Mechanism of SAG apoptosis protection under hypoxia

描述（由申请人提供）：已知缺氧会使癌细胞对放疗和化疗产生抗性，导致治疗失败。我们的长期目标是通过靶向赋予这种抗性的分子来诱导缺氧癌细胞的凋亡。SAG（对凋亡敏感的基因）或ROC 2/Rbx 2是富含半胱氨酸的抗氧化蛋白，并且是SCF（skp 1、Cullins、F-box蛋白）和可能的VCB（VHL-Cul 2-Elongin B和C）E3泛素连接酶的RING组分。我们发现SAG在缺氧条件下被诱导，并且当过表达时，在体外和体内都抑制缺氧诱导的凋亡。SAG还结合半胱天冬酶3，并在与SCF E3泛素连接酶复合时促进半胱天冬酶3泛素化。本研究的目的是阐明SAG在缺氧条件下诱导凋亡和保护凋亡的机制。中心假设是缺氧诱导HIF-1，HIF-1通过直接结合SAG启动子中的共有元件反式激活SAG表达。缺氧还诱导蛋白激酶CKII的活性，其通过磷酸化激活SAG。在其诱导和激活后，SAG与SCF和VCB E3泛素连接酶的其他组分复合，分别促进caspase 3和HIF-1 α的泛素化和降解。靶向caspase 3降解阻断caspase依赖性细胞凋亡，而靶向HIF-1 α降解通过p53稳定防止HIF-1 α诱导的细胞凋亡。拟议研究的基本原理是将SAG和CKII鉴定和表征为新型低氧癌症靶标，其最终目标是通过抑制它们使低氧癌细胞对当前癌症疗法敏感来进行药物干预。验证这一假设的具体目的是：1）阐明缺氧条件下通过HIF-1 α转录激活诱导SAG的机制; 2）阐明SAG以连接酶非依赖性方式保护细胞凋亡的机制（清除活性氧）和连接酶依赖性（半胱天冬酶3和HIF-1 α泛素化和降解），3）通过靶向SAG和CKII确定缺氧癌细胞对放射治疗的敏感性，（4）区分SAG与ROC 1/Rbx 1的应激反应。通过这项研究，我们将首次证明SAG和HIF-1 α之间的反馈回路，并揭示SAG通过促进caspase 3和HIF-1 α的泛素化/降解来抑制缺氧条件下细胞凋亡的机制。我们还将证明通过SAG沉默和CKII抑制实现的放射增敏作用，并为未来通过靶向SAG或SAG相关E3连接酶和/或CKII激酶发现新型放射增敏剂提供分子基础。

DESCRIPTION (provided by applicant): Hypoxia is known to confer resistance of cancer cells to radio- and chemo-therapies leading to treatment failure. Our long-range goal is to induce apoptosis of hypoxic cancer cells through targeting the molecules that confer such a resistance. SAG (Sensitive to Apoptosis Gene) or ROC2/Rbx2 is a cysteine-rich antioxidant protein and a RING component of SCF (skp1, Cullins, F-box proteins) and possibly VCB (VHL-Cul2-Elongin B and C) E3 ubiquitin ligases. We found that SAG is induced under hypoxia and when over-expressed, inhibits hypoxiainduced apoptosis both in vitro and in vivo. SAG also binds to caspase 3 and promotes caspase 3 ubiquitination when complexed with SCF E3 ubiquitin ligase. The objective of this application is to elucidate the mechanism of SAG induction and apoptosis protection under hypoxia. The central hypothesis is that hypoxia induces HIF-1 which transactivates SAG expression through a direct binding to its consensus elements in the SAG promoter. Hypoxia also induces activity of protein kinase CKII which activates SAG via phosphorylation. Upon its induction and activation, SAG complexes with other components of SCF and VCB E3 ubiquitin ligases to promote the ubiquitination and degradation of caspase 3 and HIF-1alpha, respectively. Targeted caspase 3 degradation blocks caspase-dependent apoptosis, whereas targeted HIF-1alpha degradation prevents HIF-1alpha induced apoptosis through p53 stabilization. The rationale for the proposed research is to identify and characterize SAG and CKII as novel hypoxic cancer targets with ultimate goal in pharmaceutical intervention through their inhibition to sensitize hypoxic cancer cells to current cancer therapies. Specific aims to test the hypothesis are 1) to elucidate the mechanism of SAG induction through HIF-1alpha transcriptional activation under hypoxia, 2) to elucidate the mechanism by which SAG protects against apoptosis in manners of ligase independence (scavenging reactive oxygen species) and ligase dependence (caspase 3 and HIF-1alpha ubiquitination and degradation), 3) to determine the sensitization of hypoxic cancer cells to radio-therapy through targeting SAG and CKII, and 4) to differentiate SAG from ROC1/Rbx1 in their stress responses. Through this proposed research, we will demonstrate, for the first time, a feedback loop between SAG and HIF-1alpha and reveal a mechanism by which SAG inhibits apoptosis under hypoxia via promoting the ubiquitination/degradation of caspase 3 and HIF-1alpha. We will also demonstrate a radio-sensitizing effect achieved by SAG silencing and CKII inhibition and provide a molecular basis for future discovery of novel radio-sensitizers through targeting SAG, or SAG-associated E3 ligases, and/or CKII kinase.