AMPK 与癌症的奇怪案例: Jekyll 医生还是 Hyde 先生


The strange case of AMPK and cancer: Dr Jekyll or Mr Hyde?



The AMP-activated protein kinase (AMPK) acts as a cellular energy sensor. Once switched on by increases in cellular AMP : ATP ratios, it acts to restore energy homeostasis by switching on catabolic pathways while switching off cell growth and proliferation. The canonical AMP-dependent mechanism of activation requires the upstream kinase LKB1, which was identified genetically to be a tumour suppressor. AMPK can also be switched on by increases in intracellular Ca2+, by glucose starvation and by DNA damage via non-canonical, AMP-independent pathways. Genetic studies of the role of AMPK in mouse cancer suggest that, before disease arises, AMPK acts as a tumour suppressor that protects against cancer, with this protection being further enhanced by AMPK activators such as the biguanide phenformin. However, once cancer has occurred, AMPK switches to being a tumour promoter instead, enhancing cancer cell survival by protecting against metabolic, oxidative and genotoxic stresses. Studies of genetic changes in human cancer also suggest diverging roles for genes encoding subunit isoforms, with some being frequently amplified, while others are mutated.

AMP活化蛋白激酶能量传感器(AMPK)作为一种细胞能量传感器。一旦通过增加细胞 AMP: ATP 比率而开启,它通过开启分解代谢途径而关闭细胞生长和增殖来恢复能量稳态。典型的 ampk 依赖机制的激活需要上游激酶 LKB1,它被鉴定为肿瘤抑制基因。AMPK 也可以通过细胞内 Ca2 + 的增加、葡萄糖饥饿和 DNA 损伤通过非正规的、与 AMPK 无关的途径被打开。对 AMPK 在小鼠癌症中作用的基因研究表明,在疾病出现之前,AMPK 作为一个肿瘤抑制基因,可以保护小鼠免受癌症的侵袭,AMPK 的激活因子如双胍苯乙双胍进一步加强了这种保护作用。然而,一旦发生癌症,AMPK 就转变成肿瘤促进剂,通过保护癌细胞免受新陈代谢、氧化和基因毒性的压力而提高癌细胞的存活率。对人类癌症基因变化的研究也表明,亚基亚单位的编码基因具有不同的作用,其中一些经常被扩增,而另一些则发生突变。

1. Introduction

1. 引言

The AMP-activated protein kinase (AMPK) is best known as a sensor of both cellular [13] and whole body [4,5] energy status. AMPK is activated when ATP bound at a key site on its γ regulatory subunit is displaced by AMP and/or ADP, causing conformational changes that trigger allosteric activation, as well as promoting net phosphorylation (and consequent activation) of the catalytic subunit by upstream kinases. As ADP rises and ATP falls during situations of cellular energy stress, the reaction catalysed by adenylate kinases (2ADP ↔ ATP + AMP) is displaced rightwards, ensuring that AMP rises to an even larger extent than ADP [6], thus activating AMPK in a very sensitive manner. AMPK is also activated by increases in intracellular Ca2+ [79], by glucose starvation [10] and by DNA damage [1113] via non-canonical, AMP/ADP-independent mechanisms. By phosphorylating downstream targets that switch on catabolic pathways, while switching off anabolic pathways and other ATP-consuming processes such as progress through the cell cycle, AMPK not only promotes ATP synthesis but also restricts cell growth and proliferation in an attempt to restore energy homeostasis and maintain cell viability.

AMPK 作为细胞和整个身体能量状态的传感器而广为人知。它是一种 AMP活化蛋白激酶传感器。当调节亚基的一个关键位点的 ATP 被 AMP 和/或 ADP 取代时,AMPK 被激活,引起构象变化,触发变构激活,同时促进催化亚基的网络磷酸化(和随后的激活)。腺苷酸激酶(adenylate kinases,adenylate kinases,2ADP something ATP + AMP)在细胞能量胁迫下,随着 ADP 的上升和 ATP 的下降,腺苷酸激酶(adenylate kinases,2ADP something ATP + AMP)的反应向右移位,使 AMP 的上升幅度大于 ADP [6] ,从而以一种非常敏感的方式激活 AMPK。AMPK 也被细胞内 Ca2 + [7-9]的增加,葡萄糖饥饿[10]和 DNA 损伤[11-13]通过非正规的、 amp/adp 独立的机制激活。通过磷酸化开启分解代谢途径的下游靶点,同时切断合成代谢途径和其他 ATP 消耗过程,例如通过细胞周期的进展,AMPK 不仅促进 ATP 合成,而且还限制细胞生长和增殖,试图恢复能量稳态和维持细胞活力。

Given this propensity to switch off cell growth and proliferation, and the discovery that the principal upstream kinase phosphorylating and activating AMPK was the well-established tumour suppressor LKB1 [1416], it seemed likely that AMPK would play a beneficial role (Dr Jekyll!) in cancer and act as a tumour suppressor. There is indeed evidence supporting this, at least in some cancer types, as well as for the obvious corollary that AMPK activators should delay tumorigenesis in those cancers. However, there is contrasting evidence that, in other contexts, the presence of AMPK may play a malevolent role (Mr Hyde!) to promote cancer, most likely by protecting transformed cells against stresses caused either when their growth rate outstrips the ability of their blood supply to deliver nutrients and oxygen or during periods of oxidative stress and/or DNA damage. In such scenarios, the presence of AMPK would increase the viability of the tumour cells and thereby potentially decrease survival of the patient, and in such cases it would be AMPK inhibitors rather than activators that might be therapeutically useful. The purpose of this review is to attempt to reconcile these two apparently conflicting roles of AMPK, and to discuss the different types of situation in which activators or inhibitors of the kinase might be efficacious.

鉴于这种关闭细胞生长和增殖的倾向,以及发现主要的上游激酶磷酸化和激活 AMPK 是公认的肿瘤抑制因子 LKB1[14-16] ,似乎 AMPK 将发挥有益的作用(Jekyll 博士!)作为肿瘤抑制剂。确实有证据支持这一观点,至少在某些类型的癌症中是这样,同时还有一个明显的推论,即 AMPK 激活剂应该延缓这些癌症的肿瘤发生。然而,有对比的证据表明,在其他情况下,AMPK 的存在可能扮演了一个恶毒的角色(海德先生!) 这些细胞的生长速度超过了它们的血液供应所能提供的营养和氧气的能力,或者在氧化应激和/或 DNA 受损期间,这些细胞的生长速度超过了血液供应所能提供的能力。在这种情况下,AMPK 的存在将增加肿瘤细胞的生存能力,从而有可能降低患者的生存率,在这种情况下,它将是 AMPK 抑制剂,而不是激活剂,可能是有治疗作用的。本综述的目的是试图调和这两个明显冲突的作用 AMPK,并讨论不同类型的情况下,激活或抑制的激酶可能是有效的。

2. AMPK—structure and regulation

2. ampk ー结构与调控

AMPK appears to exist universally as heterotrimeric complexes comprising catalytic α subunits and regulatory β and γ subunits. Genes encoding these three subunits are found in the genomes of essentially all eukaryotes, suggesting that the AMPK system evolved very early during eukaryotic evolution [2]. In mammals, there are multiple genes encoding each subunit, generating two α (α1, α2), two β (β1, β2) and three γ subunits (γ1, γ2, γ3). These paralogues appear to have arisen during the two rounds of whole genome duplication that are thought to have occurred during the early development of the vertebrates [3]. The seven gene products (not counting splice and/or start-site variants) can form up to 12 αβγ combinations that display subtle differences in regulation and in tissue and subcellular distribution [3].

AMPK 似乎普遍存在于由催化亚基和调节亚基组成的异三聚体复合物中。编码这三个亚单位的基因在基本上所有的真核生物的基因组中都有发现,这表明 AMPK 系统在真核生物进化过程中非常早期就进化了[2]。在哺乳动物中,有多个基因编码每个亚基,产生两个(1,2) ,两个(1,2)和三个亚基(1,2,3)。这些类似物似乎是在脊椎动物早期发展的两轮全基因组复制中出现的[3]。这七种基因产物(不包括剪接和/或起始位点变异)可以形成多达12个组合,它们在调节和组织及亚细胞分布方面显示出细微的差异[3]。

Crystal structures of three αβγ combinations from humans, i.e. α2β1γ1 [17], α1β1γ1 [18] and α1β2γ1 [19], as well as partial structures from mammals [20,21], budding yeast [22] and fission yeast [23,24], are now available. The generalized structure of a heterotrimeric AMPK complex is represented in a highly schematic form in figure 1. A current limitation of the existing structures of heterotrimeric complexes is that, in every case, the constructs were crystallized in active conformations, with the catalytic subunit phosphorylated at the activation site and allosteric activators bound at the regulatory sites. Due to the lack of structures in inactive conformations, we still only have a partial understanding of the conformational changes involved in the activation process.

人类的3种组合,即211[17]、111[18]和121[19]的晶体结构,以及哺乳动物的部分结构[20,21]、芽殖酵母[22]和裂殖酵母[23,24]的晶体结构,现已可以得到。异三聚体 AMPK 复合物的广义结构在图1中以高度原理图的形式表示。目前异三聚体复合物存在结构的一个局限性是,在每一种情况下,这些结构都以活性构象结晶,催化亚基在活性位点磷酸化,变构激活剂在调控位点结合。由于缺乏非活性构象的结构,我们仍然只有一个参与激活过程的构象变化的部分理解。

Figure 1.
Figure 1. Schematic diagram of the structure of AMPK heterotrimers, with the different subunits colour coded (α, yellow; β, lilac; γ blue). Based on a structure of the human α1β2γ1 complex [19], although the structures of α2β1γ1 [17] and α1β1γ1 [18] complexes are very similar.图1。AMPK 异三聚体结构示意图,不同亚基的颜色编码(,黄色; ,淡紫色; 蓝色)。基于人类121配合物[19]的结构,尽管211[17]和111[18]配合物的结构非常相似。

2.1. Structure of the α subunits

2.1. 子单元的结构

Each AMPK-α subunit (coloured yellow in figure 1) has an N-terminal kinase domain with the small N-terminal lobe and larger C-terminal lobe typical of all members of the eukaryotic protein kinase (ePK) family, with the ATP-binding catalytic site in the cleft between the two lobes. Like many other ePKs, AMPK is only significantly active after phosphorylation within the so-called ‘activation loop’ of the C-lobe. In AMPK, the target for phosphorylation is a highly conserved threonine residue, which is conventionally referred to as Thr172 [25] although the exact residue numbering varies with species and isoform (in the view of figure 1, Thr172 is located on the far side of the C-lobe). In other ePKs, phosphorylation within the activation loop changes its conformation to reorient residues involved in both catalysis and protein substrate binding, thus greatly enhancing the reaction rate [26]. The principal upstream kinase phosphorylating Thr172 on AMPK was identified in 2003 to be a complex containing LKB1 and two accessory subunits, STRAD-α or -β and MO25-α or -β [14]. Binding of STRAD-α or -β (which are pseudokinases, structurally related to protein kinases but not active) is required for the kinase activity of LKB1, whereas MO25-α or -β appear to have a structural role to stabilize the complex [27]. The gene encoding LKB1 (called STK11 in humans) had been previously identified as being involved in Peutz–Jeghers syndrome, a rare inherited cancer susceptibility; humans with this syndrome are almost always heterozygous for loss-of-function mutations in STK11 [28]. Their major clinical problem is the development of frequent but benign intestinal polyps, which appear to be caused by haploinsufficiency in STK11. However, they also have a greatly increased risk of developing malignant cancers at multiple locations due to loss of heterozygosity in STK11, and often die at a relatively early age from such malignancies [28]. Loss-of-function mutations in STK11 also frequently occur in many sporadic (i.e. non-inherited) cancers, especially in the commonest form of lung cancer, adenocarcinoma [3,29,30] (see also §6). LKB1 is therefore a classical tumour suppressor, and although its sequence showed that it was a member of the ePK family, the downstream target(s) that it phosphorylated were completely unknown until the finding that it phosphorylated and activated AMPK [1416].

腺苷酸活化蛋白激酶亚基(ampk- 亚基,图1为黄色)具有 n 端激酶结构域,n 端小,c 端大,是真核蛋白激酶(ePK)家族所有成员的典型结构,atp 结合催化位点位于两叶之间的裂隙。与其他许多 epk 一样,AMPK 只有在 c 叶的所谓“激活环”磷酸化后才显著活跃。在 AMPK,磷酸化的目标是一个高度保守的苏氨酸残基,通常被称为 Thr172[25] ,尽管确切的残基数量因物种和异构体而异(在图1中,Thr172位于 c 叶的远侧)。在其他的 ePKs 中,活化环中的磷酸化改变了其构象,使其重新定位参与催化和蛋白质底物结合的残基,从而大大提高了反应速率。2003年,人们发现主要的上游激酶能磷酸化 Thr172位于 AMPK 基因上,它是一个含有 LKB1和两个副亚基 strad- 或-和 mo25- 或-[14]的复合体。Strad- 或-(在结构上与蛋白激酶相关但不活跃的假激酶)的结合是 LKB1激酶活性的必要条件,而 mo25- 或-似乎具有稳定复合物的结构作用[27]。编码 LKB1(在人类中称为 STK11)的基因以前被认为与 Peutz-Jeghers 综合征有关,这是一种罕见的遗传性癌症易感性,在 STK11中,患有这种综合征的人几乎总是在功能缺失突变中呈杂合子状。他们的主要临床问题是常见但良性的肠息肉的发展,这似乎是由 STK11中的单一等位基因不足性引起的。然而,由于 STK11基因杂合性缺失,他们在多个部位发生恶性肿瘤的风险也大大增加,并且常常在相对较早的年龄死于这种恶性肿瘤[28]。STK11的功能缺失突变也经常发生在许多散发性(即非遗传性)癌症中,特别是在最常见的肺癌、腺癌中[3,29,30](另见6)。因此,LKB1是一个典型的肿瘤抑制因子,尽管其序列显示它是 ePK 家族的成员,但是它磷酸化的下游靶点在发现磷酸化和活化 AMPK [14-16]之前是完全未知的。

Following the kinase domain on each AMPK-α subunit (figure 1) is a compact bundle of three α-helices termed the autoinhibitory domain (αAID) [1820,24]. When ATP rather than AMP is bound at the regulatory site(s) on the γ subunit (see below), the α-AID is thought to interact with the kinase domain to clamp it in an inactive conformation [24]. The α-AID is linked to the globular C-terminal domain of the α subunit (α-CTD) by the α-linker, shown schematically as a yellow chain in figure 1. The α-linker is a region in an extended conformation that contains two conserved segments termed α-regulatory interaction motifs (αRIM1 and α-RIM2) [31]. These interact with the surface of the γ subunit containing the key regulatory adenine nucleotide-binding site (see §2.3), and movement of this linker is thought to transmit the effects of AMP or ADP binding from the regulatory γ subunit to the catalytic α subunit (see §2.4).

在每个 ampk- 亚基的激酶结构域之后(图1)是一个三螺旋结构的紧密束,称为自体抑制结构域(- aid)[18-20,24]。当 ATP 而不是 AMP 绑定在亚单位的调节位点(见下文) ,-aid 被认为是与激酶结构域相互作用,以钳制它在一个非活性构象[24]。- aid 通过-linker 连接到子单元的球状 c 末端域(- ctd) ,如图1中的黄色链所示。连接体是扩展构象中的一个区域,包含两个称为调控相互作用基序的保守片段(- rim1和-rim2)[31]。这些相互作用的亚基表面包含调节腺嘌呤核苷酸结合位点(见2.3) ,这个连接器的运动被认为是传递的影响,AMP 或 ADP 结合从调节亚基到催化亚基(见2.4)。

2.2. Structure of the β subunits

2.2. 子单元的结构

The β subunits (coloured lilac in figure 1) contain two conserved regions, the central carbohydrate-binding module (β-CBM) and the C-terminal domain (β-CTD), these being the only regions of the β subunits that are resolved in the current heterotrimer structures. The β-CBM causes a proportion of AMPK in mammalian cells to bind to glycogen particles [32,33]. One function of this may be to co-localize AMPK with glycogen synthase, the key enzyme of glycogen synthesis also found at the surface of the glycogen particle, both isoforms of which are phosphorylated and inactivated by AMPK [34,35]. The β-CBM, however, also has other functions (see §3.2 below). The β-CTD, on the other hand, plays a key structural role as the ‘core’ of the heterotrimeric complex, in that it cross-links the α-CTD and the γ subunit, via interactions that are highly conserved from fungi to mammals [2123].

