Inhibition of the PI3K but not the MEK/ERK pathway sensitizes human glioma cells to alkylating drugs

Bodo Haas,Christina Keksel,Marco Timmer,Veronika Klinger,Daniela Kiefer,Maria Wos-Maganga,Julia Walther,Julia Caspers,Gabriele Röhn,Niels Eckstein,Sandra Weickhardt,Roland Frötschl,Verena Bonigut

doi:10.1186/s12935-018-0565-4

Abstract

BackgroundIntrinsic chemoresistance of glioblastoma (GBM) is frequently owed to activation of the PI3K and MEK/ERK pathways. These signaling cascades are tightly interconnected however the quantitative contribution of both to intrinsic resistance is still not clear. Here, we aimed at determining the activation status of these pathways in human GBM biopsies and cells and investigating the quantitative impact of both pathways to chemoresistance.MethodsReceptor tyrosine kinase (RTK) pathways in temozolomide (TMZ) treatment naive or TMZ resistant human GBM biopsies and GBM cells were investigated by proteome profiling and immunoblotting of a subset of proteins. Resistance to drugs and RTK pathway inhibitors was assessed by MTT assays. Apoptotic rates were determined by Annexin V staining and DNA damage with comet assays and immunoblotting.ResultsWe analyzed activation of RTK pathways by proteome profiling of tumor samples of patients which were diagnosed a secondary GBM and underwent surgery and patients which underwent a second surgery after TMZ treatment due to recurrence of the tumor. We observed substantial activation of the PI3K and MEK/ERK pathways in both groups. However, AKT and CREB phosphorylation was reduced in biopsies of resistant tumors while ERK phosphorylation remained unchanged. Subsequent proteome profiling revealed that multiple RTKs and downstream targets are also activated in three GBM cell lines. We then systematically describe a mechanism of resistance of GBM cell lines and human primary GBM cells to the alkylating drugs TMZ and cisplatin. No specific inhibitor of the upstream RTKs sensitized cells to drug treatment. In contrast, we were able to restore sensitivity to TMZ and cisplatin by inhibiting PI3K in all cell lines and in human primary GBM cells. Interestingly, an opposite effect was observed when we inhibited the MEK/ERK signaling cascade with two different inhibitors.ConclusionsTemozolomide treatment naive and TMZ resistant GBM biopsies show a distinct activation pattern of the MEK/ERK and PI3K signaling cascades indicating a role of these pathways in resistance development. Both pathways are also activated in GBM cell lines, however, only the PI3K pathway seems to play a crucial role in resistance to alkylating agents and might serve as drug target for chemosensitization.

Highlights

Intrinsic chemoresistance of glioblastoma (GBM) is frequently owed to activation of the phosphatidylinositide 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK)/ extracellular signal-regulated kinase (ERK) pathways
We investigate the activation status of receptor tyrosine kinase (RTK) pathways in GBM tumor biopsies and GBM cell lines and subsequently elaborate the molecular mechanism of resistance to the DNA-alkylating anti-cancer drugs cisplatin and TMZ in three phosphatase and tensin homolog (PTEN) mutated GBM cell lines and primary GBM cells derived from tumor biopsies
In order to investigate the activation of RTK pathways in vivo, we performed human proteome profiler arrays with a subset of proteins of tumor biopsies of four patients, which were diagnosed a secondary GBM and underwent surgery before TMZ treatment and of four patients which underwent a second surgery later on after TMZ treatment due to recurrence of the tumor

Summary

Introduction

Intrinsic chemoresistance of glioblastoma (GBM) is frequently owed to activation of the PI3K and MEK/ ERK pathways. IDH1 mutations have been shown to be an independent positive prognostic marker of patient survival [1, 6, 7] Another important predictor of response to therapy involves O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation. (iii) GBM cells are intrinsically resistant to chemo- and radiotherapy [11] This may be owed to frequently mutated tumor suppressor genes known to mediate chemoresistance after loss of function like P53, retinoblastoma protein (RB) and phosphatase and tensin homolog (PTEN) and alterations in receptor tyrosine kinase (RTK) signaling pathways [12, 13]. Only one chemotherapy-regime results in a significant overall survival benefit: adjuvant temozolomide (TMZ) following radiotherapy [3, 4]

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