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ARNT-dependent HIF-2 transcriptional activity is not sufficient to regulate downstream target genes in neuroblastoma.
Experimental Cell Research ( IF 3.3 ) Pub Date : 2020-01-13 , DOI: 10.1016/j.yexcr.2020.111845 Camilla U Persson 1 , Kristoffer von Stedingk 2 , Elina Fredlund 3 , Daniel Bexell 1 , Sven Påhlman 1 , Caroline Wigerup 1 , Sofie Mohlin 3
Experimental Cell Research ( IF 3.3 ) Pub Date : 2020-01-13 , DOI: 10.1016/j.yexcr.2020.111845 Camilla U Persson 1 , Kristoffer von Stedingk 2 , Elina Fredlund 3 , Daniel Bexell 1 , Sven Påhlman 1 , Caroline Wigerup 1 , Sofie Mohlin 3
Affiliation
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BACKGROUND
Hypoxia-inducible factor (HIF)-2α associates with poor outcome in neuroblastoma and glioblastoma, and gain-of-function mutations in the EPAS1 gene (encoding HIF-2α) have been reported in paragangliomas and pheochromocytomas. Specific targeting of a druggable hydrophobic pocket in the HIF-2α PAS-B domain with PT2385 have demonstrated promising clinical results for clear cell renal cell carcinoma (ccRCC). Here, we investigated the effect of PT2385-mediated inhibition of ARNT dependent HIF-2 activity.
METHODS
Neuroblastoma patient-derived xenograft (PDX) cells were treated with PT2385 and analyzed for HIF-2-dependent gene expression, HIF activity, HIF-2α protein localization, response to chemotherapy and orthotopic tumor growth in vivo. Two-sided student t-test was used.
RESULTS
We detected high levels of HIF-2α protein in perivascular niches in neuroblastoma PDXs in vivo and at oxygenated conditions in PDX-derived cell cultures in vitro, particularly in the cytoplasmic fraction. Nuclear HIF-2α expression was reduced following PT2385 treatment, but surprisingly, virtually no effects on tumor growth in vivo or expression of canonical HIF downstream target genes in vitro were observed. In coherence, RNA sequencing of PT2385-treated PDX cells revealed a virtually unaffected transcriptome. Treatment with PT2385 did not affect cellular response to chemotherapy. In contrast, HIF-2α protein knockdown resulted in profound downregulation of target genes.
CONCLUSIONS
The lack of effect from PT2385 treatment in combination with high cytoplasmic HIF-2α expression at normoxia suggest that HIF-2α have additional roles than acting as an ARNT dependent transcription factor. It is important to further unravel the conditions at which HIF-2α has transcriptional and non-transcriptional roles in neuroblastoma.
中文翻译：
ARNT依赖的HIF-2转录活性不足以调节神经母细胞瘤中的下游靶基因。
背景缺氧诱导因子（HIF）-2α与神经母细胞瘤和胶质母细胞瘤的不良预后相关，据报道在副神经节瘤和嗜铬细胞瘤中，EPAS1基因（编码HIF-2α）的功能获得性突变。PT2385特异性靶向HIF-2αPAS-B域中的可药用疏水口袋，已证明对透明细胞肾细胞癌（ccRCC）很有希望。在这里，我们调查了PT2385介导的抑制ARNT依赖的HIF-2活性的影响。方法用PT2385处理成神经细胞瘤患者异种移植（PDX）细胞，分析其HIF-2依赖性基因表达，HIF活性，HIF-2α蛋白定位，对化疗的反应以及体内原位肿瘤的生长。使用双面学生t检验。结果我们在体内神经母细胞瘤PDX中和在含氧条件下在体外PDX衍生的细胞培养物中，特别是在细胞质部分中，检测到高水平的HIF-2α蛋白。PT2385处理后，核HIF-2α表达降低，但令人惊讶的是，实际上未观察到对体内肿瘤生长或体外标准HIF下游靶基因表达的影响。相干而言，经PT2385处理的PDX细胞的RNA测序揭示了实际上未受影响的转录组。PT2385的治疗不影响细胞对化学疗法的反应。相反，HIF-2α蛋白的敲低导致靶基因的大幅下调。结论PT2385治疗与正常氧水平下细胞质HIF-2α高表达的结合缺乏效果，提示HIF-2α除了充当ARNT依赖性转录因子外，还具有其他作用。重要的是进一步揭示HIF-2α在神经母细胞瘤中具有转录和非转录作用的条件。
更新日期：2020-01-14
中文翻译：
ARNT依赖的HIF-2转录活性不足以调节神经母细胞瘤中的下游靶基因。
背景缺氧诱导因子（HIF）-2α与神经母细胞瘤和胶质母细胞瘤的不良预后相关，据报道在副神经节瘤和嗜铬细胞瘤中，EPAS1基因（编码HIF-2α）的功能获得性突变。PT2385特异性靶向HIF-2αPAS-B域中的可药用疏水口袋，已证明对透明细胞肾细胞癌（ccRCC）很有希望。在这里，我们调查了PT2385介导的抑制ARNT依赖的HIF-2活性的影响。方法用PT2385处理成神经细胞瘤患者异种移植（PDX）细胞，分析其HIF-2依赖性基因表达，HIF活性，HIF-2α蛋白定位，对化疗的反应以及体内原位肿瘤的生长。使用双面学生t检验。结果我们在体内神经母细胞瘤PDX中和在含氧条件下在体外PDX衍生的细胞培养物中，特别是在细胞质部分中，检测到高水平的HIF-2α蛋白。PT2385处理后，核HIF-2α表达降低，但令人惊讶的是，实际上未观察到对体内肿瘤生长或体外标准HIF下游靶基因表达的影响。相干而言，经PT2385处理的PDX细胞的RNA测序揭示了实际上未受影响的转录组。PT2385的治疗不影响细胞对化学疗法的反应。相反，HIF-2α蛋白的敲低导致靶基因的大幅下调。结论PT2385治疗与正常氧水平下细胞质HIF-2α高表达的结合缺乏效果，提示HIF-2α除了充当ARNT依赖性转录因子外，还具有其他作用。重要的是进一步揭示HIF-2α在神经母细胞瘤中具有转录和非转录作用的条件。
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