Targeting Mitochondrial Redox Capacity to Overcome Cancer Subtype that Regrowth After Radiation

摘要（R 01 CA 251663） 放射疗法（RT）被广泛用于治疗局限性前列腺癌（PCa）。然而，癌细胞通常 通过未知的机制对RT产生耐药性，导致癌症复发。为了改善RT， 我们迫切需要发现导致细胞产生抗药性的细胞事件。我们之前已经证明， PCa异质性，特别是在具有丰富线粒体亚群的前列腺癌中， 并在RT后再生（称为放射抵抗性前列腺癌，或RR-PCa）。线粒体质量升高， 在RR-PCa细胞中获得数量、活性氧（ROS）和生物发生标志物。我们进一步 证明了线粒体生物发生调节因子TFAM（转录因子A， 线粒体），显著恢复RR-PCa细胞对RT的敏感性。因此，我们的总体假设是 RT激活的线粒体生物合成，通过ROS，是一种获得机制，驱动PCa生存后， RT，这是一个将在拟议研究中进行严格审查的前提。已知ROS直接和 通过融合、分裂、线粒体自噬和生物发生间接调节线粒体稳态。我们筛选 FDA批准的药物正在寻找对正常细胞无毒并有能力提高细胞增殖能力的化合物 PCa细胞中线粒体过氧化氢（mtH 2 O2）水平，同时阻断线粒体蛋白翻译。 我们发现阿奇霉素（AZM），一种大环内酯类抗生素，是一种有效的原型化合物， 两个属性。我们进一步证明了AZM联合RT可以增强PCa细胞的死亡， 丰富的线粒体亚群，与单独的AZM或RT相比。因此，我们建议将我们的发现 并确定有效抑制照射后癌细胞存活的机制，以提高RT 功效。具体的目的是：1）确定RT激活线粒体的分子机制， 生物发生促进细胞存活和具有丰富线粒体的PCa细胞的代谢适应， 体外和体内; 2）确定是否超载mtH 2 O2靶向固有线粒体和RT-获得性 在RT获得的线粒体中，在阻断线粒体蛋白翻译的同时， RR-PCa细胞的放射敏感性，以及3）使用mtH 2 O2发生器和线粒体蛋白来改善RT 翻译抑制剂AZM作为原型在原位小鼠异种移植模型和患者来源的异种移植中的应用 具有激活的线粒体生物合成的PCa模型。这项研究使用了最先进的平台，包括 反相蛋白质芯片，稳定同位素分辨代谢组学，全内反射荧光 使用Imaris软件进行显微镜检查、TEMPOL增强MRI成像和高分辨率O2 k-FluoRespirometer。 预计拟议的研究将揭示新的分子见解， 线粒体氧化还原能力和线粒体生物发生改善了RR-PCa治疗中的RT功效。

ABSTRACT (R01CA251663) Radiation therapy (RT) is widely used to treat localized prostate cancer (PCa). However, cancer cells often develop resistance to RT through unknown mechanisms, resulting in cancer recurrence. To improve RT, there is a dire need to uncover cellular events that cause cells to become resistant. We previously demonstrated that PCa heterogeneity, particularly in prostate cancers with an abundant mitochondria subpopulation, often survive and regrow after RT (termed radiation resistant prostate cancer, or RR-PCa). Elevation of mitochondrial mass, number, reactive oxygen species (ROS), and biogenesis markers is acquired in RR-PCa cells. We further demonstrated that knockdown of the mitochondrial biogenesis regulator, TFAM (transcription factor A, mitochondrial), significantly restored the sensitivity of RR-PCa cells to RT. Hence, our overarching hypothesis is that RT-activated mitochondrial biogenesis, via ROS, is an acquisition mechanism that drives PCa survival post- RT, a premise that will undergo stringent examination in the proposed studies. ROS are known to directly and indirectly regulate mitochondrial homeostasis through fusion, fission, mitophagy, and biogenesis. We screened FDA-approved drugs in search of compounds that are nontoxic to normal cells and have the ability to raise the level of mitochondrial hydrogen peroxide (mtH2O2) in PCa cells while blocking mitochondrial protein translation. We found azithromycin (AZM), a macrolide antibiotic, to be an effective prototype compound that possesses both properties. We further demonstrated that AZM combined with RT enhances the death of PCa cells with an abundant mitochondrial subpopulation, compared to AZM or RT alone. Thus, we propose to advance our findings and identify the mechanism(s) that effectively inhibit the survival of post-irradiated cancer cells, to improve RT efficacy. The specific aims are: 1) to define the molecular mechanism(s) by which RT-activated mitochondrial biogenesis promotes cell survival and metabolic adaptations of PCa cells with abundant mitochondria, both in vitro and in vivo; 2) to determine if overloading mtH2O2 to target inherent mitochondria and RT-acquired mitochondria while blocking mitochondrial protein translation in RT-acquired mitochondria enhances radiosensitivity of RR-PCa cells, and 3) to improve RT using a mtH2O2 generator and a mitochondrial protein translation inhibitor, AZM as prototype, in an orthotopic mouse xenograft model and a patient-derived xenograft model of PCa with activated mitochondrial biogenesis. This study uses state-of-the-art platforms including reverse phase protein array, stable isotope-resolved metabolomics, total internal reflection fluorescence microscopy with Imaris software, TEMPOL-enhanced MRI imaging, and a high resolution O2k-FluoRespirometer. The proposed studies are expected to uncover novel molecular insights by which concurrently targeting mitochondrial redox capacity and mitochondrial biogenesis improve RT efficacy in the treatment of RR-PCa.