Mechanism of SAG apoptosis protection under hypoxia

描述（由申请人提供）：已知缺氧会导致癌细胞对放疗和化疗产生耐药性，从而导致治疗失败。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 （Sensitive to Apoptosis Gene）或ROC2/Rbx2是一种富含半胱氨酸的抗氧化蛋白，是SCF （skp1, Cullins， F-box蛋白）和可能的VCB (VHL-Cul2-Elongin B和C) E3泛素连接酶的RING组分。我们发现SAG是在缺氧条件下诱导的，当其过表达时，在体外和体内均能抑制缺氧诱导的细胞凋亡。当与scfe3泛素连接酶结合时，SAG还能与caspase 3结合并促进caspase 3泛素化。本研究旨在阐明缺氧条件下SAG诱导和细胞凋亡保护的机制。中心假设是缺氧诱导HIF-1， HIF-1通过直接结合其在SAG启动子中的共识元件而激活SAG表达。缺氧还会诱导CKII蛋白激酶的活性，CKII蛋白激酶通过磷酸化激活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物分别促进caspase 3和hif -1 α的泛素化和降解。靶向caspase 3降解阻断caspase依赖的细胞凋亡，而靶向hif - 1α降解通过p53稳定阻止hif - 1α诱导的细胞凋亡。本研究的基本原理是识别和表征SAG和CKII作为新的低氧癌症靶点，并通过抑制低氧癌细胞对当前癌症治疗的敏感性，最终达到药物干预的目的。验证该假设的具体目的是：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通过连接酶独立（清除活性氧）和连接酶依赖（caspase 3和hif -1 α泛素化和降解）的方式保护细胞凋亡的机制；3)通过靶向SAG和CKII来确定缺氧癌细胞对放疗的增敏性。4)在应激反应中区分SAG与ROC1/Rbx1。通过本研究，我们将首次展示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。我们还将展示通过SAG沉默和CKII抑制实现的放射增敏效果，并为未来通过靶向SAG或SAG相关的E3连接酶和/或CKII激酶发现新的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.