磷酸甘油酸变位酶PGAM1参与DNA损伤应答机制研究

发布时间：2018-09-07 10:44

【摘要】：能量代谢异常是肿瘤细胞的重大特征之一,主要表现为依赖有氧糖酵解进行能量代谢。有氧糖酵解代谢酶是调控有氧糖酵解代谢途径的核心元件,其表达水平及活性异常与多种肿瘤的恶性化程度密切相关,是当前肿瘤代谢领域的研究重点。然而,目前对于代谢酶参与肿瘤发生发展的功能认识仍非常有限,大多停留于传统认识的肿瘤细胞恶性化增殖所必须的能量供给和生物合成两大方面。事实上,新近有证据提示,代谢酶可能贡献于肿瘤细胞恶性微环境的维持、表观遗传调控、侵袭迁移等诸多方面。全面认识代谢酶参与肿瘤发生发展的功能,深入认识其分子机制,对于揭示肿瘤代谢在肿瘤恶性化进程中的作用具有重要意义,也将为靶向肿瘤代谢的抗肿瘤策略提供重要的理论依据。磷酸甘油酸变位酶PGAM1是有氧糖酵解途径的重要代谢酶,通过催化3-磷酸甘油酸(3-PG)向2-磷酸甘油酸(2-PG)的转变,参与有氧糖酵解途径,在多种肿瘤中高表达或活性升高。新近研究报道了PGAM1通过其酶活功能同时调控磷酸戊糖和丝氨酸合成途径,支持肿瘤细胞快速增殖的重要功能。PGAM1成为肿瘤代谢领域备受关注的分子。然而,关于PGAM1的认识仍非常有限,仅停留于能量供给和生物合成两方面。本论文旨在全面认识PGAM1的生物学功能,深入探讨其分子机制,旨在为全面认识PGAM1在肿瘤发生发展中的作用提供实验依据,并为指导PGAM1抑制剂的使用及研发策略提供重要信息。首先,我们选用基于质谱的蛋白质组学技术,系统研究了干扰PGAM1功能可能影响的信号通路。结果发现,除了预期的代谢和生物合成相关通路,PGAM1干扰细胞的DNA损伤应答相关通路显著受到了影响。本文将着力对PGAM1在DNA损伤应答中可能扮演的角色进行探索。首先,为了确证PGAM1参与DNA损伤应答,我们以克隆形成等实验为指标,检测了干扰PGAM1对不同类型DNA损伤类药物敏感性的影响。结果发现PGAM1能够选择性增强细胞对喜树碱(CPT)和顺铂(CDDP)的敏感性,提示可能与CPT和CDDP造成的复制依赖的DNA双链断裂(DSBs)应答密切相关。通过研究药物处理后DSBs特异性标志物γH2AX变化、dsDNA断裂情况等多项生物学指标的动态变化情况,我们发现干扰PGAM1不影响初始DSBs损伤水平,但会导致细胞DNA修复缺陷。进一步采用DSBs两条主要修复通路,即同源重组(HR)或非同源末端连接(NHEJ)的报告基因系统,我们揭示PGAM1特异调控HR修复,而非NHEJ连接。为了研究PGAM1代谢酶活性是否贡献于HR修复,我们构建了PGAM1酶活突变体。通过对比回转野生型或酶活突变型PGAM1,我们发现PGAM1的DNA损伤修复功能是酶活依赖的。同时,使用PGAM1酶活抑制剂PGMI-004A或回补代谢产物methyl-2-PG均确认了上述结果。基于PGAM1同时参与糖酵解、磷酸戊糖和丝氨酸合成途径的已有认识,我们系统比较了上述三条通路对于HR修复功能的贡献。结果发现,PGAM1主要通过影响磷酸戊糖旁路,参与HR修复。回补磷酸戊糖途径终产物dNTP能够逆转PGAM1干扰引起的HR修复缺陷。为了进一步回答PGAM1如何影响磷酸戊糖途径参与HR修复,本研究聚焦了HR修复的多个关键步骤。结合使用免疫荧光检测修复蛋白foci形成等多项研究手段,发现干扰PGAM1影响CPT诱导的DSBs末端的单链DNA(ssDNA)暴露、阻碍RPA复合物的招募。而这一效应主要通过影响DSBs末端处理蛋白CtIP的功能实现。深入研究发现,PGAM1通过调控细胞内dNTP的水平,导致p53/p73依赖的p21转录激活,后者通过上调E3泛素连接酶APC/Cdh1活性,导致CtIP蛋白稳定性下降,蛋白水平下调。至此,本研究对PGAM1如何影响HR修复的分子机制进行了详尽的阐述。依据上述机制发现,我们对PGAM1抑制剂的临床用药策略进行探索。使用在裸小鼠皮下移植瘤模型,我们发现干扰PGAM1或联用PGAM1抑制剂能够增强Olaparib对BRCA野生型移植瘤的抑制作用,干扰PGAM1或联用PGMI-004A能显著下调肿瘤组织内CtIP蛋白水平,导致DNA损伤积累,进而诱导细胞凋亡。综上,本论文发现了PGAM1调控HR修复的新功能,并揭示了PGAM1能够通过调节胞内dNTP平衡、影响CtIP蛋白稳定性,调控HR修复通路的分子机制,为认识PGAM1在肿瘤发生发展中的功能提供了新的证据。在此基础上,本论文证实联用PGAM1抑制剂能拓展PARP抑制剂的治疗空间,为PARP抑制剂治疗BRCA野生型肿瘤带来了新契机。
[Abstract]:Abnormal energy metabolism is one of the most important characteristics of tumor cells, which mainly depends on aerobic glycolysis for energy metabolism. Aerobic glycolytic metabolic enzymes are the core elements regulating the pathway of aerobic glycolysis metabolism. The abnormal expression and activity of these enzymes are closely related to the degree of malignancy of various tumors and are the current research in the field of tumor metabolism. In fact, recent evidence suggests that metabolic enzymes may contribute to the maintenance of malignant microenvironment of tumor cells. It is of great significance to understand the role of metabolic enzymes in tumor genesis and development and their molecular mechanisms in revealing the role of tumor metabolism in the process of malignancy. It will also provide important theoretical basis for anti-tumor strategies targeting tumor metabolism. PGAM1 is an important metabolic enzyme in the aerobic glycolysis pathway. It participates in the aerobic glycolysis pathway by catalyzing the conversion of 3-phosphoglyceric acid (3-PG) to 2-phosphoglyceric acid (2-PG), and has high expression or activity in a variety of tumors. Recently, it has been reported that PGAM1 regulates pentose phosphate and serine biosynthesis pathway simultaneously through its enzyme activity. PGAM1 