DAOY-NERT2 Notch/Hypoxia Transcriptome Analysis

Hyperactivation of Notch signaling and the cellular hypoxic response are frequently observed in cancers, with increasing reports of connections to tumor initiation and progression. The two signaling mechanisms are known to intersect, but while it is well established that hypoxia regulates Notch signaling, less is known about whether Notch can regulate the cellular hypoxic response. We now report that Notch signaling specifically controls expression of HIF2a, a key mediator of the cellular hypoxic response. Transcriptional upregulation of HIF2a by Notch under normoxic conditions leads to elevated HIF2a protein levels in primary breast cancer cells as well as in human breast cancer, medulloblastoma and renal cell carcinoma cell lines. The elevated level of HIF2a protein was in certain tumor cell types accompanied by down-regulation of HIF1a protein levels, indicating that high Notch signaling may drive a HIF1a-to-HIF2a switch. At the transcriptome level, the presence of HIF2a was required for approximately 21% of all Notch-induced genes: among the 1062 genes that were upregulated by Notch in medulloblastoma cells during normoxia, upregulation was abrogated in 227 genes when HIF2a expression was knocked down by HIF2a siRNA. In conclusion, our data show that Notch signaling affects the hypoxic response via regulation of HIF2a, which may be important for future cancer therapies. DAOY-NERT2 cells, +/- Notch induction by Tamoxifen (TMX) for 48 hours, +/- hypoxia (1% O2) treatment for 48 hours, where HIF1a or HIF2a had been knocked down by siRNA, were subjected to RNA sequencing. The quality of the cDNA libraries was tested on an Agilent 2100 bioanalyzer. The libraries were sequenced on an Illumina HiSeq 2000 system, and the reads were aligned to the human genome (assembly hg19) and a transcriptome database (RefSeq and Ensembl) using bowtie. RPKM values were generated using rpkmforgenes.癌症中常可见到Notch信号通路 (Notch signaling pathway) 过度激活与细胞缺氧反应 (cellular hypoxic response) 异常，且二者与肿瘤起始及进展相关的报道日益增多。已知这两种信号机制存在交叉互作，但目前学界已明确缺氧可调控Notch信号通路，而Notch是否能够反向调控细胞缺氧反应，则尚未得到充分阐释。本研究证实，Notch信号可特异性调控细胞缺氧反应关键介质缺氧诱导因子2α (hypoxia-inducible factor 2α, HIF2α) 的表达。在常氧条件下，Notch介导的HIF2α转录上调，可使原代乳腺癌细胞、人乳腺癌细胞、髓母细胞瘤细胞及肾癌细胞系中的HIF2α蛋白水平升高。在部分肿瘤细胞类型中，HIF2α蛋白水平升高同时伴随缺氧诱导因子1α (hypoxia-inducible factor 1α, HIF1α) 蛋白表达下调，提示高活性Notch信号可能驱动HIF1α向HIF2α的表型转换。在转录组 (transcriptome) 水平，约21%的Notch诱导基因的表达依赖于HIF2α的存在：在常氧条件下的髓母细胞瘤细胞中，Notch可上调1062个基因的表达；当利用HIF2α小干扰RNA (small interfering RNA, siRNA) 敲低HIF2α的表达后，其中227个基因的上调效应被完全消除。综上，本研究数据表明，Notch信号可通过调控HIF2α影响细胞缺氧反应，这一发现对未来癌症治疗策略的开发具有重要参考价值。本研究对DAOY-NERT2细胞进行了如下分组处理：经他莫昔芬 (Tamoxifen, TMX) 诱导Notch表达48小时，或辅以1%氧浓度的缺氧处理48小时，并利用siRNA敲低HIF1α或HIF2α的表达，随后对各组细胞进行RNA测序 (RNA sequencing)。cDNA文库 (cDNA library) 的质量通过安捷伦2100生物分析仪 (Agilent 2100 bioanalyzer) 进行检测。文库在Illumina HiSeq 2000测序系统上完成测序，测序读段通过Bowtie比对至人类基因组（组装版本hg19）及转录组数据库（RefSeq与Ensembl）。利用rpkmforgenes工具生成每百万映射读段每千碱基片段数 (Reads Per Kilobase per Million mapped reads, RPKM) 值。