这些亚基(图1中的有色紫丁香)包含两个保守区域,中央碳水化合物结合模块(- cbm)和 c- 末端结构域(- ctd) ,这是目前异三聚体结构中唯一可以分解的亚基区域。- cbm 使哺乳动物细胞中的一部分 AMPK 与糖原颗粒结合[32,33]。其功能之一可能是使 AMPK 与糖原合成酶共同定位,糖原合成酶是糖原合成的关键酶,也发现在糖原颗粒表面,这两种同型的颗粒都被 AMPK [34,35]磷酸化和灭活。但是-cbm 也有其他功能(见下文3.2)。另一方面,-ctd 作为异三聚体复合体的“核心”在结构上起着关键作用,因为它通过真菌和哺乳动物之间高度保守的相互作用,将-ctd 和亚基交叉连接起来[21-23]。

2.3. Structure of the γ subunits

2.3. 子单元的结构

The γ subunits (coloured blue in figure 1) are of particular interest because they contain the regulatory adenine nucleotide-binding sites. In all species, the γ subunits contain four tandem repeats of a sequence motif of around 60 amino acids known as a CBS repeat, so-named by Bateman [36] because they are also present in the enzyme Cystathione βSynthase and invariably occur as tandem repeats. CBS repeats have been identified in around 75 proteins in the human genome [37], and are also found in archaea and bacteria. Proteins containing them usually have just two tandem repeats, but the AMPK-γ subunits are unusual in having four. A single pair of repeats (known as a Bateman domain or module) forms a pseudodimer with a cleft between the repeats that (due to the approximate twofold symmetry) can provide two ligand-binding sites, although often only one is used. Bateman modules usually bind regulatory ligands containing adenosine (e.g. AMP, ATP, S-adenosyl methionine, NAD, diadenosine polyphosphate) or, less often, guanosine [37,38]. Consistent with this, the CBS repeats in the AMPK-γ subunits provide the critical binding sites for the regulatory nucleotides AMP, ADP and ATP [38]. The four CBS repeats in every AMPK-γ subunit form two Bateman modules that assemble ‘head to head’ to form a flattened disc with the adenine nucleotide-binding sites located close together in the centre, lining a narrow aqueous channel (figure 1) [1719,21]. Given the presence of four repeats, it might have been expected that AMPK-γ subunits would bind four molecules of nucleotide, but all existing crystal structures suggest that they bind only three. These sites are now numbered according to which repeat in the linear sequence (CBS1 through CBS4) provides residues that bind the adenosine moiety of the nucleotide [39] (the phosphate groups may interact with residues from more than one repeat). Using this nomenclature, adenine nucleotides bind at CBS1, CBS3 and CBS4, while the CBS2 site appears to be always unoccupied. The CBS3 site is primarily accessible to solvent from one side of the disc of the γ subunit (facing the viewer in figure 1), and the CBS1 and CBS4 sites from the other.

这些亚基(图1中的蓝色)特别引人注目,因为它们含有调节腺嘌呤核苷酸结合位点。在所有的物种中,这些亚基包含四个串联重复序列,大约有60个氨基酸组成,称为 CBS 重复序列,贝特曼[36]之所以这样命名是因为它们也存在于胱硫酮合酶中,并且总是以串联重复的形式出现。CBS 重复序列已经在人类基因组的大约75个蛋白质中被鉴定出来,并且在古细菌和细菌中也发现了 CBS 重复序列。含有这些基因的蛋白质通常只有两个串联重复序列,但是 ampk- 亚基不同寻常地有四个。一对单重复序列(称为 Bateman 结构域或模块)形成一个假二聚体,在重复序列之间有一个间隙(由于近似的双重对称性) ,可以提供两个配体结合位点,尽管通常只使用一个。Bateman 模块通常结合含有腺苷的调节配体(例如 AMP,ATP,s- 腺苷蛋氨酸,NAD,多聚磷酸二腺苷)或鸟苷(37,38)。与此一致的是,ampk- 亚基中的 CBS 重复序列为调节性核苷酸 AMP、 ADP 和 ATP 提供了关键结合位点。Ampk- 每个亚基的四个 CBS 重复序列组成两个 Bateman 模块,头对头地组装起来形成一个扁平的圆盘,位于中心的腺嘌呤核苷酸结合位点紧密地排列在一条狭窄的水通道内(图1)[17-19,21]。由于存在四个重复序列,可以预期 ampk- 亚单位将结合四个核苷酸分子,但所有现有的晶体结构表明,他们只结合三个。这些位点现在被编号,根据线性序列中的重复序列(CBS1到 CBS4)提供的残基结合核苷酸的腺苷部分[39](磷酸基团可能与一个以上重复序列的残基相互作用)。使用这个命名法,腺嘌呤核苷酸结合在 CBS1,CBS3和 CBS4,而 CBS2位点似乎总是空的。CBS3位点主要是可以从亚单位的圆盘(面向观众在图1)的一侧获得溶剂,而 CBS1和 CBS4位点则可以从另一侧获得。

2.4. Canonical regulation of AMPK by adenine nucleotides

2.4. 腺嘌呤核苷酸对 AMPK 的规范调节

AMP-activated protein kinase received its name [40] because it is allosterically activated by 5′-AMP [41]. When the assays are performed at physiologically relevant ATP concentrations (5 mM) allosteric activation can be as much as 10-fold [42]. However, even before LKB1 was identified as the upstream kinase and Thr172 as the phosphorylation site, it was realized that increases in the AMP : ATP ratio also promoted net phosphorylation of AMPK in intact cells [43]. This is now known to occur because AMP both enhances phosphorylation by LKB1 [44,45] and inhibits dephosphorylation by protein phosphatases [46]. Both effects are due to the binding of AMP to the substrate, AMPK, and not to the upstream kinase or phosphatase; indeed the LKB1 complex appears to have a constant activity in both energy-stressed and unstressed conditions [47]. To summarize, AMP binding has three effects on AMPK: (i) promoting Thr172 phosphorylation; (ii) inhibiting Thr172 dephosphorylation; (iii) triggering allosteric activation of kinase already phosphorylated on Thr172. These three mechanisms act synergistically and make the system respond to small increases in AMP in a very sensitive manner. It was subsequently reported that the effects of AMP binding on Thr172 phosphorylation [48] and dephosphorylation [20], although not on allosteric activation, could be mimicked by ADP, at least in cell-free assays. Our group has confirmed this, but found that the effect required concentrations of ADP up to 10-fold higher than those of AMP, at least for complexes containing γ1 and γ3 (γ2-containing complexes are more sensitive to ADP) [42,45]. Overall, we believe that increases in the AMP : ATP ratio remain the most important activating signal in vivo, although increases in the ADP : ATP ratio might make a secondary contribution.

AMP活化蛋白激酶被命名为[40]是因为它被5′-AMP [41]所激活。当检测是在生理相关的 ATP 浓度(5毫米)变构激活可高达10倍[42]。然而,甚至在 LKB1被确定为上游激酶,Thr172被确定为磷酸化位点之前,人们就意识到,AMP: ATP 比例的增加也促进了完整细胞中 AMPK 的净磷酸化[43]。这是现在已知的发生,因为 AMP 既增强 LKB1[44,45]磷酸化,抑制蛋白磷酸化[46]。这两种效应都是由于 AMP 与底物 AMPK 的结合,而不是与上游的激酶或磷酸酶的结合; 事实上,LKB1复合体似乎在能量应激和非应激条件下都有一个恒定的活性[47]。综上所述,AMP 结合对 AMPK 有三种影响: (i)促进 Thr172磷酸化; (ii)抑制 Thr172脱磷酸化; (iii)触发 Thr172已磷酸化的激酶的变构激活。这三种机制协同作用,使系统以一种非常敏感的方式对 AMP 的微小增加作出反应。随后报道,AMP 结合对 Thr172磷酸化[48]和去磷酸化[20]的影响,虽然不对变构体活化,可以被 ADP 模拟,至少在无细胞实验中是这样。我们的小组已经证实了这一点,但是发现所需要的 ADP 浓度比 AMP 高10倍,至少对于含有1和3(2-含有配合物的配合物对 ADP 更敏感[42,45]。总的来说,我们认为增加 AMP: ATP 比率仍然是最重要的激活信号在体内,虽然增加 ADP: ATP 比率可能作出次要贡献。

How are these three effects of adenine nucleotide binding mediated by the three binding sites on the AMPK-γ subunits? Although it might seem tempting to propose that each effect is due to binding of nucleotides at one of the three sites, that simple model now seems to be untenable. Instead, all three effects appear to be due to binding of nucleotide at a single critical site, CBS3. The evidence supporting this may be briefly summarized as follows.

腺嘌呤核苷酸结合的三个结合位点是如何介导腺嘌呤核苷酸结合 ampk- 亚单位的这三个效应的?尽管提出每种作用都是由于核苷酸在三个位点中的一个位点结合,这个简单的模型现在看来是站不住脚的。相反,所有这三种效应似乎是由于在一个单一的关键位点,CBS3的核苷酸结合。支持这一点的证据可以简要概括如下。

(1)CBS4 normally appears to contain a tightly bound ‘non-exchangeable’ molecule of AMP [21]. Similarly, although CBS1 can bind AMP in cell-free assays, it is estimated to have a 10-fold higher affinity for ATP than AMP. Since ATP is usually present in cells at up to 100-fold higher concentrations than AMP, this suggests that, in intact cells, CBS1 would always be occupied by ATP [49]. This leaves CBS3 as the site where ATP and AMP (or ADP) could exchange with each other.CBS4通常似乎包含一个紧密结合的“非可交换”的 AMP [21]分子。类似地,虽然 CBS1可以在无细胞检测中结合 AMP,但据估计它对 ATP 的亲和力比 AMP 高10倍。由于 ATP 在细胞中的浓度通常比 AMP 高100倍,这表明在完整的细胞中,CBS1总是被 ATP 所占据。这使得 CBS3成为 ATP 和 AMP (或 ADP)相互交换的位点。
(2)The R531G mutation in the AMPK-γ2 subunit, one of up to 14 mutations that cause an inherited heart disease [50], completely blocks both allosteric activation and increased net Thr172 phosphorylation by AMP [38,51]. Although it is actually located in CBS4, the positively charged side chain of Arg531 interacts with the α-phosphate of AMP bound in CBS3 [21,49] (note that in [21] the CBS3 site was referred to as site 1; see [39] for revised nomenclature of binding sites).Ampk-2亚基的 R531G 突变是导致遗传性心脏病的多达14个突变之一,它完全阻断了变构激活和增加了 AMP [38,51]的 Thr172磷酸化。虽然它实际上位于 CBS4,带正电荷的 Arg531侧链与 CBS3[21,49]中结合 AMP 的-磷酸盐相互作用(注意,在[21]中 CBS3位点被称为位点1; 见[39]修订的结合位点命名法)。

If CBS3 is the critical site for all three effects of AMP, what are the functions of nucleotide binding at CBS1 and CBS4? All three sites are located very close together in the centre of the γ subunit, where they are likely to interact with each other. Gu 如果 CBS3是 AMP 三种作用的关键位点,那么 CBS1和 CBS4上核苷酸结合的功能是什么?所有这三个位点都位于亚基的中心非常接近,在那里他们很可能相互作用。古et al. 等等 [49] have provided evidence that binding of ATP at CBS1 alters the conformation of the neighbouring CBS4 site such that the latter binds only AMP in a non-exchangeable manner. They further propose that binding of AMP at CBS4 then enhances the affinity of AMP relative to ATP at CBS3. In particular, binding of AMP at CBS4 repositions the side chain of Arg531 such that it provides an additional positive charge to bind the two negatively charged oxygen atoms on the α-phosphate of AMP in CBS3 (note that the α-phosphates of ADP and ATP, unlike that of AMP, carry only single negative charges) [ )提供的证据表明,ATP 在 CBS1上的结合改变了相邻 CBS4位点的构象,使后者仅以非可交换的方式与 AMP 结合。他们进一步提出,AMP 在 CBS4上的结合进一步增强了在 CBS3上 AMP 相对于 ATP 的亲和力。特别是,AMP 在 CBS4上的结合重新定位了 Arg531的侧链,使其提供了一个额外的正电荷,使两个带负电荷的氧原子结合在 CBS3的 AMP 的-磷酸盐上(注意,ADP 和 ATP 的-磷酸盐不像 AMP 那样只带有单个负电荷)49]. If this is correct, constitutive binding of ATP to CBS1 and AMP to CBS4 effectively ‘tunes’ the affinity of the CBS3 site for the different nucleotides that can bind there. This model explains how AMPK achieves the difficult task of sensing changes in AMP in the presence of much higher concentrations of ATP and ADP. An additional explanation for the preference of the CBS3 site for AMP over ATP is that all three sites on the AMPK-γ subunits appear to preferentially bind free ATP ].如果这是正确的,组成型结合 ATP 的 CBS1和 AMP 的 CBS4有效地’调节’的 CBS3位点的亲和力,不同的核苷酸可以结合在一起。这个模型解释了 AMPK 如何在 ATP 和 ADP 浓度更高的情况下完成感知 AMP 变化的困难任务。另外一个解释是 CBS3位点对 AMP 的选择性高于 ATP,这是因为 ampk- 亚基上的三个位点似乎都优先绑定游离 ATP4− 4 – rather than the Mg.ATP2− 2 – complex [ 复杂[21,23,38,49], although only around 10% of ATP in cells is thought to be present in the Mg ] ,虽然只有大约10% 的 ATP 在细胞中被认为存在于 Mg 中2+ 2 +-free form. – 自由形式

How is the effect of displacement of ATP by AMP at CBS3 transmitted to the catalytic (α) subunit? Consistent with the idea that CBS3 is the critical site for activation, the heterotrimer structures show that, when AMP is bound at CBS3, the α-linker binds to that face of the γ subunit, with α-RIM1 binding across the unoccupied CBS2 site and α-RIM2 physically contacting AMP bound at CBS3 (figure 1). Although there are no crystal structures to confirm this, other biophysical approaches suggest that, when ATP displaces AMP at CBS3, the α-linker dissociates from the surface of the γ subunit containing the CBS3 site [19,52]. This is thought to release the α-AID to rotate back into its inhibitory position behind the kinase domain, with this being prevented when AMP is bound at CBS3 by the interaction of the α-linker with the CBS3 site. Consistent with this model, mutations that would affect the interactions between α-RIM1/α-RIM2 and the γ subunit abolish allosteric activation by AMP [31].

ATP 在 CBS3位置被 AMP 取代的作用是如何传递给催化亚基的?异三聚体结构与 CBS3是活化的关键位点的观点一致,表明当 AMP 结合在 CBS3上时,连接子与该亚基的表面结合,-rim1结合在 CBS2空位,-rim2物理接触结合在 CBS3上(图1)。虽然没有晶体结构来证实这一点,但其他生物物理学方法表明,当 ATP 在 CBS3位置置换 AMP 时,连接剂从含有 CBS3位点的亚基表面游离[19,52]。这被认为是释放 -aid 旋转回到其在激酶结构域后面的抑制位置,当 AMP 通过与 CBS3位点的相互作用而结合在 CBS3时,这被阻止。与该模型一致的是,影响-rim1/-rim2与亚单位相互作用的突变消除了 AMP [31]的变构激活。

While this model nicely accounts for allosteric activation by AMP, the accompanying conformational changes may also alter the exposure of Thr172 for phosphorylation and dephosphorylation, although that aspect is currently less well understood. It also remains unclear why ADP binding has effects on Thr172 phosphorylation despite the fact that, unlike AMP, it does not cause allosteric activation. Finally, as well as the ‘canonical’ activation by changes in adenine nucleotide ratios discussed above, AMPK can also be activated by several non-canonical mechanisms that will now be briefly described.

虽然这个模型很好地解释了 AMP 的变构活化,但是随之而来的构象变化也可能改变 Thr172的磷酸化和去磷酸化作用,尽管这个方面目前还不是很清楚。为什么 ADP 结合对 Thr172磷酸化有影响仍然不清楚,尽管事实上,不像 AMP,它不引起变构激活。最后,除了上面讨论的腺嘌呤核苷酸比例变化引起的“典型”激活外,AMPK 还可以被几种非典型机制激活,这些机制现在将简要描述。

2.5. Non-canonical activation by increases in intracellular Ca2+, by glucose deprivation and by DNA damage

2.5. 细胞内钙离子增加、葡萄糖剥夺和 DNA 损伤引起的非典型激活

As well as LKB1, Thr172 can also be phosphorylated by the Ca2+/calmodulin-dependent protein kinase CaMKK2 [79], which means that AMPK can be activated by increases in intracellular Ca2+ ions even in the absence of any changes in adenine nucleotide ratios. This occurs, for example, in response to hormones and agonists sensed by G protein-coupled receptors that are coupled via Gq/G11 to release inositol-1,4,5-trisphosphate (IP3) from the plasma membrane, which in turn triggers release of Ca2+ from the endoplasmic reticulum. Such agonists include, in endothelial cells, thrombin acting at protease-activated receptors and vascular endothelial cell growth factor acting at VEGF receptors [53,54] as well as, in specific neurons of the hypothalamus, ghrelin acting at GHSR1 receptors [55]. The latter effect is important in promotion of appetite during fasting [5], and the role of CaMKK2 in this pathway can explain previous findings that CaMKK2 inhibitors depress appetite in wild-type mice, although not in CaMKK2 knockouts [56].

