As a conserved molecular chaperone, heat shock protein 90 (Hsp90) maintains the stability and homeostasis of oncoproteins and helps cancer cells survive. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a pivotal role in the non-homologous end joining pathway for DNA double-strand breaks (DSB) repair. Tumor cells contain higher levels of DNA-PKcs to survive by the hostile tumor microenvironment and various antitumor therapies. Here, we showed that increased levels of Hsp90α, Hsp90β, and DNA-PKcs correlated with a poor overall survival in hepatocellular carcinoma (HCC). We revealed that Hsp90 N-terminal domain and C-terminal domain have different effects on DNA-PKcs protein and mRNA levels. The stability of DNA-PKcs depended on Hsp90α N-terminal nucleotide binding domain. Transcription factor SP1 regulates the transcription of PRKDC (gene name of DNA-PKcs) and is a client protein of Hsp90. Inhibition of Hsp90 N-terminal by STA9090 decreased the location of Hsp90α in nucleus, Hsp90α-SP1 interaction, SP1 level, and the binding of Hsp90α/SP1 at the proximal promoter region of PRKDC . Because hyperthermia induces DSBs with increases level of DNA-PKcs, combined STA9090 treatment with hyperthermia effectively delayed the tumor growth and significantly decreased DNA-PKcs levels in xenografts model. Consistently, inhibition of Hsp90 increased the number of heat shock–induced γ-H2AX foci and delayed the repair of DSBs. Altogether, our results suggest that Hsp90 inhibitor STA9090 decreases DNA-PKcs protein stability and PRKDC mRNA level, which provide a theoretical basis for the promising combination therapy of hyperthermia and Hsp90 inhibitor in HCC.

中文翻译：

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Hsp90 抑制剂 STA9090 通过抑制 DNA-PKcs 蛋白质稳定性和 mRNA 转录使肝细胞癌对高热诱导的 DNA 损伤敏感

作为一种保守的分子伴侣，热休克蛋白 90 (Hsp90) 可维持癌蛋白的稳定性和稳态，并帮助癌细胞存活。DNA 依赖性蛋白激酶催化亚基 (DNA-PKcs) 在 DNA 双链断裂 (DSB) 修复的非同源末端连接途径中起关键作用。肿瘤细胞含有更高水平的 DNA-PKcs，可以在敌对的肿瘤微环境和各种抗肿瘤疗法中生存。在这里，我们发现 Hsp90α、Hsp90β 和 DNA-PKcs 水平升高与肝细胞癌 (HCC) 的总体存活率低相关。我们发现 Hsp90 N 端结构域和 C 端结构域对 DNA-PKcs 蛋白和 mRNA 水平有不同的影响。DNA-PKcs 的稳定性取决于 Hsp90α N 端核苷酸结合结构域。转录因子 SP1 调节 PRKDC（DNA-PKcs 的基因名称）的转录，是 Hsp90 的客户蛋白。STA9090 对 Hsp90 N 末端的抑制降低了 Hsp90α 在细胞核中的位置、Hsp90α-SP1 相互作用、SP1 水平以及 Hsp90α/SP1 在 PRKDC 近端启动子区域的结合。因为热疗诱导 DSBs 增加 DNA-PKcs 水平，STA9090 与热疗相结合有效地延缓了异种移植模型中的肿瘤生长并显着降低了 DNA-PKcs 水平。一致地，抑制 Hsp90 会增加热休克诱导的 γ-H2AX 病灶的数量并延迟 DSB 的修复。总之，我们的结果表明 Hsp90 抑制剂 STA9090 降低了 DNA-PKcs 蛋白稳定性和 PRKDC mRNA 水平，