Overexpression of heat shock protein 90 (HSP90) is associated with increased tumor cell survival and radioresistance. In this study we explored the efficacy of the novel HSP90 inhibitor AT13387 and examined its radiosensitizing effects in combination with gamma-radiation in 2D and 3D structures as well as mice-xenografts. AT13387 induced effective cytotoxic activity and radiosensitized cancer cells in monolayer and tumor spheroid models, where low drug doses triggered significant synergistic effects on cell survival together with radiation. Furthermore, AT13387 treatment resulted in G2/M-phase arrest and significantly reduced the migration capacity. The expression of selected client proteins involved in DNA repair, cell-signaling and cell growth was downregulated in vitro, though the expression of most investigated proteins recurred after 8–24 h. These results were confirmed in vivo where AT13387 treated tumors displayed effective downregulation of HSP90 and its oncogenic client proteins.In conclusion, our results demonstrate that AT13387 is a potent new cancer drug and effective radiosensitizer in vitro with an excellent in vivo efficacy. AT13387 treatment has the potential to improve external beam therapy and radionuclide therapy outcomes and restore treatment efficacy in cancers that are resistant to initial therapeutic regimes.

Oncogenic client-proteins of the chaperone Heat shock protein 90 (HSP90) insure unlimited tumor growth and are involved in resistance to chemo-and radiotherapy. The HSP90 inhibitor Onalespib initiates the degradation of oncoproteins, and might also act as a radiosensitizer. The aim of this study was therefore to evaluate the efficacy of Onalespib in combination with external beam radiotherapy in an in vitro and in vivo approach. Onalespib downregulated client proteins, lead to increased apoptosis and caused DNA-double-strands. Monotherapy and combination with radiotherapy reduced colony formation, proliferation and migration assessed in radiosensitive HCT116 and radioresistant A431 cells. In vivo, a minimal treatment regimen for 3 consecutive days of Onalespib (3 × 10 mg/kg) doubled survival, whereas Onalespib with radiotherapy (3 × 2 Gy) caused a substantial delay in tumor growth and prolonged the survival by a factor of 3 compared to the HCT116 xenografted control group. Our results demonstrate that Onalespib exerts synergistic anti-cancer effects when combined with radiotherapy, most prominent in the radiosensitive cell models. We speculate that the depletion and downregulation of client proteins involved in signalling, migration and DNA repair mechanisms is the cause. Thus, individually, or in combination with radiotherapy Onalespib inhibits tumor growth and has the potential to improve radiotherapy outcomes, prolonging the overall survival of cancer patients.

Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80-95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure which is uncoupled from its essential function in DSB repair. This could have implications for the development of therapeutic strategies aiming to radiosensitize tumors by affecting the DNA-PKcs function.

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