Sirtuins蛋白处于干性、衰老和癌症的交叉路口

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Sirtuins at the crossroads of stemness, aging, and cancer

Summary

摘要

Sirtuins are stress‐responsive proteins that direct various post‐translational modifications (PTMs) and as a result, are considered to be master regulators of several cellular processes. They are known to both extend lifespan and regulate spontaneous tumor development. As both aging and cancer are associated with altered stem cell function, the possibility that the involvement of sirtuins in these events is mediated by their roles in stem cells is worthy of investigation. Research to date suggests that the individual sirtuin family members can differentially regulate embryonic, hematopoietic as well as other adult stem cells in a tissue‐ and cell type‐specific context. Sirtuin‐driven regulation of both cell differentiation and signaling pathways previously involved in stem cell maintenance has been described where downstream effectors involved determine the biological outcome. Similarly, diverse roles have been reported in cancer stem cells (CSCs), depending on the tissue of origin. This review highlights the current knowledge which places sirtuins at the intersection of stem cells, aging, and cancer. By outlining the plethora of stem cell‐related roles for individual sirtuins in various contexts, our purpose was to provide an indication of their significance in relation to cancer and aging, as well as to generate a clearer picture of their therapeutic potential. Finally, we propose future directions which will contribute to the better understanding of sirtuins, thereby further unraveling the full repertoire of sirtuin functions in both normal stem cells and CSCs.

去乙酰化酶是一种应激反应蛋白,指导多种翻译后修饰(PTMs) ,因此被认为是多种细胞过程的主要调节因子。众所周知,它们既能延长寿命,又能调节自发性肿瘤的发展。由于老化和癌症都与干细胞功能的改变有关,去乙酰化酶参与这些事件的可能性是通过它们在干细胞中的作用介导的,这值得研究。迄今为止的研究表明,sirtuin 家族成员个体可以在组织和细胞类型特异的情况下差异调节胚胎干细胞、造血干细胞以及其他成人干细胞。去乙酰化酶驱动的调节细胞分化和信号通路以前涉及干细胞维持已经被描述在下游效应器涉及决定生物学结果。同样,根据起源组织的不同,癌症干细胞(CSCs)也有不同的作用。这篇综述强调了目前将去乙酰化酶置于干细胞、衰老和癌症交叉点的知识。通过概述各种情况下与干细胞相关的角色,我们的目的是提供一个关于他们与癌症和衰老的重要性的指标,以及生成一个更清晰的图片,他们的治疗潜力。最后,我们提出了未来的方向,这将有助于更好地理解去乙酰化酶,从而进一步阐明了去乙酰化酶在正常干细胞和 CSCs 中的全部功能。

Introduction: Sirtuins

简介: Sirtuins

Sirtuins are members of the class III histone deacetylase family of enzymes that share a conserved 275‐amino acid catalytic core domain and are dependent on nicotinamide adenine dinucleotide (NAD+) for their activity (Vassilopoulos et al., 2011). Phylogenetic analysis divides the seven mammalian sirtuins (SIRT1‐7) into four classes: SIRT1‐3 are in class I, SIRT4 in class II, SIRT5 in class III, and SIRT6‐7 in class IV (Frye, 2000). Mammalian sirtuins may also be categorized according to their subcellular localization: SIRT1, 6, and 7 are present in the nucleus, SIRT3, 4, and 5 in the mitochondria, and SIRT2 is found predominately in the cytoplasm (Houtkooper et al., 2012). This diversity in subcellular location, combined with differing expression patterns and distinct substrates, contributes to the diverse biological functions of the individual family members. Although initially described as histone deacetylases in yeast, mammalian sirtuins also regulate an inestimable range of nonhistone cellular proteins through lysine deacetylation. The repertoire of sirtuin function has continued to expand since their discovery, with roles in additional PTMs being increasingly reported. In this regard, SIRT6 has been shown to regulate deacylation (Jiang et al., 2013) and, as well as SIRT4, can function as an ADP‐ribosyltransferase (Liszt et al., 2005; Haigis et al., 2006b). In addition, SIRT5 is capable of desuccinylation and demalonylation (Du et al., 2011; Park et al., 2013a).

Sirtuins 是 III 类组蛋白脱乙酰酶家族的成员,这些酶分享一个保守的275氨基酸催化核心结构域,并依赖于烟酰胺腺嘌呤二核苷酸(NAD +)的活性(Vassilopoulos et al. 2011)。系统发育分析将7种哺乳动物 sirtuins (SIRT1-7)分为4类: SIRT1-3为 i 类,SIRT4为 II 类,SIRT5为 III 类,SIRT6-7为 IV 类。哺乳动物去乙酰化酶也可以根据其亚细胞定位分类: SIRT1、6和7存在于细胞核中,SIRT3、4和5存在于线粒体中,而 SIRT2主要存在于细胞质中(Houtkooper et al. ,2012)。这种亚细胞位置的多样性,结合不同的表达模式和不同的底物,有助于个别家庭成员的不同生物功能。虽然最初在酵母中被描述为组蛋白去乙酰化酶,哺乳动物去乙酰化酶也通过赖氨酸去乙酰化来调节非组蛋白细胞蛋白的不可估量的范围。自从发现 sirtuin 功能以来,它的功能不断扩大,其他 ptm 中的角色也越来越多地被报道。在这方面,已经证明 SIRT6可以调节去乙酰化(Jiang 等人,2013年) ,和 SIRT4一样,可以作为 ADP 核糖基转移酶(Liszt 等人,2005; Haigis 等人,2006b)。此外,SIRT5能够脱 uccinylation 和 dealonylation (Du et al. ,2011; Park et al. ,2013a)。

In addition to subcellular localization and NAD+ availability, there are multiple additional mechanisms of regulation that contribute to sirtuin activity and specificity. This ensures activation of different sirtuins and consequent stimulation of distinct and diverse substrates. Transcriptional regulation including various transcription factors/repressors, miRNAs, post‐translational regulation, protein–protein interactions, and regulation by small molecules have all been described (reviewed in Houtkooper et al. (2012)). By employing such regulatory mechanisms, environmental stimuli including calorie restriction (CR) are known to control sirtuin expression and/or activity. Therefore, sirtuins are considered to be stress‐responsive enzymes that direct cellular adaptations by altering the acetylome. Although currently debated, sirtuins have been shown to regulate longevity in numerous lower organisms including yeast, nematodes, and fruit flies (Haigis & Guarente, 2006a; Burnett et al., 2011) as well as higher organisms such as mice (Kanfi et al., 2012). Consequently, mammalian sirtuin research has to date been intensely focused on their roles in aging and aging‐related diseases. As a result of the variety of proteins that can be regulated by lysine acetylation, sirtuins have been shown to be master regulators of diverse cellular activities such as gene expression, metabolism, telomere activity, cell cycle, differentiation, EMT, apoptosis, proliferation, DNA repair, senescence, and oxidative stress response. Interestingly, many of these are critical processes in the maintenance and differentiation of both normal stem cells and CSCs. In this review, we outline the roles various members of the sirtuin family play in some of these pathways and discuss the potential therapeutic implications of targeting sirtuins for the treatment of cancer and other stem cell‐related diseases.

除了亚细胞定位和 NAD + 的可用性,还有多种额外的调节机制有助于去乙酰化酶的活性和特异性。这确保激活不同的去乙酰化酶和随后刺激不同和不同的底物。转录调控包括各种转录因子/阻遏物,微 rna,翻译后调节,蛋白质-蛋白质相互作用,以及小分子的调节都已经被描述(Houtkooper 等人(2012年))。通过运用这些调节机制,包括卡路里限制在内的环境刺激可以控制 sirtuin 的表达和/或活性。因此,去乙酰化酶被认为是一种应激反应酶,通过改变乙酰基来指导细胞的适应性。虽然目前还存在争议,但去乙酰化酶已被证明能够调节许多低等生物体的长寿,包括酵母、线虫和果蝇(Haigis & Guarente,2006a; Burnett et al. ,2011)以及高等生物体,如小鼠(Kanfi et al. ,2012)。因此,迄今为止哺乳动物去乙酰化酶的研究主要集中在它们在衰老和衰老相关疾病中的作用。由于赖氨酸乙酰化可以调节多种蛋白质,sirtuins 已被证明是多种细胞活动的主要调节因子,如基因表达、代谢、端粒活性、细胞周期、分化、 EMT、凋亡、增殖、 DNA 修复、衰老和氧化应激反应。有趣的是,其中许多都是维持和分化正常干细胞和干细胞的关键过程。在这篇综述中,我们概述了 sirtuin 家族不同成员在其中一些通路中的作用,并讨论了靶向 sirtuin 治疗癌症和其他干细胞相关疾病的潜在治疗意义。

Sirtuins and stem cells

去乙酰化酶和干细胞

Embryonic stem cells and development

胚胎干细胞与发育

Histone acetylation undergoes dynamic changes during differentiation of embryonic stem cells (ESCs) and appears to play an important role in development as a result. Particularly, ESCs display higher levels of histone acetylation than lineage‐restricted and more differentiated cells (Efroni et al., 2008). Thus, it is not surprising that sirtuins have been linked to development and differentiation of ESCs. It is important to note here that early embryonic development is reported to be normal in most sirtuin‐knockout mice; however, Sirt1 knockout results in significant lethality during the fetal stage or soon after birth, with severe developmental defects (Cheng et al., 2003; Haigis et al., 2006b; Mostoslavsky et al., 2006; Lombard et al., 2007; Vakhrusheva et al., 2008; Du et al., 2011; Kim et al., 2011). As a result, SIRT1 is considered to be the most important sirtuin in these processes and is consequently the best studied in this context. As Sirt1 is highly expressed in ESCs before being downregulated by miRNAs during differentiation (Saunders et al., 2010), it is thought to play a role in maintaining stemness of ESCs and appears to be involved in developmental programs upon differentiation of ESCs (Table 1). The role SIRT1 plays in ESC differentiation differs depending on environmental conditions – loss of Sirt1 under normal conditions does not induce differentiation; however under oxidative stress, Sirt1 mediates the maintenance of stemness promoting mitochondrial over nuclear translocation of p53 and maintaining Nanog expression (Han et al., 2008; Calvanese et al., 2010). SIRT1 is a known component of Polycomb repressive complex 4 (PRC4), which represses developmental genes in ESCs (Kuzmichev et al., 2005) and also binds to the promoters of development‐associated genes in ESCs, such as TBX3 and PAX6 where it contributes to gene silencing. As a result of its ability to regulate stemness and pluripotency factors, the role of SIRT1 in cellular reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) has also been investigated. Both SIRT1 overexpression and treatment with the known sirtuin activator resveratrol have been shown to enhance the efficiency of iPSC generation, whereas Sirt1 knockdown exerts opposite action. This effect is associated with deacetylation of p53 and increased Nanogexpression (Lee et al., 2012).

