Efficacy of the HSP90 inhibitor 17-AAG in human glioma cell lines and tumorigenic glioma stem cells

Abstract

Glioblastoma multiforme (GBM) arises from genetic and signaling abnormalities in components of signal transduction pathways involved in proliferation, survival, and the cell cycle axis. Studies to date with single-agent targeted molecular therapy have revealed only modest effects in attenuating the growth of these tumors, suggesting that targeting multiple aberrant pathways may be more beneficial. Heat-shock protein 90 (HSP90) is a molecular chaperone that is involved in the conformational maturation of a defined group of client proteins, many of which are deregulated in GBM. 17-allylamino-17-demethoxygeldanamycin (17-AAG) is a well-characterized HSP90 inhibitor that should be able to target many of the aberrant signal transduction pathways in GBM. We assessed the ability of 17-AAG to inhibit the growth of glioma cell lines and glioma stem cells both in vitro and in vivo and assessed its ability to synergize with radiation and/or temozolomide, the standard therapies for GBM. Our results reveal that 17-AAG is able to inhibit the growth of both human glioma cell lines and glioma stem cells in vitro and is able to target the appropriate proteins within these cells. In addition, 17-AAG can inhibit the growth of intracranial tumors and can synergize with radiation both in tissue culture and in intracranial tumors. This compound was not found to synergize with temozolomide in any of our models of gliomas. Our results suggest that HSP90 inhibitors like 17-AAG may have therapeutic potential in GBM, either as a single agent or in combination with radiation.

Fig. 1

17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits the growth of genetically diverse glioma cell lines via degradation of heat shock protein 90 (HSP90) client proteins. (A) Assessment of phosphatase and tensin homolog (PTEN), p53, and epidermal growth factor receptor (EGFR) status of human glioma cell lines. Genomic DNA from human glioma cell lines was extracted from cells at low passage, and mutations in p53 and PTEN were determined by sequence analysis. Levels of EGFR and EGFR variant III (EGFRvIII) were assessed by Western blot analysis. (B) 17-AAG inhibits the growth of human glioma cell lines. Human glioma cell lines and a nontumorigenic fibroblast cell line were treated with increasing concentrations of 17-AAG for 4 days, and the effect on cell growth was assessed by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. The 50% inhibitory concentration (IC₅₀) of all glioma cell lines ranged from 0.05 to 0.5 μM 17-AAG, as compared to 1.5 μM for the nontumorigenic cells. (C) 17-AAG promotes the degradation of HSP90 client proteins that are involved in the pathology of glioblastoma multiforme (GBM). Glioma cell lines were treated with increasing concentrations of 17-AAG for 48 h, and protein lysates were run on Western blots. HSP90 client proteins known to be involved in gliomagenesis showed degradation while nonclient proteins remained unaffected by 17-AAG treatment. (D) The phosphorylated states of signaling proteins involved in glioma growth are also affected by 17-AAG treatment.

Fig. 2

17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits the growth of orthotopic U87 glioma cells. U87-LucNeo cells were stereotaxically implanted in the right cerebral cortices of immunodeficient mice, which were subsequently treated with either vehicle or 80 mg/kg 17-AAG for 5 days on, 2 days off, and 5 days on. (A) Tumor size was assessed weekly by luciferase bioluminescence imaging and plotted relative to bioluminescence on the first day of treatment. (B) Representative examples of luciferase bioluminescence in either vehicle-treated or 17-AAG–treated mice. (C) 17-AAG–treated animals survived significantly longer than vehicle-treated animals. (D) Proteins harvested from either vehicle- or 17-AAG–treated tumors or contralateral cortices reveal a decrease in client protein expression in the 17-AAG–treated animals.

