Enhancing radiation and cisplatin HNSCC cell killing by inhibiting mitochondrial

描述（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4 -5万例新病例，约1.2万人死于恶性鳞状细胞癌。早期疾病仅用手术或放疗治疗，而晚期HNSCC则采用顺铂和放疗联合治疗。总生存率仅为~40%，且~30%的晚期疾病有局部复发。生活在低氧微环境中的肿瘤细胞对放射治疗的抵抗力更强。顺铂联合放疗提高了细胞杀伤效果，但顺铂毒性很大，肿瘤在治疗过程中可能对顺铂产生耐药性。因此，有必要开发新的辅助疗法来改善放疗和顺铂的细胞杀伤。本研究旨在确定线粒体双链断裂（DSB）修复的破坏是否可以作为一种补充治疗来改善放疗或顺铂和放疗的治疗效果，并开发一种潜在的新分子工具，可用于增强HNSCC细胞杀伤。放疗和顺铂通过破坏细胞的DNA起作用：放疗引入dsb，顺铂引入DNA交联，可在修复过程中或通过停止复制分叉转化为dsb。线粒体基因组的损伤可导致功能性线粒体的丧失，诱导氧化应激和更大的核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导致的细胞死亡。我们已经开发出一种线粒体靶向细菌Ku蛋白（cKumyc），它可以结合dsb，但“缺少”与其他人类DNA修复蛋白连接所需的结构域。我们假设，当cKumyc靶向HNSCC细胞的线粒体时，在电离辐射或顺铂治疗后，cKumyc会与dsb结合，破坏修复，导致线粒体基因组断裂，活性氧（ROS）产生和细胞死亡。在稳定的HNSCC细胞系中，强力霉素可以诱导表达cKumyc。在放疗和/或顺铂治疗后，将进行克隆源性细胞存活、线粒体功能、ROS产生、线粒体DNA断裂和细胞死亡模式的测定。线粒体DSB修复的破坏预计会增加细胞死亡。由于缺氧在放疗抵抗中起着重要的作用，我们将开发一种仅在缺氧细胞中表达的cKumyc，其功能将使用靶1中1-5%氧气的试验进行测试。这将有助于未来靶向耐辐射缺氧肿瘤细胞。我们假设cKumyc会使缺氧的HNSCC细胞放射敏感。这些原理验证实验有可能发现一个新的靶点（线粒体DNA），也有可能为设计一种新的放疗/联合化疗-放疗的补充治疗提供新的工具，以增强癌细胞的杀伤能力。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 PUBLIC HEALTH RELEVANCE: Head and neck squamous cell carcinomas are treated with radiotherapy and/ or cisplatin. Radiotherapy kills cancer cells by producing DNA damage; the most lethal of which is the DNA double strand break. Cisplatin is a chemotherapy that introduces DNA crosslinks, which block replication forks generating double strand breaks. Double strand breaks are also repair intermediates of DNA crosslinks. This proposal aims to study the role of mitochondrial DNA double strand breaks and their repair in the survival of head and neck squamous cell carcinoma cells under normoxic and hypoxic conditions after treatment with radiation or cisplatin. A new molecular tool to disrupt mitochondrial double strand break repair has been generated and will be tested to determine whether the disruption of mitochondrial repair could be developed as a complementary therapy to radiotherapy/chemotherapy to enhance the killing of cancer cells and improve the long term survival of cancer patients.