组蛋白乙酰化在胚胎干细胞分化过程中发生动态变化,在胚胎干细胞的发育过程中起重要作用。特别是,胚胎干细胞显示更高水平的组蛋白乙酰化比谱系限制和更分化的细胞(Efroni 等人,2008年)。因此,去乙酰化酶与胚胎干细胞的发育和分化有关也就不足为奇了。需要注意的是,在大多数 sirtuin 基因敲除小鼠中,早期胚胎发育据报道是正常的; 然而,Sirt1基因敲除在胎儿期或出生后不久会导致明显的致死性,并伴有严重的发育缺陷(Cheng 等人,2003年; Haigis 等人,2006年 b; Mostoslavsky 等人,2006年; Lombard 等人,2007年; Vakhrusheva 等人,2008年; Du 等人,2011年; Kim 等人,2011年)。因此,SIRT1被认为是这些过程中最重要的 sirtuin,因此是这方面研究最好的。由于 Sirt1在胚胎干细胞中高度表达,然后在分化过程中被 mirna 下调(Saunders 等人,2010) ,它被认为在保持胚胎干细胞的稳定性方面发挥作用,并且似乎参与胚胎干细胞分化后的发育过程(表1)。在 ESC 分化中的作用取决于环境条件-在正常条件下 SIRT1的缺失不会诱导分化; 然而在氧化应激条件下,SIRT1介导了促进 p53核转位的线粒体干性的维持和 Nanog 表达的维持(Han et al. ,2008; Calvanese et al. ,2010)。SIRT1是 Polycomb 抑制复合物4(PRC4)的一个已知组成部分,它抑制胚胎干细胞中的发育基因(Kuzmichev et al. 2005) ,并且与胚胎干细胞中发育相关基因的启动子结合,例如 TBX3和 PAX6,它促进了基因的沉默。由于 SIRT1具有调节干细胞性和多能性因子的能力,人们还研究了 SIRT1在体细胞诱导多能干细胞(iPSCs)的细胞重编程中的作用。SIRT1的过度表达和已知 sirtuin 激活剂白藜芦醇的处理都显示出提高 iPSC 产生的效率,而 SIRT1击倒具有相反的作用。这种效应与 p53脱乙酰化和 Nanog 表达增加有关(Lee 等人,2012)。Table 1. 表一Sirtuin functions and mechanisms of action in stem cells 去乙酰化酶在干细胞中的作用及其机制

SirtuinAction 行动Mechanism 机制Cells/Tissue 细胞/组织References参考资料
SIRT1Maintenance of stemness保持干燥Mitochondrial translocation of p53 maintains Nanog expression P53线粒体移位维持 Nanog 的表达ESC 人事编制委员会Han 汉et al等等. (2008)
SIRT1Maintenance of stemness保持干燥Component of PRC4 represses developmental genes PRC4组分抑制发育基因ESC 人事编制委员会Kuzmichev库兹米切夫et al 等等. (2005)
SIRT1Maintenance of stemness保持干燥ROS elimination, FOXO activation, and inhibition of p53 活性氧消除、 FOXO 激活和 p53抑制HSC 高血压Matsui 松井et al 等等. (2012)
SIRT1Promotes differentiation促进分化Interacts with N‐CoR to block Notch‐Hes1 signaling 与 n-CoR 相互作用以阻断 Notch-Hes1信号NSC 国家安全委员会Hisahara 久原et al 等等. (2008)
SIRT2Promotes differentiation促进分化Negatively regulates GSK3β 负调节 gsk3ESC 人事编制委员会Si 是的et al等等. (2013)
SIRT3Maintenance of stemness保持干燥Required for HSC self‐renewal at old age, related to oxidative stress 老年时需要进行 HSC 自我更新,与氧化应激有关HSC 高血压Brown 布朗et al 等等. (2013)
SIRT6Promotes differentiation促进分化Regulates acetylation of H3K56 and H3K9 at H3K56和 H3K9的乙酰化调控Oct4 8/4 and 及Sox2 promoters促进者ESC 人事编制委员会Etchegaray et al 等等. (2015)
SIRT6Maintenance of stemness保持干燥Represses Wnt target genes by interacting with LEF1 and deacetylating histone 3 通过与 LEF1和去乙酰化组蛋白3相互作用抑制 Wnt 靶基因HSC 高血压Wang 王先生et al 等等. (2016)
SIRT7Maintenance of stemness保持干燥Regulates UPR 规管普遍定期审议mt and NRF1 及 NRF1HSC 高血压Mohrin 女名女子名et al 等等. (2015)

Although less comprehensively studied, other sirtuins have been implicated in the regulation of cell lineage specification during ESC differentiation (Table 1). Sirt2 is upregulated during mouse ESC differentiation and negatively regulates glycogen synthase kinase‐3β (GSK3β), a negative regulator of the Wnt/β‐catenin pathway. It was found that Sirt2 knockdown compromised differentiation of mouse ESCs into ectoderm while promoting mesoderm and endoderm differentiation (Si et al., 2013). Conversely, ESCs from Sirt6‐knockout mice display upregulation of ectoderm markers and downregulation of genes associated with endoderm and mesoderm, thus highlighting the sometimes opposing roles of sirtuin family members. SIRT6 controls ESC differentiation by regulating acetylation of H3K56 and H3K9 at the Oct4and Sox2 promoters. By repressing expression of these pluripotency genes, SIRT6 diminishes the expression of Tet enzymes, limits the levels of 5hmC, and allows balanced transcription of developmentally regulated genes (Etchegaray et al., 2015).

尽管对去乙酰化酶的研究还不够全面,但其他去乙酰化酶已经牵涉到 ESC 分化过程中细胞谱系规范的调控(表1)。Sirt2在小鼠胚胎干细胞分化过程中上调,负性调节 wnt/-catenin 通路的负性调节因子糖原合成酶激酶 -3(gsk3)。结果发现 Sirt2抑制小鼠胚胎干细胞向外胚层的分化,同时促进中胚层和内胚层的分化(Si et al. 2013)。相反,Sirt6基因敲除小鼠的胚胎干细胞表现出外胚层标记的上调和内胚层和中胚层相关基因的下调,从而突出了 sirtuin 家族成员有时相反的作用。SIRT6通过调控 Oct4和 Sox2启动子上 H3K56和 H3K9的乙酰化来控制 ESC 分化。通过抑制这些多能性基因的表达,SIRT6减少了 Tet 酶的表达,限制了5hmC 的水平,并允许发育调节基因的平衡转录(Etchegaray et al. ,2015)。

Hematopoietic stem cells

造血干细胞

As the first family member to be discovered, SIRT1 is also the best studied in other types of stem cells. In particular, its role in hematopoietic stem cells (HSCs), where it is expressed in both human and mouse cells of all lineages and stages of maturation, is well understood. A number of in vivo studies that utilize Sirt1−/− mice have demonstrated that SIRT1 positively regulates stemness in HSCs (Table 1). In embryonic hematopoietic development, Sirt1−/− ESC formed fewer mature blast cell colonies, with defective hematopoietic potential associated with delayed deactivation of Oct4, Nanog, and Fgf5 expression (Ou et al., 2011). Consistent with a role of SIRT1 in mouse hematopoiesis and differentiation, another study demonstrated that SIRT1 does contribute to the maintenance of the HSC pool as murine bone marrow c‐KithighSca‐1+Lineage cells isolated from Sirt1−/− mice more readily differentiate and lose stem cell characteristics than wild‐type HSC. The mechanism behind SIRT1 maintenance of hematopoietic cell stemness was found to involve ROS elimination, FOXO activation, and inhibition of p53 (Matsui et al., 2012). According to these previous findings, it would be expected that SIRT1 is indispensable for normal function of HSCs. However, a recent in vivo study showed that Sirt1 deletion had no effect on the production of mature blood cells, lineage distribution within hematopoietic organs, and frequencies of the most primitive HSC populations (Leko et al., 2012). Specific hematopoietic cell‐knockout and inducible Sirt1‐knockout mouse models have also contributed to the understanding of SIRT1 function in HSCs while overcoming the experimental challenges related to the developmental defects and perinatal death of Sirt1‐knockout mice. Following tamoxifen‐induced Sirt1 deletion, a gradual increase in the total number and the frequency of HSCs as well as an expansion of the myeloid lineage at the expense of lymphoid cells were observed (Rimmelé et al., 2014). As above, this study also identified FOXO3 as an important mediator of the homeostatic control by SIRT1 in HSCs. Results obtained regarding the role of SIRT1 in regulating HSCs under stressful conditions serve to highlight the significant role the extracellular context may play in directing sirtuin function in stem cells. In the fraction of Sirt1−/− mice that survive postnatally, loss of SIRT1 is associated with decreased hematopoietic progenitors particularly under hypoxic conditions (Ou et al., 2011). This is consistent with a recent study showing that deletion of SIRT1 specifically in hematopoietic cells, after crossing Sirt1‐floxed mice with vav‐iCre transgenic mice, promotes aberrant HSC expansion and exhaustion but only under conditions of hematopoietic stress (Singh et al., 2013).

作为第一个被发现的家族成员,SIRT1在其他类型的干细胞研究中也是最好的。特别是,它在造血干细胞(hsc)中的作用,在所有系列和成熟阶段的人类和小鼠细胞中都有表达,这一点已经被很好地理解。许多利用 SIRT1-/-小鼠的体内研究表明,SIRT1对造血干细胞的干性有积极的调节作用(表1)。在胚胎造血发育过程中,Sirt1-/-ESC 形成的成熟细胞集落较少,具有与 Oct4、 Nanog 和 Fgf5表达延迟失活相关的造血功能缺陷(Ou 等,2011)。与 SIRT1在小鼠造血和分化中的作用一致,另一项研究表明,SIRT1确实有助于维持 HSC 库,因为从 SIRT1/-小鼠分离的小鼠骨髓 c-KithighSca-1 + 谱系细胞比野生型 HSC 更容易分化和失去干细胞特性。SIRT1维持造血细胞干性的机制被发现涉及 ROS 消除、 FOXO 激活和 p53抑制(Matsui 等,2012)。根据以往的研究结果,可以预期 SIRT1对于造血干细胞的正常功能是必不可少的。然而,最近的一项体内研究表明,Sirt1缺失对成熟血细胞的产生、造血器官内的谱系分布以及最原始的 HSC 人群的频率没有影响(Leko 等人,2012年)。在克服 SIRT1基因敲除小鼠发育缺陷和围产期死亡的实验挑战的同时,特异性的造血细胞敲除和可诱导的 SIRT1基因敲除小鼠模型也有助于了解 SIRT1基因敲除小鼠在造血干细胞中的功能。在他莫西芬诱导的 Sirt1缺失后,观察到造血干细胞的总数和频率逐渐增加,以及以淋巴细胞为代价的髓系扩张(rimmel é 等人,2014年)。同时,本研究还证实 FOXO3是 SIRT1调控造血干细胞内环境稳定的重要调节因子。在应激条件下 SIRT1在调节造血干细胞中的作用的结果突出了细胞外环境在干细胞去乙酰化酶功能中可能发挥的重要作用。在出生后存活的 SIRT1-/-小鼠中,SIRT1的丢失与造血祖细胞减少有关,特别是在低氧条件下(Ou 等人,2011)。这与最近的一项研究相一致,该研究表明,在 SIRT1-floxed 小鼠与 vav-iCre 转基因小鼠杂交后,造血细胞特异性 SIRT1缺失,促进 HSC 异常扩张和疲劳,但仅在造血应激条件下(Singh 等人,2013年)。

A similar experimental in vivo approach has been followed to uncover the role of SIRT6 in HSCs (Table 1). Using Sirt6fl/fl Vav‐Cre mice for hematopoietic‐specific deletion, a pIpC‐inducible mouse model (Sirt6fl/fl Mx1‐Cre), and Sirt6fl/fl ERT2‐Cre mice for inducible deletion in adult HSCs, it has been shown that Sirt6 deficiency results in a significant increase in the number of immunophenotypically defined HSCs (Wang et al., 2016). However, SIRT6‐deficient HSCs exhibited a remarkable decrease in the long‐term multilineage repopulating activity, which is similar to the previously described effect of Sirt1 loss. The phenotypic expansion and functional decline of SIRT6‐deficient HSCs is associated with an abnormal hyperproliferation induced by aberrant activation of Wnt signaling pathway.

