Notch信號通過乳酸鹽代謝調控髓系細胞分化和成熟在腫瘤進展中的作用和機制研究
發(fā)布時間：2018-08-30 13:28
【摘要】：腫瘤作為危害人類健康的頭號殺手,對其的研究日益增多。惡性增殖的腫瘤細胞具有復制永生化、轉移與擴散、凋亡抵抗、多發(fā)突變和免疫逃逸等特征,這使得大多數(shù)腫瘤難以被單純的手術,或者放、化療以及靶向藥物治愈。隨著腫瘤發(fā)生發(fā)展過程被認識的更加深刻,腫瘤炎性微環(huán)境逐漸成為腫瘤的主要特征之一。腫瘤組織中大量浸潤免疫細胞,如腫瘤相關巨噬細胞(tumor-associated macrophages,TAMs)、腫瘤相關粒細胞(tumor-associated neutrophils,TANs)、不成熟的樹突狀細胞(immature DCs,iDCs)、髓系來源的抑制性細胞(myeloid-derived suppressor cells,MDSCs)和調節(jié)性T淋巴細胞(regulatory T cells,Tregs)。這些細胞相互調節(jié),構成免疫抑制網絡,是調節(jié)腫瘤組織免疫耐受微環(huán)境、促進腫瘤進展的主要因素。而在這些細胞中,髓系細胞發(fā)揮了尤其重要的作用。其不僅可以通過旁分泌途徑直接促進慢性損傷部位的炎-癌轉化、調控腫瘤干細胞自我更新、幫助腫瘤細胞轉移和促進腫瘤血管新生,還可以通過影響NK細胞和殺傷性T細胞應答,減少對腫瘤細胞的殺傷和清除。因此如何改善腫瘤微環(huán)境,阻斷腫瘤相關髓系細胞的免疫抑制活性,并對其進行再教育,成為腫瘤治療所要探討和研究的重要方向和策略。與正常發(fā)育分化形成的髓系細胞相對比,腫瘤相關髓系細胞具有較低的成熟度,其調控細胞發(fā)育和功能的多種轉錄因子和信號通路也有較大的差異。Notch信號作為個體發(fā)育中起到精密調控作用的通路之一,在髓系細胞增殖和凋亡、分化命運選擇和干性維持中均發(fā)揮重要的作用。Notch信號在后生動物中高度保守,通過相鄰細胞表面的配體分子與其細胞表面受體結合,引起受體胞內段入核,介導轉錄復合物的形成進而激活靶基因的轉錄。在髓系細胞的發(fā)育和功能調控中,Notch信號的作用顯著,但具體功能及作用階段尚存爭議,調控機制也未被闡明。一方面,Notch信號可通過GATAs抑制髓系細胞向成熟粒細胞分化,并保持未分化或低分化狀態(tài);而同時,Notch信號也可調控髓系發(fā)育轉錄因子PU.1表達,促進髓系細胞向成熟細胞分化。這很大程度上是由于所采用的研究模型不同,存在不同的細胞因子環(huán)境。本人前期研究表明Notch信號可通過一系列miRNA簇調控髓系細胞的發(fā)育和分化(未發(fā)表)。同時,髓系細胞終末分化后同樣存在功能的可塑性調控,巨噬細胞會在不同的刺激物的誘導下行使完全不同的功能。所在課題組第一次證實巨噬細胞可塑性選擇依賴于Notch信號的激活(Wang YC et al,Cancer Res.2010);后續(xù)研究指出巨噬細胞中的Notch信號通過不同的下游分子,在肝纖維化中也發(fā)揮重要作用(He F et al,Hepatology.2015),提示Notch信號可通過多種分子機制調控巨噬細胞活化或功能。本人前期工作也證實Notch-miR125a調控軸可以對促腫瘤功能的TAMs進行再教育,使其發(fā)揮抗腫瘤功能(Zhao JL et al,Cancer Res.2016)。以上工作基礎均證實Notch信號在髓系細胞發(fā)育和TAM功能調控中發(fā)揮重要作用。除了細胞自主性的調控之外,腫瘤組織復雜的微環(huán)境也會對髓系細胞發(fā)育起到非自主性調節(jié)。腫瘤細胞為了滿足其快速增殖,需要大量的生物能量和生物原料,因此即使在有氧條件下,也進行糖酵解作為能量供應。