model it will be possible to further study the role of EGFR and EGFRvIII, and response to targeted therapy, in GBM., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We hypothesized that co-targeting the epidermal growth factor receptor (EGFR) and Src with the EGFR inhibitor gefitinib and the Src inhibitor AZD0530 would increase growth inhibition and impede migration. Cells overexpressing EGFR were more sensitive to gefitinib than cells expressing mutated EGFR or normal levels of wild-type EGFR. Furthermore, cells with mutated EGFR responded to low doses of gefitinib with increased proliferation. AZD0530 was an effective inhibitor of proliferation and migration, irrespective of EGFR status. These results suggest that co-targeting EGFR and Src might be a valuable treatment approach for malignancies associated with altered expression of EGFR, EGFRvIII, and/or Src.

Recently, we reported that epidermal growth factor receptor (EGFR) induce expression of a module of genes known to be inducible by interferons and particularly interferon-gamma. Here we show that the module is tightly regulated by EGFR in the 2 human cancer cell lines that overexpress EGFR, A431 and HN5. The module of genes included the tumor suppressor IRF-1, which was used as a prototypical member to further investigate the regulation and function of the module. Ligand-activated EGFR induce expression of IRF-1 via phosphorylation of STAT1 and STAT3. In contrast, cells expressing the constitutively active cancer specific receptor EGFRvIII are unable to mediate phosphorylation of these STATs and thereby incapable of inducing IRF-1. We also demonstrate that IRF-1 is expressed in an EGF dose-dependent manner, which correlates with inhibition of cell proliferation, and that the regulation of IRF-1 is partially dependent on intracellular Src family kinase activity. Treatment with the dual specific Abl/c-Src kinase inhibitor AZD0530 significantly reduces the growth inhibitory effect of high EGF concentrations, signifying that EGFR induced IRF-1 is responsible for the observed growth inhibition. In addition, we show that media from these EGF treated cancer cells upregulate the activation marker CD69 on both B-cells and T-cells in peripheral blood. Taken together, these results suggest that cells acquiring sustained high activity of oncogenes such as EGFR are able to activate genes, whose products mediate growth arrest and activate immune effector cells, and which potentially could be involved in alerting the immune system in vivo leading to elimination of the transformed cells., (Copyright 2007 Wiley-Liss, Inc.)

Background: Epidermal growth factor receptor (EGFR) analysis by traditional immunohistochemistry does not provide clinicians with a reliable tool for the selection of patients to EGFR-targeted treatment in colorectal cancer (CRC). Alternative methods and further understanding of the EGFR signaling network are being investigated and mutations in the EGFR gene have been identified. The type III epidermal growth factor receptor, a tumour-specific, ligand independent, constitutively activated form of EGFR, might contribute to the malignant phenotype in CRC and may be a potential target for anticancer therapy. The aim of the present study was to investigate the presence of EGFRvIII in CRC by PCR and protein analysis., Materials and Methods: The study included 79 colorectal cancer patients for PCR analysis and 50 patients for protein analysis by Western blots, in two different laboratories., Results: No type III mutations were detected in our material., Conclusion: The EGFRvIII mutations are rare in colorectal adenocarcinomas and overall probability does not appear to contribute to the malignant phenotype of this disease.

We examined whether changes in the frequency of contact with the social network (partner, children, grandchildren, other relatives, friends and colleagues) in a group of colorectal cancer patients were associated with survival. The study was based on a questionnaire survey from 1991. The study population consisted of 770 Danish colorectal cancer patients diagnosed in the period 1985-1990, and followed up for survival until March 2002. Using Cox regression analysis we found a significantly higher mortality among patients who had lost their partner before the operation compared to patients cohabiting with the same partner as before the operation (rate ratio (RR)=1.4, 95% confidence interval (CI): 1.1-1.8) possibly because of less active cancer treatment. This needs investigation in future studies. Contrary to expectations we also found a significantly higher mortality among patients reporting increased contact with their children compared to patients reporting unchanged contact frequency (RR=1.7, 95% CI: 1.3-2.4). However, a low physical functioning of the cancer patient may have confounded the latter result.

Overexpression or expression of activating mutations of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. The present study employed Affymetrix oligonucleotide arrays to profile genes induced by ligand-activated EGFR with the receptor either moderately expressed or overexpressed at an in-itself transforming level. These changes were compared to those induced by the naturally occurring constitutively active variant EGFRvIII. This study provides novel insight on the activities and mechanisms of EGFRvIII and EGFR mediated transformation, as genes encoding proteins with functions in promoting cell proliferation, invasion, antiapoptosis, and angiogenesis featured prominently in the EGFRvIII- and EGFR-expressing cells. Surprisingly, it was found that ligand-activated EGFR induced the expression of a large group of genes known to be inducible by interferons. Expression of this module was absent in the EGFRvIII-expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor-mediated events. Furthermore, activation of this module correlated with activation of STAT1 and STAT3. The results thus demonstrate that ligand-activated EGFR at different expression levels results in different kinetics of signaling and induction of gene expression. In addition, the constitutively active variant EGFRvIII seems to activate only a subset of signal pathways and induce a subset of genes as compared to the ligand-activated EGFR., (Copyright (c) 2005 Wiley-Liss, Inc.)