与 LKB1一样,Thr172也能被 Ca2 +/钙调素依赖的蛋白激酶 CaMKK2[7-9]磷酸化,这意味着即使在腺嘌呤核苷酸比率没有任何变化的情况下,AMPK 也能被细胞内 Ca2 + 离子的增加所激活。例如,g 蛋白偶联受体感受到的激素和激动剂通过 Gq/G11从质膜释放肌醇-1,4,5-三磷酸(IP3) ,从而触发内质网释放 Ca2 + ,这些激素和激动剂通过 Gq/G11偶联从质膜释放肌醇-1,4,5-三磷酸(IP3)。这些激动剂包括内皮细胞中作用于蛋白酶激活受体的凝血酶和作用于血管内皮细胞生长因子[53,54] ,以及下丘脑特定神经元中作用于 GHSR1受体的 ghrelin。后一种效应在禁食期间促进食欲方面很重要,而 CaMKK2在这一通路中的作用可以解释先前的发现,即 CaMKK2抑制剂可以抑制野生型小鼠的食欲,尽管在 CaMKK2击倒小鼠中并非如此。

It has been known for many years that glucose deprivation of mammalian cells activates AMPK [57], and this treatment is often used to switch on AMPK in cultured cells. In fact, genes encoding the budding yeast orthologue of AMPK (the SNF1 complex) were originally identified via mutations that prevented the normal changes in gene expression in response to glucose deprivation [58]. For many years, it was assumed that glucose deprivation activated AMPK by interfering with catabolic ATP production, and thus activated AMPK via the canonical, AMP-dependent mechanism (§2.4). This does indeed seem to be the case in some established tumour cell lines, perhaps because they are highly glycolytic and have a high dependency on glucose for ATP production. However, in other cells such as immortalized mouse embryo fibroblasts (MEFs) it has been found that glucose deprivation activates AMPK without changing AMP : ATP or ADP : ATP ratios, as long as an alternative carbon source such as glutamine is available; similar AMP/ADP-independent activation is also observed in rat liver during starvation in vivo[10]. In such cases, activation is thought to occur via a complex mechanism involving the direct sensing of the glycolytic intermediate fructose-1,6-bisphosphate (FBP) by FBP aldolase, and the recruitment of AMPK to a ‘super-complex’ on the lysosomal membrane involving the vacuolar-ATPase, the Ragulator complex, Axin, LKB1 and AMPK. Although this mechanism may operate in tumour cells that are dependent on rapid glucose uptake, a full discussion of it is beyond the scope of this article and interested readers are referred to the original papers [10,59,60] or a recent review [2].

众所周知,多年来,哺乳动物细胞的葡萄糖剥夺激活 AMPK [57] ,这种处理往往是用来开启 AMPK 在培养细胞。事实上,编码 AMPK (SNF1复合体)的芽殖酵母正交基因最初是通过突变来识别的,突变阻止了葡萄糖剥夺后基因表达的正常变化。多年来,人们认为葡萄糖剥夺通过干扰分解代谢 ATP 的产生来激活 AMPK,因此通过典型的 AMPK 依赖机制激活 AMPK (2.4)。这似乎确实是一些已经建立的肿瘤细胞系的情况,也许是因为它们是高度糖酵解的,并且高度依赖于葡萄糖来产生 ATP。然而,在其他细胞如永生化小鼠胚胎成纤维细胞(MEFs)中发现,只要有谷氨酰胺等替代碳源,葡萄糖剥夺可激活 AMPK 而不改变 AMP: ATP 或 ADP: ATP 比例,在体内饥饿的大鼠肝脏中也观察到类似的 AMP/ADP 非依赖性激活[10]。在这种情况下,激活被认为是通过一种复杂的机制发生的,包括 FBP 醛缩酶直接感应糖酵解中间体1,6-二磷酸果糖(FBP) ,以及 AMPK 在溶酶体膜上的一个“超复合体”招募,包括液泡 atp 酶、 Ragulator 复合体、 Axin、 LKB1和 AMPK。尽管这种机制可能在依赖于快速葡萄糖摄取的肿瘤细胞中发挥作用,但对它的充分讨论超出了本文的范围,感兴趣的读者可参考原始论文[10,59,60]或最近的一篇综述[2]。

A third type of non-canonical activation of AMPK occurs in response to DNA damage. This was originally reported to occur in response to the topoisomerase II inhibitor etoposide [12], and was later observed following treatment of cells with ionizing radiation [13]. Both treatments cause double-strand breaks in DNA, and are often used in cancer treatment. Double-strand DNA breaks are known to be detected by ATM, a member of the phosphatidylinositol 3-kinase-like kinase (PIKK) family, and the effects of etoposide to activate AMPK were originally claimed to be dependent on ATM, because the effects appeared to be reduced in ATM-deficient cells [12]. In addition, ATM is known to phosphorylate LKB1 at Thr366 [61], and it was reported that AMPK activation by etoposide in cells was reduced by siRNA-mediated knockdown of either ATM or LKB1 [12,62], suggesting the existence of a kinase cascade from ATM to LKB1 to AMPK. However, this cannot be the primary mechanism, because both etoposide [12] and ionizing radiation [13] still activate AMPK in LKB1-null tumour cells. Moreover, our laboratory showed that AMPK activation by etoposide was not blocked by the ATM inhibitor KU-55993, despite the fact that the inhibitor did block phosphorylation of known ATM substrates [11]. We went on to show that AMPK activation by etoposide in LKB1-null cells was mediated by Thr172 phosphorylation catalysed by CaMKK2, and that this was associated with increases in Ca2+ within the nucleus. Interestingly, only AMPK complexes within the nucleus containing the α1 isoform were activated, even though α2 was also expressed in the cells under study. Perhaps most interesting of all, activating AMPK in LKB1-null cells (using the Ca2+ ionophore A23187 to activate CaMKK2) provided significant protection against cell death induced by etoposide. The most likely mechanism to explain this was that A23187 caused a G1 cell cycle arrest, thus restricting entry of cells into S phase where they are particularly susceptible to DNA damage. This hypothesis was supported by the fact that the G1 cyclin-dependent kinase inhibitor palbociclib caused a very similar degree of protection against cell death in those cells where it caused G1 arrest, but not in those where it did not [11]. These results are significant, because they suggest that genotoxic treatments such as etoposide and ionizing radiation might be more effective for cancer treatment if they were combined with inhibitors that prevent AMPK activation, and the consequent protection that AMPK can provide against genotoxic stress. This point is addressed further in §6 below.

AMPK 的第三种非正规激活是对 DNA 损伤的反应。最初报道这是由于拓扑异构酶 II 抑制剂依托泊苷[12]引起的,后来在用电离辐射治疗细胞后观察到。这两种治疗方法都会导致 DNA 双链断裂,而且常用于癌症治疗。已知双链 DNA 断裂可以被 ATM 检测到,它是磷脂酰肌醇3激酶样激酶(PIKK)家族的一员,而依托泊苷激活 AMPK 的效应最初被认为是依赖于 ATM,因为这种效应在 ATM 缺陷细胞中似乎减弱了[12]。另外,ATM 已知在 Thr366[61]磷酸化 LKB1,并且据报道,依托泊苷在细胞中的 AMPK 活化被 sirna 介导的 ATM 或 LKB1[12,62]击倒,提示存在一个从 ATM 到 LKB1到 AMPK 的激酶级联。然而,这不可能是主要机制,因为依托泊苷[12]和电离辐射依然激活 LKB1-null 肿瘤细胞中的 AMPK。此外,我们的实验室表明,尽管 ATM 抑制剂 KU-55993阻断了已知 ATM 底物的磷酸化,但是依托泊苷对 AMPK 的活化并没有被阻断。我们进一步证明,依托泊苷在 LKB1-null 细胞中的活化是由 CaMKK2催化的 Thr172磷酸化介导的,这与细胞核内 Ca2 + 的增加有关。有趣的是,在细胞核内只有包含1亚型的 AMPK 复合物被激活,尽管2也在研究中的细胞中表达。也许最有趣的是,激活 LKB1-null 细胞中的 AMPK (使用 Ca2 + 离子载体 A23187激活 CaMKK2)对足叶乙甙诱导的细胞死亡具有显著的保护作用。最有可能的解释机制是 A23187导致 G1细胞周期阻滞,从而限制细胞进入 s 期,在那里它们特别容易受到 DNA 损伤。这一假说得到了以下事实的支持: g 1周期蛋白依赖性激酶抑制剂 palbociclib 在 g 1阻滞的细胞中对细胞死亡起到了非常类似的保护作用,但是在没有 g 1阻滞的细胞中却没有。这些结果意义重大,因为他们表明,基因毒性治疗,如依托泊苷和电离辐射,如果他们与抑制剂结合,防止 AMPK 激活,以及随之产生的保护 AMPK 可以提供对基因毒性应激更有效的癌症治疗。这一点将在下文6中进一步讨论。

3. Pharmacological activation and inhibition of AMPK

3. AMPK 的药理活性和抑制作用

The realization that AMPK acts as a metabolic master switch, which transforms cellular metabolism from an anabolic to a catabolic state, originally suggested that activators of AMPK might be useful in treating disorders of energy balance such as obesity and type 2 diabetes [63]. Similarly, the discoveries that AMPK inhibited both cell growth and cell proliferation suggested that activators might also be useful in the treatment of cancer [64]. Over the past 20 years, scores of compounds that pharmacologically activate AMPK have been described, a few of which are shown in figure 2. These are discussed in §§3.1–3.3 according to their likely modes of action. There has been much less emphasis on the development of inhibitors, but these are briefly discussed in §3.4.

认识到 AMPK 作为一个代谢主开关,它将细胞代谢从合成代谢状态转变为分解代谢状态,最初表明 AMPK 的激活因子可能在治疗能量平衡失调如肥胖和2型糖尿病上有用。同样,AMPK 抑制细胞生长和细胞增殖的发现表明激活剂也可能在治疗癌症方面有用。在过去的20年中,已经描述了大量的药理活性 AMPK 的化合物,其中一些如图2所示。这些在3.1-3.3中根据它们可能的行动模式进行了讨论。关于抑制剂的研究还没有得到足够的重视,但是在3.4中对这些进行了简要的讨论。

Figure 2.
Figure 2. Structures of a number of AMPK activators. They have been classified according to their mechanisms of activation of AMPK (see §§3.1–3.3). (a) Pro-drugs that are converted inside cells to AMP analogues. (b) Compounds that bind in the allosteric drug and metabolite (ADaM) site. (c) Compounds that activate indirectly by inhibiting mitochondrial ATP synthesis.图2。多种 AMPK 激活剂的结构。根据 AMPK 的激活机制对它们进行了分类(见3.1-3.3)。(a)在细胞内转化为 AMP 类似物的前药物。(b)在变构药物和代谢产物(亚当)位点结合的化合物。(c)通过抑制线粒体 ATP 合成而间接激活的化合物。

3.1. Pro-drugs that are converted inside cells to AMP analogues

3.1. 在细胞内转化为 AMP 类似物的前药

The first compound shown to activate AMPK in intact cells was the adenosine analogue 5-aminoimidazole-4-carboxamide riboside (AICAR), which is taken up into cells via adenosine transporters [65] and converted by adenosine kinase into the equivalent monophosphorylated nucleotide, ZMP [6668] (figure 2a). ZMP mimics all three of the effects of AMP described in §2.4 [66], and AICAR has been much used as an experimental tool to activate AMPK in intact cells and in vivo. It should be noted, however, that ZMP is much less potent as an AMPK activator than AMP, and AICAR only activates AMPK in intact cells because intracellular ZMP accumulates to millimolar concentrations, even higher than the external concentrations of AICAR [66]. The use of AICAR is no longer recommended by the present authors, because ZMP has known off-target effects (e.g. it also mimics the effects of AMP to activate skeletal muscle glycogen phosphorylase [69] and inhibit hepatic fructose-1,6-bisphosphatase [67,70]), and because much more specific activators are now available. One such is C13, a derivative of another adenosine analogue termed C2 that has been esterified on two oxygen atoms of its phosphonate group to make it more cell permeable [71]. C13 is indeed readily taken up by cells, but is then converted into C2 by cellular esterases (figure 2a). Remarkably, C2 is an even more potent activator of AMPK than AMP itself, although it should be noted that it is specific for AMPK complexes containing the α1 isoform, and is inactive on α2 complexes [72]. The high affinity of C2 may arise because it binds, unexpectedly, to the AMPK-γ subunits in a somewhat different orientation than AMP [73].

第一个在完整细胞中激活 AMPK 的化合物是腺苷类似物5- 氨基咪唑 -4- 羧酰胺核苷(AICAR) ,它通过腺苷转运体[65]被吸收到细胞中,并被腺苷激酶转化为相应的单磷酸化核苷酸 ZMP [66-68](图2a)。ZMP 模拟了2.4[66]中描述的 AMP 的所有三种作用,AICAR 已被广泛用作激活完整细胞和活体内 AMPK 的实验工具。值得注意的是,ZMP 作为 AMPK 激活剂的作用比 AMP 弱得多,AICAR 只能激活完整细胞中的 AMPK,因为细胞内的 ZMP 积累到毫摩尔浓度,甚至高于 AICAR 的外部浓度[66]。本文作者不再推荐使用 AICAR,因为 ZMP 具有已知的非靶向效应(例如,它还模仿 AMP 激活骨骼肌糖原磷酸化酶[69]和抑制肝脏果糖 -1,6-二磷酸酶[67,70]的效应) ,并且因为现在有更多的特异性激活剂可用。其中一种是 C13,它是另一种类似物 C2的衍生物,这种腺苷被酯化在其磷酸盐基团的两个氧原子上,使其更具细胞渗透性[71]。C13确实很容易被细胞吸收,但随后又被细胞酯酶转化为 C2(图2a)。值得注意的是,C2是比 AMP 本身更有效的 AMPK 激活剂,虽然它是 AMPK 复合物的特异性激活剂,包含1个亚型,并且在2个复合物[72]上是无活性的。C2的高亲和力可能是因为它以与 AMP [73]略有不同的方向与 ampk- 亚基结合。

3.2. Compounds that bind in the allosteric drug and metabolite (ADaM) site

3.2. 在变构药物和代谢产物(ADaM)位点结合的化合物

In the structures of AMPK heterotrimers containing either β1 [17,18] or β2 [19], the β-CBM interacts with the N-lobe of the kinase domain of the α subunit via the surface opposite to its glycogen-binding site (figure 1). The cleft between these domains forms the binding site for novel ligands acting on AMPK, which in most cases came out of high-throughput screens that searched libraries of synthetic chemicals for allosteric activators of AMPK. The first to be discovered was the thienopyridone A-769662 [74] but at least 10 have now been reported, including PF-739 [75] and MK-8722 [76] (figure 2b). All activate β1 complexes with higher potency than β2 complexes, and this makes some of them (including A-769662 [77]) highly selective for the former. As well as causing allosteric activation, binding of these compounds also inhibits Thr172 dephosphorylation in cell-free assays [78,79], although in intact cells the predominant effect appears to be allosteric, since large changes in phosphorylation of the AMPK target acetyl-CoA carboxylase in response to these agonists are usually only accompanied by modest changes in Thr172 phosphorylation [78].

在含有1[17,18]或2[19]的 AMPK 异三聚体结构中,-cbm 通过与其糖原结合位点相对的表面与亚单位激酶结构域的 n- 叶相互作用(图1)。这些结构域之间的间隙形成了作用于 AMPK 的新配体的结合位点,在大多数情况下,这些结合位点是通过高通量筛选来寻找 AMPK 变构活化剂的合成化学物质库。第一个被发现的是噻吩吡啶酮 A-769662[74] ,但至少有10个已报告,包括 PF-739[75]和 MK-8722[76](图2b)。所有配合物均能活化1个高于2个配合物的配合物,这使得其中一些配合物(包括 A-769662[77])对前者具有高度选择性。除了引起变构激活,这些化合物的结合也抑制 Thr172脱磷酸化在无细胞实验中[78,79] ,虽然在完整的细胞中,主要的作用似乎是变构,因为针对这些激动剂的 AMPK 目标乙酰辅酶A羧化酶磷酸化的大量改变通常只伴随着 Thr172磷酸化的轻微改变。

One of the curious features of the ligands currently known to bind at this site is that almost all of them are synthetic chemicals rather than natural products. However, many in the field believe that these compounds may be mimicking the effect of some natural metabolite that binds to this site, which is why it has been termed the ‘allosteric drug and metabolite’ (ADaM) site [80]. The only natural product currently known to bind to this site is salicylate, a compound made by plants that acts as a hormone signalling infection by pathogens [81]. In the form of extracts of willow bark, salicylates have been used by humans as medicines since ancient times, and they are still in very wide use as the synthetic derivative acetyl salicylic acid (ASA or aspirin), which is rapidly broken down to salicylate once it enters the circulation. Although aspirin itself is a potent irreversible inhibitor of the cyclo-oxygenases involved in biosynthesis of prostanoids such as thromboxanes [82], salicylate activates AMPK by direct binding at the ADaM site, which occurs at concentrations reached in plasma of patients taking high doses of aspirin and other salicylate-based drugs [81]. Interestingly, regular use of aspirin, usually taken to reduce the formation of blood clots via inhibition of thromboxane synthesis, is associated with a reduced incidence of cancer [83]. Whether this can be explained entirely by inhibition of cyclo-oxygenases, or whether it involves some other target such as AMPK, currently remains unclear.