Fig. 3

17-allylamino-17-demethoxygeldanamycin (17-AAG) radiosensitizes but does not chemosensitize glioma cell lines in vitro and in vivo. (A) The ability of 17-AAG to radiosensitize U87 glioma cells in vitro was assessed by clonogenic cell survival assays, in which the cells plated at clonal density were pretreated for 16 h with two doses of 17-AAG and then exposed to increasing doses of γ-irradiation. Colonies containing greater than 50 cells were counted after 6 days of growth, and sensitizer enhancement ratios were determined by calculating a ratio of observed versus predicted colony number (inset). (B) The ability of 17-AAG to radiosensitize in vivo was assessed by orthotopic implantation of U87 cells expressing the firefly luciferase gene, followed by treatment of tumor-bearing animals with suboptimal doses of either 17-AAG, radiation, or a combination of both treatment agents. Tumor growth was assessed over time with luciferase bioluminescence. The combination of 17-AAG and radiation therapy (RT) yielded a statistically significant effect on tumor growth inhibition relative to treatment with single agents. (C) Efficacy of either concurrent or sequential treatment of temozolomide (TMZ) and 17-AAG in U87 glioma cells in vitro was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assays. Interaction ratios were calculated to determine synergy between these compounds. This ratio is calculated as the observed growth inhibition seen with both compounds relative to the amount of predicted growth inhibition if either compound is acting independently. A ratio of 1 reveals additive growth inhibition, a ratio greater than 1 demonstrates synergistic growth inhibition, while a ratio less than 1 suggests subadditive effects on growth inhibition. (D) The effect of 17-AAG on the combination of TMZ and RT in vivo was assessed using orthotopically implanted U87-LucNeo cells in immunodeficient mice. Mice were treated with either vehicle, RT, and TMZ (2.5 Gy radiation × 2 days and 5mg/kg TMZ × 2 days), or radiation, TMZ, and 17-AAG (2.5 Gy radiation × 2 days, 5 mg/kg TMZ × 2 days, and 80 mg/kg 17-AAG on days 1–5), and tumor growth was monitored by luciferase bioluminescence. Addition of 17-AAG to TMZ and RT does not provide additional efficacy on growth inhibition of orthotopic glioma tumors.

Fig. 4

17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits the growth of genetically diverse tumorigenic glioma stem cells via degradation of heat shock protein (HSP90) client proteins. (A) Phosphatase and tensin homolog (PTEN), p53, and epidermal growth factor receptor variant III (EGFRvIII) status of human glioma stem cell lines. Genomic DNA was extracted from neuro-sphere cultures plated from dissociated subcutaneous tumors, and mutations in p53 and PTEN were determined by sequence analysis. Levels of EGFRvIII were assessed by Western blot analysis. (B) Stem cells were treated with increasing concentrations of 17-AAG, and the effect on cell growth was assessed by 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-zolium, inner salt (MTS) cell viability assays. In the murine model, cell viability in the tumorigenic Ink4a^{−/ −} EGFRvIII was significantly lower than in their wild-type counterparts, consistent with the ability of 17-AAG to preferentially target tumorigenic cells over nontumorigenic cells. Dose-dependent growth inhibition was also seen in all human glioma stem cells tested. (C) 17-AAG results in degradation of HSP90 client proteins in glioma stem cells. Murine neural stem cells (NSCs) and human BT37 glioma stem cells were treated with increasing concentrations of 17-AAG, and protein lysates were harvested after 48 h. In both models, proteins involved in glioma growth show a dose-dependent decrease in expression levels, with no change in expression levels of proteins that are not HSP90 client proteins.

Fig. 5

Transient exposure to 17-allylamino-17-demethoxygeldana-mycin (17-AAG) in vitro significantly reduces the growth of human glioma stem cells in vivo. Human glioma stem cells were treated for 24 h in vitro with either 10 μM 17-AAG or dimethyl sulfoxide, then 20,000 viable cells were orthotopically implanted in immunodeficient mice, and survival of mice was determined over time. Mice implanted with the 17-AAG–treated glioma stem cells survived significantly longer than control animals.