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. 2019 Nov 14;25(1):49.
doi: 10.1186/s10020-019-0116-z.

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

Bryan G Harder  1 , Sen Peng  2 , Christopher P Sereduk  1 , Andrej M Sodoma  1 , Gaspar J Kitange  3 , Joseph C Loftus  4 , Jann N Sarkaria  3 , Nhan L Tran  5
Affiliations

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

Bryan G Harder et al. Mol Med. .

Abstract

Background: Temozolomide (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.

Methods: Using 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.

Results: Here, 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.

Conclusions: The 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.

Keywords: Autophagy; Bafilomycin; Glioblastoma (GBM); PI3K; PX-866; Temozolomide (TMZ).

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Temozolomide induces autophagic flux in glioma cells. a T98G cells were treated with increasing doses of TMZ for 48 h and only living (attached) cells were collected for analysis. Western blotting shows an increase in LC3 II protein levels and a decrease in p62 protein levels. GAPDH was used as a loading control. b The LC3 flux assay was performed on T98G cells. T98G cells were treated with vehicle or TMZ (500 μM) for 48 h containing either DMSO or Baf (10 nM) and only living (adherent) cells were collected. Western blotting for LC3 indicates an increase in LC3 II for the TMZ + Baf lane compared to Baf alone, proving TMZ-induced autophagosome formation. The data (LC3 II/GAPDH) was quantified and graphed to mark this increase. Data represents three independent experiments
Fig. 2
Fig. 2
TMZ-resistant GBM-PDX tumors display increased LC3 II expression. Parental TMZ-sensitive (P) and TMZ-resistant (T) GBM-PDX subcutaneous tumors that were derived from either (a) primary tumor resections or (b) recurrent tumor resections were harvested and lysed for western blot analysis. LC3 protein levels were examined by western blotting and α-tubulin was used as a loading control. c Densitometry was used to quantify the western blot results
Fig. 3
Fig. 3
MGMT expression correlates with PI3K-AKT pathway activation. a Total protein from parental GBM-PDX subcutaneous tumor lysates was used to assess the differential expression of MGMT and relative activation of p-AKT between the samples. b Densitometry was used to quantify the western blot results and all PDX tumors were normalized to GBM5. c Gene set variation analysis (GSVA) was used to determine gene set enrichment scores of the PI3K-AKT signaling pathway according to the methylation status of the MGMT promoter in clinical samples
Fig. 4
Fig. 4
PX-866 blocks autophagy in glioma cells. a T98G cells were treated with increasing doses of PX-866 for 24 h and then total protein was isolated. b T98G cells were subjected to single treatment with 3-MA (2.5 mM) or co-treatment with either PX-866 (1 μM) or Baf (10 nM) for 24 h, according to the treatment legend. c The LC3 flux assay was performed on T98G cells at 4 h and 24 h using Baf (10 nM), PX-866 (1 μM) or a combination of the two. Data represents three independent experiments
Fig. 5
Fig. 5
TMZ and PX-866 cooperate to diminish survival. a T98G (EGFR WT) and U373vIII (EGFRvIII) were treated with TMZ (500 μM) and increasing doses of PX-866 for 48 h (co-treatment). Both floating and remaining adherent cells were collected for analysis and western blotting for PARP and LC3 was used to assess apoptosis and autophagy, respectively. The triangle representing the dose escalation for PX-866 indicates: 0.125, 0.25, 0.5, and 1 μM. b T98G and U373vIII cells were treated with TMZ (500 μM) for 24 h, followed by the addition of PX-866 for another 24 h (sequential treatment). The triangle representing the dose escalation for PX-866 indicates: 0.125, 0.25, 0.5, and 1 μM. T98G cells (c) and U373vIII cells (d) were treated with the indicated treatments for 24 h. Cells were trypsinized 24 h after drug treatment and re-plated in 35 mm dishes in triplicate and allowed to form colonies. After about 9–10 days, colonies were fixed (6% glutaraldehyde) and stained (0.5% crystal violet). The number of colonies were counted and presented as a bar graph. Values are the mean ± the standard deviation of three separate measurements, *, P < 0.05 and, **, P < 0.01. Data represents the mean and S.D. from three independent experiments with each experiment conducted in triplicates
Fig. 6
Fig. 6
Schematic of the proposed mechanism of how PX-866 cooperates with TMZ to enhance apoptosis through inhibition of TMZ-induced autophagy
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