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.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.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.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.

Viability assay

Sprouting

10.1186/s12935-016-0309-2

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Level of Notch activation determines the effect on growth and stem cell-like features in glioblastoma multiforme neurosphere cultures

Cancer Biology & Therapy (2013)

Karina Kristoffersen Mette Villingshøj Hans Skovgaard Poulsen Marie‐Thérése Stockhausen

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.

Neurosphere

Notch 1

10.4161/cbt.24595

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Citations (39)

Ubiquilin-1 is a novel HASH-1-complexing protein that regulates levels of neuronal bHLH transcription factors in human neuroblastoma cells

International Journal of Oncology (2004)

Paula Persson Marie‐Thérése Stockhausen Sven Påhlman Håkan Axelson

The basic helix-loop-helix (bHLH) transcription factor mammalian achaete-scute homologue-1 (MASH-1 in mouse and HASH-1 in humans) is expressed in specific subsets of embryonic neuronal precursors of both the peripheral and central nervous systems. This gene is essential for development of olfactory and most peripheral autonomic neurons. Neuro-blastoma is a pediatric malignancy derived from sympathetic nervous system precursors and HASH-1 is expressed in a majority of neuroblastoma tumors and cell lines, indicating the immature phenotype of these cells. Using a human neuroblastoma cDNA library and the yeast two-hybrid system to identify novel HASH-1-interacting proteins, we isolated ubiquilin-1 (DA41, hPLIC-1), a gene that contains multiple ubiquitin-related domains. Further analyses showed that ubiquilin-1 interacts not only with HASH-1, but also with other tissue-specific bHLH proteins, including HES-1. Overexpression of ubiquilin-1 led to accumulation of HASH-1 and HES-1. Moreover, ubiquilin-1 was translocated from the cytoplasm to the nucleus upon co-expression with HASH-1. These results indicate that ubiquilin-1 plays an active role in the precise regulation of HASH-1 and of other tissue-specific bHLH proteins.

10.3892/ijo.25.5.1213

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Maintenance of EGFR and EGFRvIII expressions in an in vivo and in vitro model of human glioblastoma multiforme

Experimental Cell Research (2011)

Marie‐Thérése Stockhausen Helle Broholm Mette Villingshøj Maria Kirchhoff Tommy Gerdes

EGFR Inhibitors

10.1016/j.yexcr.2011.04.001

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Citations (49)

Cell adhesion and EGFR activation regulate EphA2 expression in cancer

Cellular Signalling (2009)

Alice Bjerregaard Larsen Marie‐Thérése Stockhausen Hans Skovgaard Poulsen

EPH receptor A2

10.1016/j.cellsig.2009.11.018

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Citations (55)

36The function of normal and mutated epidermal growth factor receptors in Glioblastoma Multiforme - Establishment of anin vivoandin vitromodel

Apmis (2008)

Marie‐Thérése Stockhausen Mette Villingshøj Mogens Spang‐Thomsen Helle Broholm Michael Kosteljanetz

10.1111/j.1600-0463.2008.001165_45.x

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Evaluation of 4-[18F]fluorobenzoyl-FALGEA-NH2 as a positron emission tomography tracer for epidermal growth factor receptor mutation variant III imaging in cancer

Nuclear Medicine and Biology (2011)

Charlotte Lund Denholt Tina Binderup Marie‐Thérése Stockhausen Hans Skovgaard Poulsen Mogens Spang‐Thomsen

Radiosynthesis

Radioligand

10.1016/j.nucmedbio.2010.11.003

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Differentiation of glioblastoma multiforme stem-like cells leads to downregulation of EGFR and EGFRvIII and decreased tumorigenic and stem-like cell potential

Cancer Biology & Therapy (2013)

Marie‐Thérése Stockhausen Karina Kristoffersen Louise Stobbe Hans Skovgaard Poulsen

Glioblastoma multiforme (GBM) is the most common and devastating primary brain tumor among adults. Despite recent treatment progress, most patients succumb to their disease within 2 years of diagnosis. Current research has highlighted the importance of a subpopulation of cells, assigned brain cancer stem-like cells (bCSC), to play a pivotal role in GBM malignancy. bCSC are identified by their resemblance to normal neural stem cells (NSC), and it is speculated that the bCSC have to be targeted in order to improve treatment outcome for GBM patients. One hallmark of GBM is aberrant expression and activation of the epidermal growth factor receptor (EGFR) and expression of a deletion variant EGFRvIII. In the normal brain, EGFR is expressed in neurogenic areas where also NSC are located and it has been shown that EGFR is involved in regulation of NSC proliferation, migration, and differentiation. This led us to speculate if EGFR and EGFRvIII are involved in the regulation of bCSC. In this study we use GBM neurosphere cultures, known to preserve bCSC features. We demonstrate that EGFR and EGFRvIII are downregulated upon differentiation and moreover that when EGFR signaling is abrogated, differentiation is induced. Furthermore, we show that differentiation leads to decreased tumorigenic and stem cell-like potential of the neurosphere cultures and that by specifically inhibiting EGFR signaling it is possible to target the bCSC population. Our results suggest that differentiation therapy, possibly along with anti-EGFR treatment would be a feasible treatment option for patients with GBM, by targeting the bCSC population.

Neurosphere

10.4161/cbt.26736

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Citations (33)