目前已知在这个位点结合的配体的一个奇怪特征是,它们几乎都是合成化学物质,而不是天然产物。然而,该领域的许多人认为,这些化合物可能是模仿与这个位点结合的某些天然代谢物的作用,这就是为什么它被称为“变构药物和代谢物”(ADaM)位点[80]。目前已知唯一与这个部位结合的天然产物是水杨酸盐,这是一种由植物制成的化合物,作为被病原体感染的激素信号。自古以来,柳树皮提取物中的水杨酸盐就被人类用作药物,它们作为人工合成的衍生物乙酰水杨酸(ASA 或阿司匹林)仍在广泛使用,一旦进入流通环节,水杨酸盐就迅速分解为水杨酸盐。虽然阿司匹林本身是参与血栓素[82]等前列腺素生物合成的环氧合酶的强有力的不可逆抑制剂,但水杨酸通过在 ADaM 位点直接结合激活 AMPK,这种结合发生在高剂量阿司匹林和其他水杨酸类药物患者血浆中达到的浓度时[81]。有趣的是,经常服用阿司匹林,通常是通过抑制血栓素合成来减少血栓的形成,可以降低癌症的发病率[83]。这是否可以完全用抑制环氧化酶来解释,或者它是否涉及其他一些目标,如 AMPK,目前还不清楚。

3.3. Compounds, including biguanides, that activate AMPK indirectly by inhibiting mitochondrial ATP synthesis

3.3. 化合物,包括双胍,通过抑制线粒体 ATP 合成间接激活 AMPK

Metformin and phenformin (figure 2c) are synthetic biguanides derived from galegine(isoprenyl guanidine) [84], a natural product from the plant goat’s rue or Galega officinalis, which was well known as a herbal remedy in seventeenth century England [85]. Both biguanides were introduced for treatment of type 2 diabetes in the 1950s, although phenformin was withdrawn in most countries in the 1970s because its use was associated with the rare but life-threatening side effect of lactic acidosis. The risk of lactic acidosis is much lower with metformin, which has subsequently become the drug of first choice in the treatment of type 2 diabetes worldwide. Although biguanides have been used since the 1950s, the first clues to their mechanism of action did not emerge until 2000, when they were reported to inhibit complex I of the mitochondrial respiratory chain, thus explaining the risk of lactic acid accumulation [86,87]; they have subsequently also been shown to inhibit the mitochondrial ATP synthase [88]. Clearly, inhibition of mitochondrial ATP synthesis would be expected to increase cellular ADP : ATP and AMP : ATP ratios and thus activate AMPK by the canonical mechanism. Indeed, activation of AMPK by biguanides in intact cells and in vivo was reported in 2001 [89], and it was subsequently confirmed that this was caused by increases in AMP and/or ADP [51], although metformin may also activate AMPK via the non-canonical lysosomal pathway [90]. Metformin has two major clinical effects: (i) inhibiting glucose production by the liver and (ii) enhancing insulin sensitivity of tissues such as liver and skeletal muscle. Surprisingly, studies with liver-specific double AMPK (α1−/− α2−/−) knockout mice showed that the rapid effects of metformin on liver glucose production were AMPK independent, despite the fact that they were accompanied by increases in cellular AMP : ATP ratios [91]. These acute effects of metformin now appear to be due to direct allosteric inhibition of the gluconeogenic enzyme fructose-1,6-bisphosphatase by AMP [70]. Despite this, studies of mice with double knock-in mutations of the single serine residues that are targeted by AMPK in ACC1 (S79A) and ACC2 (S212A) suggested that the longer term insulin-sensitizing effects of metformin are indeed mediated by AMPK [92]. These mice, in which AMPK no longer acutely inhibits fatty acid synthesis or activates fatty acid oxidation, accumulate excess di- and tri-glycerides in liver and muscle, which is accompanied by insulin resistance. Although wild-type mice developed a similar degree of insulin resistance when placed on a high-fat diet, insulin sensitivity in the knock-in mice did not deteriorate further, possibly because they were already synthesizing so much fat. However, when the high-fat-fed mice were treated with metformin for six weeks, this reversed the insulin resistance of the wild-type mice but had no effect in the knock-in mice. Thus, the longer term effects of metformin on insulin sensitivity, although not its short-term effects on hepatic glucose production, are due to modulation of lipid metabolism by AMPK, most likely by reducing the excessive storage of lipids in tissues such as liver and skeletal muscle [92].

二甲双胍和苯乙双胍(图2 c)是人工合成的双胍类化合物,它们来自于 galegine (异戊二烯基胍)[84] ,这是一种天然产物,来自于植物山羊的芸香或山羊豆,在17世纪的英格兰作为一种草药药物而闻名于世。这两种双胍类药物都是在20世纪50年代用于治疗2型糖尿病的,尽管苯乙双胍在20世纪70年代在大多数国家被停用,因为它的使用与乳酸性酸中毒的罕见但危及生命的副作用有关。二甲双胍的乳酸性酸中毒风险要低得多,后来二甲双胍成为世界范围内治疗2型糖尿病的首选药物。虽然双胍类化合物自20世纪50年代以来就已被使用,但直到2000年才首次发现其作用机制的线索,当时有报告称双胍类化合物抑制线粒体呼吸链的复合物 i,从而解释了乳酸积累的风险[86,87] ; 随后也证明它们抑制线粒体 ATP 合酶[88]。显然,抑制线粒体 ATP 合成可望增加细胞 ADP: ATP 和 AMP: ATP 比例,从而激活 AMPK 的规范机制。事实上,双胍类药物在完整细胞和体内激活 AMPK 的报道在2001年[89] ,随后证实这是由 AMP 和/或 ADP [51]的增加引起的,虽然二甲双胍也可能通过非正规溶酶体途径激活 AMPK [90]。二甲双胍有两个主要的临床作用: (i)抑制肝脏葡萄糖生成和(ii)提高肝脏和骨骼肌等组织的胰岛素敏感性。令人惊讶的是,对肝特异性双 AMPK (1-/-2-/-)基因敲除小鼠的研究表明,二甲双胍对肝葡萄糖生成的快速作用是独立的,尽管它们伴随着细胞内 AMP: ATP 比值的增加[91]。二甲双胍的这些急性效应现在看来是由于 AMP [70]对葡萄糖异生酶果糖 -1,6-二磷酸酶的直接变构抑制所致。尽管如此,对 ACC1(S79A)和 ACC2(S212A)中 AMPK 靶向的单丝氨酸残基双重敲入突变小鼠的研究表明,二甲双胍的长期胰岛素增敏作用确实是由 AMPK [92]介导的。这些小鼠,其中 AMPK 不再急性抑制脂肪酸合成或激活脂肪酸氧化,积累过多的二和三甘油酯肝脏和肌肉,并伴有胰岛素抵抗。虽然野生型小鼠在高脂肪饮食中产生了类似程度的胰岛素抵抗,但是敲入型小鼠的胰岛素敏感性并没有进一步恶化,可能是因为它们已经合成了如此多的脂肪。然而,当高脂肪喂养的小鼠用二甲双胍治疗六周后,这逆转了野生型小鼠的胰岛素抵抗,但对敲入小鼠没有影响。因此,二甲双胍对胰岛素敏感性的长期影响,尽管不是对肝脏葡萄糖产生的短期影响,是由于 AMPK 对脂质代谢的调节,最有可能是通过减少脂质在肝脏和骨骼肌等组织中的过度储存。

Following the initial findings that AMPK was activated by biguanides [89], and that the tumour suppressor LKB1 acted upstream of AMPK [14], the question of whether biguanide use had any influence on cancer was addressed. Retrospective studies suggested that the use of metformin in patients with type 2 diabetes in the Tayside region of Scotland was associated with a significant (around 30%) reduction in the incidence of cancer [93]. This association has since been confirmed in studies of many other diabetic cohorts [9496], although its validity has been challenged due to the possibility of time-related biases [97] and it remains just a correlation, with no proof of direct causation. In addition, even if the association is valid, it does not necessarily imply that metformin acts directly on AMPK within the tumours themselves, rather than indirectly via AMPK-dependent or -independent effects on other tissues or organs. For example, since metformin is currently only used to treat type 2 diabetes, we do not know whether its use would be associated with reduced cancer incidence in subjects without diabetes (although there have been small trials in patients with breast or endometrial cancer, these were only ‘window-of-opportunity’ trials to assess various markers in the short period prior to surgery [98101]). Note also that the different cancer incidence in patients with type 2 diabetes taking metformin is observed when comparing with those on other medications [9396]. Metformin enhances insulin sensitivity and thus reduces insulin release, but many of the other commonly used medications, such as sulfonylureas and glucagon-like peptide-1 agonists, work in part by enhancing insulin secretion, while some subjects are even treated directly with insulin. Insulin is, of course, a growth factor that promotes proliferation of cells by activating the Akt pathway. One explanation of the apparent protective effect of metformin against cancer in patients with diabetes is therefore that, unlike most other treatments, it reduces rather than increases the levels of insulin, with high insulin levels being responsible for increased cancer incidence in patients on other medications [102]. Indeed, a related phenomenon is seen in patients with cancer who are treated with phosphatidyl-inositol 3-kinase (PI3 K) inhibitors, who often secrete extra insulin to compensate for the insulin resistance induced by the drugs, thus reducing their anti-cancer efficacy. Experiments with mouse models suggest that this effect can be overcome by additional dietary or pharmacological treatments that reverse the insulin resistance induced by these drugs [103].

随着最初的发现,双胍激活 AMPK [89] ,肿瘤抑制因子 LKB1作用于 AMPK [14]的上游,双胍的使用是否对癌症有任何影响的问题得到了解决。回顾性研究表明,在苏格兰泰赛德地区2型糖尿病患者中使用二甲双胍与显著(约30%)降低癌症发病率有关[93]。这种联系已经在许多其他糖尿病患者的研究中得到证实[94-96] ,尽管其有效性受到质疑,因为可能存在与时间有关的偏差[97] ,而且它仍然只是一种相关性,没有直接因果关系的证据。此外,即使这种联系是有效的,也不一定意味着二甲双胍直接作用于肿瘤内部的 AMPK,而不是间接作用于其他组织或器官的 AMPK 依赖性或独立性效应。例如,由于二甲双胍目前只用于治疗2型糖尿病,我们不知道它的使用是否会降低非糖尿病患者的癌症发病率(尽管已经有一些乳腺癌或子宫内膜癌的小型试验,这些只是评估手术前短期内各种标志物的‘机会窗口’试验[98-101])。还要注意,服用二甲双胍的2型糖尿病患者的癌症发病率与服用其他药物的患者相比有所不同[93-96]。二甲双胍增强胰岛素敏感性,从而减少胰岛素释放,但许多其他常用药物,如磺脲类和胰高血糖素样肽-1激动剂,部分通过促进胰岛素分泌起作用,而一些受试者甚至直接接受胰岛素治疗。当然,胰岛素是一种生长因子,通过激活 Akt 途径促进细胞增殖。因此,二甲双胍对糖尿病患者癌症的明显保护作用的一种解释是,与大多数其他治疗方法不同,它降低而不是增加胰岛素水平,高胰岛素水平是增加其他药物治疗患者癌症发病率的原因[102]。事实上,在使用磷脂酰肌醇3- 激酶(pi3k)抑制剂治疗的癌症患者中也出现了一种相关现象,这些患者经常分泌额外的胰岛素以补偿药物引起的胰岛素抵抗,从而降低其抗癌效果。用小鼠模型进行的实验表明,这种效应可以通过额外的饮食或药物治疗来克服,以逆转这些药物所诱导的胰岛素抵抗[103]。

There are many other compounds that activate AMPK by inhibiting mitochondrial ATP synthesis, one example being resveratrol [51], which inhibits the mitochondrial ATP synthase [104]. Another is sorafenib, originally developed as an inhibitor of receptor-linked tyrosine kinases such as the VEGF and platelet-derived growth factor (PDGF) receptors and used to treat some liver, kidney and thyroid cancers [105]. However, it also activates AMPK at therapeutically relevant concentrations by inhibiting the respiratory chain [106]. Remarkably, more than 100 natural products derived from traditional Asian medicines have within the last few years also been shown to activate AMPK in intact cells [107], and the effects of at least two of them, i.e. berberine [51] and arctigenin [108], appear to be due to inhibition of complex I of the mitochondrial respiratory chain. We suspect that many of the others may also work through inhibition of either complex I or the ATP synthase, which are both large, membrane-bound complexes containing no less than 44 and 14 protein subunits, respectively. It is perhaps not surprising that many hydrophobic compounds might find inhibitory binding sites within these complexes. This class of AMPK activator is particularly diverse in structure (e.g. those in figure 2c), indicating that they may interact with distinct sites. Many of the natural products that activate AMPK may be produced by plants to provide a chemical defence to deter grazing by insects or other animals, or infection by pathogens, and poisoning of complex I or the ATP synthase would seem to represent good ways to achieve those aims. Interestingly, many of these toxic plant products are stored within the plants that synthesize them either in the vacuole or in the cell wall [109], where they would not come into contact with the plant’s own mitochondria.

还有许多其他化合物通过抑制线粒体 ATP 合成激活 AMPK,例如白藜芦醇[51] ,它抑制线粒体 ATP 合酶[104]。另一种是索拉非尼,最初是作为一种受体连接的酪氨酸激酶抑制剂开发的,如 VEGF 和血小板衍生生长因子受体(PDGF) ,用于治疗一些肝癌、肾癌和甲状腺癌。然而,它也通过抑制呼吸链在治疗相关浓度激活 AMPK [106]。值得注意的是,在过去几年中,有100多种来自传统亚洲药物的天然产品也被证明能够激活完整细胞中的 AMPK,其中至少有两种产品,即小檗碱[51]和牛蒡苷元[108] ,似乎是由于线粒体呼吸链复合物 i 的抑制作用。我们怀疑其他许多物质也可能通过抑制复合物 i 或 ATP 合成酶而发挥作用,这两种物质都是大型的膜结合复合物,分别包含不少于44个和14个蛋白质亚单位。许多疏水化合物可能在这些复合物中发现抑制性结合位点,这也许并不令人惊讶。这类 AMPK 激活剂在结构上特别多样化(如图2c 所示) ,表明它们可能与不同的位点相互作用。许多激活 AMPK 的天然产物可能是由植物产生的,以提供一种化学防御来阻止昆虫或其他动物的吃草,或者阻止病原体的感染,而对复杂的 i 或 ATP 合成酶的中毒似乎是实现这些目标的好方法。有趣的是,许多这些有毒植物产品储存在合成它们的植物体内,或者储存在液泡中,或者储存在细胞壁中,在那里它们不会与植物自身的线粒体接触。

3.4. AMPK inhibitors

3.4. AMPK 抑制剂

At present, no specific AMPK inhibitors are available. The only AMPK inhibitor that has been widely used in the literature is compound C (also known as dorsomorphin). Although developed as an AMPK inhibitor, the claim that it was specific for AMPK came from the original report that it did not inhibit a panel of just five other protein kinases [89]. However, in a screen of 70 protein kinases, nine were inhibited to a greater extent than AMPK [110], while in a more recent screen of 120 kinases documented in the MRC Kinase Inhibitor Database ( no less than 30 were inhibited to a greater extent than AMPK. The use of compound C cannot therefore be recommended, even as an experimental tool. Other AMPK inhibitors have been reported [111,112], but have not yet been widely used.

目前,还没有特异性的 AMPK 抑制剂。唯一的 AMPK 抑制剂,已被广泛使用的文献是化合物 c (也称为背寄生)。虽然发展成为一种 AMPK 抑制剂,声称它是专为 AMPK 来自最初的报告,它没有抑制一个小组只有五个其他蛋白质激酶[89]。然而,在70个蛋白激酶的筛选中,9个蛋白激酶的抑制程度大于 AMPK [110] ,而在最近的 MRC 激酶抑制剂数据库(MRC Kinase Inhibitor Database)中记录的120个激酶的筛选中,不少于30个激酶的抑制程度大于 AMPK。因此,不能推荐使用化合物 c,即使是作为一种实验工具。其他 AMPK 抑制剂已被报道[111,112] ,但尚未得到广泛应用。

4. Downstream targets of AMPK

4. AMPK 的下游目标

Once activated, AMPK phosphorylates numerous downstream proteins, with at least 60 being identified as well-established targets in a recent review [113]. The core recognition motif for AMPK is well defined: it requires a basic residue (R, K or H) either three or four residues N-terminal to the phosphorylated serine/threonine (referred to as the P-3 and P-4 positions) as well as hydrophobic residues (L, M, I, F or V) at P-5 and P+4 [113115]. The ACC1 isoform of acetyl-CoA carboxylase, which is a particularly good substrate for AMPK, has additional specificity determinants N-terminal to this core motif, which are not present in all downstream targets. These are another basic residue at P-6, and an amphipathic α-helix running from P-5 to P-16 that binds in a hydrophobic groove on the surface of the C-lobe of the AMPK kinase domain [116]. We discuss some of these targets below, focusing on those that may be particularly relevant to the role of AMPK in cancer.

