GLUT1 and TUBB4 in Glioblastoma Could be Efficacious Targets

Abstract

Glioblastoma multiforme (GBM) is the most aggressive and deadly brain tumor, portending a median 13-month survival even following gross total resection with adjuvant chemotherapy and radiotherapy. This prognosis necessitates improved therapies for the disease. A target of interest for novel chemotherapies is the Warburg Effect, which describes the tumor's shift away from oxidative phosphorylation towards glycolysis. Here, we elucidate GLUT1 (Glucose transporter 1) and one of its associated binding partners, TUBB4 (Tubulin 4), as potentially druggable targets in GBM. Using data mining approach, we demonstrate that GLUT1 is overexpressed as a function of tumor grade in astrocytoma's and that its overexpression is associated with poorer prognosis. Using both mass spectrometry performed on hGBM (human glioblastoma patient specimen) and in silico modeling, we show that GLUT1 interacts with TUBB4, and more accurately demonstrates GLUT1's binding with fasentin. Proximity ligation assay (PLA) and immunoprecipitation studies confirm GLUT1 interaction with TUBB4. Treatment of GSC33 and GSC28 cells with TUBB4 inhibitor, CR-42-24, reduces the expression of GLUT1 however, TUBB4 expression is unaltered upon fasentin treatment. Using human pluripotent stem cell antibody array, we demonstrate reduced levels of Oct3/4, Nanog, Sox2, Sox17, Snail and VEGFR2 (Vascular endothelial growth factor receptor 2) upon CR-42-24 treatment. Overall, our data confirm that silencing TUBB4 or GLUT1 reduce GSC tumorsphere formation, self-renewal and proliferation in vitro. These findings suggest GLUT1 and its binding partner TUBB4 as druggable targets that warrant further investigation in GBM.

Keywords: CR-42-24; GLUT1; TUBB4; fasentin; homology modeling.

Figure 1

GLUT1 expression in The Cancer Genome Analysis (TCGA) and Repository for Molecular Brain Neoplasia Data (REMBRANDT) cohort. (A) GLUT1 expression is high in the mesenchymal subtype compared to other glioblastoma multiforme (GBM) subtypes. (B) Kaplan-Meier curves show increased GLUT1 corresponds to decreased survival. (C) GLUT1 is highly expressed in GBM relative to lower grade glioma (p < 0.0001). (D) Survival analysis of long-term survivors from the REMBRANDT dataset show that the patients with high GLUT1 expression show shorter overall survival.

Figure 2

GLUT1 is highly expressed in human glioblastoma (hGBM) surgical biopsies. (A) Immunohistochemical staining for GLUT1 with anti-GLUT1 antibody (brown, diaminobenzidine; light blue, nuclear counter stain with DAPI; GS = glioblastoma specimen). (B) RT-PCR analysis of GLUT1 from hGBM specimens. GAPDH is used as a loading control.

Figure 3

GLUT1 associate and interact with TUBB4 in GSC. (A) Around 400 μg of total protein was extracted from hGBM specimen (GS6-4). The lysates were immunoprecipitated with GLUT1 antibody and the eluates were subjected to LC/MS analysis. LS/MS spectra showed the high relative intensity of reporter ions for TUBB4. The spectra acquisition is mentioned in the methods section. (B) Annotation of the spectral data using Pantherdb software. (C) Immunohistochemical analysis for TUBB4 using an anti-TUBB4 antibody (brown, diaminobenzidine; light blue, nuclear counter stain with DAPI; GS = glioblastoma specimen). (D) TCGA analysis of TUBB4. (E) The proximity ligation assay (PLA) confirmed the association of GLUT1 and TUBB4 in GSC33 cells. The representative micrograph of GLUT1 + TUBB4 shows the positive PLA. Red spots confirmed the association. Single antibody stain with TUBB4 and GLUT1 showed negative results. Scale = 10 µm. (F) Immunofluorescence analysis show a TUBB4-GLUT1 association in GSC28 control cells. CR-42-24 treatment diminished their association. Blue = DAPI; red = GLUT1; green = TUBB4; yellow color in the overlay shows the association. Scale = 10 µm. (G) Immunoprecipitation of GSC28 control and 1 uM CR-42-24 treated cells confirmed the interaction of GLUT1-TUBB4. IgG is shown as a loading control.

Figure 4

Docking studies of GLUT1-TUBB4. (A) Alpha spheres (grey and red spheres) obtained by applying molecular operating environment’s (MOE’s) Site Finder tool on the crystal structure of the human GLUT1 (Deng et al. 2014; PDB ID 4PYP) accounting for the most probable binding pocket. The binding region includes 69 residues, many of them being deeply buried within the channel. Contact atoms of the protein are highlighted in pink. (B1,B2) The molecular surface representation of the TUBB4-GLUT1 complex after 50 ns. TUBB4 is shown in green, the cytoplasmic domain of GLUT1 is red and both the transmembrane and extracellular domains of GLUT1 are shown in grey. Residues at the top channel opening are shown in blue while residues at the opening near the transmembrane domain are colored in cyan. At this predicted position, TUBB4 (green) occludes the top opening in the GLUT1 channel. (C) Most probable poses of fasentin as predicted from the 1st (green), 2nd (yellow) and 3rd (pink) most populated clusters. (D) Interaction diagrams of the most probable poses of fasentin as predicted from the 1st (top) and 3rd (bottom) most populated clusters. (E) Docking model of CR-42-24 and TUBB.

Figure 5

Targeting GLUT1 and TUBB4 reduces GSC cell growth and proliferation. (A) MTT analysis of increasing concentration of fasentin and CR-42-24 on GSC33 and GSC28. (B) Immunoblot analysis of GLUT1 and TUBB4 in GSC33 and GSC28 cells treated with 1µM CR-42-24 and 50 µM fasentin. GAPDH was used as a loading control. (C) Limiting dilution assays were performed on GSC33 cells treated with fasentin and CR-42-24 with increasing concentrations and increased number of cells. (D) Tumorsphere formation assay in GSC33 and GSC28 cells. (E) GSC33 cells treated with 1 µM CR-42-24 showed reduced levels of various stem cell factors when compared to the untreated cells. A human pluripotent stem cell antibody array (ARY010) was used in this study.