Enhancing radiation and cisplatin HNSCC cell killing by inhibiting mitochondrial

描述（由申请人提供）：头颈部鳞状细胞癌（HNSCC）是全球第九大常见癌症。在美国，每年有40- 50，000例新发病例和约12，000例因HNSCC而死亡。早期疾病仅用手术或放疗治疗，而晚期HNSCC用顺铂和放疗的组合治疗。总生存率仅为~ 40%，且~30%的晚期疾病局部复发。生活在低氧微环境中的肿瘤细胞对放射治疗更有抵抗力。联合顺铂与放射治疗可以提高细胞杀伤力，但顺铂毒性很强，肿瘤在治疗过程中可能对顺铂产生耐药性。因此，需要开发新的补充疗法以改善放射和顺铂的细胞杀伤。该提案旨在确定线粒体双链断裂（DSB）修复的破坏是否可以用作补充治疗，以改善放射治疗或顺铂和放射治疗的治疗结果，并开发一种潜在的新分子工具，可用于增强HNSCC细胞杀伤。放射疗法和顺铂通过破坏细胞的DNA起作用：放射疗法引入DSB，顺铂引入DNA交联，DNA交联可以在修复过程中或通过阻止复制叉转化为DSB。线粒体基因组的损伤可导致功能性线粒体的丧失、氧化应激的诱导和更大的核DNA损伤。核和线粒体DNA修复机制都存在，尽管对线粒体修复知之甚少。这项工作旨在更多地了解线粒体损伤引起的细胞死亡。我们已经开发了一种靶向肠道的细菌Ku蛋白（cKumyc），它可以结合DSB，但“缺失”了与其他人类DNA修复蛋白连接所需的结构域。我们假设cKumyc在靶向HNSCC细胞中的线粒体时，将在用电离辐射或顺铂处理后结合DSB，破坏修复，引起线粒体基因组片段化、活性氧（ROS）产生和细胞死亡。在稳定的HNSCC细胞系中，将产生可以用多西环素诱导以表达cKumyc的细胞系。将进行测定以确定用放射和/或顺铂处理后的克隆形成细胞存活、线粒体功能、ROS产生、线粒体DNA片段化和细胞死亡模式。线粒体DSB修复的破坏预期会增强细胞死亡。由于缺氧在对放射治疗的抗性中起着特定的目的1的重要作用，因此我们将开发仅在缺氧细胞中表达的cKumyc，并且将使用目的1中的测定在1-5%氧气下测试其功能。这将有助于未来靶向放射抗性缺氧肿瘤细胞。我们推测cKumyc在缺氧条件下对HNSCC细胞具有放射增敏作用。这些原理验证实验有可能发现一个新的靶点（线粒体DNA），以及一种新的工具，用于设计一种新的放射治疗/联合化疗-放射治疗的补充治疗，以增强癌细胞的杀伤。具体目标2

DESCRIPTION (provided by applicant): Head and neck squamous cell carcinoma (HNSCC) is the ninth most common cancer worldwide. In the US there are 40-50,000 new cases a year and ~12,000 deaths due to HNSCC. Early stage disease is treated with surgery or radiotherapy alone, while advanced stage HNSCC is treated with a combination of cisplatin and radiotherapy. The overall survival rate is only ~40%, and ~30% of advanced stage disease has locoregional recurrence. Tumor cells living in low oxygen microenvironments are more resistant to radiotherapy. Combining cisplatin with radiation has improved cell killing, but cisplatin is very toxic and tumors can become resistant to cisplatin during treatment. Therefore there is a need to develop new complementary therapies to improve cell killing by radiation and cisplatin. This proposal aims to determine whether disruption of mitochondrial double strand break (DSB) repair can be used as a complementary treatment to improve the therapeutic outcome of radiotherapy, or cisplatin and radiotherapy, and to develop a potential new molecular tool that can be used to enhance HNSCC cell killing. Radiotherapy and cisplatin work by damaging the cell's DNA: radiotherapy introduces DSBs, and cisplatin introduces DNA crosslinks that can be converted to DSBs during repair or by stalling replication forks. Damage to the mitochondrial genome can result in loss of functional mitochondria, an induction of oxidative stress and greater nuclear DNA damage. Both nuclear and mitochondrial DNA repair mechanisms exist, although less is known about mitochondrial repair. This work aims to understand more about cell death from mitochondrial damage. We have developed a mitochondrial-targeted bacterial Ku protein (cKumyc) that can bind DSBs but is "missing" the domains required to link with other human DNA repair proteins. We hypothesize that the cKumyc when targeted to the mitochondria in HNSCC cells will bind to DSBs following treatment with ionizing radiation or cisplatin, disrupting repair, causing mitochondrial genome fragmentation, reactive oxygen species (ROS) production and cell death. In stable HNSCC cell lines will be generated that can be induced with doxycycline to express cKumyc. Assays will be performed to determine clonogenic cell survival, mitochondrial function, ROS production, mitochondrial DNA fragmentation and mode of cell death after treatment with radiation and/ or cisplatin. Disruption of mitochondrial DSB repair is expected to enhance cell death. Since hypoxia plays a Specific Aim 1 significant role in resistance to radiotherapy, in we will develop a cKumyc that is expressed only in hypoxic cells and its function will be tested using the assays in aim 1 at 1-5% oxygen. This will aid future targeting of radioresistant hypoxic tumor cells. We hypothesize that cKumyc will radiosensitize the HNSCC cells under hypoxia. These proof-of-principle experiments have the potential of uncovering a new target (mitochondrial DNA) as well as a new tool for the design of a novel complementary treatment for radiotherapy/ combined chemo-radiotherapy to enhance cancer cell killing. Specific Aim 2