一个类似的体内实验方法已经被跟踪,以揭示 SIRT6在造血干细胞中的作用(表1)。使用 sirt6fl/flvav-Cre 小鼠进行造血特异性缺失,使用 pIpC 诱导的小鼠模型(sirt6fl/flmx1-Cre) ,使用 sirt6fl/flert2-Cre 小鼠进行成年造血干细胞诱导性缺失,结果表明 Sirt6缺陷导致免疫特异性定义造血干细胞的数量显著增加(Wang 等人,2016)。然而,SIRT6缺陷的造血干细胞在长期多谱系再繁殖活性方面显著下降,这与先前描述的 Sirt1缺失的影响相似。SIRT6缺陷造血干细胞的表型扩增和功能下降与 Wnt信号通路异常激活所致的异常增殖有关。

SIRT3 and SIRT7 are also involved in HSC maintenance through the regulation of mitochondrial homeostasis (Table 1). Although SIRT3 seems to be dispensable for HSC maintenance at a young age, Sirt3 deficiency results in a reduced HSC pool at an old age and compromised HSC self‐renewal upon serial transplantation stress (Brown et al., 2013). These phenotypes are attributed to increased oxidative stress due to decreased antioxidant activity of acetylated MnSOD upon Sirt3 loss. Interestingly, Sirt7 genetic inactivation also results in compromised regenerative capacity of HSCs, in this instance by failing to alleviate mitochondrial protein folding stress. Sirt7−/− bone marrow cells or purified HSCs display a 40% reduction in long‐term reconstitution of the recipients’ hematopoietic system compared with their Sirt7+/+ counterparts. Even though Sirt7 loss does not affect HSC frequency in the bone marrow under steady‐state conditions, a 50% reduction in the frequency of Sirt7−/− HSCs upon transplantation is observed coupled with increased apoptosis (Mohrin et al., 2015).

SIRT3和 SIRT7也通过调节线粒体内稳态参与 HSC 的维持(表1)。虽然 SIRT3在年轻时似乎是可有可无的 HSC 维持,但 SIRT3缺乏导致老年时 HSC 库减少,并损害了 HSC 在连续移植压力下的自我更新(Brown 等人,2013)。这些表型是由于 Sirt3丢失后乙酰化 MnSOD 的抗氧化活性降低而导致氧化应激增加。有趣的是,Sirt7基因的失活也导致造血干细胞的再生能力受损,在这种情况下,它不能缓解线粒体蛋白质折叠的压力。与 Sirt7 +/+ 细胞相比,Sirt7-/-骨髓细胞或纯化的造血干细胞长期重建受者造血系统的能力下降40% 。即使 Sirt7的丢失在稳定状态下并不影响骨髓中 HSC 的频率,移植后 Sirt7-/-HSC 的频率减少了50% ,伴随着凋亡的增加(Mohrin 等人,2015)。

Cell differentiation

细胞分化

Given that the balance between cell differentiation and self‐renewal is critical for adult stem cell maintenance and tissue regeneration, studies have revealed a role for sirtuins as regulators of differentiation in several cell types. In normal differentiation of neural stem cells (NSCs), SIRT1 translocates to the nucleus where it interacts with the nuclear receptor corepressor (N‐CoR) to block Notch‐Hes1 signaling and promote neuronal differentiation (Hisahara et al., 2008) (Table 1). However, stress conditions appear to particularly affect SIRT1 functions in stem cell differentiation pathways, even within the same cell type. Therefore, mild oxidation causes SIRT1 to bind to Hes1 and directs NSC differentiation toward the astroglial lineage rather than neuronal (Prozorovski et al., 2008), which might facilitate astrogliosis and healing in response to brain and spinal cord injuries. In further support of its role in regulating differentiation pathways above, SIRT1 has been reported to be involved in muscle differentiation. Under fasting conditions, which are known to activate sirtuins, SIRT1 responds to the altered [NAD(+)]/[NADH] ratio to inhibit muscle differentiation through deacetylation of PCAF and MyoD (Fulco et al., 2003). SIRT1 has also been shown to suppress differentiation in iPSCs as well. During the generation of NSCs from mouse iPSCs, levels of Sirt1 have been observed to decrease, whereas miRNA‐34a, an inhibitor of SIRT1, increases. Furthermore, pharmacologic inhibition of SIRT1 using nicotinamide (NAM) enhanced the generation of NSCs and mature nerve cells (Hu et al., 2014a). Although it is generally considered a SIRT1 suppressor, mi‐R34a positively regulates SIRT1 during smooth muscle cell (SMC) differentiation from pluripotent stem cells. In this cell‐specific context, SIRT1 positively regulates differentiation by promoting the expression of transcription factors that regulate SMC genes by inhibiting H3K9 methylation (Yu et al., 2015).

鉴于细胞分化和自我更新之间的平衡对于成体干细胞维持和组织再生至关重要,研究已经揭示了 sirtuins 作为几种细胞类型的分化调节因子的作用。在神经干细胞(neural stem cells,NSCs)的正常分化过程中,SIRT1转移到细胞核,在那里它与核受体辅阻遏因子(n-CoR)相互作用,阻断 Notch-Hes1信号传导,促进神经元的分化(Hisahara et al. ,2008)。然而,应激条件似乎特别影响干细胞/细胞分化通路中的 SIRT1功能,即使是在同一类型的细胞中。因此,轻度氧化导致 SIRT1与 Hes1结合,并将 NSC 分化引向星形胶质细胞而非神经元(Prozorovski et al. 2008) ,这可能有助于星形胶质细胞增生和对大脑和脊髓损伤的愈合。为了进一步支持其在上述调节分化通路的作用,SIRT1已被报道参与肌肉分化。在已知能激活去乙酰化酶的禁食条件下,SIRT1对[ NAD (+)]/[ NADH ]比值的改变作出反应,通过去乙酰化 PCAF 和 MyoD 抑制肌肉分化(Fulco et al. 2003)。SIRT1也被证明可以抑制 iPSCs 的分化。在小鼠诱导多能干细胞产生神经干细胞过程中,SIRT1的水平下降,而 SIRT1的抑制剂 miRNA-34a 的水平上升。此外,烟酰胺(NAM)对 SIRT1的药理抑制作用增强了神经干细胞和成熟神经细胞的生成(Hu 等,2014a)。Mi-R34a 在平滑肌细胞向多能干细胞分化过程中对 SIRT1具有正向调节作用。在这种细胞特异性的背景下,SIRT1通过抑制 H3K9甲基化,促进调节 SMC 基因的转录因子的表达,从而积极调节分化(Yu 等,2015)。

SIRT2 also plays complex roles in both promoting and suppressing differentiation depending on the tissue studied. SIRT2 may positively regulate differentiation of keratinocytes – loss of Sirt2 is associated with increased expression of epidermal stem cell markers keratin‐5, keratin‐19, and CD34, as well as decreased expression of loricrin, a marker of terminal keratinocyte differentiation (Ming et al., 2014). Focusing on adipogenesis and consistent with a negative role described for SIRT1 in this process (Picard et al., 2004), SIRT2 has been shown to inhibit preadipocyte differentiation in 3T3‐L1 cells. Downregulation of Sirt2 increases acetylation of FOXO1, thereby affecting FOXO1 phosphorylation, nuclear/cytoplasmic localization, and ultimately activity, resulting in adipogenesis (Jing et al., 2007).

SIRT2还根据所研究的组织在促进和抑制分化方面发挥着复杂的作用。SIRT2可能正向调节角质形成细胞的分化—— SIRT2的缺失与表皮干细胞标志物角蛋白5、角蛋白19和 CD34的表达增加有关,同时也与角质形成细胞终末分化标志物氯蛋白的表达减少有关(Ming 等人,2014)。SIRT2专注于脂肪生成,并与 SIRT1在这一过程中的负作用相一致(Picard 等人,2004) ,已被证明能抑制3T3-L1细胞的前脂肪细胞分化。Sirt2的下调增加 FOXO1的乙酰化,从而影响 FOXO1的磷酸化,核/质定位,并最终活性,导致脂肪生成(Jing 等,2007)。

Given that unique expression patterns and substrate specificity may dictate cellular functions regulated by the different members of the sirtuin family, SIRT3 appears to be required for the differentiation of brown adipocytes in contrast to the negative effect of both SIRT1 and SIRT2 in adipogenesis. The coordinated action of the transcriptional coactivator peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) with the orphan nuclear receptor estrogen‐related receptor‐α induces Sirt3 gene expression in white adipocytes and embryonic fibroblasts. This seems to be required for the induction of a brown adipose tissue‐specific pattern of gene expression, as evidenced by the finding that PGC‐1α fails to fully induce brown adipose tissue‐specific gene expression in cells lacking Sirt3 (Giralt et al., 2011).

鉴于独特的表达模式和底物特异性可能决定了 sirtuin 家族不同成员所调节的细胞功能,SIRT3似乎是棕色脂肪细胞分化所必需的,而 SIRT1和 SIRT2在脂肪形成中都有负面影响。转录辅激活剂过氧化物酶体增殖物激活受体辅激活剂1(PGC-1)与孤儿核受体雌激素相关受体-诱导白细胞和胚胎成纤维细胞 Sirt3基因表达。这似乎是需要诱导一个褐色脂肪组织特异性的基因表达模式,证据是发现 PGC-1不能完全诱导缺乏 Sirt3细胞的褐色脂肪组织特异性基因表达(Giralt 等人,2011年)。

Sirtuins in stem cell signaling pathways

干细胞信号转导途径中的去乙酰化酶

Signaling pathways that regulate stem cell function are crucial for normal embryonic development and adult tissue homeostasis. Pathways such as Hedgehog, Wnt, and Notch, among others, are critical players in controlling the intricate balance of properties that define a stem cell, including self‐renewal and differentiation, even though their significance may vary in different stem cell populations. These pathways are strictly controlled by epigenetic regulation such as DNA methylation and histone modification (Toh et al., 2017). Furthermore, epigenetic changes in response to environmental signals, including nutrient stress, are known to alter stem cell pathway function (Brunet & Rando, 2017). It comes as no surprise therefore that sirtuins, particularly SIRT1, have been shown to interact with various components of these signaling networks (Fig. 1). Even though not all studies mentioned below that outline interactions between sirtuins and these pathways have been performed in stem cells, it is clear that members of the sirtuin family are actively involved in these signaling pathways with the notion that the underlying biology remains to be further explored in a stem cell‐specific context.

调节干细胞功能的信号通路对正常胚胎发育和成体组织内环境稳定至关重要。Hedgehog、 Wnt 和 Notch 等通路在控制包括自我更新和分化在内的干细胞特性的复杂平衡方面起着关键作用,尽管它们的重要性在不同的干细胞群体中可能有所不同。这些通路是严格控制表观遗传调控,如 DNA 甲基化和组蛋白修饰(Toh 等人,2017年)。此外,表观遗传变化对环境信号的反应,包括营养应激,已知改变干细胞通路功能(Brunet & Rando,2017年)。因此,去乙酰化酶,特别是 SIRT1,已经被证明与这些信号网络的各种成分相互作用就不足为奇了(图1)。尽管下面提到的概述去乙酰化酶与这些通路之间的相互作用的研究并非都是在干细胞中进行的,但很明显去乙酰化酶家族的成员积极参与了这些信号通路,其基本生物学机制仍有待于在干细胞特异性的背景下进一步探索。

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Figure 1 图1Open in figure viewer 打开图形查看器PowerPoint 简报Schematic representation of sirtuin roles in Hedgehog, Wnt/β‐catenin, and Notch stem cell signaling pathways. By interacting with BCL6 and BCOR, Sirt1 represses Sonic Hedgehog effectors Gli1 and Gli2. Sirt1 promotes Wnt/β‐catenin signaling by deacetylating β‐catenin and FOXO transcription factors and by suppressing Wnt pathway antagonists SFRP2 and DACT1. Sirt2, however, inhibits β‐catenin signaling and downregulates expression of Wnt target genes, while Sirt6 represses Wnt target genes by interacting with LEF1 and deacetylating histone 3. With the focus on Notch pathway, Sirt1 deacetylates and destabilizes the NICD. Sirt1 also cooperates with LSD1 to repress Notch target genes. ADAMs, a disintegrin and metalloproteases; APC, adenomatous polyposis coli; CK1α, casein kinase 1α; CSL/RBPJ, CBF1 Suppressor of Hairless LAG‐1/recombination signal binding protein for immunoglobulin κ J region; Dvl, Disheveled; GSK3β, glycogen synthase kinase‐3β; LEF/TCF, lymphoid enhancer factor/T‐cell factor; LSD1, lysine demethylase 1A; LRP5/6, low‐density lipoprotein‐related proteins 5 and 6; NICD, Notch intracellular domain; SHH, Sonic Hedgehog; β‐cat, β‐catenin; γ‐sec, γ‐secretase. Sirtuin 在 Hedgehog、 wnt/-catenin 和 Notch 干细胞信号通路中作用的图式表达。通过与 BCL6和 BCOR 的相互作用,Sirt1抑制了 Sonic Hedgehog 效应因子 Gli1和 Gli2。Sirt1通过去乙酰化-连环素和 FOXO 转录因子以及抑制 Wnt 通路拮抗剂 SFRP2和 DACT1促进 Wnt/连环素信号转导。而 Sirt2则通过与 LEF1和去乙酰化组蛋白3相互作用,抑制 Wnt 靶基因的表达并下调 Wnt 靶基因的表达。随着对 Notch 路径的重点,Sirt1去乙酰化和不稳定的 NICD。Sirt1还与 LSD1合作抑制 Notch 靶基因。去整合素和金属蛋白酶; APC,腺瘤性息肉病; ck1,酪蛋白激酶1; 无毛 LAG-1/重组信号结合蛋白;Dvl,disheed; gsk3,糖原合成酶激酶 -3; LEF/TCF,淋巴增强因子/t 细胞因子; LSD1,赖氨酸去甲基化酶1A; LRP5/6,低密度脂蛋白相关蛋白5和6; NICD,Notch 胞内结构域; SHH,Sonic Hedgehog;-cat,-catenin;-sec,-secretase

