Epichaperome Inhibition by PU-H71-Mediated Targeting of HSP90 Sensitizes Glioblastoma Cells to Alkylator-Induced DNA Damage

Abstract

Background: Targeted therapies have been largely ineffective against glioblastoma (GBM) owing to the tumor's heterogeneity and intrinsic and adaptive treatment resistance. Targeting multiple pro-survival pathways simultaneously may overcome these limitations and yield effective treatments. Heat shock protein 90 (HSP90), an essential component of the epichaperome complex, is critical for the proper folding and activation of several pro-survival oncogenic proteins that drive GBM biology.

Methods: Using a panel of biochemical and biological assays, we assessed the expression of HSP90 and its downstream targets and the effects of PU-H71, a highly specific and potent HSP90 inhibitor, on target modulation, downstream biochemical alterations, cell cycle progression, proliferation, migration, and apoptosis in patient-derived glioma stem-like cells (GSCs) with molecular profiles characteristic of GBM, as well as commercial glioma cell lines and normal human astrocytes (NHAs).

Results: HSP90 inhibition by PU-H71 in GSCs significantly reduced cell proliferation, colony formation, wound healing, migration, and angiogenesis. In glioma cells, but not NHAs, potent PU-H71-mediated HSP90 inhibition resulted in the downregulation of pro-survival client proteins such as EGFR, MAPK, AKT, and S6. This reduction in pro-survival signals increased glioma cells' sensitivity to temozolomide, a monofunctional alkylator, and the combination of PU-H71 and temozolomide had greater anticancer efficacy than either agent alone.

Conclusions: These results confirm that HSP90 is a strong pro-survival factor in molecularly heterogeneous gliomas and suggest that epichaperome inhibition with HSP90 inhibitors warrants further investigation for the treatment of gliomas.

Keywords: HSP70; HSP90 effector proteins; PU-H71; heat shock protein 90 (HSP90); malignant glioma.

Figure 1

PU-H71 inhibits glioma cell proliferation. (a) Percentage cell proliferation rates of GSC11, GSC23, GSC20, GSC262, GSC811, GSC272 (b) LN229, T98G, U251-HF (c) and NHA after 0, 24, 48, and 72 h of treatment with the indicated concentrations of PU-H71. (d) IC50 values for the indicated glioma cell lines and NHAs after 72 h of PU-H71 treatment.

Figure 2

PU-H71 induces programmed cell death in glioma cells. (a) Logarithmic displays of flow cytometry data for U251-HF and GSC811 cells treated with vehicle or with 0.25 or 1.0 µM PU-H71 for 48 or 72 h and stained with annexin V–FITC and propidium iodide. (b) Proportions of live (blue), preapoptotic (red), apoptotic (green), and necrotic (purple) cells after 48 and 72 h of treatment with vehicle or 0.25 or 1.0 µM PU-H71.

Figure 3

PU-H71 inhibits the biological functions of glioma cells. (a) Gelcount^TM images show the colony-forming ability of LN229, T98G, and U251-HF cells exposed to vehicle or PU-H71. The graphs show the numbers of colonies. (b) Brightfield microscopy images show the wound-healing capability of LN229, T98G, and U251-HF cells exposed to vehicle or PU-H71. The graphs show the percentages of wound areas not enclosed by the cells after 0 and 24 h of PU-H71 treatment. (c) Brightfield microscopy images show the migration ability of HBMECs exposed to vehicle or PU-H71 in a transmembrane assay. The graph shows the numbers of migrated cells at 48 and 72 h of treatment with vehicle or 0.25 or 1.0 µM PU-H71. *** p < 0.001, **** p < 0.0001.

Figure 4

PU-H71 induces programmed cell death in glioma cells specifically. Immunoblotting analysis of the expression of HSP90, HSP70 (a molecular marker of HSP90 inhibition), cleaved PARP (a marker of cell death), and GAPDH (loading control) in vehicle- or PU-H71-treated NHAs and GSC262 and GSC811 cells. Raw images of these data are available in Supplementary Figure S1.

Figure 5

PU-H71 downregulates the expression of HSP90 effector proteins. (a,b) Immunoblotting analysis showed the dose-dependent (a) and time-dependent (b) impact of HSP90 inhibition on EGFR, p-AKT, AKT, p-MAPK, MAPK, pS6, S6, and cleaved PARP. GAPDH was used as the loading control. Raw images of Figure 5a and Figure 5b are available in Supplementary Figures S2 and S3, respectively.

Figure 6

PU-H71 sensitizes glioma cells to temozolomide. (a) Dose–response curve for PU-H71. (b) Dose–response curve for temozolomide (TMZ). (c) Matrix representing the dose-dependent inhibition of cell growth. (d) Bliss energy model of the synergistic index for PU-H71 plus temozolomide.