一旦激活,AMPK 磷酸化许多下游蛋白质,至少有60个被确定为最近的审查[113]。AMPK 的核心识别模体定义明确: 它需要磷酸化丝氨酸/苏氨酸(P-3和 P-4位点)的3个或4个 n 末端基本残基(r,k 或 h) ,以及 P-5和 p + 4[113-115]处的疏水残基(l,m,i,f 或 v)。乙酰辅酶A羧化酶的 ACC1亚型是 AMPK 的一个特别好的底物,对这个核心序列有额外的特异性 n 末端决定因子,这在所有的下游目标中都不存在。这些是 P-6的另一个基本残基,以及一个从 P-5到 P-16的两亲性螺旋结合在 AMPK 激酶结构域 c 叶表面的疏水沟中[116]。我们在下面讨论其中的一些目标,重点讨论那些可能与 AMPK 在癌症中的作用特别相关的目标。

4.1. Proteins and genes involved in catabolic pathways

4.1. 参与分解代谢途径的蛋白质和基因

Catabolic processes switched on by AMPK are summarized in figure 3. In many cell types, depending on the expression of specific glucose transporters (GLUTs), AMPK activation enhances glucose uptake. In skeletal muscle, AMPK acutely promotes translocation of vesicles containing GLUT4 from intracellular vesicles to the plasma membrane, in part by a mechanism involving phosphorylation of the Rab-GAP protein TBC1D1 [117]. In the longer term, AMPK also increases expression of GLUT4 protein via a mechanism that may involve direct phosphorylation of class IIa histone deacetylases (e.g. HDAC5) [118], which appears to cause their exclusion from the nucleus [119] and therefore promotes net acetylation and transcriptional activation at the GLUT4 promoter. AMPK activation also acutely activates glucose transport by the more widely expressed glucose transporter GLUT1 [120], in part via phosphorylation and consequent degradation of TXNIP, an α-arrestin family member that appears to promote internalization of GLUT1 as well as reduced levels of its mRNA [121]. In some but not all cells, AMPK acutely stimulates glycolytic flux via a mechanism involving direct phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, the enzyme that makes and breaks down fructose-2,6-bisphosphate via distinct domains of a bienzyme polypeptide [122]. Phosphorylation by AMPK increases the kinase activity, thus increasing the cellular concentration of fructose-2,6-bisphosphate, a potent allosteric activator of the glycolytic enzyme 6-phosphofructo-1-kinase [122]. However, this mechanism is limited to specific cell types, because only the PFKFB2 [123] and PFKFB3 [124] isoforms are targets for AMPK. PFKFB2 is expressed in cardiac myocytes and some other tissues, while alternative splicing or differential promoter usage yields two main isoforms of PFKFB3 that differ by a short C-terminal sequence; these are the so-called ubiquitous (or constitutive) isoform and the inducible isoform [122]. The expression of the inducible form is very low in most adult tissues, but is increased by pro-inflammatory stimuli in monocytes and macrophages [124] and it is constitutively expressed in many tumour cells [125].

分解代谢过程开启 AMPK 总结在图3。在许多细胞类型中,根据特异性葡萄糖转运蛋白(glut)的表达,AMPK 活化增强葡萄糖摄取。在骨骼肌中,AMPK 通过磷酸化 Rab-GAP 蛋白 TBC1D1[117]促进含有 GLUT4的囊泡从细胞内囊泡向细胞膜的转运。从长远来看,AMPK 还通过一种可能涉及 IIa 类组蛋白去乙酰化酶(如 HDAC5)[118]直接磷酸化的机制增加 GLUT4蛋白的表达,这似乎导致它们被细胞核排除在外[119] ,因此促进 GLUT4启动子的净乙酰化和转录激活。AMPK 激活也可以通过更广泛表达的葡萄糖转运蛋白 GLUT1[120]激活葡萄糖转运,部分通过磷酸化和 TXNIP 的随之降解,TXNIP 是一种甲状腺素家族成员,似乎促进 GLUT1的内在化,以及降低其 mRNA [121]的水平。AMPK 通过直接磷酸化6- 磷酸果糖 -2- 激酶/果糖 -2,6- 二磷酸酶的机制刺激糖酵解通量,6- 磷酸果糖 -2- 激酶/果糖 -2,6- 二磷酸酶是通过双酶多肽[122]的不同结构域产生和分解果糖 -2,6- 二磷酸的酶。AMPK 的磷酸化增加了激酶的活性,从而增加了细胞中果糖 -2,6- 二磷酸,糖酵解酶6- 磷酸果糖 -1- 激酶的一种强力的变构激活剂的浓度。然而,这种机制仅限于特定的细胞类型,因为只有 PFKFB2[123]和 pffb3[124]亚型是 AMPK 的靶标。PFKFB2在心肌细胞和其他一些组织中表达,而启动子选择性剪接或差异启动子的使用产生两种主要的 PFKFB3亚型,它们之间的差别是一个短的 c 末端序列,这些是所谓的普遍存在(或组成型)亚型和诱导型亚型[122]。诱导型的表达在大多数成年组织中非常低,但是在单核细胞和巨噬细胞中的促炎性刺激增加,并且在许多肿瘤细胞中组成性表达。

Figure 3.
Figure 3. A ‘wheel’ of downstream targets and the pathways they regulate, focusing on catabolic processes that are activated by AMPK.图3。一个下游目标和他们调节的路径的“车轮” ,重点是分解代谢过程是由 AMPK 激活。

Although AMPK can therefore acutely activate ATP production by glycolysis in some cell types, in the longer term it tends to promote mitochondrial oxidative metabolism instead, which is much more efficient in terms of ATP production per glucose consumed (≈36 ATP per glucose by oxidative metabolism, as opposed to only two by glycolysis). Oxidative metabolism is, however, less compatible with providing precursors for cell growth, so it tends to be used to a greater extent in quiescent rather than proliferating cells [126]. In the short term, AMPK activates the uptake of fatty acids into mitochondria via phosphorylation of the acetyl-CoA carboxylase isoform ACC2 [127]. While ACC1, the first AMPK target to be identified, is thought to produce the cytoplasmic malonyl-CoA used in fatty acid synthesis, ACC2 localizes to mitochondria [128] and is thought to produce the mitochondrial malonyl-CoA that inhibits uptake of fatty acids into mitochondria via the carnitine:palmitoyl transferase system. Phosphorylation of ACC2 lowers malonyl-CoA and therefore relieves inhibition of carnitine:palmitoyl-CoA transferase-1 (CPT1), thus causing acute promotion of mitochondrial fatty acid oxidation [127].

虽然 AMPK 可以通过糖酵解激活某些细胞类型的 ATP 产生,但从长远来看,它倾向于促进线粒体唿吸作用的产生,从每消耗一个葡萄糖的角度来看,线粒体唿吸作用产生 ATP 的效率要高得多(与糖酵解只产生2个葡萄糖相比,糖酵解产生36个 ATP)。然而,唿吸作用不能提供细胞生长的前体,所以它往往更多地用于静止细胞而不是增殖细胞[126]。短期内,AMPK 通过乙酰辅酶A羧化酶异构体 ACC2的磷酸化活化线粒体对脂肪酸的摄取。虽然 ACC1,第一个被确定的 AMPK 靶点,被认为是产生细胞质丙二酰辅酶 a 用于脂肪酸合成,ACC2定位于线粒体[128] ,并被认为是产生线粒体丙二酰辅酶 a,抑制通过肉碱进入线粒体的脂肪酸摄取: 棕榈酰转移酶系统。ACC2的磷酸化降低丙二酰辅酶 a,从而减轻肉碱: 棕榈酰辅酶 a 转移酶 -1(CPT1)的抑制,从而导致急性促进线粒体脂肪酸氧化[127]。

In the longer term, AMPK activation has several effects on mitochondria that enhance their capacity to produce ATP at a rapid rate. Firstly, AMPK activation promotes mitochondrial biogenesis itself, involving increased replication of mitochondrial DNA as well as expression of many nuclear-encoded mitochondrial proteins, by activating the transcriptional co-activator PGC-1α [129]. This is effected either by direct phosphorylation of PGC-1α [130] or by increasing the cellular concentration of NAD+, a cofactor required for deacetylation and activation of PGC-1α by SIRT1 [131]. Secondly, being the major site of cellular production of reactive oxygen species, mitochondrial components are particularly prone to oxidative damage, and if this affects their function mitochondria need to be removed and their contents recycled by the targeted form of autophagy known as mitophagy. Relevant to this, AMPK has been shown to promote both autophagy and mitophagy either by phosphorylation of the protein kinase that triggers autophagy, ULK1 [132,133], or by phosphorylation of the Ca2+/calmodulin-dependent kinase DAPK, generating a Ca2+/calmodulin-independent form that phosphorylates the key autophagy protein Beclin-1 [134]. Thirdly, mitochondria are now known to exist, especially in quiescent cells, not as small separate organelles, but as branching networks of tubules that can be almost as long as the cell containing them [135]. If any regions of such a network become damaged, they need to be segregated off from healthy regions via the process of mitochondrial fission, so that they become small enough to be recycled by mitophagy. Intriguingly, AMPK activation has been shown to promote mitochondrial fission by direct phosphorylation of mitochondrial fission factor(MFF) [136]. These findings are consistent with one aspect of the phenotype of skeletal muscle-specific double AMPK knockouts (either α1 and α2 [137] or β1 and β2 [138]), in which muscle accumulates abnormally shaped and apparently malfunctioning mitochondria. Overall, AMPK appears to play several crucial roles in mitochondrial homeostasis. Since mitochondria are the main source of cellular ATP in most cells, this makes perfect sense for a signalling pathway that is activated by energy stress and/or glucose deprivation.

从长远来看,AMPK 的激活对线粒体有几种影响,这些影响可以提高线粒体快速产生 ATP 的能力。首先,AMPK 激活通过激活转录辅助激活因子 pgc-1[129] ,促进线粒体自身的生物发生,包括增加线粒体脱氧核糖核酸的复制以及许多核编码的线粒体蛋白的表达。这可能是通过直接磷酸化 pgc-1[130]或通过增加细胞内 NAD + 浓度来影响的,NAD + 是 SIRT1去乙酰化和激活 pgc-1所需的辅助因子。其次,作为细胞产生活性氧类的主要场所,线粒体成分特别容易受到氧化损伤,如果这影响到它们的功能,线粒体需要被去除,它们的内容通过被称为噬细胞的靶向自噬形式被循环利用。与此相关的是,AMPK 已被证明可以通过引发自噬的蛋白激酶 ULK1[132,133]的磷酸化促进自噬和吞噬,或者通过磷酸化 Ca2 +/钙调素依赖性激酶 DAPK,生成一种 Ca2 +/钙调素无关的形式,磷酸化关键的自噬蛋白 Beclin-1[134]。第三,现在我们知道线粒体是存在的,特别是在静止的细胞中,不是作为一个小的分离的细胞器,而是作为小管的分支网络,几乎和包含它们的细胞一样长[135]。如果这样一个网络的任何一个区域遭到破坏,它们需要通过线粒体分裂过程与健康区域隔离开来,这样它们就变得足够小,可以被食肉者回收利用。有趣的是,AMPK 的激活已被证明可以通过直接磷酸化线粒体分裂因子(MFF)来促进细胞凋亡。这些发现与骨骼肌特异性双 AMPK 基因敲除(1和2[137]或1和2[138])表型的一个方面是一致的,在这种表型中,肌肉聚集形状异常,线粒体明显功能障碍。总的来说,AMPK 似乎在线粒体内环境稳态中扮演着几个重要的角色。由于线粒体是大多数细胞中 ATP 的主要来源,这对于能量压力和/或葡萄糖缺乏所激活的信号通路来说是完全合理的。

4.2. Proteins and genes involved in anabolic pathways

4.2. 蛋白质和基因参与合成途径

As well as switching on catabolic pathways that generate ATP, AMPK also switches off almost all major anabolic pathways (figure 4). AMPK was originally defined via its ability to phosphorylate and inactivate both acetyl-CoA carboxylase (ACC1) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key regulatory enzymes of fatty acid and sterol synthesis, respectively [139,140]. Fatty acid synthesis is a significant energy-consuming pathway in rapidly dividing tumour cells, being a major consumer of both ATP (consumed in the ACC1 reaction) and NADPH (consumed in the two reductive steps catalysed by the fatty acid synthase complex). Indeed, ACC1 remains one of most rapidly phosphorylated substrates for AMPK, and the phosphorylation of Ser80 (human numbering) on ACC1, monitored using a phosphospecific antibody, remains the most reliable and widely used cellular marker of AMPK function.

除了开关分解代谢途径产生 ATP,AMPK 也开关几乎所有主要的合成代谢途径(图4)。AMPK 最初的定义是通过其分别磷酸化和灭活乙酰辅酶A羧化酶和羟甲基戊二酸单酰辅酶A还原酶的能力,后者是脂肪酸和甾醇合成的关键调节酶[139,140]。脂肪酸合成是肿瘤细胞快速分裂的重要能量消耗途径,是 ATP (ACC1反应中消耗的)和 NADPH (脂肪酸合酶复合物催化的两个还原步骤中消耗的)的主要消耗者。实际上,ACC1仍然是 AMPK 最快速磷酸化的底物之一,并且使用磷酸化特异性抗体监测 ACC1上 Ser80(人类编码)的磷酸化,仍然是 AMPK 功能最可靠和广泛使用的细胞标记物。

Figure 4.
Figure 4. A ‘wheel’ of downstream targets and the pathways they regulate, focusing on anabolic and other processes that are inhibited by AMPK.图4。一个下游目标和他们调节的路径的’车轮’ ,重点是合成代谢和其他过程,被 AMPK 抑制。

As well as acutely inhibiting fatty acid synthesis by direct phosphorylation of ACC1 (which catalyses the first two steps of fatty acid synthesis from acetyl-CoA), AMPK activation also downregulates expression of the genes encoding ACC1 (ACACA) as well as the gene (FASN) encoding the fatty acid synthase complex, a dimeric multienzyme polypeptide that catalyses the remaining seven reactions leading to a saturated C16 fatty acid (palmitate). The AMPK targets responsible for these effects may be the transcription factors sterol response element binding protein-1c (SREBP1c) [141] and/or the carbohydrate response element binding protein (ChREBP) [142], which have both been reported to be directly phosphorylated by AMPK.

除了通过直接磷酸化 ACC1(催化乙酰辅酶 a 中的脂肪酸合成的前两个步骤)来抑制脂肪酸合成,AMPK 激活还下调了 ACC1(ACACA)基因的表达,以及编码脂肪酸合酶复合物的基因(FASN)的表达,这是一种二聚体多酶多肽,催化剩下的7个反应导致饱和的 C16脂肪酸(棕榈酸酯)。AMPK 的作用靶点可能是转录因子甾醇反应元件结合蛋白 -1c (SREBP1c)[141]和/或糖反应元件结合蛋白(ChREBP)[142] ,这两种蛋白都被 AMPK 直接磷酸化。

As well as inhibiting de novo synthesis of fatty acids, AMPK inhibits synthesis of triglyceride and phospholipid synthesis by inactivating the first enzyme (glycerol phosphate acyl transferase, GPAT) involved in the synthesis of the common intermediate diacylglycerol [143], although whether this is due to direct phosphorylation of the enzyme remains unclear. Two direct targets for AMPK are the muscle (GYS1) [34] and liver (GYS2) [35] isoforms of glycogen synthase, which catalyse the transfer of glucose from UDP-glucose to the growing non-reducing ends of the glycogen particle. Both are inactivated by phosphorylation at equivalent N-terminal sites by AMPK, although this inactivation is over-ridden by high concentrations of the feed-forward allosteric activator of glycogen synthase, glucose-6-phosphate [144]. The need to co-localize AMPK with glycogen synthase may be one reason why AMPK-β subunit isoforms in all species carry a carbohydrate-binding module (β-CBM; see figure 1).

