Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond

Abstract

Radiotherapy is an essential component of multi-modality treatment of glioblastoma (GBM). However, treatment failure and recurrence are frequent and give rise to the dismal prognosis of this aggressive type of primary brain tumor. A high level of inherent treatment resistance is considered to be the major underlying reason, stemming from constantly activated DNA damage response (DDR) mechanisms as a consequence of oncogene overexpression, persistent replicative stress, and other so far unknown reasons. The molecular chaperone heat shock protein 90 (HSP90) plays an important role in the establishment and maintenance of treatment resistance, since it crucially assists the folding and stabilization of various DDR regulators. Accordingly, inhibition of HSP90 represents a multi-target strategy to interfere with DDR function and to sensitize cancer cells to radiotherapy. Using NW457, a pochoxime-based HSP90 inhibitor with favorable brain pharmacokinetic profile, we show here that HSP90 inhibition at low concentrations with per se limited cytotoxicity leads to downregulation of various DNA damage response factors on the protein level, distinct transcriptomic alterations, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in glioblastoma cells in vitro. In vivo, HSP90 inhibition by NW457 improved the therapeutic outcome of fractionated CBCT-based irradiation in an orthotopic, syngeneic GBM mouse model, both in terms of tumor progression and survival. Nevertheless, in view of the promising in vitro results the in vivo efficacy was not as strong as expected, although apart from the radiosensitizing effects HSP90 inhibition also reduced irradiation-induced GBM cell migration and tumor invasiveness. Hence, our findings identify the combination of HSP90 inhibition and radiotherapy in principle as a promising strategy for GBM treatment whose performance needs to be further optimized by improved inhibitor substances, better formulations and/or administration routes, and fine-tuned treatment sequences.

Integrative analysis of therapy resistance and transcriptomic profiling data in glioblastoma cells identifies sensitization vulnerabilities for combined modality radiochemotherapy

“…Irradiation of cells was performed with an RS-225 X-ray cabinet (X-strahl, Camberley, Great Britain) at 200 kV and 10 mA (Thoraeus filter, 1 Gy in 243 s) as described [ 18 ].…”

“…Quantification of mRNA expression levels of DNA damage response (DDR) genes was performed by quantitative real-time RT-PCR (qRT-PCR) as previously described [ 18 – 20 ]. Briefly, total RNA was extracted from cells using the NucleoSpin RNA II extraction kit (Macherey & Nagel, Dueren, Germany).…”

“…Primer sequences have been previously described [ 19 , 20 ]. Expression values of LN229 and T98G cells had been published before [ 18 ] and were re-analyzed for this study.…”

“…Reducing gradient SDS-PAGE and western blot analyses were performed as previously described [ 18 ]. Briefly, cells from cryostocks were thawed, washed with PBS, and lysed with lysis buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% Triton X-100 (v/v, all from Sigma-Aldrich), 1 × EDTA-free protease inhibitor cocktail (Roche)) for 30 min on ice.…”

Senolytics: Eliminating Senescent Cells and Alleviating Intervertebral Disc Degeneration

“…Studies have found that HSP90 can activate Akt through phosphorylation and inhibit the apoptosis of senescent cells ( Chen et al, 2017 ). In addition, according to what we have introduced before, DNA damage caused by radiation or external and internal factors is an important part of cell senescence and even apoptosis ( Dote et al, 2006 ), and the expression of HSP90 increases during this process, which stabilizes the DDR reactive proteins in senescent cells ( Solier et al, 2012 ; Orth et al, 2021 ).…”