Notch信号通过乳酸盐代谢调控髓系细胞分化和成熟在肿瘤进展中的作用和机制研究
发布时间：2018-08-30 13:28
【摘要】：肿瘤作为危害人类健康的头号杀手,对其的研究日益增多。恶性增殖的肿瘤细胞具有复制永生化、转移与扩散、凋亡抵抗、多发突变和免疫逃逸等特征,这使得大多数肿瘤难以被单纯的手术,或者放、化疗以及靶向药物治愈。随着肿瘤发生发展过程被认识的更加深刻,肿瘤炎性微环境逐渐成为肿瘤的主要特征之一。肿瘤组织中大量浸润免疫细胞,如肿瘤相关巨噬细胞(tumor-associated macrophages,TAMs)、肿瘤相关粒细胞(tumor-associated neutrophils,TANs)、不成熟的树突状细胞(immature DCs,iDCs)、髓系来源的抑制性细胞(myeloid-derived suppressor cells,MDSCs)和调节性T淋巴细胞(regulatory T cells,Tregs)。这些细胞相互调节,构成免疫抑制网络,是调节肿瘤组织免疫耐受微环境、促进肿瘤进展的主要因素。而在这些细胞中,髓系细胞发挥了尤其重要的作用。其不仅可以通过旁分泌途径直接促进慢性损伤部位的炎-癌转化、调控肿瘤干细胞自我更新、帮助肿瘤细胞转移和促进肿瘤血管新生,还可以通过影响NK细胞和杀伤性T细胞应答,减少对肿瘤细胞的杀伤和清除。因此如何改善肿瘤微环境,阻断肿瘤相关髓系细胞的免疫抑制活性,并对其进行再教育,成为肿瘤治疗所要探讨和研究的重要方向和策略。与正常发育分化形成的髓系细胞相对比,肿瘤相关髓系细胞具有较低的成熟度,其调控细胞发育和功能的多种转录因子和信号通路也有较大的差异。Notch信号作为个体发育中起到精密调控作用的通路之一,在髓系细胞增殖和凋亡、分化命运选择和干性维持中均发挥重要的作用。Notch信号在后生动物中高度保守,通过相邻细胞表面的配体分子与其细胞表面受体结合,引起受体胞内段入核,介导转录复合物的形成进而激活靶基因的转录。在髓系细胞的发育和功能调控中,Notch信号的作用显著,但具体功能及作用阶段尚存争议,调控机制也未被阐明。一方面,Notch信号可通过GATAs抑制髓系细胞向成熟粒细胞分化,并保持未分化或低分化状态;而同时,Notch信号也可调控髓系发育转录因子PU.1表达,促进髓系细胞向成熟细胞分化。这很大程度上是由于所采用的研究模型不同,存在不同的细胞因子环境。本人前期研究表明Notch信号可通过一系列miRNA簇调控髓系细胞的发育和分化(未发表)。同时,髓系细胞终末分化后同样存在功能的可塑性调控,巨噬细胞会在不同的刺激物的诱导下行使完全不同的功能。所在课题组第一次证实巨噬细胞可塑性选择依赖于Notch信号的激活(Wang YC et al,Cancer Res.2010);后续研究指出巨噬细胞中的Notch信号通过不同的下游分子,在肝纤维化中也发挥重要作用(He F et al,Hepatology.2015),提示Notch信号可通过多种分子机制调控巨噬细胞活化或功能。本人前期工作也证实Notch-miR125a调控轴可以对促肿瘤功能的TAMs进行再教育,使其发挥抗肿瘤功能(Zhao JL et al,Cancer Res.2016)。以上工作基础均证实Notch信号在髓系细胞发育和TAM功能调控中发挥重要作用。除了细胞自主性的调控之外,肿瘤组织复杂的微环境也会对髓系细胞发育起到非自主性调节。肿瘤细胞为了满足其快速增殖,需要大量的生物能量和生物原料,因此即使在有氧条件下,也进行糖酵解作为能量供应。这样的代谢过程被称之为瓦尔堡效应(Warburg effect)。这直接导致肿瘤微环境中营养物质的耗竭和代谢废物的堆积。很多报道均指出代谢环境的改变可以影响髓系细胞的表型和功能,尤其是肿瘤进展过程中,髓系细胞代谢方式的转变往往伴随着“抗肿瘤-促肿瘤”功能的变化。不同极化状态的巨噬细胞利用完全不同的糖代谢方式。促炎的M1型极化巨噬细胞利用糖酵解作为主要供能方式;而拮抗炎症的M2型巨噬细胞则会选择氧化磷酸化途径。除此之外,不同功能极化巨噬细胞在精氨酸代谢和脂代谢的选择上也有较大不同。MDSC的代谢方式同样调控其功能和活化,同脾脏和骨髓相比,肿瘤微环境中MDSC会加速摄取和消耗脂肪酸,同时观察到线粒体氧化磷酸化水平的升高,这主要是肿瘤组织中葡萄糖缺乏的微环境所致。同时肿瘤驱动的高浓度乳酸盐也可以促进MDSC的积累,并抑制NK细胞的杀伤活性,然而调节机制仍有待进一步研究。基于以上事实,肿瘤代谢微环境的改变是癌症进展的一个重要标志,不仅对肿瘤细胞本身的细胞学行为造成影响,对微环境中的其他免疫细胞也起到重要的调控作用。那么髓系细胞的自主性和非自主性调控是否存在相互串扰,共同参与其分化发育和功能选择?最近有多项研究指出,Notch信号的异常可导致代谢器官功能性病变及发育异常。作为糖脂代谢中枢器官的肝脏,其胚胎发育和成体后的再生都被Notch信号精确的调控。同时,Notch信号还参与到脂肪细胞的分化过程,以及白色和米色脂肪细胞的转换。在髓系细胞中,Notch信号同样可以通过代谢重编程,促进线粒体ROS释放,从而调控M1极化巨噬细胞功能重塑。然而关于Notch信号调控髓系细胞代谢调控的分子机制,仍需进一步的深入研究。综上所述,在肿瘤发生发展过程中,肿瘤代谢微环境和髓系细胞中Notch信号通路均会影响髓系细胞的分化发育及功能应答,并反之作用于肿瘤的进展。然而关于其细胞自主性和非细胞自主性因素的相互串扰及分子机制还所知甚少。基于上述问题,本学位论文拟研究Notch信号通过乳酸盐代谢重编程调控肿瘤相关髓系细胞发育及功能的分子机制。目前所得结论如下:1、在肿瘤发生发展过程中,发挥免疫抑制性功能的G-MDSC逐渐积累,发挥抗肿瘤功能的成熟M1-样的TAM减少;2、Notch信号激活后,通过调控M-MDSC分化命运,促进其分化形成成熟巨噬细胞、抑制分化形成G-MDSC,从而抑制肿瘤生长;3、分子机制研究提示,Notch信号通过抑制髓系细胞摄取肿瘤微环境中乳酸盐,进而调控M-MDSC分化方向和TAM成熟;4、乳酸盐进入髓系细胞内,可以和c-Jun相互作用形成非共价结合,进而竞争性抑制c-Jun与FBW7结合,阻断其在细胞核中的泛素化降解,维持转录活性;Notch信号的激活可以消除乳酸盐对髓系细胞的影响。通过上述实验,我们证实了“Notch信号通路抑制乳酸盐胞内信号出口调控髓系细胞分化,进而抑制肿瘤生长”的调控通路,为建立靶向肿瘤相关MDSC和TAM进行抗肿瘤免疫治疗提供新的思路和靶点,具有重要的理论意义和潜在的临床应用价值。
[Abstract]:Tumors, as the leading killer of human health, are increasingly being studied. Malignant proliferative tumor cells have the characteristics of replication immortalization, metastasis and diffusion, apoptosis resistance, multiple mutations and immune escape, which make it difficult for most tumors to be cured by simple surgery, radiotherapy, chemotherapy and targeted drugs. Tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), and immature dendritic cells (immature DCs) are abundant in tumor tissues. IDCs, myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). These cells regulate each other and form an immunosuppressive network. These cells are the main factors that regulate the immune tolerance microenvironment of tumor tissue and promote tumor progression. It can not only directly promote the inflammation-cancer transformation of chronic injury sites through paracrine pathways, regulate the self-renewal of tumor stem cells, help tumor cells metastasis and promote tumor angiogenesis, but also reduce the killing and clearance of tumor cells by influencing NK cells and killer T cell responses. Improving the tumor microenvironment, blocking the immunosuppressive activity of tumor-associated myeloid cells and re-educating them have become an important direction and strategy for tumor therapy. Notch signaling, as