Metabolic targeting of EGFRvIII/PDK1 axis in temozolomide resistant glioblastoma

Abstract

Glioblastomas are characterized by amplification of EGFR. Approximately half of tumors with EGFR over-expression also express a constitutively active ligand independent EGFR variant III (EGFRvIII). While current treatments emphasize surgery followed by radiation and chemotherapy with Temozolomide (TMZ), acquired chemoresistance is a universal feature of recurrent GBMs. To mimic the GBM resistant state, we generated an in vitro TMZ resistant model and demonstrated that dichloroacetate (DCA), a metabolic inhibitor of pyruvate dehydrogenase kinase 1 (PDK1), reverses the Warburg effect. Microarray analysis conducted on the TMZ resistant cells with their subsequent treatment with DCA revealed PDK1 as its sole target. DCA treatment also induced mitochondrial membrane potential change and apoptosis as evidenced by JC-1 staining and electron microscopic studies. Computational homology modeling and docking studies confirmed DCA binding to EGFR, EGFRvIII and PDK1 with high affinity. In addition, expression of EGFRvIII was comparable to PDK1 when compared to EGFR in GBM surgical specimens supporting our in silico prediction data. Collectively our current study provides the first in vitro proof of concept that DCA reverses the Warburg effect in the setting of EGFRvIII positivity and TMZ resistance leading to GBM cytotoxicity, implicating cellular tyrosine kinase signaling in cancer cell metabolism.

Keywords: DCA; EGFR; EGFRvIII; glioblastoma; homology modeling.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest exists with this manuscript.

Figures

Figure 1. Detection of EGFR, EGFRvIII and PDK1 in hGBM clinical specimens

A. Immunohistochemical staining of a primary human GBM with EGFR-, EGFRvIII-, or PDK1-specific antibody (top) on consecutive sections is shown (brown, diaminobenzidine; light blue, nuclear counterstain with DAPI; Bar=100 μm) B. Dual immunohistochemical staining for co-localization was conducted with anti-EGFRvIII and anti-PDK1 antibodies followed by the secondary antibodies conjugated with fluorophores for red (PDK1), green (EGFRvIII) and blue (DAPI) fluorescence, respectively. Representative merged images show the cells expressing PDK1 and EGFRvIII. Bar=100 μm. C. Western blot analysis of EGFR, EGFRvIII and PDK1 protein expression in hGBM specimens D. Immunoprecipitation experiments were conducted on hGBM patient specimen tissue lysates by using PDK1 antibody. Western blotting analysis was performed on these immuno-precipitated samples using EGFRvIII antibody. IgG probing was done to confirm equal loading.

Figure 2. Binding energies and Protein Ligand Interaction fingerprint (PLIF) diagrams

A. Binding Energies and Protein Ligand Interaction fingerprint (PLIF) diagrams showing atoms of DCA interacting with residues of A. EGFR B. EGFRvIII C. PDK1. Binding energies and structure of best pose for D. PDK1-EGFR complex E. PDK1-EGFRvIII complex. F. Top conformations of DCA docked in site close to TYR869 in case of wild type EGFR whereas this site does not anchor DCA conformations if TYR869 is mutated to Phenylalanine.

Figure 3. Development and validation of temozolomide resistant model

A. Schematic representation depicting generation of U373vIII cells with 150μM TMZ resistance (U373vIIIR). Micrographs in lower panel demonstrate the timeline and development of U373vIIIR cells; Panel B1=confluent cells, B2=150 μM TMZ block, B3= TMZ resistant cells, B4=TMZ resistance cells growing to near confluence B. Immunofluorescence staining for the presence of GFAP was conducted on the U373, U373vIII and U373vIIIR cells followed by the secondary antibodies conjugated with green fluorophore. DAPI was used to stain the nuclei. Representative merged images show the cells expressing GFAP Bar=100 μm C. FACS analysis was done to analyze the cell cycle in U373vIII and U373vIIIR cells when treated with 150μM TMZ D. Western blot analysis of EGFR, EGFRvIII, pEGFR and PDK1 protein expression in U373, U373vIII and U373vIIIR cells. GAPDH was used as a loading control E. Semi-quantitative qRT-PCR analysis for EGFR, EGFRvIII and PDK1 in U373 and its EGFRvIII counterparts F. U373vIII or U373vIIIR cells were cultured with 1mM DCA and cell proliferation was measured by MTT assay G. Cells were stained for apoptosis using TUNEL assay. Results represent the mean ± SD of three experiments performed in triplicate.

Figure 4. Differential expression of key genes involved in glucose metabolism; si-PDK1/DCA induce mitochondrial apoptosis and ΔΨm change

A. Heat map demonstrating the differential expression of various key genes involved in GBM glucose metabolism. Red=strong downregulation, Orange=modest downregulation, Yellow=modest upregulation and Green=strong upregulation B. Quantification of HIF1α gene expression with response to 1mM DCA treatment on U373vIII/U373vIIIR cells (n = 4; p = 0.005). The data presented is normalized to loading control GAPDH C. GBM cells were transfected with siRNA for PDK1. After 72h of transfections, both control and treated cells were monitored for ΔΨm change using JC-1 dye and was analyzed by fluorescence microscopy (Red= J aggregation (live cells); Green=JC-1 monomer (dead cells) D. Identification of mitochondrial morphologies by electron microscopy. Bar=200 nm.

Figure 5. Measurement of bioenergetic parameters of U373vIII/U373vIIIR cells using Seahorse assays

DCA treatment activates U373vIII A. U373vIIIR B. cells towards energetic phase. DCA treatment is believed to increase the aerobic potential as shown by the difference in stressed OCR between the control (blue) and treated (red) values. The assay was performed in triplicate. Effect of DCA on mitochondrial respiration and phenotype. U373vIII C. and U373vIIIR D. cells were treated with 1mM DCA 24 h. Oxygen consumption rate measured under basal conditions, following the addition of the oligomycin (mitochondrial F1-F0-ATPase inhibitor), FCCP (uncoupler) and rotenone (complex I inhibitor). Oxygen consumption rate (OCR) was measured using the Seahorse™ XFp Extracellular Flux analyzer. Each data point is the average of five independent measurements. Error bars indicate ±S.E.M.

Figure 6. DCA treatment reduced EGFRvIII/EGFRvIIIR- induced tumor growth in mice

A. Dual immunohistochemical staining for colocalization was conducted on the U373vIIIR cells with anti-EGFRvIII antibody and mitotracker followed by the secondary antibodies conjugated with fluorophores for green (EGFRvIII) and mitotracker (red) fluorescence, respectively. Representative merged images show the cells expressing EGFRvIII in colocalized in the mitochondria (Bar=100mm) B. Whole cell lysates of U373vIIIR and DCA treated cells were subjected to EGFR phosphorylation array and its representative images were presented C. Immuno-histochemical analysis of EGFRvIII and PDK1 expression in U373vIII/ U373vIIIR- induced tumors and their respective DCA treatment with 100gm/kg body weight. Representative H&E staining is seen in the insets of left panel (Bar=100mm) (n = 3) D. Kaplan–Meier survival curves from mice bearing intracranial EGFRvIII/EGFRvIIIR- induced tumors. DCA treatment group had an increased survival compared with the control group with a median survival 3-5 weeks. E. Immuno-fluorescent staining for PDK1 (red), EGFRvIII (green), COX2 (blue), demonstrating intense PDK1-EGFR co-localization (yellow) in U373vIIIR cells.