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. 2017 May;133(1):47-57.
doi: 10.1007/s11060-017-2422-z. Epub 2017 Apr 21.

Expression and function of ABCG2 and XIAP in glioblastomas

Affiliations

Expression and function of ABCG2 and XIAP in glioblastomas

Ivette F Emery et al. J Neurooncol. 2017 May.

Abstract

Despite multimodal treatment that includes surgery, radiation and chemotherapy, virtually all glioblastomas (GBM) recur, indicating that these interventions are insufficient to eradicate all malignant cells. To identify potential new therapeutic targets in GBMs, we examined the expression and function of proteins that are associated with therapy resistance and cancer cell survival. We measured the expression of eight such proteins in 50 GBM samples by immunohistochemistry and analyzed patient survival. We report that GBM patients with high expression of ABCG2 (also called BCRP) or XIAP at the protein level had worse survival than those with low expression. The adjusted hazard ratio for ABCG2 was 2.35 and for XIAP was 2.65. Since glioma stem cells (GSCs) have been shown to be more resistant than bulk tumor cells to anti-cancer therapies and to express high levels of these proteins, we also sought to determine if ABCG2 and XIAP have functional roles in GSCs. We used small molecule inhibitors to treat patient-derived GBM tumorspheres in vitro and observed that inhibitors of ABCG2, Ko143 and fumitremorgin, significantly reduced self-renewal. These results suggest that ABCG2 and XIAP proteins may be useful indicators of patient survival and that inhibition of ABCG2 may be a promising therapeutic strategy in GBMs.

Keywords: ABCG2; Glioblastoma; Glioma stem cells; Ko143; XIAP.

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Conflict of interest statement

Conflict of Interest: This work was funded by unrestricted research grants from the Maine Medical Center Neuroscience Institute, the Northern New England Clinical Oncology Society, and the Schering-Plough Corporation (grant ID XX-3903). These sponsors had no involvement in study design, data collection, analysis or interpretation, manuscript writing, or the decision to submit the article for publication. The authors report no conflicts of interest beyond the direct research support detailed above.

Figures

Fig. 1
Fig. 1. ABGC2 and XIAP protein expression levels are associated with GBM patient survival
a) Box plot analyses of the immunohistochemical (IHC) scores of paraffin-embedded, formalin-fixed tumor samples from GBM patients after monoclonal antibodies were used to visualize the expression of 8 protein targets (ABCG2, XIAP, MGMT, MSH2, pATM, pTP53, pAKT, and NESTIN). Each box represents the IHC scores between the 1st and the 3rd quartile. Lines represent the median IHC score ( formula image−) and the maximum and minimum IHC scores ( formula image−). b,c) Kaplan-Meier analyses of overall survival in months among GBM patients whose tumor samples had low expression by IHC formula image(− −) versus high expression ( formula image−−−) of ABCG2 (b) or XIAP (c) proteins.
Fig. 2
Fig. 2. Isolation of primary GBM tumorspheres that are self-renewing, multipotential, and tumorigenic
a) Tumorspheres derived from patient samples express a neural stem cell marker (NESTIN). They can be differentiated to express a neuronal marker (β-III-tubulin) and a glial marker (GFAP). b) Three independent patient tumorsphere lines, MMC1, MMC10, and MMC11, were tested for secondary sphere formation in vitro at a clonal density for multiple passages. Approximately 0.5–3% of tumorsphere cells self-renew long term and can be passaged for >20 passages (not shown). The number of passages is denoted below each graph. c) Tumorsphere cells express GSC markers, CD133 and CD15/SSEA1, as determined by FACS analyses. d) Microscopic images of tissue sections stained with hematoxylin and eosin at high and low magnifications. Solid arrows point to mitoses and open arrows point to atypical cells.
Fig. 3
Fig. 3. ABCG2 and XIAP expression in matching patient tumors and tumorsphere lines
a) Immunofluoresence analyses of patient-derived tumorsphere lines MMC1, MMC10 and MMC11 and U87 cells stained with antibodies against ABCG2 (red), and XIAP (green). All nuclei are stained with DAPI (blue). b) immunohistochemistry (IHC) images of MMC1, MMC10, and MMC11 patient tumors using antibodies against ABCG2 and XIAP. IHC scores for MMC1, MMC10 and MMC11 were 40, 160 and 180 respectively for ABCG2, and 100, 140 and 100 respectively for XIAP.
Fig. 4
Fig. 4. Inhibition of ABCG2 suppresses GBM tumorsphere self-renewal
Secondary sphere formation assay with low passage (p<10) MMC1, MMC10, and MMC11 cells in the presence of (a) 10μM Embelin (XIAP inhibitor) or (b) 10μM Ko143 (ABCG2 inhibitor) alone or, in combination with 15μM Temozolomide (TMZ). Error bars: SEM. c) GUAVA viability measurement of MMC11 cells showing live/dead cells at 72 hours post 10μM Ko143 treatment. Error bars= STME. d) Viability of U87 cells treated with control (DMSO) or 10μM Ko143. Error bars: STME.

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