Radiosensitization of HSF-1 Knockdown Lung Cancer Cells by Low Concentrations of Hsp90 Inhibitor NVP-AUY922.

Annett Kühnel,Melissa Schwab,Daniela Schilling,Stephanie E Combs,Bernhard Haller,Gabriele Multhoff

doi:10.3390/cells8101166

Abstract

The inhibition of heat shock protein 90 (Hsp90) a molecular chaperone for multiple oncogenic client proteins is considered as a promising approach to overcome radioresistance. Since most Hsp90 inhibitors activate HSF-1 that induces the transcription of cytoprotective and tumor-promoting stress proteins such as Hsp70 and Hsp27, a combined approach consisting of HSF-1 knockdown (k.d.) and Hsp90 inhibition was investigated. A specific HSF-1 k.d. was achieved in H1339 lung cancer cells using RNAi-Ready pSIRENRetroQ vectors with puromycin resistance. The Hsp90 inhibitor NVP-AUY922 was evaluated at low concentrations—ranging from 1–10 nM—in control and HSF-1 k.d. cells. Protein expression (i.e., Hsp27/Hsp70, HSF-1, pHSF-1, Akt, ß-actin) and transcriptional activity was assessed by western blot analysis and luciferase assays and radiosensitivity was measured by proliferation, apoptosis (Annexin V, active caspase 3), clonogenic cell survival, alkaline comet, γH2AX, 53BP1, and Rad51 foci assays. The k.d. of HSF-1 resulted in a significant reduction of basal and NVP-AUY922-induced Hsp70/Hsp27 expression levels. A combined approach consisting of HSF-1 k.d. and low concentrations of the Hsp90 inhibitor NVP-AUY922 reduces the Hsp90 client protein Akt and potentiates radiosensitization, which involves an impaired homologous recombination mediated by Rad51. Our findings are key for clinical applications of Hsp90 inhibitors with respect to adverse hepatotoxic effects.

Highlights

Lung cancer is one of the most commonly diagnosed cancer in Western societies and the leading cause of cancer-related deaths worldwide [1,2]
A combined approach consisting of heat shock factor-1 (HSF-1) k.d. and low concentrations of the heat shock protein 90 (Hsp90) inhibitor NVP-AUY922 reduces the Hsp90 client protein Akt and potentiates radiosensitization, which involves an impaired homologous recombination mediated by Rad51
HSF-1 k.d. in H1339 cells was verified by a drastic reduction in the total amount of non-phosphorylated (HSF-1) and phosphorylated HSF-1 protein (Figure 1A), and a significant downregulation of the basal and NVP-AUY922-induced transcriptional activity of HSF-1, as compared to control cells (Figure 1B)

Summary

Introduction

Lung cancer is one of the most commonly diagnosed cancer in Western societies and the leading cause of cancer-related deaths worldwide [1,2]. Despite novel therapeutic approaches including immune checkpoint inhibitor blockade to which only a proportion of patients showed beneficial responses, the 5-year survival rate of late stage tumor diseases remains poor with approximately. There is a high unmet medical need for combined therapeutic approaches that significantly improve the radiosensitivity of lung cancer cells. (HSF-1) are frequently overexpressed in many tumor cell types [5,6,7], including lung carcinoma. Hsp is an attractive therapeutic target [10,11,12,13,14] and its inhibition affects many oncogenic driver proteins, which are involved in cell cycle regulation, apoptosis, and DNA repair [15,16,17].

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Conclusion