Luo Ang, Gong Yao, Kim Hyungjin, Chen Yue
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota at Twin Cities, Minneapolis, MN 55455, USA.
Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA.
NAR Cancer. 2020 Jun;2(2):zcaa010. doi: 10.1093/narcan/zcaa010. Epub 2020 Jun 29.
Mechanistic understanding of hypoxia-responsive signaling pathways provides important insights into oxygen- and metabolism-dependent cellular phenotypes in diseases. Using SILAC-based quantitative proteomics, we provided a quantitative map identifying over 6300 protein groups in response to hypoxia in prostate cancer cells and identified both canonical and novel cellular networks dynamically regulated under hypoxia. Particularly, we identified SDE2, a DNA stress response modulator, that was significantly downregulated by hypoxia, independent of HIF (hypoxia-inducible factor) transcriptional activity. Mechanistically, hypoxia treatment promoted SDE2 polyubiquitination and degradation. Such regulation is independent of previously identified Arg/N-end rule proteolysis or the ubiquitin E3 ligase, CDT2. Depletion of SDE2 increased cellular sensitivity to DNA damage and inhibited cell proliferation. Interestingly, either SDE2 depletion or hypoxia treatment potentiated DNA damage-induced PCNA (proliferating cell nuclear antigen) monoubiquitination, a key step for translesion DNA synthesis. Furthermore, knockdown of SDE2 desensitized, while overexpression of SDE2 protected the hypoxia-mediated regulation of PCNA monoubiquitination upon DNA damage. Taken together, our quantitative proteomics and biochemical study revealed diverse hypoxia-responsive pathways that strongly associated with prostate cancer tumorigenesis and identified the functional roles of SDE2 and hypoxia in regulating DNA damage-induced PCNA monoubiquitination, suggesting a possible link between hypoxic microenvironment and the activation of error-prone DNA repair pathway in tumor cells.
对缺氧反应信号通路的机制性理解为深入了解疾病中氧依赖和代谢依赖的细胞表型提供了重要见解。利用基于稳定同位素标记氨基酸的细胞培养物定量蛋白质组学技术，我们提供了一张定量图谱，确定了前列腺癌细胞在缺氧反应中有超过6300个蛋白质组，并鉴定了在缺氧条件下动态调节的经典和新的细胞网络。特别地，我们鉴定出SDE2，一种DNA应激反应调节剂，它在缺氧条件下显著下调，且不依赖于缺氧诱导因子（HIF）的转录活性。从机制上讲，缺氧处理促进了SDE2的多聚泛素化和降解。这种调节独立于先前确定的精氨酸/N端规则蛋白水解或泛素E3连接酶CDT2。SDE2的缺失增加了细胞对DNA损伤的敏感性并抑制了细胞增殖。有趣的是，SDE2的缺失或缺氧处理均增强了DNA损伤诱导的增殖细胞核抗原（PCNA）单泛素化，这是跨损伤DNA合成的关键步骤。此外，敲低SDE2会使细胞脱敏，而SDE2的过表达则保护了DNA损伤时缺氧介导的PCNA单泛素化调节。综上所述，我们的定量蛋白质组学和生化研究揭示了与前列腺癌肿瘤发生密切相关的多种缺氧反应途径，并确定了SDE2和缺氧在调节DNA损伤诱导的PCNA单泛素化中的功能作用，提示缺氧微环境与肿瘤细胞中易出错的DNA修复途径激活之间可能存在联系。
