Inhibition of phosphatidylinositol 3-kinase by PX-866 suppresses temozolomide-induced autophagy and promotes apoptosis in glioblastoma cells

Bryan G Harder,Joseph C Loftus,Nhan L Tran,Andrej M Sodoma,Christopher P Sereduk,Sen Peng,Jann N Sarkaria,Gaspar J Kitange

doi:10.1186/s10020-019-0116-z

Bryan G Harder, Joseph C Loftus + Show 6 more

Open Access

https://doi.org/10.1186/s10020-019-0116-z

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Abstract

BackgroundTemozolomide (TMZ) is the most commonly used chemotherapeutic agent used to treat glioblastoma (GBM), which causes significant DNA damage to highly proliferative cells. Our observations have added to accumulating evidence that TMZ induces stress-responsive cellular programs known to promote cell survival, including autophagy. As such, targeting these survival pathways may represent new vulnerabilities of GBM after treatment with TMZ.MethodsUsing the T98G human glioma cell line, we assessed the molecular signaling associated with TMZ treatment, the cellular consequences of using the pan-PI3K inhibitor PX-866, and performed clonogenic assays to determine the effect sequential treatment of TMZ and PX-866 had on colony formation. Additionally, we also use subcutaneous GBM patient derived xenograft (PDX) tumors to show relative LC3 protein expression and correlations between survival pathways and molecular markers which dictate clinical responsiveness to TMZ.ResultsHere, we report that TMZ can induce autophagic flux in T98G glioma cells. GBM patient-derived xenograft (PDX) tumors treated with TMZ also display an increase in the autophagosome marker LC3 II. Additionally, O6-methylguanine-DNA-methyltransferase (MGMT) expression correlates with PI3K/AKT activity, suggesting that patients with inherent resistance to TMZ (MGMT-high) would benefit from PI3K/AKT inhibitors in addition to TMZ. Accordingly, we have identified that the blood-brain barrier (BBB) penetrant pan-PI3K inhibitor, PX-866, is an early-stage inhibitor of autophagic flux, while maintaining its ability to inhibit PI3K/AKT signaling in glioma cells. Lastly, due to the induction of autophagic flux by TMZ, we provide evidence for sequential treatment of TMZ followed by PX-866, rather than combined co-treatment, as a means to shut down autophagy-induced survival in GBM cells and to enhance apoptosis.ConclusionsThe understanding of how TMZ induces survival pathways, such as autophagy, may offer new therapeutic vulnerabilities and opportunities to use sequential inhibition of alternate pro-survival pathways that regulate autophagy. As such, identification of additional ways to inhibit TMZ-induced autophagy could enhance the efficacy of TMZ.

Highlights

Glioblastoma (GBM) is the most common and deadly primary adult brain tumor in adults with a median survival time of about 15 months from diagnosis (Thomas et al 2014; Siegel et al 2018)
Temozolomide induces autophagic flux in glioma cells Previous research has shown that treatment with TMZ has an effect on autophagy in various GBM cell lines
To verify autophagy flux is induced by TMZ, we measured the protein level of p62, a receptor for cargo destined to be degraded by autophagy, and itself is degraded upon autophagy induction

Summary

Introduction

Glioblastoma (GBM) is the most common and deadly primary adult brain tumor in adults with a median survival time of about 15 months from diagnosis (Thomas et al 2014; Siegel et al 2018) Numerous factors such tumor heterogeneity, invasiveness, and the blood-brain barrier (BBB) make treating this disease extremely challenging without compromising healthy brain tissue (Sottoriva et al 2013; Patel et al 2014; Xie et al 2014; van Tellingen et al 2015). Our observations have added to accumulating evidence that TMZ induces stress-responsive cellular programs known to promote cell survival, including autophagy As such, targeting these survival pathways may represent new vulnerabilities of GBM after treatment with TMZ

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