丙酮酸脱氢酶在线粒体-核逆向调控肿瘤细胞放射敏感性中的作用

In the previous study, we found that mitochondrial dysfunction is associated with mitochondrial retrograde signaling（ROS/NF-κB） pathway activation in the radiation resistant cell lines. Further studies showed that, in the radiation resistant cell lines with a higher expression of PDK1（PDH kinase）, inhibition of PDK1 can increase radiosensitivity. The finding indicated that the PDK1/PDH may be related with regulation of radiosensitivity, however the specific signal pathway between the metabolic enzyme and radiosensitivity was not clear at present. In our furture study, the model of cancer cell with mitochondrial dysfunction will be constructed by decreasing of mtDNA content and respiratory chain enzyme dysfunction, then the mitochondrial ransmembrane potential, ROS content and radiosensitivity be detected to investigate the relationship between mitochondrial dysfunction and radiosensitivity. Furthermore, the change of nucleus and mitochondrial PDH localization and content, histone acetylation levels, DNA damage and repair will be detected in vivo and vitro model with or without suppressing the expression of translocated protein Hsp70 and the acetylation of histone H3 to investigate the effect of PDH content and translocation in mitochondrial retrograde signaling for the regulation of radiosensitivity, simultaneously, to reveal the possible trigger of RTG(Retrograde signaling). This project aims to clarify the non-metabolic signaling pathway.induced by the metabolism reprogramming and its radiobiological function in cancer cells. This study will provide supports to an novel theory and strategy for predicting radiosensitivity, and a potential molecular target for radiosensitizing.

前期研究发现辐射诱导的肿瘤放抗细胞株中线粒体功能障碍、线粒体-核逆向信号通路（ROS/NF-κB）活化，进一步研究发现放抗株中糖酵解调节酶丙酮酸脱氢酶（PDH）激酶PDK1升高，且抑制PDK1后，细胞放射敏感性增加，提示PDK1/PDH与放射敏感性有关，然而其影响放射敏感性的机制及相关逆向信号通路尚不明晰。本项目拟通过不同方法构建线粒体功能障碍模型（mtDNA含量改变、呼吸链酶功能障碍），检测线粒体跨膜电位、ROS含量及放射敏感性的变化，探讨线粒体功能障碍对肿瘤细胞放射敏感性的影响；观察胞核及线粒体中PDH定位及含量变化、组蛋白乙酰化水平、DNA损伤修复能力改变，并在体内体外抑制转位蛋白Hsp70的表达及组蛋白H3的乙酰化，研究以PDH核转位为核心的线粒体-核逆向调控放射敏感性的机制及触发点，阐明肿瘤代谢重编程的非代谢功能及其调节信号通路，为肿瘤放射敏感性预测及干预新策略奠定新的理论基础。

线粒体逆向调控通路在肿瘤重编程和肿瘤细胞对放射线敏感性上发挥重要作用。线粒体功能障碍引起线粒体逆向调控通路的激活。之前的研究发现，磷酸化的丙酮酸脱氢酶(PDH)和丙酮酸激酶-1(PDK-1)，参与无氧糖酵解，与肿瘤对放射线的抵抗密切相关。但它们在线粒体逆向调控通路种的作用未知。在本次研究中，对放射线抗拒的结直肠癌细胞系转录组分析，发现呼吸链复合体I在线粒体功能障碍模型中下调。在呼吸链复合体I功能障碍的结直肠细胞模型中，发现磷酸化丙酮酸脱氢酶高表达，并诱导肿瘤细胞放射抵抗。通过对呼吸链复合体I功能障碍的结直肠细胞模型钙离子浓度测定，发现呼吸链复合体I障碍通过 [Ca2+]m-PDP1-PDH轴，降低PDH在胞质和细胞核内的浓度。细胞核内PDH水平下降通过抑制组蛋白乙酰化，促进DNA损伤修复。同时，过表达呼吸链复合体I重要元件，NDUFS1,可提高呼吸链复合体I功能，在体外核体内中逆转糖酵，增加肿瘤细胞对放射线的敏感性。在新辅助放疗直肠癌样本中，低表达NDUFS1和PDH证明与差肿瘤缓解率相关。由线粒体呼吸链复合体I缺陷激活的[Ca2+]m-PDP1-PDH-组蛋白乙酰化轴促进肿瘤细胞对放射线的抵抗性。这为理解线粒体逆向调控通路提出新思路。NDUFS1首次被证明可以做为预测结直肠癌复合体I功能和肿瘤放射敏感性的靶点。. 此外通过免疫组化在宫颈癌根治性手术标本检测丙酮酸脱氢酶激酶1(PDK1)、磷酸化的丙酮酸脱氢酶 (p-PDH)和丙酮酸激酶 M2 (PKM2)的水平，发现PDK1high/p-PDHhigh/PKM2high亚型宫颈癌患者预后和术后放疗无复发生存差，此亚型患者可能为 I-IIB期宫颈癌预后不良和术后放疗易复发的高危人群，为宫颈癌的分层治疗提供新思路。