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)是全球第九大最常见的癌症。在美国，每年有40-50,000个新病例和大约12,000人死于HNSCC。早期疾病仅用手术或放射治疗，而晚期HNSCC则用顺铂和放射治疗的组合治疗。总存活率仅为~40%，约30%的晚期疾病局部区域复发。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。顺铂与放射联合使用可以提高细胞杀伤率，但顺铂毒性很大，治疗过程中肿瘤会对顺铂产生抗药性。因此，有必要开发新的补充疗法，以改善辐射和顺铂对细胞的杀伤作用。本研究旨在确定破坏线粒体双链断裂(DSB)修复是否可以作为辅助治疗来改善放疗或顺铂和放疗的疗效，并开发一种潜在的新的分子工具，可用于增强HNSCC细胞的杀伤作用。放射治疗和顺铂通过破坏细胞的DNA起作用：放射治疗引入DSB，而顺铂引入DNA交联物，在修复过程中或通过停止复制叉子可转化为DSB。线粒体基因组的损伤可能导致功能线粒体的丧失、氧化应激的诱导和更大的核DNA损伤。细胞核和线粒体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的细菌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。我们推测，当cKumyc靶向HNSCC细胞的线粒体时，会在电离辐射或顺铂处理后与DSB结合，破坏修复，导致线粒体基因组碎裂，产生活性氧(ROS)和细胞死亡。在稳定的HNSCC细胞系中，可以用多西环素诱导表达cKumyc。在放射和/或顺铂治疗后，将进行克隆细胞存活率、线粒体功能、ROS产生、线粒体DNA片段化和细胞死亡模式的检测。线粒体DSB修复的中断预计会加剧细胞死亡。由于低氧在放射治疗抵抗中扮演着特定的目标1的重要角色，我们将开发一种仅在低氧细胞中表达的cKumyc，并将使用目标1中在1-5%氧气中的检测来测试其功能。这将有助于将来靶向抗辐射的低氧肿瘤细胞。我们推测cKumyc对缺氧条件下的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)，以及一个新的工具，用于设计一种新的放射治疗补充疗法/联合化疗-放射治疗以增强癌细胞杀伤力。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