除了抑制脂肪酸的从头合成外,AMPK 还通过失活第一种酶(甘油磷酸酰基转移酶,GPAT)抑制甘油三酯的合成和磷脂的合成,这种酶参与了甘油二酰基的合成[143] ,尽管这是否是由于这种酶的直接磷酸化还不清楚。AMPK 的两个直接作用靶点是肌肉(GYS1)[34]和肝脏(GYS2)[35]糖原合成酶亚型,它们催化葡萄糖从 udp 葡萄糖转移到生长中的糖原颗粒的非还原末端。两者都被 AMPK 在相同的 n 端位点磷酸化灭活,虽然这种灭活是由高浓度的前馈变构糖原合成酶激活剂,葡萄糖-6-磷酸[144]。需要与糖原合成酶共同定位 AMPK 可能是为什么所有物种的 AMPK- 亚基异构体携带碳水化合物结合模块的原因之一(- cbm; 见图1)。

Another key pathway in growing cells is biosynthesis of nucleotides. The ribose or deoxyribose components of nucleotides are derived from ribose-5-phosphate generated in the pentose phosphate pathway. It has recently been reported that PRPS1 and PRPS2, the two major isoforms of phosphoribosyl pyrophosphate synthetase that metabolize ribose-5-phosphate in the first step of nucleotide biosynthesis, are phosphorylated and inactivated by direct phosphorylation by AMPK [145]. The pentose phosphate pathway also generates NADPH that is used for fatty acid biosynthesis, as well as for regenerating reduced glutathione used to combat oxidative stress. The large requirement for NADPH and nucleotide biosynthesis in rapidly proliferating cells may be one reason why they exhibit rapid glucose uptake to provide input of glucose into the pentose phosphate pathway.

细胞生长的另一个关键途径是核苷酸的生物合成。核苷酸的核糖或脱氧核糖组分来源于磷酸戊糖途径产生的5- 磷酸核糖。最近有报道说,PRPS1和 PRPS2是磷酸核糖焦磷酸合成酶的两个主要亚型,在核糖 -5- 磷酸合成的第一步代谢过程中,它们被 AMPK [145]磷酸化而磷酸化和失活。磷酸戊糖途径还产生用于脂肪酸生物合成的 NADPH,以及用于再生用于对抗氧化应激的谷胱甘肽。快速增殖的细胞对 NADPH 和核苷酸生物合成的巨大需求可能是它们快速摄取葡萄糖为磷酸戊糖途径提供葡萄糖输入的原因之一。

Nucleotides are, of course, the building blocks for RNA and DNA. In rapidly proliferating thymocytes, the addition of actinomycin D (a general inhibitor of RNA synthesis) reduces oxygen uptake by as much as 15% [146], suggesting that RNA synthesis accounts for at least that percentage of total ATP turnover. Since up to 80% of the total RNA in a typical cell is ribosomal RNA (rRNA), synthesis of the latter is a major anabolic pathway and consumer of energy in proliferating cells. Consistent with this, AMPK activation has been found to inhibit rRNA synthesis by direct phosphorylation of the transcription factor for RNA polymerase I, TIF-1A (encoded by the RRN3 gene) [147].

当然,核苷酸是构建 RNA 和 DNA 的基石。在快速增殖的胸腺细胞中,添加放线菌素 d (一种 RNA 合成的一般抑制剂)可以减少多达15% 的氧摄取,这表明 RNA 合成至少占 ATP 总周转量的百分比。由于一个典型的细胞中80% 的总 RNA 是核糖体 RNA (rRNA) ,后者的合成是细胞增殖过程中主要的合成途径和能量消耗。与此相一致的是,AMPK 的激活被发现通过直接磷酸化转录因子的 RNA聚合酶I,TIF-1A (由 RRN3基因编码)抑制 rRNA 的合成。

Arguably the most important biosynthetic pathway in proliferating cells is translation (protein synthesis). Over 50% of the dry weight of most cells is protein while, in the proliferating thymocyte system mentioned above, inhibition of protein synthesis reduced oxygen uptake by more than 20% [146]. AMPK switches off translation by at least two mechanisms. Firstly, it inactivates the target of rapamycin complex-1 (TORC1), which is known to promote the initiation step of ribosomal protein synthesis by triggering the phosphorylation of multiple proteins, including eukaryotic initiation factor-4E binding protein-1 (EIF4EBP1) and ribosomal protein S6 kinase-1 (RPS6KB1) [148]. Phosphorylation of EIF4EBP1 leads to selective translation of mRNAs containing 5′-terminal oligopyrimidine (5′-TOP) sequences, which often encode mRNAs encoding proteins required for rapid cell growth, including most ribosomal proteins as well as other proteins involved in translation [149]. AMPK inactivates mTORC1 by at least two mechanisms, i.e. inhibitory phosphorylation of the Raptor subunit that targets the complex to downstream targets and to the lysosome where it is activated, and activatory phosphorylation of TSC2, which forms a key part of the TSC1:TSC2:TBC1D7 complex. The latter has a Rheb:GTPase activator protein (Rheb:GAP) domain on TSC2 that converts the mTORC1-activating G protein Rheb to its inactive GDP-bound form [150]. Secondly, AMPK inhibits the elongation step of ribosomal protein synthesis by promoting phosphorylation of elongation factor-2 at Thr56. This residue is phosphorylated not by AMPK itself but by elongation factor-2 kinase (EF2 K), a member of the atypical protein kinase (aPK) family that is activated by Ca2+/calmodulin. AMPK appears to activate EF2 K in part by direct phosphorylation [151] and in part by inactivating mTORC1, with EF2 K being phosphorylated and inactivated by p70S6K1 downstream of mTORC1 [152,153].

可以说,增殖细胞中最重要的生物合成途径是翻译(蛋白质合成)。大多数细胞干重的50% 以上是蛋白质,而在上述增殖的胸腺细胞系统中,抑制蛋白质合成使氧摄取减少了20% 以上[146]。AMPK 至少通过两个机制关闭平移。首先,它通过触发多种蛋白质的磷酸化,包括真核起始因子4 e 结合蛋白1(EIF4EBP1)和核糖体蛋白质 S6激酶1(RPS6KB1) ,从而抑制雷帕霉素复合物1(TORC1)的活性。磷酸化的 EIF4EBP1导致选择性地翻译含有5′端寡核苷酸(5′-TOP)序列的 mRNAs,这些序列通常编码细胞快速生长所需的 mRNAs 蛋白,包括大多数核糖体蛋白以及其他参与翻译的蛋白[149]。AMPK 至少通过两种机制使 mTORC1失活,即抑制作用于复合体下游靶点和溶酶体的 Raptor 亚基的磷酸化,以及作用于 TSC1: TSC2: TBC1D7复合体关键部分的 TSC2的激活磷酸化。后者在 TSC2上有一个 Rheb: GTPase 激活蛋白(Rheb: GAP)结构域,可将 mtorc1激活 g 蛋白 Rheb 转化为其非活性 gdp- 结合形式[150]。其次,AMPK 通过促进伸长因子2在 Thr56的磷酸化,抑制核糖体蛋白质合成的伸长步骤。这种残基不是通过 AMPK 本身磷酸化,而是通过延伸因子 -2激酶(ef2k)磷酸化,ef2k 是非典型蛋白激酶(aPK)家族的一员,被 Ca2 +/钙调蛋白激活。AMPK 部分通过直接磷酸化[151]激活 EF2 k,部分通过灭活 mTORC1激活 EF2 k,其中 EF2 k 被 mTORC1下游的 p70S6K1磷酸化并灭活[152,153]。

4.3. Progress through the cell cycle

4.3. 在细胞周期中进步

As well as inhibiting most major biosynthetic pathways, AMPK activation can also cause cell cycle arrest (figure 4). As long ago as 2001, it was reported that the AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR) caused arrest in the G1 phase of the cell cycle in HepG2 cells, which was attributed to phosphorylation of the transcription factor p53 at Ser15, and consequent increased expression of the G1 cyclin-dependent kinase inhibitor p21CIP1 (CDKN1A) [154]. This was followed by a demonstration that both AICAR and low glucose caused cell cycle arrest in MEFs [155]. These effects were at least partially dependent upon p53, because they were reduced in p53−/− MEFs. Although both AICAR and glucose deprivation can have off-target, AMPK-independent effects, the effects of low glucose also appeared to be AMPK dependent because they were reduced by expression of a dominant negative AMPK mutant (a kinase-inactive AMPK-α mutant that inhibits endogenous AMPK-α subunits by competing for available β and γ subunits). This group also reported that an activated kinase domain construct of AMPK could directly phosphorylate p53 at Ser15 [155]. However, the sequence around Ser15 is not a good fit to the AMPK recognition motif, lacking a basic residue at P-4 or P-3 (in fact, with an acidic residue at P-4 instead). It seems more likely that the phosphorylation of p53 observed in response to AMPK activation is indirect.

与抑制大多数主要的生物合成途径一样,AMPK 激活也能导致细胞周期阻滞(图4)。早在2001年,就有报道说 AMPK 激活剂5- 氨基咪唑 -4- 羧基核糖苷(AICAR)在 HepG2细胞周期的 g 1期引起阻滞,这是由于在 Ser15位的转录因子 p53磷酸化,以及随之而来的 g 1周期蛋白依赖性激酶抑制剂 p21CIP1(CDKN1A)[154]的表达增加。接下来的实验证明 AICAR 和低葡萄糖都能引起 MEFs 的细胞周期阻滞。这些效应至少部分依赖于 p53,因为 p53-/-MEFs 降低了这些效应。虽然 AICAR 和葡萄糖剥夺都可以有非靶向的 AMPK 独立效应,但是低葡萄糖的效应似乎也是 AMPK 依赖性的,因为它们被显性负 AMPK 突变体(一个激酶失活突变体,通过竞争可用的和亚基抑制内源性 kamp- 亚基)的表达而降低。这个小组还报道了 AMPK 的活化激酶结构域可以直接磷酸化位于 Ser15[155]的 p53蛋白。然而,Ser15周围的序列并不适合 AMPK 识别序列,在 P-4或 P-3缺乏碱性残基(事实上,在 P-4有酸性残基)。更有可能的是,观察到的 p53磷酸化对 AMPK 活化的反应是间接的。

Another potential mechanism by which AMPK causes G1 arrest involves phosphorylation of another cyclin-dependent kinase inhibitor, p27WAF1 (CDKN1B). In breast cancer cells (MCF-7), p27 was found to be phosphorylated at its C-terminal residue (Thr198), and this appeared to stabilize the protein, thus increasing its expression and causing cell cycle arrest as well as appearing to promote autophagy. Phosphorylation of Thr198 increased in response to AICAR treatment or glucose starvation of cells. Although some evidence was presented that Thr198 is directly phosphorylated by AMPK, the site is not a perfect fit to the AMPK recognition motif (for example, being the C-terminal residue, there is no hydrophobic residue at P+4), and mutation of Thr198 only had a modest effect on phosphorylation by AMPK in cell-free assays [156]. Further studies are therefore required to confirm that this is a direct effect of AMPK.

AMPK 导致 G1期阻滞的另一个潜在机制涉及另一种周期蛋白依赖性激酶抑制剂 p27WAF1(CDKN1B)的磷酸化。在乳腺癌细胞(MCF-7)中,发现 p27在其 c 末端残基(Thr198)处被磷酸化,这似乎稳定了蛋白质,从而增加了其表达,导致细胞周期停滞,并且似乎促进了自噬。在 AICAR 处理或细胞葡萄糖饥饿状态下,Thr198的磷酸化增加。虽然有证据表明 Thr198直接被 AMPK 磷酸化,但该位点并不完全适合 AMPK 识别模体(例如,作为 c 末端残基,在 p + 4处没有疏水性残基) ,Thr198的突变对 AMPK 磷酸化的影响不大[156]。因此需要进一步的研究来证实这是 AMPK 的直接作用。

Although AMPK activation by both AICAR [14] and low glucose [60] requires the presence of LKB1, AMPK could still cause G1 arrest in three different LKB1-deficient tumour cell lines if it was activated by the addition of a Ca2+ ionophore to activate the alternative upstream kinase, CaMKK2. This effect was abolished either by expression of a dominant negative AMPK-α2 mutant or by a double knockout of AMPK-α1 and -α2 [157]. Thus, AMPK can cause G1 arrest even in the absence of its tumour suppressor upstream kinase, LKB1. Interestingly, treatment of cells with the Ca2+ ionophore A23187 caused G1 arrest without affecting the expression of CDKN1A or CDKN1B, despite the fact that the overall expression of both was reduced by expression of the dominant negative mutant or the double knockout [157]. Thus, in this case changes in CDKN1A or CDKN1B expression cannot be the sole explanation for cell cycle arrest.

虽然 AICAR [14]和低葡萄糖[60]激活 AMPK 都需要 LKB1的存在,但是如果加入 Ca2 + 离子载体激活替代的上游激酶 CaMKK2,AMPK 仍然可以在三种 LKB1缺陷的肿瘤细胞系中引起 G1的阻滞。这种效应可以通过表达显性负性 ampk-2突变体或双重敲除 ampk-1和 -2[157]来消除。因此,即使在缺乏肿瘤抑制基因上游激酶 LKB1的情况下,AMPK 也能导致 G1停滞。有趣的是,用 Ca2 + 离子载体 A23187处理细胞,虽然显性阴性突变或双基因敲除降低了 CDKN1A 或 CDKN1B 的整体表达,但却导致 G1期阻滞,而不影响 CDKN1A 或 CDKN1B 的表达。因此,在这种情况下,CDKN1A 或 CDKN1B 表达的改变不能作为细胞周期阻滞的唯一解释。

5. AMPK and cancer—evidence from mouse models

5. 腺苷酸活化蛋白激酶与癌症ーー小鼠模型的证据

We will now discuss the evidence that, depending upon the context, AMPK can act either as a tumour suppressor or as a tumour promoter in mouse models.

我们现在将讨论的证据,取决于上下文,AMPK 可以作为肿瘤抑制或作为肿瘤启动子在小鼠模型。

5.1. AMPK—a tumour suppressor?

5.1. ampk ー一种肿瘤抑制因子?

With the discovery that AMPK activation requires the tumour suppressor LKB1, the realization that AMPK inhibits cell growth and proliferation, and the epidemiological evidence that the AMPK activator, metformin, provides protection against cancer, it seemed increasingly likely that AMPK would also be a tumour suppressor. One caveat was that, soon after the discovery that LKB1 acted upstream of AMPK, LKB1 was found to be required for the phosphorylation and activation of at least 12 other kinases closely related to AMPK (now referred to as the AMPK-related kinase or ARK family), which share very similar sequences within their activation loops [158,159]. Although none of the ARKs (unlike AMPK) are known to inhibit cell growth and proliferation, knockdown of LKB1 using RNAi was reported to enhance expression of SNAIL, a protein that promotes the epithelial-to-mesenchymal transition, and hence metastasis of tumour cells, by reducing the phosphorylation of DIXDC1 by two of the ARKs, MARK1 and MARK4 [160]. It therefore remains possible that at least some of the tumour suppressor effects of LKB1 might be mediated by one or more of the ARKs, rather than AMPK.

随着 AMPK 激活需要肿瘤抑制基因 LKB1的发现,AMPK 抑制细胞生长和增殖的认识,以及流行病学证据表明 AMPK 激活剂二甲双胍能够提供对抗癌症的保护,似乎 AMPK 也越来越有可能成为肿瘤抑制基因。一个警告是,在发现 LKB1作用于 AMPK 的上游后不久,就发现 LKB1需要磷酸化和激活至少12个与 AMPK (现在称为与 AMPK 相关的激酶或 ARK 家族)密切相关的其他激酶,这些激酶在其激活循环中具有非常相似的序列[158,159]。尽管 ARKs (不同于 AMPK)都不能抑制细胞生长和增殖,但据报道利用 rna 干扰击倒 LKB1可以通过减少两个 ARKs MARK1和 MARK4的磷酸化作用,增强 SNAIL 的表达,这是一种促进上皮细胞向间充质细胞转化的蛋白质,因此肿瘤细胞会转移。因此,LKB1的一些肿瘤抑制作用仍然有可能是由一个或多个 ARKs 介导的,而不是 AMPK。

Confirmation of a tumour suppressor role for AMPK in vivo required the study of tumorigenesis in AMPK knockout mice. However, there are two isoforms of the catalytic subunit (α1 and α2) and a global double knockout is embryonic lethal [161], thus necessitating the use of tissue-specific double knockouts. While this approach is now possible, it is also very time-consuming. However, a shortcut arose with the realization that cells of the haematopoietic lineage, including lymphocytes, exclusively express AMPK-α1 [162]. Thus, to study the role of AMPK in lymphomas and/or leukaemias, it was only necessary to knock out a single gene, i.e. the Prkaa1 gene encoding AMPK-α1.