has become an important molecule in the field of tumor metabolism. However, the understanding of PGAM1 is still very limited, only in the aspects of energy supply and biosynthesis. The role of PGAM1 inhibitors in biological development provides experimental evidence and important information for guiding the use and development of PGAM1 inhibitors. First, we used mass spectrometry-based proteomics techniques to systematically study the possible signaling pathways that interfere with PGAM1 function. In this paper, we will explore the possible role of PGAM1 in DNA damage response. Firstly, to confirm the role of PGAM1 in DNA damage response, we examined the effects of PGAM1 interference on the sensitivity of different types of DNA damage drugs by clone formation and other experiments. It was found that PGAM1 could selectively enhance the cell sensitivity to CPT and CDDP, suggesting that PGAM1 might be closely related to the replication-dependent DNA double strand breaks (DSBs) response induced by CPT and CDDP. We found that interfering with PGAM1 did not affect the initial damage level of DSBs, but resulted in DNA repair deficiencies. Further, we revealed that PGAM1 specifically regulates HR repair, not NHEJ junction, through two major repair pathways, homologous recombination (HR) or non-homologous end-joining (NHEJ) reporter gene system. By comparing PGAM1 with wild-type or mutant PGAM1, we found that the DNA damage repair function of PGAM1 was enzyme-dependent. Meanwhile, PGMI-004A or methyl-2-PG, the enzyme activity inhibitor of PGAM1, were used to confirm the above results. The results showed that PGAM1 participated in HR repair mainly by affecting the pentose phosphate pathway. The end product of the pentose phosphate pathway, dNTP, could reverse the defect of HR repair induced by PGAM1 interference. In this study, we focused on several key steps in HR repair. Combining with immunofluorescence detection of repair protein foci formation, we found that interfering with PGAM1 affected CPT-induced single-stranded DNA (ssDNA) exposure at the end of DSBs, which hindered the recruitment of RPA complexes. In-depth study found that PGAM1 regulated intracellular dNTP levels, resulting in p53/p73-dependent p21 transcription activation, the latter by up-regulating the activity of E3 ubiquitin ligase APC/Cdh1, resulting in decreased stability of CtIP protein, protein level down-regulation. Based on the above mechanism, we explored the clinical strategy of PGAM1 inhibitor. Using subcutaneous transplantation tumor model in nude mice, we found that interfering with PGAM1 or combining with PGAM1 inhibitor could enhance the inhibitory effect of Olaparib on BRCA wild-type transplanted tumor, and interfering with PGAM1 or PGMI-004A significantly. To sum up, we have found that PGAM1 regulates the repair of HR by regulating the level of CtIP protein in tumor tissues, leading to the accumulation of DNA damage and inducing apoptosis. On this basis, this study confirmed that the combination of PGAM1 inhibitors can expand the therapeutic space of PARP inhibitors, and provide a new opportunity for PARP inhibitors to treat BRCA wild-type tumors.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R96

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