DEAH-BOX HELICASE 8 IS A REGULATOR OF HEAT SHOCK FACTOR 1

Heat Shock Factor 1 (HSF1) is a key transcription factor mediating the cellular stress response and promoting cancer cell survival. We identify DEAH-box helicase 8 (DHX8) as a crucial regulator of HSF1 pre-mRNA processing. Silencing DHX8 leads to the accumulation of HSF1 transcripts with retained introns and reduced HSF1 protein. Intron retention is also detected in canonical heat shock and cancer-signature gene sets regulated by HSF1, and in mitosis, G2M checkpoint, cMYC and E2F-associated gene sets. Consistent with intron retention and the role DHX8 in the later stages of splicing, eCLIP analysis finds significant enrichment of DHX8 binding between the lariat branch site and 3' splice junction. DHX8 silencing induces apoptosis in cancer cells to a greater extent than in non-tumorigenic cells. We show that the ATPase activity of DHX8 is essential for function, and propose DHX8 as a therapeutic target on pathways that protect cancer cells from oncogene-induced stress. Overall design: H1299 non-small cell lung cancer cells DHX8-dTAG cells were treated for 6 hours with 250 nM dTAG-V1 heterobifunctional small molecule to induce degradation of the DHX8-dTAG fusion protein. H1299 cells were also treated with 10nM siTOOLs DHX8 or scramble control pooled siRNA for 72 hours. HCT116 colorectal cancer cells were transduced with a lentiviral construct encoding doxycyline-inducible human DHX8 (DHX8 wild-type, DHX8-K549 ATPase-dead or DHX8-R647G/F648G RNA binding mutant). Cells were treated with DHX8 siRNA to remove endogenous DHX8 or scramble control siRNA or mock transfection conditions. Treatments were run in the absence or presence of doxycycline to assess the impact of inducing the exogenous wild-type or mutant DHX8. Four independent biological repeats were run for each condition

热休克因子1（Heat Shock Factor 1，HSF1）是介导细胞应激反应并促进癌细胞存活的关键转录因子。本研究鉴定出DEAH盒解旋酶8（DEAH-box helicase 8，DHX8）为HSF1前体mRNA（pre-mRNA）加工过程的关键调控因子。沉默DHX8会导致携带滞留内含子的HSF1转录本积累，并使HSF1蛋白水平降低。在HSF1调控的经典热休克基因集与癌症特征基因集中，以及有丝分裂、G2/M检查点、cMYC及E2F相关基因集中，均检测到内含子滞留现象。鉴于内含子滞留现象与DHX8在剪接后期的作用，增强型交联免疫沉淀（enhanced Cross-Linking and Immunoprecipitation，eCLIP）分析发现，DHX8在套索分支位点与3'剪接位点之间存在显著结合富集。沉默DHX8对癌细胞的凋亡诱导作用显著强于非致瘤细胞。本研究证实DHX8的ATP酶活性对其功能至关重要，并提出DHX8可作为保护癌细胞免受癌基因诱导应激的通路的治疗靶点。
实验设计概述：将H1299非小细胞肺癌DHX8-dTAG细胞系用250 nM的dTAG-V1异双功能小分子处理6小时，以诱导DHX8-dTAG融合蛋白降解。同时将H1299细胞用10 nM的siTOOLs合成的DHX8混合siRNA或乱序阴性对照siRNA处理72小时。将HCT116结直肠癌细胞通过慢病毒载体转导，携带可被多西环素诱导表达的人源DHX8（包括DHX8野生型、DHX8-K549 ATP酶失活突变体及DHX8-R647G/F648G RNA结合突变体）。各组细胞分别用DHX8 siRNA敲除内源性DHX8，或用乱序阴性对照siRNA处理，或设置空白转染对照组。所有处理均设置多西环素存在与缺失两组，以评估诱导表达外源性野生型或突变型DHX8的影响。每个实验条件均设置4次独立生物学重复。