Enhancing radiation and cisplatin HNSCC cell killing by inhibiting mitochondrial

通过抑制线粒体增强放射和顺铂对 HNSCC 细胞的杀伤作用

• 批准号: 8305841
• 负责人: LYNN HARRISON
• 金额: $ 18.49万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8305841
• 关键词: Aftercare; Animal Model; Apoptosis; Binding; Biological Assay; Cancer Patient; Cancer cell line; Cell Culture Techniques; Cell Death; Cell Line; Cell Nucleus; Cell Survival; Cells; Cessation of life; Chimeric Proteins; Cisplatin; Collaborations; Complementary therapies; Cytochromes; DNA; DNA Damage; DNA Double Strand Break; DNA Fragmentation; DNA Repair; DNA crosslink; DNA repair protein; DNA-PKcs; Disease; Dominant-Negative Mutation; Double Strand Break Repair; Doxycycline; Fibrinogen; Frequencies; Future; G22P1 gene; Growth; HIF1A gene; Head and Neck Squamous Cell Carcinoma; Human; Hypoxia; Hypoxia Inducible Factor; Ionizing radiation; Ku Protein; Lasers; Letters; Life; Link; Malignant Epithelial Cell; Malignant Neoplasms; Mitochondria; Mitochondrial DNA; Modeling; Molecular; Monitor; Mus; Mycobacterium tuberculosis; Normal Cell; Nuclear; Operative Surgical Procedures; Oral; Outcome; Oxidases; Oxidation-Reduction; Oxidative Stress Induction; Oxygen; Patients; Play; Production; Radiation; Radiation therapy; Reactive Oxygen Species; Recurrence; Resistance; Role; Staging; Survival Rate; System; Testing; Therapeutic; Time; Viral; Work; Xenograft Model; Xenograft procedure; cancer cell; cell injury; cell killing; chemotherapy; crosslink; design; effective therapy; functional loss; gene therapy; homologous recombination; human DNA; hypoxia inducible factor 1; improved; keratinocyte; killings; mitochondrial dysfunction; mitochondrial genome; neoplastic cell; novel; oxidative damage; prevent; repaired; research study; stable cell line; subcutaneous; tool; tumor

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.

描述（由申请人提供）：头部和颈部鳞状细胞癌（HNSCC）是全球第九个最常见的癌症。在美国，每年有40-50,000例新病例，HNSCC造成的死亡约为12,000例。早期疾病仅通过手术或放疗治疗，而晚期HNSCC则通过顺铂和放射疗法的组合进行治疗。总体存活率仅约40％，大约30％的晚期疾病患有局部复发。生活在低氧气微环境中的肿瘤细胞对放射疗法更具耐药性。将顺铂与辐射结合起来可以改善细胞的杀伤，但顺铂具有非常毒性，在治疗过程中肿瘤对顺铂具有抗性。因此，有必要开发新的补充疗法来改善辐射和顺铂的杀伤。该提案旨在确定线粒体双链断裂（DSB）修复的破坏可以用作补充治疗方法，以改善放射疗法的治疗结果，或顺铂和放射疗法，并开发可用于增强HNSCC细胞杀死的潜在新分子工具。放射疗法和顺铂通过损害细胞的DNA来起作用：放射疗法引入DSB，顺铂引入DNA交叉链接，可以在修复过程中或通过停止复制叉在修复过程中转换为DSB。线粒体基因组的损害会导致功能性线粒体的损​​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的细胞死亡。我们已经开发了一种靶向线粒体的细菌KU蛋白（CKUMYC），该细菌蛋白可以结合DSB，但“缺少”与其他人DNA修复蛋白链接所需的结构域。我们假设ckumyc靶向HNSCC细胞中的线粒体时，用电离辐射或顺铂治疗后将与DSB结合，从而破坏修复，导致线粒体基因组碎片化，活性氧，活性氧（ROS）生产（ROS）生产和细胞死亡。在稳定的HNSCC细胞系中，将生成可以用强力霉素诱导以表达ckumyc的。将进行测定以确定克隆原性的存活，线粒体功能，ROS产生，线粒体DNA碎片和用辐射和/或顺铂治疗后细胞死亡的模式。线粒体DSB修复的破坏有望增强细胞死亡。由于缺氧在对放射疗法的耐药性中起特定的目标1的重要作用，因此我们将开发仅在低氧细胞中表达的ckumyc，其功能将在1-5％氧气中的AIM 1中测试。这将有助于将来靶向放射耐药性低氧肿瘤细胞。我们假设Ckumyc将在缺氧下放射敏感性HNSCC细胞。这些原则实验具有揭示新靶标（线粒体DNA）的潜力，以及一种新的工具，用于设计新型的放射治疗/联合化学疗法治疗，以增强癌细胞杀伤。具体目标2 公共卫生相关性：用放射治疗和/或顺铂治疗头部和颈部鳞状细胞癌。放射疗法通过产生DNA损伤杀死癌细胞。其中最致命的是DNA双链断裂。顺铂是一种化学疗法，它引入了DNA交联，该化疗阻断了复制叉会产生双链断裂。双链断裂也是DNA交联的修复中间体。该提案旨在研究线粒体DNA双链断裂的作用，并在辐射或顺铂治疗后在常氧和低氧条件下的头颈部鳞状细胞癌细胞的存活中修复。已经产生了一种破坏线粒体双链断裂修复的新分子工具，并将进行测试，以确定是否可以开发线粒体修复的破坏，作为对放射疗法/化学疗法的补充治疗，以增强癌细胞的杀伤并改善癌症患者的长期存活。