LRP16钝化肿瘤细胞放疗敏感性的分子机制及其阻抗研究

The excessive activation of NF-kB, which plays crucial roles for blunting ionizing radiation-induced cell apoptosis, accounts for the acquisition of radioresistance frequently occurred in tumor patients. However, the mechanisms that trigger NF-kB overactivation at the early phase of DNA damage remain obscure so far. LRP16, initially cloned by our group, is a special member of macrodomain protein superfamily. Overexpression or strong nuclear staining of LRP16 was frequently observed in several types of tumor cells. Further clinicpathological investigation suggested the poor prognosis of patients with LRP16 overexpression. Recently, our findings demonstrated that LRP16 translocated swiftly from nucleus into cytoplasm upon DNA damage induced by IR and transiently interacted with PKR, IKKb, NEMO in cytoplasm, and then returned back to the nucleus. Inhibition of LRP16 remarkably suppressed IKK activation and NF-kB activity induced by genotoxic agents, and sensitized tumor cells to apoptosis significantly; whereas,overexpression of LRP16 showed the reverse results. Our preliminary data suggested that LRP16 should confer the radioresistance of tumor cells through mediating the activation of NF-kB induced by DNA damage. In this study, we will: 1) dissect the molecular mechanisms by which LRP16 confers the early-phase activation of NF-kB induced by ionizing radiation in tumor cells; 2) investigate the possible relevance of LRP16 expression in tumor samples and the radioresistance of patients; 3) By screening of small molecular compound library and the use of animal models with tumor xenograft, we expect to identify the small molecular compounds that can effectively block ionizing radiation-induced nuclear export of LRP16, and subsequently suppress NF-kB activation, and finally sensitize tumor cells to apoptosis. The accomplishment of this program will not only reveal a new molecule pathway mediating the NF-kB activation at the early phase of ionizing radiation-induced DNA damage, but will also provide a helpful basis for developing novel radiotherapy sensitizers.

NF-kB过度激活是导致肿瘤细胞产生放疗抵抗的关键因素，然而导致NF-kB过度激活的分子机制尚不完全明确。LRP16在多种肿瘤组织中高表达,并且预示预后不良。近期，我们的研究结果显示电离辐射等诱导DNA损伤后，LRP16快速的由细胞核穿梭到细胞浆，然后再回到核，并在胞浆内与PKR、IKKb、NEMO等分子相互作用；抑制LRP16显著抑制了DNA损伤剂诱导的IKK及NF-kB活性，增加了肿瘤细胞对凋亡的敏感性，过表达时则相反。提示LRP16通过介导DNA损伤诱导的NF-kB的激活呈递肿瘤细胞的辐射抵抗。本项目将：1）从分子水平深入阐明LRP16介导电离辐射诱导NF-kB早期激活的分子机制；2）研究多种癌组织中LRP16表达与临床放疗抵抗的关联性；3）从细胞与动物水平研究小分子化合物对电离辐射诱导的LRP16出核阻滞及辐射增敏效应。本项目的完成将为新的肿瘤放疗增敏途径的识别奠定基础。

NF-κB过度激活是导致肿瘤细胞产生放疗抵抗的关键因素，然而导致NF-κB过度激活的分子机制尚不完全明确。LRP16在多种肿瘤组织中高表达，并且预示预后不良。我们的早期研究结果显示电离辐射等诱导DNA损伤后，LRP16快速的由细胞核穿梭到细胞浆，然后再回到核，并在胞浆内与PKR、IKΚB、IKKγ等分子相互作用；抑制LRP16显著抑制了DNA损伤剂诱导的IKK及NF-κB活性，增加了肿瘤细胞对凋亡的敏感性，过表达时则相反。提示LRP16通过介导DNA损伤诱导的NF-κB的激活呈递肿瘤细胞的辐射抵抗。本项目从三个部分进行了深入研究：第一部分：从分子水平深入阐明LRP16介导电离辐射诱导NF-κB早期激活的分子机制；第二部分：研究多种癌组织中LRP16表达与临床放疗抵抗的关联性；第三部分：从细胞与动物水平研究小分子化合物对电离辐射诱导的LRP16出核阻滞及辐射增敏效应。结果显示，在细胞核内，LRP16可以组成性的与PARP1、IKKγ相互作用，当细胞发生双链DNA损伤时，这种组成性的作用可介导PARP1、IKKγ及PIASy形成复合体，进而促进IKKγ的修饰反应和NF-κB激活。该功能作用依赖于双链DNA断裂感应分子Ku70/Ku80。在细胞浆内，LRP16可选择性地与PKR相互作用，在DNA损伤剂刺激下，LRP16作为支架蛋白募集PKR与IKKβ，进而促进PAR依赖的NF-κB激活，最终导致肿瘤细胞产生获得性放化疗抵抗。我们前期识别的小分子化合物MRS2578可以显著抑制LRP16与PKR、IKKβ的相互作用，逆转LRP16在放化疗抵抗中的功能作用。并且，MRS2578可以与依托泊苷协同作用发挥抗肿瘤作用。该研究不仅为DNA损伤诱导NF-κB激活的分子机制提供了新的认识，也为如何增强肿瘤放化疗效果提供了新的策略。主要内容发表在《Nucleic Acids Research》和《Elife》上。