Hedgehog

刺猬

The Hedgehog pathway controls normal development and organ patterning during embryogenesis and maintains adult tissue homeostasis by controlling cellular proliferation and differentiation (Matsui, 2016). Upon Sonic Hedgehog (Shh) binding to the Patched receptor, the transmembrane protein Smoothened is activated and allows the release and nuclear translocation of Gli transcription factors to mediate expression of Hedgehog target genes. SIRT1 has been identified in vivo as a negative regulator of this pathway in neuron precursors. In complex with BCL6/BCOR, SIRT1 epigenetically represses Shh signaling effectors Gli1 and Gli2, which are responsible for the expression of genes required for normal cerebellar development. Of note, this same mechanism of epigenetic regulation also acts as a tumor suppressor in medulloblastoma where the Hedgehog pathway is activated by genetic mutations. This suggests that activation of the BCL6/BCOR/SIRT1 complex may be exploited therapeutically in Sonic Hedgehog‐dependent tumors (Tiberi et al., 2014). Although not reported in a stem cell context, SIRT6 appears to regulate expression of another Hedgehog ligand, Indian hedgehog (Ihh), and its downstream genes. Chondrocytes from Sirt6−/− mice exhibit decreased expression of Ihh, impaired proliferation and differentiation, and a senescent phenotype (Piao et al., 2013).

Hedgehog 通路控制胚胎发育过程中的正常发育和器官分化,通过控制细胞生长和分化来维持成年组织的稳态(Matsui,2016)。在 Sonic Hedgehog (Shh)与受体 Patched 结合后,跨膜蛋白被激活,允许 Gli 转录因子释放和核转位介导 Hedgehog 靶基因的表达。SIRT1在体内已被证实为神经元前体细胞这一通路的负调节因子。在 BCL6/BCOR 复合体中,SIRT1表观抑制 Shh 信号效应因子 Gli1和 Gli2,这些效应因子负责正常小脑发育所需的基因表达。值得注意的是,这种相同的表观遗传调控机制也在髓母细胞瘤中起抑癌作用,而 Hedgehog 通路是由基因突变激活的。这表明 BCL6/BCOR/SIRT1复合物的激活可能被用于治疗 Sonic Hedgehog 依赖性肿瘤(Tiberi 等人,2014)。虽然没有在干细胞方面的报道,但 SIRT6似乎可以调节另一种 Hedgehog 配体印度刺猬(Ihh)及其下游基因的表达。Sirt6-/-小鼠的软骨细胞表现出 Ihh 表达减少,增殖和分化受损,以及衰老表型(Piao 等人,2013年)。

Wnt

The canonical Wnt pathway activates gene transcription via β‐catenin. Upon Wnt ligand binding to Frizzled and/or lipoprotein‐related (LRP) 5 and 6 coreceptors, the degradation complex that inactivates β‐catenin is disassembled, allowing stabilization and nuclear translocation of accumulated β‐catenin. The canonical Wnt pathway regulates proliferation and survival and alterations in epigenetic control of the pathway are associated with a variety of cancers (Toh et al., 2017). Increasing evidence suggests that SIRT1 promotes Wnt/β‐catenin signaling in both normal progenitor and cancer cells by a variety of mechanisms. These include both activation of essential pathway components, including β‐catenin itself, and inhibition of Wnt pathway antagonists. SIRT1 is known to deacetylate β‐catenin. This promotes nuclear accumulation of β‐catenin and the transcription of Wnt/β‐catenin target genes in both adipogenesis and osteogenesis (Feng et al., 2016; Zhou et al., 2016b). In osteoblast progenitors, deacetylation of FOXO transcription factors by SIRT1 promotes Wnt signaling and bone formation by preventing sequestration of β‐catenin by FOXOs (Iyer et al., 2014). In mesenchymal stem cells, SIRT1 activates Wnt/β‐catenin signaling and promotes myogenic differentiation by suppressing expression of the Wnt pathway antagonists SFRP2 and DACT1 (Zhou et al., 2015). Like SIRT1, SIRT2 has been shown to bind directly to β‐catenin. However while SIRT1 activates β‐catenin, SIRT2 has been shown to inhibit the Wnt signaling pathway, again highlighting the sometimes opposing actions of different members of the sirtuin family. SIRT2 binding to β‐catenin occurs particularly in response to oxidative stress by ionizing radiation. This interaction impairs expression of Wnt target genes such as survivin, cyclin D1, and c‐myc (Nguyen et al., 2014). SIRT6 also inhibits transcription of Wnt target genes. Unlike SIRT2, repression is not a result of interaction between SIRT6 and Wnt but rather direct binding of SIRT6 with the transcription factor LEF1 and deacetylation of histone 3. Notably, this mechanism of epigenetic regulation of Wnt signaling by SIRT6 was described in HSCs in vivo and plays a role in HSC homeostasis whereby SIRT6 is required to maintain HSC self‐renewal ability as discussed above (Wang et al., 2016).

经典 Wnt 通路通过-catenin 激活基因转录。Wnt 配体与 Frizzled 和/或脂蛋白相关(LRP)5和6受体结合后,失活-连环蛋白的降解复合体被分解,允许累积-连环蛋白的稳定和核移位。经典 Wnt 通路调节增殖、生存和表观遗传控制的改变与多种癌症有关(Toh 等人,2017)。越来越多的证据表明,SIRT1通过多种机制促进正常祖细胞和癌细胞的 wnt/-catenin 信号转导。这些包括激活必需的途径组件,包括-连环素本身,和抑制 Wnt 途径拮抗剂。SIRT1已知有脱乙酰化连环素(deacetylcatenin)。这促进了-catenin 的核聚集和 wnt/-catenin 靶基因在成脂和成骨过程中的转录(Feng 等,2016; Zhou 等,2016b)。在成骨细胞前体中,SIRT1对 FOXO 转录因子的去乙酰化通过阻止 FOXOs 对-catenin 的隔离促进 Wnt 信号和骨形成(Iyer et al. ,2014)。在间充质干细胞中,SIRT1通过抑制 Wnt 通路拮抗剂 SFRP2和 DACT1的表达,激活 Wnt/-catenin 信号转导,促进肌源性分化。与 SIRT1一样,SIRT2也被证明可以直接与-catenin 结合。然而,当 SIRT1激活-catenin 时,SIRT2已经被证明抑制了 Wnt信号通路,再次突出了 sirtuin 家族不同成员有时相反的行为。2与-catenin 的结合特别发生在氧化应激对电离辐射的反应中。这种相互作用损害 Wnt 靶基因的表达,如 survivin、 cyclin D1和 c-myc (Nguyen 等人,2014)。SIRT6还抑制 Wnt 靶基因的转录。与 SIRT2不同,阻遏不是 SIRT6和 Wnt 相互作用的结果,而是 SIRT6与组蛋白3的去乙酰化和转录因子 LEF1的直接结合。值得注意的是,这种 SIRT6 Wnt 信号的表观遗传调控机制在体内的造血干细胞中被描述,并在造血干细胞内稳态中发挥作用,正如上面讨论的那样,SIRT6需要维持造血干细胞的自我更新能力(Wang 等人,2016)。

Notch

凹口

Notch ligands and receptors are transmembrane proteins that mediate cell contact‐dependent signaling. Ligand binding causes cleavage and nuclear translocation of the Notch intracellular domain (NICD) where it activates transcription of target genes. Notch signaling activity is known to be regulated by epigenetic control of several components of the Notch pathway, including Notch itself. Although a role for sirtuins in Notch signaling in stem cells has not been described, SIRT1 in particular is capable of regulating the activity of components of the Notch pathway. In fact, SIRT1 appears to be both a direct and indirect negative regulator of Notch signaling, particularly in endothelial cells where research has focused to date. SIRT1 directly regulates Notch1 by deacetylating conserved lysines in the NICD. This destabilizes Notch1 and has been shown to limit Notch signaling in endothelial cells (Guarani et al., 2011). SIRT1 also inhibits Notch pathway signaling indirectly via its H4K16 deacetylation activity. SIRT1 and lysine demethylase 1A (LSD1) interact directly and play conserved and concerted roles in H4K16 deacetylation and H3K4 demethylation to repress Notch target genes (Mulligan et al., 2011).

Notch 配体和受体是介导细胞接触依赖信号的跨膜蛋白。配体结合引起缺口细胞内域(NICD)的卵裂和核移位,在那里它激活目标基因的转录。已知 Notch 信号活动受 Notch 通路的几个组成部分的表观遗传控制调节,包括 Notch 通路本身。虽然去乙酰化酶在干细胞 Notch 信号转导中的作用还没有被描述,但是 SIRT1特别能够调节 Notch 信号通路成分的活性。事实上,SIRT1似乎是 Notch 信号的直接和间接的负调节因子,特别是在内皮细胞中,这是迄今为止研究的重点。SIRT1通过去乙酰化 NICD 中保守的 lysine 来直接调控 Notch1。这不稳定的 Notch1,并已被证明限制 Notch 信号在内皮细胞(瓜拉尼等人,2011年)。SIRT1还通过 H4K16脱乙酰化活性间接抑制 Notch 信号通路。SIRT1和赖氨酸去甲基化酶1A (LSD1)直接相互作用,在 H4K16去乙酰化和 H3K4去甲基化中发挥保守和协同作用,抑制 Notch 靶基因(Mulligan 等,2011)。

Other members of the sirtuin family have been found to regulate Notch signaling pathways in the context of cancer cells. SIRT3 suppresses both mRNA and protein expression of NOTCH1 in gastric cancer cells. This inhibition was associated with decreased proliferation and colony formation (Wang et al., 2015). Similarly, SIRT6 has been shown to inhibit proliferation of ovarian cancer cells by downregulating NOTCH3 mRNA and protein levels (Zhang et al., 2015).

Sirtuin 家族的其他成员已经被发现在癌细胞中调节 Notch 信号通路。SIRT3抑制胃癌细胞 NOTCH1的 mRNA 和蛋白表达。这种抑制与减少增殖和集落形成有关(Wang 等人,2015)。类似地,SIRT6已被证明通过下调 NOTCH3 mRNA 和蛋白质水平来抑制卵巢癌细胞的增殖(Zhang et al. 2015)。

Sirtuins and cancer stem cells

去乙酰化酶与癌症干细胞

The stem cell signaling pathways discussed above are often aberrantly activated or suppressed in cancer due to deregulation of epigenetic control. As a result, sirtuins may be implicated in the generation of a population of cancer cells capable of self‐renewal and differentiation which drive tumor growth. Evidence also suggests that, at least in some tissues, distinct stem cell programs exist in CSCs and normal stem cells of the corresponding tissue (Ye et al., 2015). As a result, sirtuins may play unique roles in CSCs in addition to those observed in normal stem cells.