在体内确认 AMPK 的肿瘤抑制作用需要在 AMPK 基因敲除小鼠中进行肿瘤发生的研究。然而,催化亚基有两种亚型(1和2) ,而一个全球性的双基因敲除是胚胎致死的[161] ,因此需要使用组织特异性的双基因敲除。虽然这种方法现在是可行的,但也非常耗时。然而,随着实现造血系的细胞,包括淋巴细胞,专门表达 ampk- 1[162] ,出现了一条捷径。因此,要研究 AMPK 在淋巴瘤和/或白血病中的作用,只需敲除一个基因,即编码 AMPK- 1的 Prkaa1基因。

The first study to suggest that AMPK was a tumour suppressor used a Eµ-Myc model [163], in which B-cell lymphoma is induced by transgenic over-expression of the Myconcogene from a B-cell-specific promoter. Consistent with the idea that AMPK-α1 is a tumour suppressor, loss of both alleles of Prkaa1 in this model markedly accelerated development of B-cell lymphomas, whereas loss of a single allele had an intermediate effect. Eµ-Myc lymphoma cells and other tumour cells expressing shRNAs targeted at AMPK-α1 were also studied in vitro. In general, the AMPK knockdown cells exhibited mTORC1 hyper-activation and increased glucose uptake and lactate production compared with controls, and this appeared to be due to increased expression of hypoxia-inducible transcription factor-1α (HIF) [163]. The 5′-UTR of mRNA encoding HIF-1α contains 5′-TOP sequences [164] and their translation is thus enhanced by mTORC1 activation ([165]; see §4.2). Thus, loss of AMPK in the tumour progenitor cells enhances glucose uptake and glycolysis even under normoxic conditions. This is an example of the well-known ‘Warburg effect’, in which tumour cells display high levels of glucose consumption in order to generate precursors for biosynthesis derived from the pentose phosphate pathway and glycolysis. For example, ribose-5-phosphate for nucleotide biosynthesis and NADPH for lipid synthesis are generated via the pentose phosphate pathway, while serine (required for one-carbon metabolism used in purine nucleotide biosynthesis) is generated by a pathway that branches off from the glycolytic intermediate 3-phosphoglycerate [126].

第一项研究表明 AMPK 是一种肿瘤抑制剂使用 e-Myc 模型[163] ,其中 b 细胞淋巴瘤是由转基因过度表达的 Myc 癌基因从 b 细胞特异性启动子诱导。与 ampk- 1是肿瘤抑制基因的观点一致,在这个模型中,Prkaa1的两个等位基因的丢失明显加速了 b 细胞淋巴瘤的发展,而单个等位基因的丢失具有中等效果。E-myc 淋巴瘤细胞和其他肿瘤细胞表达靶向 ampk-1的 shRNAs 也在体外进行了研究。与对照组相比,AMPK 击倒细胞表现出 mTORC1高活化、葡萄糖摄取和乳酸生成增加,这可能是由于缺氧诱导转录因子 -1(hif-1)[163]的表达增加所致。编码 hif-1的 mRNA 的5′-UTR 包含5′-TOP 序列[164] ,因此 mTORC1的激活增强了它们的翻译(见4.2)。因此,即使在常氧条件下,肿瘤祖细胞中 AMPK 的缺失也会促进葡萄糖的摄取和糖酵解。这是著名的“ Warburg 效应”的一个例子,其中肿瘤细胞表现出高水平的葡萄糖消耗,以便生成来自磷酸戊糖途径和糖酵解的生物合成前体。例如,用于核苷酸生物合成的核糖 -5- 磷酸和用于脂质合成的 NADPH 是通过磷酸戊糖途径合成生成的,而丝氨酸(用于嘌呤核苷酸生物合成的一碳代谢所需的丝氨酸)是通过从糖酵解中间体3-磷酸甘油酸分支生成的途径生成的[126]。

A drawback with this B-cell lymphoma model was that Prkaa1 was knocked out globally [163], so it was not possible to conclude that the effect was due to a cell-autonomous loss of AMPK-α1 in the B-cell progenitors themselves, rather than an indirect effect of loss of AMPK-α1 in some other cell type. In an attempt to address this, wild-type mice were irradiated to inactivate their endogenous immune system, and were then reconstituted with haematopoietic stem cells from either Eµ-Myc/Prkaa1−/− or Eµ-Myc/Prkaa1+/+ mice. Interestingly, all of the mice receiving AMPK knockout cells developed lymphomas, but only 20% of those receiving the AMPK wild-type cells [163], thus supporting the idea that the effect of AMPK loss was at least partly cell autonomous.

这种 b 细胞淋巴瘤模型的一个缺点是 Prkaa1全部被淘汰[163] ,因此不可能得出结论认为这种效应是由于 b 细胞前体细胞自身 ampk-1的缺失,而不是其他细胞类型 ampk-1缺失的间接影响。为了解决这个问题,野生型小鼠被放射灭活其内源性免疫系统,然后用 e-myc/prkaa1-/-或 e-myc/prkaa1 +/+ 小鼠的造血干细胞重组。有趣的是,所有接受 AMPK 基因敲除细胞的小鼠都发生了淋巴瘤,但只有20% 的接受 AMPK 野生型细胞的小鼠发生了淋巴瘤,这就支持了 AMPK 基因敲除的影响至少部分是细胞自主性的观点。

Another study involved crossing mice with global knockouts of the genes encoding p53 (Trp53) and AMPK-β1 (Prkab1), the latter being the principal β subunit isoform expressed in T-cell precursors in the thymus [166]. Knockout of Prkab1 caused earlier onset of T-cell lymphomas in both homozygous and heterozygous p53 knockouts, suggesting that β1 had a tumour suppressor role in T-cell lymphoma. However, once again the knockout of Prkab1 in this model was global rather than T-cell specific, so it was not possible to conclude whether this was a cell-intrinsic effect on AMPK in the tumour progenitor cells themselves.

另一项研究涉及将编码 p53(Trp53)和 ampk-1(Prkab1)的基因全球敲除的小鼠杂交,后者是胸腺 t 细胞前体表达的主要亚基[166]。Prkab1基因敲除引起纯合子和杂合子 p53基因敲除的 t 细胞淋巴瘤提前发生,提示1在 t 细胞淋巴瘤中具有肿瘤抑制作用。然而,在这个模型中,Prkab1基因再次被敲除是全球性的,而不是 t 细胞特异性的,所以不可能得出这是否是肿瘤祖细胞自身对 AMPK 的细胞内在影响。

A specific loss of AMPK in the tumour progenitor cells has recently been achieved using a model of T-cell acute lymphoblastic leukaemia/lymphoma (T-ALL) [167]. As reported previously [168], mice with a T-cell-specific knockout of PTEN (using Cre recombinase expressed from the Lck promoter) started to develop lymphomas at about 50 days of age, and essentially all of the mice had developed T-ALL by 150 days. While knocking out the Prkaa1 gene using the same Lck-Cre system had no effect on its own, when combined with PTEN knockout the lymphomas arose earlier and overall tumour-free survival was greatly reduced (figure 5) [167]. These results suggested that basal AMPK activity in developing T cells is sufficient to provide protection against T-ALL. However, this model also provided an excellent opportunity to test whether treatment with biguanides would protect against this type of cancer (see §3.3). Since the expression of AMPK-α1 would be absent in lymphoma cells and their progenitors but normal everywhere else, it would also be possible to determine whether any effect of biguanides was a cell-intrinsic effect to activate AMPK in the tumour progenitor cells themselves. Rather disappointingly, metformin had no effect, which correlated with a lack of AMPK activation and a failure to detect metformin by liquid chromatography–mass spectrometry (LC:MS) in the thymus of mice with lymphomas. By contrast, phenformin significantly enhanced tumour-free survival, and this correlated with AMPK activation, and detection of phenformin by LC:MS, in the thymus of mice with lymphoma. Intriguingly, protection against T-ALL by phenformin was only observed when the tumours expressed AMPK, with no effect in the AMPK knockouts (figure 5). Thus, protection against T-ALL by phenformin was dependent upon the expression of AMPK in the tumour progenitor cells, and was cell autonomous, while the failure of metformin to provide protection was due to lack of uptake of the drug by thymocytes. Phenformin has also been shown recently to slow growth of murine breast cancer cells in vivo in a mouse allograft model, although the role of AMPK was not examined [169].

使用 t 细胞急性淋巴细胞白血病/淋巴瘤(T-ALL)模型,肿瘤祖细胞中 AMPK 的特异性丢失最近已经实现。正如以前的报道[168] ,经 t 细胞特异性敲除 PTEN (利用 Lck 启动子表达的 Cre 重组酶)的小鼠在50天龄时开始出现淋巴瘤,基本上所有的小鼠在150天时都出现 t 淋巴瘤。虽然使用同样的 Lck-Cre 系统敲除 Prkaa1基因本身没有效果,但是当与 PTEN 基因敲除联合使用时,淋巴瘤出现得更早,整体无瘤生存率大大降低(图5)[167]。这些结果表明,发育中的 t 细胞中基本的 AMPK 活性足以提供对 T-ALL 的保护。然而,这个模型也提供了一个极好的机会来测试双胍类药物治疗是否可以预防这种类型的癌症(见3.3)。由于 AMPK-1的表达在淋巴瘤细胞及其前体细胞中不存在,但在其他任何地方都是正常的,因此也有可能确定双胍类化合物的任何影响是否是激活肿瘤前体细胞自身 AMPK 的细胞内在效应。令人相当失望的是,二甲双胍没有作用,这与缺乏 AMPK 活化和无法通过液相色谱-质谱法(LC: MS)检测淋巴瘤小鼠胸腺中的二甲双胍有关。相比之下,苯乙双胍显著提高了淋巴瘤小鼠的无瘤存活率,这与 AMPK 激活和 LC: MS 检测苯乙双胍有关。有趣的是,仅当肿瘤表达 AMPK 时才观察到苯乙双胍对 T-ALL 的保护作用,而 AMPK 敲除没有影响(图5)。因此,苯乙双胍对 T-ALL 的保护依赖于肿瘤祖细胞中 AMPK 的表达,并且是细胞自主的,而二甲双胍未能提供保护是由于胸腺细胞对药物的摄取不足。苯乙双胍最近在小鼠移植物模型中也被证明可以减缓小鼠乳腺癌细胞的生长,虽然 AMPK 的作用没有被检测到[169]。

Figure 5.
Figure 5. Effect of T-cell knockout of AMPK (AMPK KO) and oral phenformin on tumour-free survival in mice bearing T-cell knockout of PTEN (PTEN KO). Where indicated, phenformin was administered in drinking water starting from 30 days of age. Original data from [167].图5。腺苷酸活化蛋白激酶(AMPK) t 细胞基因敲除和口服苯乙双胍对 PTEN 基因敲除小鼠无瘤存活的影响。如有需要,从出生30天开始在饮用水中给予苯乙双胍。原始数据来自[167]。

Another mouse model suggesting a tumour suppressor role for AMPK used prostate epithelial-specific knockouts of the Pten and Prkab1 genes [170]. Although the knockout of Prkab1 as well as Pten did not affect prostate size, it did result in a higher proliferative index and pathological grade. A drawback with this model was that the prostate gland also expresses AMPK-β2, which might have partially compensated for lack of β1 and might be why the effects on tumorigenesis were relatively modest.

另一个小鼠模型表明 AMPK 的肿瘤抑制作用使用了前列腺上皮特异敲除 Pten 和 Prkab1基因[170]。虽然 Prkab1和 Pten 基因敲除不影响前列腺大小,但确实导致了较高的增殖指数和病理分级。这个模型的一个缺点是前列腺也表达 ampk-2,这可能部分弥补了缺乏1的缺陷,这可能是为什么对肿瘤发生的影响相对较小的原因。

Other evidence supporting the idea that AMPK is a tumour suppressor comes from studies of ubiquitin ligases involved in cellular degradation of AMPK subunits. MAGE-A3/-A6 are closely related members of the melanoma antigen family of proteins, which are normally only expressed in testis but become re-expressed in many tumours, hence their designation as tumour antigens [171]. MAGE-A3/-A6 bind to the ubiquitin E3 ligase TRIM28, and a screen revealed AMPK-α1 to be a target for polyubiquitylation by this complex, with consequent proteasomal degradation. Consistent with this, knockdown of MAGE-A3/A6 or TRIM28 in tumour cells increased the expression of AMPK-α1 and triggered the expected changes in metabolism and signalling, including inhibition of mTORC1. Finally, various human tumour cells that express MAGE-A3/-A6 have reduced levels of AMPK-α1 protein [171].

其他证据支持的想法,AMPK 是一个肿瘤抑制来自泛素连接酶参与细胞降解 AMPK 亚单位的研究。MAGE-A3/-A6是黑色素瘤抗原家族蛋白质中密切相关的成员,这种蛋白质通常只在睾丸中表达,但在许多肿瘤中重新表达,因此它们被称为肿瘤抗原[171]。MAGE-A3/-A6与泛素 E3连接酶 TRIM28结合,筛选结果显示 ampk-1是泛素 E3连接酶 TRIM28聚泛素化的靶标,由此产生蛋白酶体降解。与此相一致的是,在肿瘤细胞中击倒 MAGE-A3/A6或 TRIM28可增加 ampk-1的表达,并触发代谢和信号的预期变化,包括抑制 mTORC1。最后,各种表达 MAGE-A3/-A6的人类肿瘤细胞的 ampk-1蛋白水平降低[171]。

Another cancer-associated ubiquitin ligase, UBE2O, targets degradation of α2, the other catalytic subunit isoform of AMPK [172]. Knockout of Ube2o attenuated tumour development in mouse models of both breast and prostate cancer, supporting the idea that the protein has tumour-promoting functions. A search identified AMPK-α2 as an UBE2O-interacting protein that is targeted for polyubiquitylation and proteasomal degradation, and the levels of α2 but not α1 were upregulated in tissues from Ube2o−/−mice. A human colon carcinoma cell line also grew less rapidly in mouse xenografts when UBE2O was knocked down using shRNA, and this was reversed by concurrent knockdown of AMPK-α2 but not -α1. The UBE2O gene is located in humans at 17q25, a region amplified in up to 20% of breast, bladder, liver and lung carcinomas. Using immunohistochemistry of human breast tumours, there was a negative correlation between expression of UBE2O and AMPK-α2, but a positive correlation between UBE2O expression and S6 phosphorylation, a marker for the mTORC1 pathway [172].

另一种与癌症相关的泛素连接酶,UBE2O,目标降解2,另一种 AMPK 的催化亚单位亚型[172]。Ube2o 基因的敲除减弱了乳腺癌和前列腺癌小鼠模型中的肿瘤发展,支持了这种蛋白质具有促进肿瘤生长的功能的观点。研究表明 ampk-2是一种与 Ube2o 相互作用的蛋白,靶向于泛泛素化和蛋白酶体降解,并且在 Ube2o-/-小鼠组织中,ampk-2的水平被上调,而不是1。当使用 shRNA 击倒 UBE2O 时,人结肠癌细胞系在小鼠异种移植物中的生长速度也较慢,这种情况可以通过同时击倒 ampk-2而不是 -1来逆转。UBE2O 基因位于人类体内17q25,该区域在乳腺癌、膀胱癌、肝癌和肺癌的扩增率高达20% 。使用人类乳腺肿瘤的免疫组织化学,UBE2O 和 ampk- 2的表达之间呈负相关,但是 UBE2O 的表达和 S6磷酸化之间呈正相关,S6磷酸化是 mTORC1通路的标志[172]。

5.2. AMPK—a tumour promoter?

5.2. ampk ーー肿瘤促进剂?

Despite the evidence discussed in the previous section that AMPK-α1 and -β1 are tumour suppressors that protect against the development of B- and T-cell lymphomas as well as prostate cancer, other studies suggest that AMPK may protect the tumour cells (rather than the patient), and thus promote tumour formation, at least when disease is already established. Rathmell’s group used a different model of T-ALL in which oncogenic NOTCH1 was expressed in vitro in murine haematopoietic stem cells that carried a floxed AMPK-α1 gene and Cre recombinase driven by a tamoxifen-inducible promoter. These were multiplied in irradiated mice, and then injected into irradiated secondary recipient mice. After a period of 10 days to allow disease to become established, the mice were then treated with tamoxifen to acutely delete AMPK-α1 in the T-ALL cells. In this model, knocking out AMPK-α1 reduced the recovery of T-ALL cells in spleen, lymph nodes and bone marrow, and enhanced survival of the mice [173]. Thus, once T-ALL tumour cells have developed the presence of AMPK-α1 appears to enhance T-ALL cell viability and reduce mouse survival. While AMPK therefore acts as a tumour suppressor during the development of T-ALL [167], once the tumours have occurred it appears to paradoxically switch to being a tumour promoter instead.