這樣的代謝過程被稱之為瓦爾堡效應(Warburg effect)。這直接導致腫瘤微環(huán)境中營養(yǎng)物質的耗竭和代謝廢物的堆積。很多報道均指出代謝環(huán)境的改變可以影響髓系細胞的表型和功能,尤其是腫瘤進展過程中,髓系細胞代謝方式的轉變往往伴隨著“抗腫瘤-促腫瘤”功能的變化。不同極化狀態(tài)的巨噬細胞利用完全不同的糖代謝方式。促炎的M1型極化巨噬細胞利用糖酵解作為主要供能方式;而拮抗炎癥的M2型巨噬細胞則會選擇氧化磷酸化途徑。除此之外,不同功能極化巨噬細胞在精氨酸代謝和脂代謝的選擇上也有較大不同。MDSC的代謝方式同樣調控其功能和活化,同脾臟和骨髓相比,腫瘤微環(huán)境中MDSC會加速攝取和消耗脂肪酸,同時觀察到線粒體氧化磷酸化水平的升高,這主要是腫瘤組織中葡萄糖缺乏的微環(huán)境所致。同時腫瘤驅動的高濃度乳酸鹽也可以促進MDSC的積累,并抑制NK細胞的殺傷活性,然而調節(jié)機制仍有待進一步研究。基于以上事實,腫瘤代謝微環(huán)境的改變是癌癥進展的一個重要標志,不僅對腫瘤細胞本身的細胞學行為造成影響,對微環(huán)境中的其他免疫細胞也起到重要的調控作用。那么髓系細胞的自主性和非自主性調控是否存在相互串擾,共同參與其分化發(fā)育和功能選擇?最近有多項研究指出,Notch信號的異常可導致代謝器官功能性病變及發(fā)育異常。作為糖脂代謝中樞器官的肝臟,其胚胎發(fā)育和成體后的再生都被Notch信號精確的調控。同時,Notch信號還參與到脂肪細胞的分化過程,以及白色和米色脂肪細胞的轉換。在髓系細胞中,Notch信號同樣可以通過代謝重編程,促進線粒體ROS釋放,從而調控M1極化巨噬細胞功能重塑。然而關于Notch信號調控髓系細胞代謝調控的分子機制,仍需進一步的深入研究。綜上所述,在腫瘤發(fā)生發(fā)展過程中,腫瘤代謝微環(huán)境和髓系細胞中Notch信號通路均會影響髓系細胞的分化發(fā)育及功能應答,并反之作用于腫瘤的進展。然而關于其細胞自主性和非細胞自主性因素的相互串擾及分子機制還所知甚少。基于上述問題,本學位論文擬研究Notch信號通過乳酸鹽代謝重編程調控腫瘤相關髓系細胞發(fā)育及功能的分子機制。目前所得結論如下:1、在腫瘤發(fā)生發(fā)展過程中,發(fā)揮免疫抑制性功能的G-MDSC逐漸積累,發(fā)揮抗腫瘤功能的成熟M1-樣的TAM減少;2、Notch信號激活后,通過調控M-MDSC分化命運,促進其分化形成成熟巨噬細胞、抑制分化形成G-MDSC,從而抑制腫瘤生長;3、分子機制研究提示,Notch信號通過抑制髓系細胞攝取腫瘤微環(huán)境中乳酸鹽,進而調控M-MDSC分化方向和TAM成熟;4、乳酸鹽進入髓系細胞內,可以和c-Jun相互作用形成非共價結合,進而競爭性抑制c-Jun與FBW7結合,阻斷其在細胞核中的泛素化降解,維持轉錄活性;Notch信號的激活可以消除乳酸鹽對髓系細胞的影響。通過上述實驗,我們證實了“Notch信號通路抑制乳酸鹽胞內信號出口調控髓系細胞分化,進而抑制腫瘤生長”的調控通路,為建立靶向腫瘤相關MDSC和TAM進行抗腫瘤免疫治療提供新的思路和靶點,具有重要的理論意義和潛在的臨床應用價值。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R730.2
，
本文編號：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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R730.2
，
本文編號：2213217
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