one of the precise regulatory pathways in ontogeny, plays an important role in the proliferation and apoptosis of myeloid cells, the selection of differentiation fate and the maintenance of dry matter. Notch signaling plays an important role in the development and functional regulation of myeloid cells, but its specific function and stage of action are still controversial and the regulatory mechanism is not clarified. Inhibition of myeloid cell differentiation into mature granulocytes and maintenance of undifferentiated or poorly differentiated status; at the same time, Notch signal can also regulate the expression of myeloid development transcription factor PU.1, promote myeloid cell differentiation into mature cells. This is largely due to the use of different research models, there are different cytokine environment. It is suggested that Notch signaling can regulate the development and differentiation of myeloid cells through a series of microRNAs (unpublished). At the same time, there is also functional plasticity regulation in myeloid cells after terminal differentiation. Macrophages can perform completely different functions under different stimuli induction. Neotch signaling in macrophages also plays an important role in liver fibrosis through different downstream molecules (He F et al, Hepatology. 2015), suggesting that Notch signaling can regulate macrophage activation or function through a variety of molecular mechanisms. It is also confirmed that Notch-microRNA125a regulatory axis can re-educate TAMs to promote tumor function and make them play an anti-tumor role (Zhao JL et al, Cancer Res. 2016). The above work has confirmed that Notch signaling plays an important role in the development of myeloid cells and the regulation of TAM function. The environment also plays an involuntary role in the development of myeloid cells. Tumor cells require a large amount of bioenergy and biomass to proliferate rapidly, so glycolysis is performed even under aerobic conditions as an energy supply. This metabolic process is known as the Warburg effect, which leads directly to tumor micropropagation. Nutrient depletion and accumulation of metabolic wastes in the environment. Many reports have shown that changes in the metabolic environment can affect the phenotype and function of myeloid cells, especially in the course of tumor progression. Metabolic changes in myeloid cells are often accompanied by changes in "anti-tumor-promoting" function. Utilization of macrophages in different polarized states Different glycometabolism patterns were observed. Type M1 pro-inflammatory polarized macrophages used glycolysis as the main energy supply, while type 2 anti-inflammatory macrophages chose the oxidative phosphorylation pathway. Compared with spleen and bone marrow, MDSC in tumor microenvironment accelerates the uptake and consumption of fatty acids, and increases the level of mitochondrial oxidative phosphorylation, which is mainly due to glucose deficiency in tumor tissues. Based on the above facts, the change of tumor metabolic microenvironment is an important marker of cancer progression. It not only affects the cytological behavior of tumor cells, but also plays an important role in the regulation of other immune cells in the microenvironment. Does autonomic and involuntary regulation interact with each other and participate in their differentiation, development and functional selection? Recently, several studies have shown that abnormal Notch signaling can lead to functional lesions and developmental abnormalities in metabolic organs. Notch signaling is also involved in the differentiation of adipocytes and the conversion of white and beige adipocytes. In myeloid cells, Notch signaling also promotes the release of mitochondrial ROS through metabolic reprogramming, thereby regulating the functional remodeling of M1-polarized macrophages. In conclusion, both the microenvironment of tumor metabolism and Notch signaling pathways in myeloid cells can affect the differentiation and development of myeloid cells and the functional response of myeloid cells, and vice versa, affect the progress of tumors. Based on these problems, this dissertation intends to study the molecular mechanism of Notch signaling regulating the development and function of tumor-associated myeloid cells through lactate metabolism reprogramming. The current conclusions are as follows: 1. After activation of Notch signal, it can promote the differentiation of M-MDSC into mature macrophages and inhibit the formation of G-MDSC, thus inhibiting tumor growth. 