Combined EGFR- and notch inhibition display additive inhibitory effect on glioblastoma cell viability and glioblastoma-induced endothelial cell sprouting in vitro

Mikkel Staberg; Signe Regner Michaelsen; Louise Stobbe Olsen; Mette Kjølhede Nedergaard; Mette Villingshøj; Marie-Thérése Stockhausen; Petra Hamerlik; Hans Skovgaard Poulsen

doi:10.1186/s12935-016-0309-2

Combined EGFR- and notch inhibition display additive inhibitory effect on glioblastoma cell viability and glioblastoma-induced endothelial cell sprouting in vitro

Mikkel Staberg, Signe Regner Michaelsen, Louise Stobbe Olsen, Mette Kjølhede Nedergaard, Mette Villingshøj, Marie-Thérése Stockhausen, Petra Hamerlik, Hans Skovgaard Poulsen

5 Citations (Scopus)

Abstract

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.

Original language	English
Article number	34
Journal	Cancer Cell International
Volume	16
ISSN	1475-2867
DOIs	https://doi.org/10.1186/s12935-016-0309-2
Publication status	Published - 26 Apr 2016

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Staberg, M., Michaelsen, S. R., Olsen, L. S., Nedergaard, M. K., Villingshøj, M., Stockhausen, M-T., Hamerlik, P., & Poulsen, H. S. (2016). Combined EGFR- and notch inhibition display additive inhibitory effect on glioblastoma cell viability and glioblastoma-induced endothelial cell sprouting in vitro. Cancer Cell International, 16, [34]. https://doi.org/10.1186/s12935-016-0309-2

Combined EGFR- and notch inhibition display additive inhibitory effect on glioblastoma cell viability and glioblastoma-induced endothelial cell sprouting in vitro. / Staberg, Mikkel; Michaelsen, Signe Regner; Olsen, Louise Stobbe et al.

In: Cancer Cell International, Vol. 16, 34, 26.04.2016.

Research output: Contribution to journal › Journal article › Research › peer-review

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title = "Combined EGFR- and notch inhibition display additive inhibitory effect on glioblastoma cell viability and glioblastoma-induced endothelial cell sprouting in vitro",

abstract = "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.",

author = "Mikkel Staberg and Michaelsen, {Signe Regner} and Olsen, {Louise Stobbe} and Nedergaard, {Mette Kj{\o}lhede} and Mette Villingsh{\o}j and Marie-Th{\'e}r{\'e}se Stockhausen and Petra Hamerlik and Poulsen, {Hans Skovgaard}",

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doi = "10.1186/s12935-016-0309-2",

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T1 - Combined EGFR- and notch inhibition display additive inhibitory effect on glioblastoma cell viability and glioblastoma-induced endothelial cell sprouting in vitro

AU - Staberg, Mikkel

AU - Michaelsen, Signe Regner

AU - Olsen, Louise Stobbe

AU - Nedergaard, Mette Kjølhede

AU - Villingshøj, Mette

AU - Stockhausen, Marie-Thérése

AU - Hamerlik, Petra

AU - Poulsen, Hans Skovgaard

PY - 2016/4/26

Y1 - 2016/4/26

N2 - 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.

AB - 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.

U2 - 10.1186/s12935-016-0309-2

DO - 10.1186/s12935-016-0309-2

M3 - Journal article

C2 - 27118928

SN - 1475-2867

VL - 16

JO - Cancer Cell International

JF - Cancer Cell International

M1 - 34

ER -

Combined EGFR- and notch inhibition display additive inhibitory effect on glioblastoma cell viability and glioblastoma-induced endothelial cell sprouting in vitro

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