上面讨论的干细胞信号通路通常在癌症中被异常激活或抑制,这是由于放松了表观遗传控制。因此,去乙酰化酶可能与能够自我更新和分化从而促进肿瘤生长的癌细胞群的产生有关。证据还表明,至少在一些组织中,CSCs 和相应组织的正常干细胞中存在不同的干细胞程序(Ye 等人,2015)。因此,去乙酰化酶除了在正常干细胞中观察到的作用外,在 CSCs 中也可能发挥独特的作用。

As in normal stem cells, SIRT1 is the best studied sirtuin in CSCs. SIRT1 expression has been shown to be upregulated in a variety of CSCs both in vitro and in vivo, including glioma (Lee et al., 2015), breast (Ma et al., 2015), colorectal (Chen et al., 2014b), and leukemia (Li et al., 20122014). SIRT1 is required for both oncogenic transformation and maintenance of stemness in glioma cells (Lee et al., 2015). Consequently, CD133+ glioma stem cells express high levels of SIRT1 compared to CD133 non‐stem cells. Notably, knockdown of SIRT1increased the radiosensitivity of CD133+ cells both in vitro and in vivo (Chang et al., 2009). High levels of SIRT1, as well as low expression of its direct regulator miR‐34a, have been identified in CD44+/CD24 breast cancer stem cells (BCSCs). Both overexpression of miR‐34a and knockdown of SIRT1 were found to decrease BCSCs, tumorsphere formation, and expression of CSC markers including ALDH1 and Nanog (Ma et al., 2015). In colorectal cancer cells, SIRT1 is also overexpressed and colocalizes with the colorectal CSC marker CD133. Knockdown of SIRT1 reduced CD133+ cells, decreased sphere formation, and attenuated tumorigenicity in vivo. Furthermore, the expression of several stem cell markers, including OCT4, NANOG, and TERT was also found to be decreased (Chen et al., 2014b). SIRT1 is highly expressed in NanogPOS liver CSCs but decreases during differentiation. Consequently, it has been shown to be responsible in vitro for the maintenance of self‐renewal in liver CSCs by epigenetic regulation of the SOX2 promoter (Liu et al., 2016). It is clear therefore that SIRT1 is required for the maintenance of CSCs. Although the precise downstream mechanisms involved may vary, loss of SIRT1 is associated with decreased sphere formation, reduced expression of CSC markers, and increased sensitivity to treatment. Outside of its specific roles identified in CSCs, SIRT1 is also known to regulate the stemness‐associated Wnt signaling pathway in several non‐stem cancer cell contexts. It mediates epigenetic silencing of Wnt antagonists SFRP1, SFRP2, and DKK1 in addition to positively regulating levels of all three mammalian Disheveled (Dvl) proteins (Pruitt et al., 2006; Hussain et al., 2009; Holloway et al., 2010). In breast cancer cells, inhibition of SIRT1/2 decreased Frizzled7 (FZD7) protein expression, as well as β‐catenin and c‐Jun binding to the FZD7 promoter (Simmons et al., 2014). Similarly, inhibition of SIRT1 in acute lymphoblastic leukemia (ALL) also inhibits the Wnt/β‐catenin signaling pathway resulting in elimination of ALL stem/progenitor cells (Jin et al., 2015).

与正常干细胞一样,SIRT1是 CSCs 中研究最多的 sirtuin。SIRT1表达在各种肿瘤干细胞的体内外均有上调,包括胶质瘤(Lee 等人,2015年)、乳腺癌(Ma 等人,2015年)、结直肠癌(Chen 等人,2014b)和白血病(Li 等人,2012年,2014年)。SIRT1在神经胶质瘤细胞的致癌转化和干细胞保持中都是必需的(Lee et al. 2015)。因此,CD133 + 胶质瘤干细胞表达高水平的 SIRT1相比,CD133-非干细胞。值得注意的是,击倒 SIRT1增加了 CD133 + 细胞在体外和体内的放射敏感性(Chang et al. ,2009)。在 CD44 +/CD24-乳腺癌干细胞(BCSCs)中发现高水平的 SIRT1及其直接调节因子 miR-34a 的低表达。miR-34a 的过表达和 SIRT1的敲除均可降低 BCSCs、肿瘤球形成和 CSC 标志物 ALDH1和 Nanog 的表达(Ma 等,2015)。在大肠癌细胞中,SIRT1也过度表达并与结直肠癌 CSC 标记 CD133共定位。SIRT1基因敲除可降低体内 CD133 + 细胞数量,减少球形细胞数量,减弱体内致瘤性。此外,几个干细胞标志物,包括 OCT4,NANOG,和 TERT 的表达也被发现减少(Chen 等人,2014b)。SIRT1在 NanogPOS 肝脏 CSCs 中高表达,但在分化过程中减少。因此,它已被证明在体外负责维持肝脏 CSCs 的自我更新的 SOX2启动子的表观遗传调控(刘,2016年)。因此,显然需要 SIRT1来维持 CSCs。虽然精确的下游机制可能有所不同,SIRT1的缺失与球形减少,CSC 标记的表达减少,以及对治疗的敏感性增加有关。除了在 CSCs 中确定的特定作用外,SIRT1还被认为在几种非干细胞中调节干性相关的 Wnt信号通路。它介导 Wnt 拮抗剂 SFRP1,SFRP2和 DKK1的表观遗传沉默,除此之外,它还积极调节所有三种哺乳动物的 disheded (Dvl)蛋白的水平(Pruitt 等人,2006; Hussain 等人,2009; Holloway 等人,2010)。在乳腺癌细胞中,SIRT1/2的抑制降低了 Frizzled7(FZD7)蛋白的表达,以及-catenin 和 c-Jun 与 FZD7启动子的结合(Simmons 等人,2014)。类似地,抑制急性淋巴性白血病中的 SIRT1也会抑制 wnt/-catenin 信号通路,从而导致 ALL 干/祖细胞的消失(Jin 等,2015)。

In addition to the regulatory role of sirtuins in distinct stem cell programs, it has been shown that they may directly regulate the activity of CSC markers themselves. In this regard, ALDH1A1 activity is a commonly used marker for CSCs and evidence suggests that it may be involved in CSC maintenance or differentiation. SIRT2 has recently been shown to post‐translationally regulate ALDH1A1. NOTCH signaling induces SIRT2 to deacetylate ALDH1A1, leading to increased ALDH activity, CSC populations, and CSC self‐renewal in breast cancer (Zhao et al., 2014). On the other hand, SIRT2 activity has been reported to exert an opposite effect in the context of glioblastoma. There, SIRT2 activation mediates the antiproliferative function of resveratrol specifically on glioblastoma stem cells (Sayd et al., 2014), thus underlining the importance of cellular context in determining specific roles for sirtuins.

除了去乙酰化酶在不同的干细胞程序中的调节作用外,已经证明它们可以直接调节 CSC 标记本身的活性。在这方面,ALDH1A1活性是 CSCs 常用的标志物,有证据表明它可能参与了 CSC 的维持或分化。SIRT2最近被证明可以在翻译后调节 ALDH1A1。NOTCH 信号诱导 SIRT2去乙酰化 ALDH1A1,导致乳腺癌中 ALDH 活性、 CSC 群体和 CSC 自我更新增加(赵等人,2014)。另一方面,据报道 SIRT2活性在胶质母细胞瘤中具有相反的作用。在那里,SIRT2激活介导白藜芦醇特异性对胶质母细胞瘤干细胞的抗增殖功能(Sayd 等人,2014) ,从而强调了细胞背景在决定 sirtuins 的特定作用中的重要性。

Consistent with the previously described tumor suppressive role for SIRT6, its overexpression results in suppressed CSC activity in breast, lung, and colorectal cancer cells with PI3K activation as evidenced by the decreased tumorsphere‐forming capacity and size of ALDH+ cell population which are established readouts of CSCs (Ioris et al., 2017). These results were further confirmed in vivo after cell injection into the flanks of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice as well as after monitoring tumor growth in transgenic mice expressing polyomavirus middle T oncogene (PyMT) under the mouse mammary tumor virus promoter. In both cases, SIRT6 overexpression delays tumor growth, which is associated with decreased ALDH expression and enzymatic activity, altered glucose and lipid metabolism, and reduced cancer stemness. From a mechanistic point of view, enhanced SIRT6 dampens phosphoinositide 3‐kinase (PI3K) signaling at the transcriptional level counteracting the positive role of PI3K signaling in survival and maintenance of CSCs.

与先前描述的 SIRT6的肿瘤抑制作用相一致,其过度表达导致抑制乳腺、肺和大肠癌细胞的 CSC 活性,并伴随 PI3K 的激活,这可以从肿瘤形成能力和 ALDH + 细胞数量的减少中得到证实。这些结果在非肥胖糖尿病/重度联合免疫缺陷(NOD/SCID)小鼠侧翼细胞注射后,以及在小鼠乳腺肿瘤病毒启动子下监测表达多瘤病毒中间 t 基因(PyMT)的转基因小鼠肿瘤生长后,在体内得到进一步证实。在这两种情况下,SIRT6的过度表达延迟了肿瘤的生长,这与 ALDH 表达和酶活性的降低,葡萄糖和脂质代谢的改变,以及减少癌症干性有关。从机制上看,增强的 SIRT6在转录水平抑制了磷脂酰肌醇3- 激酶(PI3K)信号,抵消了 PI3K 信号在 CSCs 存活和维持中的积极作用。

Epithelial–Mesenchymal Transition (EMT)

上皮-间充质转变(EMT)

The EMT program consists of multiple transitional states in which cells move through between epithelial and mesenchymal phenotypes (Nieto et al., 2016). During EMT, epithelial cells reorganize their cytoskeleton, lose apical–basal polarity and cell–cell adhesion, and attain an increased capacity for cell mobility. Importantly, activation of EMT programs confers stem‐like traits on both normal and neoplastic cells (Mani et al., 2008). Several reports have outlined roles for various sirtuins in the promotion of EMT. However, consistent with their characterization as either tumor promoters or suppressors depending on cellular context, sirtuins have also been described as both enhancers and repressors of EMT (Fig. 2). The actual functions then may differ between individual sirtuins, tissues of origin, microenvironments, and cellular contexts. Furthermore, the biochemical mechanisms that underpin sirtuin regulation of EMT remain unclear and it is not yet known whether all of these effects involve deacetylase activity.