3. Molecular mechanism studies suggest that Notch signal can regulate M-MDSC by inhibiting the uptake of lactate by myeloid cells in tumor microenvironment. 4. Lactate can interact with c-Jun to form non-covalent binding, and then competently inhibit the binding of c-Jun to FBW7, blocking its ubiquitination degradation in the nucleus and maintaining transcriptional activity; Notch signal activation can eliminate the effect of lactate on myeloid cells. We confirm that Notch signaling pathway inhibits lactate intracellular signal export to regulate myeloid cell differentiation and then inhibits tumor growth, which provides new ideas and targets for establishing tumor-related MDSC and TAM for anti-tumor immunotherapy, and has important theoretical significance and potential clinical application value.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.2
，
本文编号：2213217
[Abstract]:Tumors, as the leading killer of human health, are increasingly being studied. Malignant proliferative tumor cells have the characteristics of replication immortalization, metastasis and diffusion, apoptosis resistance, multiple mutations and immune escape, which make it difficult for most tumors to be cured by simple surgery, radiotherapy, chemotherapy and targeted drugs. Tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), and immature dendritic cells (immature DCs) are abundant in tumor tissues. IDCs, myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). These cells regulate each other and form an immunosuppressive network. These cells are the main factors that regulate the immune tolerance microenvironment of tumor tissue and promote tumor progression. It can not only directly promote the inflammation-cancer transformation of chronic injury sites through paracrine pathways, regulate the self-renewal of tumor stem cells, help tumor cells metastasis and promote tumor angiogenesis, but also reduce the killing and clearance of tumor cells by influencing NK cells and killer T cell responses. Improving the tumor microenvironment, blocking the immunosuppressive activity of tumor-associated myeloid cells and re-educating them have become an important direction and strategy for tumor therapy. Notch signaling, as one of the precise regulatory pathways in ontogeny, plays an important role in the proliferation and apoptosis of myeloid cells, the selection of differentiation fate and the maintenance of dry matter. Notch signaling plays an important role in the development and functional regulation of myeloid cells, but its specific function and stage of action are still controversial and the regulatory mechanism is not clarified. Inhibition of myeloid cell differentiation into mature granulocytes and maintenance of undifferentiated or poorly differentiated status; at the same time, Notch signal can also regulate the expression of myeloid development transcription factor PU.1, promote myeloid cell differentiation into mature cells. This is largely due to the use of different research models, there are different cytokine environment. It is suggested that Notch signaling can regulate the development and differentiation of myeloid cells through a series of microRNAs (unpublished). At the same time, there is also functional plasticity regulation in myeloid cells after terminal differentiation. Macrophages can perform completely different functions under different stimuli induction. Neotch signaling in macrophages also plays an important role in liver fibrosis through different downstream molecules (He F et al, Hepatology. 