EMT 计划包括多种过渡状态,细胞在上皮和间充质表型之间移动(Nieto 等,2016)。在 EMT 过程中,上皮细胞重组其细胞骨架,失去顶基极性和细胞-细胞粘附,增加细胞活动能力。重要的是,EMT 程序的激活给正常细胞和肿瘤细胞带来了干样特征(Mani et al. ,2008)。一些报告已经概述了各种去乙酰化酶在促进 EMT 中的作用。然而,与其作为肿瘤促进剂或抑制剂的角色塑造一致,去乙酰化酶也被描述为 EMT 的增强子和抑制子(图2)。实际的功能在不同的去乙酰化酶、起源组织、微环境和细胞环境之间可能有所不同。此外,去乙酰化酶调节 EMT 的生化机制仍不清楚,目前尚不清楚所有这些效应是否都涉及去乙酰化酶活性。

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Figure 2 图2Open in figure viewer 打开图形查看器PowerPoint 简报Schematic representation of positive and negative regulation of EMT by sirtuins. Positive: TGF‐β signaling is associated with an increase in Sirt1. Sirt1 is recruited by Zeb1 to the E‐cadherin promoter and causes transcriptional repression. Similarly, Sirt1 interacts with Twist and MBD1 to silence the E‐cadherin promoter. Sirt1 may also recruit Sirt7 to repress E‐cadherin expression. Sirt2 activates Akt/GSK3β/β‐catenin signaling to promote EMT. Deacetylation of Slug by Sirt2 promotes Slug protein stability and repression of Slug target genes, including E‐cadherin. Negative: Sirt1 inhibits TGF‐β signaling by deacetylating Smad4. This decreases MMP transcription and E‐cadherin degradation. Sirt4 upregulates E‐cadherin expression via its repression of glutamine metabolism. E‐cad, E‐cadherin; GSK3β, glycogen synthase kinase‐3β; MBD1, methyl‐CpG binding domain protein‐1; MMP7, metalloproteinase 7; TGF‐βR, transforming growth factor‐β receptor; β‐cat, β‐catenin. 去乙酰化酶对 EMT 正负调节的图式表征。正面: TGF-信号与 Sirt1的增加有关。Sirt1被 Zeb1招募到 e-cadherin 启动子并引起转录抑制。类似地,Sirt1与 Twist 和 MBD1相互作用使 e-cadherin 启动子沉默。Sirt1也可能会招募 Sirt7来抑制 e-cadherin 的表达。Sirt2激活 akt/gsk3/-catenin 信号促进 EMT。Sirt2对蛞蝓蛋白去乙酰化作用的研究促进蛞蝓蛋白的稳定性和对 e- 钙粘蛋白等靶基因的阻遏作用。否定: Sirt1通过去乙酰化 Smad4抑制 TGF 信号传导。这减少了基质金属蛋白酶的转录和 e-钙粘蛋白的降解。Sirt4通过抑制谷氨酰胺代谢上调 e- 钙粘蛋白的表达。E-cad,e-cadherin; gsk3,糖原合成酶激酶3; MBD1,甲基 CpG 结合域蛋白1; MMP7,金属蛋白酶7; TGF-r,转化生长因子受体;-cat,-catenin

SIRT1 has been shown to be a positive regulator of EMT in prostate cancer, through its deacetylase activity. SIRT1 is recruited to the E‐cadherin promoter by the zinc finger transcription factor ZEB1. Here, it deacetylates histone H3, reduces binding of RNA polymerase II ultimately causing transcriptional repression of E‐cadherin. As a result, loss of SIRT1 decreases prostate cancer cell migration and metastasis, independent of any effects on cell survival (Byles et al., 2012). Similarly, SIRT1 interacts with Twist and methyl‐CpG binding domain protein‐1 (MBD1) to silence the E‐cadherin promoter in pancreatic cancer (Xu et al., 2013). Furthermore, EMT in pancreatic cancer cells, induced by transforming growth factor‐β (TGF‐β), is associated with upregulation of SIRT1, while inhibition of SIRT1 induced mesenchymal–epithelial transition (Deng et al., 2014). On the other hand, deacetylation of Smad4 by SIRT1 was identified as a mechanism of reducing EMT by repressing the effects of the TGF‐β signaling pathway in both transformed primary human mammary epithelial cells and kidney epithelial cells (Simic et al., 2013). Moreover, SIRT1 has been identified as an EMT repressor in oral squamous cell carcinoma, lung cancer, and ovarian cancer (Sun et al., 2013a,b; Chen et al., 2014a).

SIRT1通过其去乙酰化酶活性被证明是前列腺癌中 EMT 的一个积极调节因子。SIRT1被锌指转录因子 ZEB1招募到 e-cadherin 启动子上。在这里,它去乙酰化组蛋白 h 3,减少 RNA聚合酶Ⅱ的结合,最终导致 e-cadherin 的转录抑制。因此,SIRT1的缺失降低了前列腺癌细胞的迁移和转移,而不受细胞存活率的影响(Byles 等人,2012)。类似地,SIRT1与 Twist 和 methyl-CpG binding domain protein-1(MBD1)相互作用使 e-cadherin 启动子在胰腺癌中沉默(Xu et al. ,2013)。此外,胰腺癌细胞的 EMT,由转化生长因子-(TGF -)诱导,与 SIRT1的上调有关,同时抑制 SIRT1诱导的间充质上皮转变(Deng et al. ,2014)。另一方面,SIRT1对 Smad4的去乙酰化作用被认为是通过抑制转化的人原代乳腺上皮细胞和肾上皮细胞中 TGF-信号通路的作用来减少 EMT 的机制(Simic et al. ,2013)。此外,SIRT1已被确定为口腔鳞状细胞癌、肺癌和卵巢癌的 EMT 抑制因子(Sun 等人,2013a,b; Chen 等人,2014a)。

Despite the reduced expression of E‐cadherin observed in SIRT2−/− MEFs (Nguyen et al., 2014), SIRT2 has been shown to positively regulate EMT in the context of cancer. SIRT2 expression is upregulated in hepatocellular carcinoma where it promotes EMT by deacetylating and activating protein kinase B to target the Akt/GSK3β/β‐catenin signaling pathway (Chen et al., 2013). Consistent with its positive role, a recently published study showed that SIRT2 maintains Slug protein levels through deacetylation‐mediated increased protein stability. Furthermore, it was shown that elevated Slug protein caused by SIRT2 overexpression corresponded to stronger repression of the Slug transcriptional targets, epithelial cell adhesion molecule, and E‐cadherin, implying that SIRT2 might regulate EMT‐related phenotypes such as aggressiveness and invasion specifically in triple‐negative basal‐like breast cancer (Zhou et al., 2016a).

尽管在 SIRT2-/-MEFs 中观察到 e-cadherin 的表达减少(Nguyen 等人,2014年) ,但是已经证明 SIRT2在癌症中对 EMT 有积极的调节作用。SIRT2表达在肝细胞性肝癌中上调,通过去乙酰化和激活蛋白激酶 b 来促进 EMT,靶向 akt/gsk3/-catenin 信号通路(Chen et al. ,2013)。与其积极作用相一致,最近发表的一项研究表明,SIRT2通过去乙酰化增加蛋白质的稳定性来维持蛞蝓蛋白的水平。此外,SIRT2过表达导致 Slug 蛋白升高,对应于 Slug 转录靶点、上皮细胞细胞粘附分子和 e-cadherin 的强烈抑制,这意味着 SIRT2可能调节 EMT 相关表型,如三阴性基底样乳腺癌的侵袭性和特异性侵袭(Zhou et al. 2016a)。

With emphasis on the diverse functions regulated by the different members of the sirtuin family, SIRT4 has been described as a negative regulator of EMT. SIRT4 expression is associated with upregulation of E‐cadherin expression and decreased vimentin expression in colorectal cancer cells. Under these experimental conditions, SIRT4 suppresses glutamine metabolism by repressing the enzymatic activity of glutamate dehydrogenase. Given that α‐ketoglutarate, an important product of glutamine metabolism, inhibited the upregulation of E‐cadherin expression by SIRT4, it was suggested that regulation of E‐cadherin expression occurs via inhibition of glutamine metabolism (Miyo et al., 2015).

由于 sirtuin 家族不同成员所调节的不同功能,SIRT4被描述为 EMT 的负面调节者。SIRT4的表达与 e-cadherin 表达上调和降低大肠癌细胞的 vimentin 表达有关。在这些实验条件下,SIRT4通过抑制谷氨酸脱氢酶的酶活性来抑制谷氨酰胺的代谢。鉴于-酮戊二酸,谷氨酰胺代谢的一个重要产物,抑制了上调的 e-钙粘蛋白的表达 SIRT4,这表明,调节 e-钙粘蛋白的表达是通过抑制谷氨酰胺代谢(Miyo 等人,2015年)。

SIRT7 expression has been found to correlate inversely with E‐cadherin in prostate cancer. Furthermore, loss of SIRT7 expression in prostate carcinoma cell lines caused a reversal of the EMT phenotype with upregulation of E‐cadherin and decreased vimentin and Slug expression. Interestingly, it has been suggested that SIRT1 mediates the recruitment of SIRT7 to the E‐cadherin promoter and that this interplay between SIRT1 and ‐7 is responsible for promoting EMT (Malik et al., 2015). Additionally, overexpression of SIRT7 in colorectal cancer is associated with downregulation of epithelial markers, including E‐cadherin, and upregulated expression of mesenchymal markers (Yu et al., 2014).

在前列腺癌中,SIRT7的表达与 e-cadherin 呈负相关。此外,SIRT7在前列腺癌细胞系中的表达缺失导致了 e-cadherin 表达上调的 EMT 表型的逆转,并降低了 vimentin 和 Slug 的表达。有趣的是,有人认为 SIRT1介导了 e-cadherin 启动子上 SIRT7的补充,而这种 SIRT1和 -7之间的相互作用促进了 EMT (Malik 等人,2015)。此外,SIRT7在大肠癌中的过度表达与上皮标志物的下调有关,包括 e-cadherin,以及间质标志物的上调表达(Yu 等,2014)。

Sirtuins as therapeutic targets for CSCs

去乙酰化酶作为 CSCs 的治疗靶点

Given that sirtuins can act as both cancer promoters and suppressors, both activators and inhibitors of sirtuins have been developed in recent years. Interestingly, inhibition of sirtuins has attracted more interest as a potential therapeutic anticancer strategy. To date, several sirtuin inhibitors have been developed which differ based on their mechanism of action and structural features (Hu et al., 2014b). Two classes of sirtuin inhibitors, NAM and thioacyllysine‐containing compounds, can be considered as mechanism‐based inhibitors, whereas other sirtuin inhibitors, including sirtinol and its analogues, splitomicin and its derivatives, indole derivatives as well as tenovin and its analogues, presumably work by noncovalent binding to the sirtuin active site and blocking substrate binding. With regard to anticancer effects, sirtuin inhibition has been found to induce growth arrest or cell death in various cancer cell lines from a wide range of tissues (Table 2) (Heltweg et al., 2006; Ota et al., 2006; Lara et al., 2009; Rotili et al., 2012b). Importantly, decreasing sirtuin activity has been shown to be effective in specifically targeting CSCs that are resistant to standard therapy. Tenovin‐6 (a small‐molecule inhibitor of SIRT1 and SIRT2) treatment yielded a significant loss of imatinib‐resistant chronic myeloid leukemia (CML) CD34+ stem cells in vivo. This effect was caused mainly by SIRT1 inhibition resulting in elevated acetylated and total p53 levels (Li et al., 2012). Similarly, it has been shown to eliminate CD133+ ALL stem cells and also decrease ALDH+ cells and tumorsphere formation in uveal melanoma cell lines (Jin et al., 2015; Dai et al., 2016). Furthermore, benzodeazaoxaflavin SIRT1/2 inhibitors have demonstrated antiproliferative activity in spheroidal cell cultures from both colon carcinoma and glioblastoma multiforme (Rotili et al., 2012a). In addition to antiproliferative potency against leukemia and breast cancer cell lines, the SIRT1/2 inhibitor salermide reduces viability in spheroidal cultures of colorectal CSC cell lines (Rotili et al., 2012b). In the same study, the SIRT2‐selective inhibitor AGK2 displayed antiproliferative activity against glioblastoma multiforme tumorspheres. It is worth noting, though, that in accordance with the previously reported opposing roles played by the different members of the sirtuin family and the undeniable significance of biological and contextual factors, a recent study suggested that sirtuin activation, rather than inhibition, could be a therapeutic strategy to target CSCs with activating PI3K mutations. More specifically, SIRT6 overexpression suppresses PI3K signaling at the transcriptional level and antagonizes tumorsphere formation. This implies that SIRT6 activation may be exploited therapeutically to hinder stemness of tumors with PIK3CA gene mutations or PTEN loss (Ioris et al., 2017).

鉴于去乙酰化酶既可以作为癌症促进剂又可以作为抑制剂,近年来研究人员开发了去乙酰化酶的激活剂和抑制剂。有趣的是,去乙酰化酶的抑制作为一种潜在的抗癌治疗策略吸引了更多的兴趣。迄今为止,几种去乙酰化酶抑制剂已经被开发出来,它们的作用机制和结构特征不同(Hu 等人,2014b)。2类去乙酰化酶抑制剂,NAM 和巯基赖氨酸类化合物,可以作为机制性抑制剂,而其他去乙酰化酶抑制剂,包括去乙酰化酶及其类似物、分裂霉素及其衍生物、吲哚衍生物以及替诺霉素及其类似物,可能通过与去乙酰化酶活性中心的非共价结合和阻断底物结合起作用。关于抗癌作用,已经发现去乙酰化酶抑制剂可以诱导来自各种组织的各种癌细胞系的生长停滞或细胞死亡(表2)(Heltweg 等人,2006; Ota 等人,2006; Lara 等人,2009; Rotili 等人,2012b)。重要的是,减少去乙酰化酶的活性已被证明是有效的,特别是针对对标准治疗耐药的 CSCs。Tenovin-6(一种 SIRT1和 SIRT2的小分子抑制剂)治疗在体内产生了伊马替尼耐药的 CD34 + 干细胞的明显流失。这种效应主要是由 SIRT1抑制导致乙酰化和总 p53水平升高(李等人,2012年)。类似地,它已经被证明可以消除 CD133 + ALL 干细胞,并且还可以减少葡萄膜黑色素瘤细胞系中 ALDH + 细胞和肿瘤球的形成(Jin 等人,2015; Dai 等人,2016)。此外,苯甲脱氮草黄素 SIRT1/2抑制剂已证明在结肠癌和胶质母细胞瘤的球状细胞培养中具有抗增殖活性。除了对白血病和乳腺癌细胞株的抗增殖能力外,SIRT1/2抑制剂 salermide 还降低了结直肠癌 CSC 细胞株的球形培养中的活性(Rotili 等,2012b)。在同一项研究中,SIRT2选择性抑制剂 AGK2显示了对胶质母细胞瘤肿瘤球的抗增殖活性。但是值得注意的是,根据先前报道的 sirtuin 家族不同成员发挥的相对作用以及生物学和环境因素的不可否认的重要性,最近的一项研究表明 sirtuin 的激活,而不是抑制,可能是一种治疗策略,目标 CSCs 激活 PI3K 突变。更具体地说,SIRT6过表达在转录水平上抑制 PI3K 信号转导并拮抗肿瘤小球的形成。这意味着 SIRT6的激活可能被用于治疗阻碍 PIK3CA 基因突变或 PTEN 缺失的肿瘤的干性(Ioris 等人,2017)。Table 2. 表二Sirtuin inhibitors with anticancer activity 具有抗癌活性的去乙酰化酶抑制剂

Inhibitors 抑制剂Target 目标Anti‐CSC activity 反 CSC 活动
Tenovin‐6 Tenovin-6SIRT1/2CML, AML 慢性粒细胞白血病
BDF4‐1, ‐2a, ‐2b, ‐2d BDF4-1,-2a,-2b,-2dSIRT1/2Colon, glioblastoma 结肠,胶质母细胞瘤
AGK2SIRT2Glioblastoma 胶质母细胞瘤
SalermideSIRT1/2Colorectal 结直肠
Inhibitors 抑制剂Target 目标Anticancer activity 抗癌活性
Nicotinamide 烟酰胺SIRT1/2/3/5/6Leukemia, oral, prostate 白血病,口腔,前列腺
Sirtinol 去乙酰化酶SIRT2Breast, lung, prostate, oral 乳房,肺,前列腺,口腔
SalermideSIRT1/2Lung, breast, colon 肺,乳房,结肠
JGB‐1741 JGB-1741SIRT1Breast 乳房
Cambinol 女名女子名SIRT1/2/5Burkitt lymphoma 伯基特淋巴瘤
EX527SIRT1Leukemia 白血病
AC‐93253 AC-93253SIRT1/2/3Prostate, pancreas, lung 前列腺,胰腺,肺
InauhzinSIRT1Lung, colon 肺,结肠
Tenovin‐1 Tenovin-1Unknown 未知Burkitt lymphoma, melanoma 伯基特淋巴瘤黑色素瘤
  • The upper part shows inhibitors that have been reported to exert an anti‐CSC‐specific effect (Li 上半部分显示了已报道的能够发挥抗 CSC 特异性作用的抑制剂(Liet al 等等., 2012; Rotili et al 等等., 2012a,b; Hu ; 胡et al 等等., 2014b; Jin et al 等等., 2015; Dai 戴et al 等等., 2016).

Sirtuins, aging, and stem cells

去乙酰化酶、衰老和干细胞

Sirtuins have long been recognized as regulators of aging – overexpression of sirtuins has been shown to extend lifespan in several organisms (Tissenbaum & Guarente, 2001; Kanfi et al., 2012). Sirtuin function in aging has to date been reported to be related to their roles in regulation of energy metabolism, response to calorie restriction (CR), control of cell death, and circadian rhythms (Araki et al., 2004; Chang & Guarente, 2013; Guarente, 2013). A new mechanism of lifespan modulation by sirtuins has been gaining attention, related to their potential roles in cellular and mitochondrial protein homeostasis networks. Recent developments have highlighted the close relationship between healthy aging and protein homeostasis, or proteostasis (Kaushik & Cuervo, 2015; Walther et al., 2015). A gradual loss of proteostasis is associated with age (Labbadia & Morimoto, 2015) and the longest living organisms are known to have more stable proteasomes and active proteostasis networks (Perez et al., 2009; Treaster et al., 2014). Most importantly, enhancing the functionality of proteostasis networks has been shown to extend both lifespan and healthspan of certain organisms (Morimoto & Cuervo, 2014; Vilchez et al., 2014a; Labbadia & Morimoto, 2015). Given that sirtuins are well‐known lifespan modulators whose deficiencies have been linked to a higher incidence of age‐related diseases, the investigation of their roles in proteostasis networks would appear to be warranted. In fact, a relationship between sirtuins and ER stress appears to be conserved from C. elegans to mammals, indicating a crucial link between sirtuins and proteostasis (Viswanathan et al., 2005). SIRT1 is a known negative regulator of ER stress responses through deacetylating IRE‐1‐generated active XBP1 and subsequent inhibition of its transcriptional activity to promote ER stress‐induced apoptosis (Wang et al., 2011). SIRT1 also suppresses pERK‐eIF2α‐dependent translational inhibition (Ghosh et al., 2011). In breast cancer cells, the unfolded protein response (UPR) triggered by the accumulation of misfolded proteins in the mitochondria (UPRmt) requires the activation of SIRT3 together with CHOP and estrogen receptor alpha (ERα). By orchestrating both the antioxidant machinery and mitophagy in a CHOP‐ and ERα‐independent manner, SIRT3 contributes to overcoming proteotoxic stress and mitochondrial stress, which may represent an essential mechanism of adaptation of cancer cells (Papa & Germain, 2014).

Sirtuins 长期以来一直被认为是衰老的调节因子—— Sirtuins 的过度表达已被证明能延长一些生物体的寿命(Tissenbaum & Guarente,2001; Kanfi 等人,2012)。Sirtuin 在衰老中的功能迄今为止已被报道与它们在调节能量代谢、响应卡路里限制反应、控制细胞死亡和昼夜节律中的作用有关(Araki 等人,2004; Chang & Guarente,2013; Guarente,2013)。抗衰老蛋白调节寿命的新机制已经引起人们的关注,这与它们在细胞和线粒体蛋白质稳态网络中的潜在作用有关。最近的发展强调了健康老化和蛋白质内稳态或蛋白质内稳态之间的密切关系(Kaushik & Cuervo,2015; Walther et al. ,2015)。蛋白质平衡的逐渐丧失与年龄有关(Labbadia & Morimoto,2015) ,已知最长寿的有机体拥有更稳定的蛋白质平衡体和活跃的蛋白质平衡网络(Perez et al. ,2009; Treaster et al. ,2014)。最重要的是,增强蛋白抑制网络的功能已被证明可以延长某些有机体的寿命和健康寿命(Morimoto & Cuervo,2014; Vilchez et al. ,2014a; Labbadia & Morimoto,2015)。鉴于去乙酰化酶是众所周知的寿命调节剂,其缺陷与年龄相关疾病的高发病率有关,研究它们在蛋白抑制网络中的作用似乎是必要的。事实上,从秀丽隐杆线虫到哺乳动物,去乙酰化酶和内质网应激之间的关系似乎是保守的,这表明去乙酰化酶和蛋白质平衡之间存在重要联系(Viswanathan et al. ,2005)。SIRT1是一个已知的 ER 应激反应负调节剂,通过去乙酰化 IRE-1产生的活性 XBP1和随后抑制其转录活性促进 ER 应激诱导的凋亡(Wang 等人,2011)。SIRT1还抑制 pERK-eif2依赖的翻译抑制(Ghosh 等人,2011)。在乳腺癌细胞中,由于线粒体中错误折叠的蛋白质堆积而引发的未折叠蛋白反应,需要 SIRT3与 CHOP 和雌激素受体 α 一起被激活。通过协调抗氧化机制和吞噬机制,以一种与 CHOP 和 er 无关的方式,SIRT3有助于克服蛋白毒性应激和线粒体应激,这可能代表了癌细胞适应的基本机制(Papa & Germain,2014)。

Looking more closely into stem cells, they seem to have increased mechanisms to protect their proteasomes, and proteostasis networks impact their function (Vilchez et al., 2014b). This would appear to be related to the stem cell theory of aging, which suggests that a progressive decline in the self‐renewal of adult stem cells and their potential to differentiate into specific cell types in order to replenish the tissues of an organism underlie the mechanistic basis for aging. Although the age‐dependent loss of function of different types of adult stem cells has been reported, we are just now starting to understand the molecular mechanisms involved in this process. With the focus on sirtuins, SIRT7‐mediated alleviation of mitochondrial protein folding stress plays a critical role in modulating the aging process by regulating HSC quiescence and tissue maintenance (Mohrin et al., 2015). SIRT7 functions as a stress sensor in proliferating, metabolically active HSCs and reduces the expression of the mitochondrial translation machinery through repressing activity of the master regulator of mitochondria, nuclear respiratory factor 1 (NRF1), which is necessary to alleviate mitochondrial protein folding stress. Of note, rescue of the impaired reconstitution capacity in aged HSCs upon SIRT7 overexpression or NRF1 inactivation underscores the significance of sirtuin‐regulated proteostasis in maintaining stemness. Interestingly, decreased Sirt3expression in aged HSCs is associated with a concomitant repression of mitochondrial protective programs (Brown et al., 2013), which might result in compromised function of the previously described SIRT3‐directed UPRmt pathway. Certainly, further studies need to address whether similar mechanisms are involved in other adult stem cells and tissues. Also, it would be interesting to see whether these mechanisms are crucial for self‐renewal and differentiation of CSCs based on the fact that CSCs resemble a proliferating, metabolically active normal stem cell. Furthermore, even though SIRT7 and SIRT3 cross at mitochondrial regulation, they do activate these protective mechanisms through their function in nucleus and mitochondria, respectively. As similar protective programs might be orchestrated by other sirtuins, it remains to be determined whether SIRT2, which is the main cytoplasmic sirtuin strongly downregulated in aged HSCs as well (Chambers et al., 2007), is involved in stem cell maintenance, and possibly, new pathways crucial for stem cell maintenance remain to be identified.