2015), suggesting that Notch signaling can regulate macrophage activation or function through a variety of molecular mechanisms. It is also confirmed that Notch-microRNA125a regulatory axis can re-educate TAMs to promote tumor function and make them play an anti-tumor role (Zhao JL et al, Cancer Res. 2016). The above work has confirmed that Notch signaling plays an important role in the development of myeloid cells and the regulation of TAM function. The environment also plays an involuntary role in the development of myeloid cells. Tumor cells require a large amount of bioenergy and biomass to proliferate rapidly, so glycolysis is performed even under aerobic conditions as an energy supply. This metabolic process is known as the Warburg effect, which leads directly to tumor micropropagation. Nutrient depletion and accumulation of metabolic wastes in the environment. Many reports have shown that changes in the metabolic environment can affect the phenotype and function of myeloid cells, especially in the course of tumor progression. Metabolic changes in myeloid cells are often accompanied by changes in "anti-tumor-promoting" function. Utilization of macrophages in different polarized states Different glycometabolism patterns were observed. Type M1 pro-inflammatory polarized macrophages used glycolysis as the main energy supply, while type 2 anti-inflammatory macrophages chose the oxidative phosphorylation pathway. Compared with spleen and bone marrow, MDSC in tumor microenvironment accelerates the uptake and consumption of fatty acids, and increases the level of mitochondrial oxidative phosphorylation, which is mainly due to glucose deficiency in tumor tissues. Based on the above facts, the change of tumor metabolic microenvironment is an important marker of cancer progression. It not only affects the cytological behavior of tumor cells, but also plays an important role in the regulation of other immune cells in the microenvironment. Does autonomic and involuntary regulation interact with each other and participate in their differentiation, development and functional selection? Recently, several studies have shown that abnormal Notch signaling can lead to functional lesions and developmental abnormalities in metabolic organs. Notch signaling is also involved in the differentiation of adipocytes and the conversion of white and beige adipocytes. In myeloid cells, Notch signaling also promotes the release of mitochondrial ROS through metabolic reprogramming, thereby regulating the functional remodeling of M1-polarized macrophages. In conclusion, both the microenvironment of tumor metabolism and Notch signaling pathways in myeloid cells can affect the differentiation and development of myeloid cells and the functional response of myeloid cells, and vice versa, affect the progress of tumors. Based on these problems, this dissertation intends to study the molecular mechanism of Notch signaling regulating the development and function of tumor-associated myeloid cells through lactate metabolism reprogramming. The current conclusions are as follows: 1. After activation of Notch signal, it can promote the differentiation of M-MDSC into mature macrophages and inhibit the formation of G-MDSC, thus inhibiting tumor growth. 3. Molecular mechanism studies suggest that Notch signal can regulate M-MDSC by inhibiting the uptake of lactate by myeloid cells in tumor microenvironment. 4. Lactate can interact with c-Jun to form non-covalent binding, and then competently inhibit the binding of c-Jun to FBW7, blocking its ubiquitination degradation in the nucleus and maintaining transcriptional activity; Notch signal activation can eliminate the effect of lactate on myeloid cells. We confirm that Notch signaling pathway inhibits lactate intracellular signal export to regulate myeloid cell differentiation and then inhibits tumor growth, which provides new ideas and targets for establishing tumor-related MDSC and TAM for anti-tumor immunotherapy, and has important theoretical significance and potential clinical application value.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.2
，
本文编号：2213217
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