更仔细地观察干细胞,他们似乎有更多的机制来保护他们的蛋白酶体,和蛋白郁积网络影响他们的功能(Vilchez 等人,2014b)。这似乎与衰老的干细胞理论有关,该理论认为,成体干细胞的自我更新能力及其分化为特定细胞类型以补充有机体组织的潜力逐渐下降,是衰老的机制基础。虽然不同类型的成体干细胞功能的年龄依赖性丧失已有报道,但我们现在才刚刚开始了解这一过程中涉及的分子机制。随着对去乙酰化酶的关注,SIRT7介导的减轻线粒体蛋白质折叠应激在调节 HSC 静止和组织维持的老化过程中发挥了关键作用(Mohrin 等人,2015)。SIRT7作为应激传感器参与细胞增殖、代谢活跃的造血干细胞,通过抑制线粒体主要调节因子核呼吸因子1(nuclear respiratory factor 1,NRF1)的活性降低线粒体翻译机制的表达,这是缓解线粒体蛋白折叠应激所必需的。值得注意的是,通过 SIRT7过度表达或 NRF1失活来挽救衰老造血干细胞受损的重建能力强调了去乙酰化酶调节蛋白抑制在维持干燥状态中的重要性。有趣的是,SIRT3在老年人造血干细胞中的表达减少与线粒体保护程序的抑制相关(Brown 等人,2013) ,这可能导致以前描述的 SIRT3定向的 UPRmt 通路的功能受损。当然,进一步的研究需要解决其他成体干细胞和组织中是否存在类似的机制。另外,基于 CSCs 类似于一个增殖的、代谢活跃的正常干细胞,研究这些机制是否对 CSCs 的自我更新和分化至关重要也是很有意义的。此外,即使 SIRT7和 SIRT3在线粒体调节上交叉,它们也通过分别在细胞核和线粒体中的功能激活这些保护机制。由于类似的保护程序可能由其他 sirtuins 组织,SIRT2,这个主要的细胞质 sirtuin 在老年造血干细胞中也强烈下调,是否参与干细胞维持,可能,对干细胞维持至关重要的新途径仍有待确定。

Calorie restriction and stem cells

卡路里限制和干细胞

CR is one of the most potent dietary interventions for increasing lifespan and delays the onset of age‐related diseases including cancer (Wanagat et al., 1999; Longo & Fontana, 2010; Colman et al., 2014). It is accepted that its beneficial effects might relate, at least in some significant part, to epigenetically reprogramming stemness while prolonging the capacity of stem‐like cell states to proliferate, differentiate, and replace mature cells in adult aging tissues. This is based on studies showing that CR may maintain stem cell function of HSCs (Ertl et al., 2008), enhance stem cell availability and activity in the muscle of young and old animals (Cerletti et al., 2012), and increase hippocampal neural stem and progenitor cell proliferation in aging mice (Park et al., 2013b). Considering that sirtuins are NAD‐dependent protein deacetylases directly activated by CR, it could be proposed that they may mediate some of the beneficial effects of CR on normal stem cells in adult somatic tissues. However, this is a relatively unexplored area of research and there is lack of experimental evidence to either support or counter this hypothesis. Only very recently, it was reported that SIRT1 is necessary for the expansion of intestinal stem cells (ISCs) upon CR. More specifically, CR results in deacetylation of p70 ribosomal S6 kinase due to SIRT1 activation, which consequently promotes its phosphorylation by mammalian target of rapamycin complex 1 (mTORC1). This signal‐response mechanism mediates the increase in both protein synthesis and number of ISCs, even as mTOR signaling is turned down by CR in more differentiated cells (Igarashi & Guarente, 2016). Similarly, little is known about the role of sirtuins as mediators of the CR‐induced effect on tumorigenesis. As previously mentioned, cancer was among the age‐related diseases which exhibited a delayed onset in response to CR in several early studies (Hursting et al., 1994; Berrigan et al., 2002; Mai et al., 2003). Thus, it is rather surprising that there is a lack of experimental data to address the contribution of sirtuins in the inhibitory effect of CR on tumorigenesis. Regarding SIRT1, which is the only sirtuin studied so far, its overexpression failed to influence the anticancer effects of every‐other‐day fasting (a variation in CR), suggesting that SIRT1 may play a limited role in the effects of CR on cancer (Herranz et al., 2011). Undoubtedly, future studies are necessary to check more thoroughly the role of sirtuins under this setting. However, it seems a very intriguing question to ask whether sirtuin‐directed functions may regulate either CSCs or non‐CSCs, given that cancer is now viewed as a stem cell disease. This is further supported by recent evidence highlighting the effect of CR on unique characteristics of CSCs such as EMT (Dunlap et al., 2012), protein synthesis (Lamb et al., 2015), metabolic plasticity (Peiris‐Pages et al., 2016), as well as the importance of the HIF pathway in regulating metabolism, cellular responses to hypoxia and stemness (Lim et al., 2010; Zhong et al., 2010; Yun & Lin, 2014), which are all processes previously shown to be regulated by sirtuins. It is hoped that future research will shed light on mechanisms underlying the interplay between CR, sirtuins, and stem cells.

CR 是延长寿命和延缓包括癌症在内的年龄相关疾病发作的最有效的饮食干预措施之一(Wanagat 等人,1999; Longo & Fontana,2010; Colman 等人,2014)。人们认为,它的有利影响可能与表观遗传学重编程干细胞的能力,同时延长干细胞状态的增殖,分化和取代成熟细胞的能力在成熟组织中,至少在某些重要的部分。这是基于研究表明 CR 可以维持造血干细胞的干细胞功能(Ertl et al. ,2008) ,提高幼年和老年动物肌肉中干细胞的可用性和活性(Cerletti et al. ,2012) ,并增加老龄小鼠海马神经干细胞和祖细胞的增殖(Park et al. ,2013b)。考虑到 sirtuins 是 CR 直接激活的 NAD 依赖性蛋白质去乙酰化酶,可能介导了 CR 对成体组织正常干细胞的一些有益作用。然而,这是一个相对未开发的研究领域,并且缺乏实验证据来支持或反驳这一假设。直到最近,才有报道说 SIRT1对于小肠干细胞在 CR 上的扩增是必要的。更具体地说,由于 SIRT1的激活,CR 导致 p70核糖体 S6激酶脱乙酰化,从而促进其通过哺乳动物靶蛋白雷帕霉素复合物1(mTORC1)磷酸化。这种信号反应机制介导了蛋白质合成和 ISCs 数量的增加,即使 mTOR 信号在更多分化的细胞中被 CR 所抑制(Igarashi & Guarente,2016)。类似地,关于 sirtuins 作为 CR 诱导的肿瘤发生作用的介质的作用知之甚少。如前所述,癌症是一种与年龄有关的疾病,在一些早期研究中表现出缓慢发作的 CR 反应(Hursting 等人,1994; Berrigan 等人,2002; Mai 等人,2003)。因此,相当令人惊讶的是,缺乏实验数据来说明去乙酰化酶在 CR 抑制肿瘤发生中的作用。至于 SIRT1,这是迄今为止研究的唯一的 sirtuin,它的过度表达不能影响每隔一天禁食的抗癌效果(CR 的一种变异) ,这表明 SIRT1在 CR 对癌症的影响中可能发挥有限的作用(Herranz 等人,2011)。毫无疑问,未来的研究有必要更彻底地检查去乙酰化酶在这种情况下的作用。然而,这似乎是一个非常有趣的问题,考虑到癌症现在被视为一种干细胞疾病,去乙酰化酶定向的功能是否可以调节 CSCs 或非 CSCs。这是进一步支持最近的证据突出的影响 CSCs 的独特性,如 EMT (Dunlap 等人,2012年) ,蛋白质合成(Lamb 等人,2015年) ,新陈代谢可塑性(Peiris-Pages 等人,2016年) ,以及 HIF 通路在调节新陈代谢,细胞对缺氧和干性反应的重要性(Lim 等人,2010年; 钟等人,2010年; Yun & Lin,2014年) ,这些都是过去被证明由 sirtuins 调节的过程。希望未来的研究能够阐明 CR、去乙酰化酶和干细胞之间相互作用的机制。

Conclusion/Future directions

结语/未来方向

Emerging evidence suggests that sirtuins could be placed at the crossroads of stemness, aging, and cancer. This is based on the plethora of functions they regulate both in normal stem cells and in CSCs. However, it is clear that we are just starting to appreciate the importance of identifying specific processes regulated by the different members of the sirtuin family in a tissue‐, cell type‐, and genetic‐specific context. This might be necessary in order to gain a better understanding of their role and fill current knowledge gaps in the field. With this in mind, it is worth mentioning that most of the previous studies, including the published papers presented in this review article, have followed a targeted approach regarding elucidation of mechanisms regulated by sirtuins. To do so, they were focused on either unraveling how sirtuins regulate signaling pathways/processes already implicated in stemness or exploring whether previously well‐established functions of sirtuins play a significant role in stem cells. Toward this direction, it could be proposed that implementation of unbiased high‐throughput experimental approaches would provide more mechanistic insights. Proteomics have been employed in the past to identify sirtuin‐specific interacting proteins and substrates. The regulatory role of SIRT2 on anaphase‐promoting complex (APC/C) during mitosis was identified based on a proteomics approach that revealed its interaction with proteins of the complex including the APC activator proteins Cdc20 and Cdh1 (Kim et al., 2011). Recently, proteomics were used to elucidate the mitochondrial sirtuin protein interaction landscape showing that this experimental approach can uncover novel functions and/or substrates (Yang et al., 2016). Thus, it could be suggested that similar approaches on stem and progenitor cells or CSCs would identify novel functions of sirtuins. Furthermore, recent advances in high‐resolution mass spectrometry‐based proteomics have enabled the study of the acetylome under different experimental conditions establishing acetylation as an equally widespread PTM as phosphorylation (Kim et al., 2006; Choudhary et al., 20092014). Given that similar approaches have enabled the identification of sirtuin‐specific deacetylation targets (Hebert et al., 2013; Vassilopoulos et al., 2014), it would be reasonable to suggest that studying the acetylome in the context of stem cells/progenitors or CSCs would reveal novel functions/substrates regulated at the post‐translational level. In a similar way, a detailed characterization of target genes epigenetically regulated by sirtuins in specific subcellular populations could help shape new directions in this field and complement previous comprehensive studies focused on the analysis of the transcriptome, DNA methylome, and histone modifications (Sun et al., 2014) in stem cells. Collectively, such studies will provide novel insights into both aging and cancer.

新出现的证据表明去乙酰化酶可能处于干性、衰老和癌症的交叉点。这是基于它们在正常干细胞和 CSCs 中调节的过多功能。然而,很明显,我们刚刚开始意识到在组织、细胞类型和遗传特异性的背景下,鉴定由 sirtuin 家族不同成员调节的特定过程的重要性。这可能是必要的,以便更好地了解他们的作用,并填补该领域目前的知识空白。考虑到这一点,值得一提的是,以前的大多数研究,包括本文中提出的已发表的论文,在阐明 sirtuins 规范的机制方面都采用了有针对性的方法。为了做到这一点,他们要么专注于阐明去乙酰化酶如何调节已经牵涉到干细胞的信号通路/过程,要么专注于探索先前已经确立的功能是否在干细胞中发挥了重要作用。朝着这个方向,可以提出无偏见的高通量实验方法的实施将提供更多的机理性见解。蛋白质组学在过去已被用来鉴定 sirtuin 特异性相互作用蛋白和底物。SIRT2在有丝分裂后期促进复合物(APC/C)中的调控作用是基于蛋白质组学方法,该方法揭示了它与复合物的蛋白质相互作用,包括 APC 激活蛋白 Cdc20和 Cdh1(Kim 等人,2011)。最近,蛋白质组学被用来阐明线粒体 sirtuin 蛋白质相互作用的景观,表明这种实验方法可以揭示新的功能和/或底物(Yang 等人,2016)。因此,这可能表明,类似的方法对干细胞和祖细胞或 CSCs 将确定新的功能去乙酰化酶。此外,基于质谱法的高分辨率蛋白质组学的最新进展使得在不同实验条件下乙酰化的研究成为可能,乙酰化作为一种同样广泛的 PTM 磷酸化作用(Kim et al. ,2006; Choudhary et al. ,2009,2014)。鉴于类似的方法已经能够鉴定 sirtuin 特异性去乙酰化靶点(Hebert 等人,2013; Vassilopoulos 等人,2014) ,有理由认为,在干细胞/祖细胞或 CSCs 的背景下研究乙酰基组将揭示在翻译后水平调节的新功能/底物。同样,在特定的亚细胞群体中,由 sirtuins 调控的靶基因表观遗传学的详细角色塑造可以帮助形成这一领域的新方向,并补充以前的综合研究,重点分析干细胞中的转录组、 DNA 甲基化和组蛋白修饰(Sun 等人,2014)。总的来说,这些研究将为衰老和癌症提供新的见解。

Funding

资金

A. Vassilopoulos was supported by R01CA182506‐01A1 and the Lynn Sage Foundation.

Vassilopoulos 得到了 R01CA182506-01A1和 Lynn Sage 基金会的支持。

Conflict of interest

利益冲突

The authors have no conflict of interests to declare.

作者可以宣布。没有利益冲突

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