尽管上一节讨论的证据表明 AMPK-1和-1是肿瘤抑制剂,可以防止 b 细胞和 t 细胞淋巴瘤以及前列腺癌的发展,但其他研究表明,AMPK 可以保护肿瘤细胞(而不是病人) ,从而促进肿瘤的形成,至少在疾病已经确定的情况下。Rathmell 的小组使用了不同的 T-ALL 模型,其中致癌性 NOTCH1在体外表达于携带有 ampk-1基因的小鼠造血干细胞,以及由它莫昔芬诱导的启动子驱动的 Cre 重组酶。这些细胞在受辐射的小鼠体内繁殖,然后注射到受辐射的第二受体小鼠体内。经过10天的时间,让疾病建立,然后用他莫昔芬处理小鼠,急性删除在 T-ALL 细胞 ampk- 1。在这个模型中,敲除 ampk-1降低了脾脏、淋巴结和骨髓中 T-ALL 细胞的恢复,并提高了小鼠的存活率[173]。因此,一旦 T-ALL 肿瘤细胞发展出 ampk-1的存在,似乎可以提高 T-ALL 细胞的活力并降低小鼠的存活率。虽然 AMPK 因此作为肿瘤抑制剂在 T-ALL [167]的发展过程中,一旦肿瘤发生似乎矛盾地转变为肿瘤促进剂而不是。

Another study using a mouse model of acute myeloid leukaemia (AML) also concluded that AMPK acted as a tumour promoter [174]. Here, mice carrying floxed alleles of Prkaa1and Prkaa2, as well as Cre recombinase expressed from the Mx1 promoter, were injected with poly(I:C) to delete AMPK-α1 and -α2 from haematopoietic cells, with mice lacking Mx1-Cre as controls. Haematopoietic progenitor cells from these mice were then transduced with retroviruses expressing three different cancer-promoting gene fusions (MLL-AFP, MOZ-TIF2 or BCR-ABL) and were then transplanted into irradiated recipient mice. The absence of AMPK from the leukaemia-initiating cells either delayed the onset of disease (BCR-ABL) or enhanced mouse survival (MLL-AFP or MOZ-TIF1). Thus, the presence of AMPK was required to maintain full leukaemogenic potential of the cells in these models. Evidence was provided that this was because the lack of AMPK increased the recovery of reactive oxygen species (ROS) in leukaemia-initiating cells from bone marrow, correlating with decreased ratios of reduced : oxidized NADP and glutathione, and increased DNA damage. This was ascribed to a reduced glucose uptake via GLUT1, which is regulated by AMPK via phosphorylation of TXNIP (see §4.1). The authors also proposed that the leukaemia-initiating cells lacking AMPK were particularly vulnerable to stress in the bone marrow, because the glucose concentrations were lower than in peripheral blood, especially under conditions of dietary restriction of the mice [174].

另一项使用小鼠急性髓系白血病(AML)模型的研究也得出结论,AMPK 作为肿瘤促进剂[174]。本研究以缺乏 Mx1-Cre 基因的小鼠为对照,将 Prkaa1和 Prkaa2等位基因以及 Mx1启动子表达的 Cre 重组酶注射到造血细胞中去除 ampk-1和 -2。然后用逆转录病毒转化来自这些小鼠的造血祖细胞,表达三种不同的促癌基因融合(MLL-AFP,MOZ-TIF2或 BCR-ABL) ,然后移植到辐射受体小鼠体内。白血病起始细胞中缺乏 AMPK 可能延迟了疾病的发生(BCR-ABL)或提高了小鼠的存活率(MLL-AFP 或 MOZ-TIF1)。因此,AMPK 的存在是必要的,以保持充分的白血病潜力的细胞在这些模型。有证据表明,这是因为缺乏 AMPK 增加了骨髓中白血病起始细胞中活性氧类的恢复,与减少氧化 NADP 和谷胱甘肽的比率以及增加 DNA 损伤有关。这是由于葡萄糖摄取通过 GLUT1减少,这是由 AMPK 通过磷酸化 TXNIP (见4.1)调节。作者还提出,缺乏 AMPK 的白血病起始细胞特别容易受到骨髓应激的影响,因为葡萄糖浓度低于外周血,特别是在限制饮食的条件下[174]。

Consistent with these findings, reduced survival of AMPK-deficient human tumour cells undergoing stress has been observed in several in vitro studies. For example, LKB1-null tumour cells, or LKB1-expressing tumour cells with AMPK-α1 knocked down using shRNA, were more susceptible to cell death induced by glucose starvation or extracellular matrix detachment, suggesting that AMPK activation protected against these insults [175]. In another example, a synthetic lethal siRNA screen was carried out to detect protein kinases required for survival of U2OS cells that over-expressed the Myc oncogene from a tamoxifen-inducible promoter. One of the top hits was AMPK-α1, which was also shown to be activated during Myc over-expression [176].

与这些发现一致的是,在一些体外研究中已经观察到缺乏 ampk 的人类肿瘤细胞在承受压力时存活率降低。例如,LKB1-null 肿瘤细胞,或 lkb1-表达 AMPK- 1的肿瘤细胞使用 shRNA 击倒,更容易受到葡萄糖饥饿或细胞外间质分离诱导的细胞死亡,这表明 AMPK 的激活对这些损伤有保护作用[175]。在另一个例子中,人工合成的致死性 siRNA 筛选被用来检测 U2OS 细胞生存所需的蛋白激酶,这些蛋白激酶从它莫西芬诱导的启动子中过度表达 Myc 癌基因。其中最热门的是 ampk- 1,它也被证明在 Myc 过度表达时被激活[176]。

Evidence that AMPK can promote tumours was also obtained recently using a mouse model of lung cancer in which the tumours develop in situ at their site of origin, and in which the authors had ‘bitten the bullet’ by knocking out both AMPK-α1 and -α2. Here, mice expressing Lox-STOP-Lox alleles of the KRASG12D oncogene and firefly luciferase were crossed with mice expressing floxed alleles of Tp53 (encoding p53) and/or Stk11 and/or Prkaa1 plus Prkaa2. To model non-small cell lung carcinoma, Cre-recombinase was delivered to the lungs by nasal inhalation of lentiviral vectors. This procedure triggers recombination at twin loxP sites in a small subset of lung epithelial cells, in which expression of KRASG12D and luciferase would be switched on, and p53 and/or LKB1 and/or AMPK-α1/-α2 would be knocked out; expression of luciferase also allowed tumours to be imaged by bioluminescence, and thus their growth to be monitored in vivo. Knockout of LKB1 enhanced growth in tumours expressing mutant K-Ras as reported previously [177] but, by contrast, knockout of both AMPK-α1 and -α2 was found to cause reductions in the size and number of lung tumours, especially in tumours expressing mutant K-Ras and lacking p53. Overall, these results confirmed that LKB1 is a tumour suppressor in non-small cell lung cancer as expected, while the presence of either AMPK-α1 or -α2 promoted tumour growth [178].

最近,研究人员还利用小鼠肺癌模型获得了 AMPK 可以促进肿瘤生长的证据,在该模型中,肿瘤原位生长,研究人员敲除了 AMPK-1和-2,从而“咬掉了子弹”。本研究将表达 KRASG12D 癌基因和萤火虫荧光素酶 lox 等位基因的小鼠与表达 Tp53(编码 p53)和/或 Stk11和/或 Prkaa1加 Prkaa2的小鼠进行杂交。为了建立非小细胞肺癌模型,鼻吸入慢病毒载体将 cre 重组酶转染肺组织。这一过程触发了肺上皮细胞小亚群中双 loxP 位点的重组,其中 KRASG12D 和荧光素酶的表达将被打开,p53和/或 LKB1和/或 ampk-1/-2将被淘汰; 荧光素酶的表达也允许通过生物发光体成像,从而在体内监测它们的生长。LKB1基因敲除可以促进表达突变型 K-Ras 基因的肿瘤的生长,正如先前报道的[177] ,但是,相比之下,ampk-1和 -2基因敲除可以导致肺肿瘤的大小和数量减少,特别是在表达突变型 K-Ras 基因和缺乏 p53基因的肿瘤中。总的来说,这些结果证实 LKB1是一个肿瘤抑制非小细胞肺癌的预期,而存在 ampk-1或 -2促进肿瘤生长[178]。

6. Evidence from analysis of human cancer genomes

6. 人类癌症基因组分析的证据

Although most of the evidence discussed in §5 was obtained in mouse models of cancer, comparison of genetic alterations in genes encoding the LKB1-AMPK pathway in biopsies of human cancers, compared with normal tissue, can also provide useful clues about roles of the pathway in human cancer. The cBioPortal database ( [179,180]) provides a particularly user-friendly way to analyse the many studies of human cancer of this type that have been performed to date. Figure 6 summarizes genetic changes in the STK11 gene encoding LKB1, and all seven genes encoding subunit isoforms of AMPK, extracted from cBioPortal in April 2019. Each vertical bar represents an individual cancer genome project, with the height of the bar representing the percentage of cases where genetic alterations were seen (only studies with changes in greater than or equal to 3% of cases are shown). Since LKB1 is a known tumour suppressor, one would expect to observe mainly mutations (green bars) or deletions (blue bars) when analysing STK11. This is indeed generally the case (figure 6a), although there are some anomalous cancer studies where gene amplification was observed instead (red bars), particularly in pancreatic and prostate cancers. By contrast, changes in the PRKAA1 gene, encoding AMPK-α1, were mostly amplifications (note preponderance of red bars in figure 6b), which is more consistent with the idea that AMPK-α1 can act as a tumour promoter. An important caveat is that gene amplifications in cancer usually involve whole segments of chromosomes rather than individual genes. It was therefore possible that the PRKAA1 gene is located close to an oncogene for which amplification was being selected, with PRKAA1 simply accompanying it as an innocent bystander. However, an argument against that possibility comes from analysis of concurrent genetic changes in STK11 and PRKAA1 in single cancer studies, such as the 230 cases of lung adenocarcinoma in The Cancer Genome Atlas (figure 7) [181]. In that study, the STK11 gene was either deleted or mutated (mostly truncations or missense mutations predicted to cause loss of function) in 43 cases (19%) and PRKAA1 was amplified in 22 (10%). However, these changes never coincided (p = 0.005), which would be expected to occur by random chance if they were occurring independently. The most frequent mutations in this study of lung adenocarcinoma were in the KRAS (36%) and TP53 genes (47%), encoding K-Ras and p53. Interestingly, amplification of PRKAA1 was almost mutually exclusive with mutations in KRAS (p = 0.005), but co-occurred with mutations in TP53 (p < 0.001).

尽管5中讨论的大多数证据都是在小鼠癌症模型中获得的,但是在人类癌症活检中对编码 LKB1-AMPK 途径的基因改变进行比较,与正常组织相比,也可以提供有关该途径在人类癌症中作用的有用线索。cBioPortal 数据库(数据库[179,180])提供了一种特别方便用户的方式来分析迄今为止进行的许多此类人类癌症的研究。图6总结了编码 LKB1的 STK11基因的遗传变化,以及2019年4月从 cbiporortal 中提取的所有7个编码 AMPK 亚基亚单位亚型的基因。每个竖条代表一个单独的癌症基因组项目,竖条的高度代表发现基因改变的病例的百分比(只有变化大于或等于3% 的病例的研究才显示)。由于 LKB1是一个已知的肿瘤抑制基因,人们在分析 STK11时可能会主要观察到突变(绿条)或缺失(蓝条)。这确实是一个普遍的情况(图6a) ,虽然有一些异常的癌症研究观察到基因扩增代替(红条) ,特别是在胰腺癌和前列腺癌。相比之下,编码 ampk-1的 PRKAA1基因的改变主要是扩增(注意图6b 中的红条优势) ,这更符合 ampk-1可以作为肿瘤启动子的想法。一个重要的警告是,癌症中的基因扩增通常涉及整个染色体片段,而不是单个基因。因此,PRKAA1基因可能位于被选择进行扩增的癌基因附近,PRKAA1只是作为一个无辜旁观者陪伴着它。然而,反对这种可能性的观点来自于对 STK11和 PRKAA1在单一癌症研究中同时发生的基因变化的分析,例如《癌症基因图谱》中的230例肺腺癌(图7)[181]。在这项研究中,43例(19%) STK11基因缺失或突变(主要是截断或错义突变导致功能丧失) ,22例(10%) PRKAA1基因扩增。然而,这些变化从未重合(p = 0.005) ,如果它们是独立发生的,那么这些变化可能是随机发生的。这项肺腺癌研究中最常见的突变是编码 K-Ras 和 p53的 KRAS 基因(36%)和 TP53基因(47%)。PRKAA1基因扩增与 KRAS 基因突变几乎相互排斥(p = 0.005) ,与 TP53基因突变共同发生(p < 0.001)。

Figure 6.
Figure 6. Summary of genetic alterations in human cancer in genes encoding (a) LKB1 (STK11), and (b–h) the seven genes encoding AMPK subunit isoforms. Based on analysis of the ‘curated set of non-redundant studies’ in the cBioPortal database in early April 2019, using the gene names shown.图6。人类肿瘤中编码(a) LKB1(STK11)和(b-h)7个 AMPK 亚基亚单位亚型基因的遗传变异综述。基于对2019年4月初 cbiportal 数据库中“精选的一组非冗余研究”的分析,使用了所示的基因名称。
Figure 7.
Figure 7. Co-occurrence or mutual exclusion of genetic alterations in the PRKAA1 (AMPK-α1), STK11 (LKB1), KRAS (K-Ras) and TP53 (p53) genes in human lung adenocarcinoma. Results were generated using cBioPortal from the results of a single study [181]. Each column of vertically aligned bars represents a single case; cases with no alterations in any of the genes are not shown.图7。肺腺癌中 PRKAA1(ampk- 、 STK11(LKB1)、 KRAS (K-Ras)和 TP53(p53)基因的共存或共互斥锁改变。结果是使用 cBioPortal 从一个单一的研究结果[181]。每一列垂直对齐的条形图代表一种情况; 没有显示任何基因改变的情况。

Why should amplification of the AMPK-α1 gene be mutually exclusive with mutations in the LKB1 gene? The answer to this seems obvious, because there would be little point in over-expressing AMPK-α1 if LKB1 was not present to phosphorylate and activate it. These considerations suggest that PRKAA1 amplification is being selected for, rather than just being an innocent bystander. However, why amplification of the AMPK-α1 gene should co-occur with mutations in p53 is less obvious. The classical role of p53 [182] is to become stabilized or activated in response to DNA damage, and to cause a G1 cell cycle arrest in order to allow time for the damage to be repaired, which it achieves by inducing transcription of genes such as the G1 cyclin-dependent kinase inhibitor p21CIP1 (CDKN1A). Intriguingly, as already discussed in §2.5, AMPK complexes containing α1 are also activated by genotoxic agents such as etoposide, and can trigger a similar G1 cell cycle arrest [11]. It therefore seems possible that PRKAA1 amplification may be selected for in p53-null tumours because over-expression of AMPK-α1 can compensate to some extent for p53 loss, and could thus enhance survival of p53-null tumour cells undergoing genotoxic stress.

为什么 ampk-1基因的扩增应该与 LKB1基因的突变相互排斥?这个问题的答案似乎很明显,因为如果 LKB1没有被磷酸化和激活,那么过度表达 ampk-1就没有什么意义。这些考虑表明 PRKAA1基因的扩增正在被选中,而不仅仅是一个无辜的旁观者。然而,为什么 ampk-1基因的扩增应该与 p53突变共同发生的原因不太明显。P53[182]的经典作用是在 DNA 损伤时变得稳定或激活,并引起 g 1细胞周期阻滞,以便有时间修复损伤,这是通过诱导 g 1周期蛋白依赖性激酶抑制剂 p21CIP1(CDKN1A)等基因转录实现的。有趣的是,正如在2.5中已经讨论过的,含有1的 AMPK 复合物也被基因毒性剂如依托泊苷激活,并且可以触发类似的 G1细胞周期阻滞[11]。因此,在 p53缺失的肿瘤中,PRKAA1基因的扩增似乎是可能的,因为过度表达 ampk-1可以在一定程度上弥补 p53缺失的损失,从而可以提高 p53缺失的肿瘤细胞在基因毒性应激下的存活率。

In marked contrast to the frequent amplification of the PRKAA1 gene in cancer, the PRKAA2 gene encoding AMPK-α2 is much more often mutated (note preponderance of green bars in figure 6c). Interestingly, all six of the cancer studies where the gene was most frequently mutated (10–23% of cases) were of skin cancer or melanoma. The reasons for this are not clear, but separate analysis showed that in all of the skin cancer/melanoma studies listed in cBioPortal there were 80 mutations affecting AMPK-α2 and just 10 affecting α1. Although it is not yet clear how many of the former cause loss of function in α2 complexes, these results suggest that AMPK-α2 may play a tumour suppressor role in skin cancer and melanoma.

与癌症中 PRKAA1基因的频繁扩增形成鲜明对比的是,编码 ampk-2的 PRKAA2基因更容易发生突变(注意图6c 中的绿条优势)。有趣的是,所有六项癌症研究中基因最常发生突变的(10-23% 的病例)都是皮肤癌或黑色素瘤。其原因尚不清楚,但是单独的分析表明,在 cBioPortal 列出的所有皮肤癌/黑色素瘤研究中,有80个突变影响 ampk-2,只有10个影响1个。虽然目前还不清楚有多少前者导致2个复合体的功能丧失,但这些结果表明 ampk-2可能在皮肤癌和黑色素瘤中发挥肿瘤抑制作用。

When it comes to the AMPK-β subunits, there was a striking difference between the behaviour in human cancers of the PRKAB1 and PRKAB2 genes, encoding β1 and β2 (figure 6d,e). While genetic changes in PRKAB1 were detected in just a very small number of cancer studies and were quite variable in genetic type, the PRKAB2 gene was frequently amplified in numerous different cancers (note preponderance of red bars in figure 6e), suggesting, if anything, a tumour promoter role. Since the C-terminal domain of the β subunit (β-CTD) forms the ‘core’ of the heterotrimeric AMPK complex (see §2.2), over-expression of β2 may perhaps help to stabilize and increase expression of the α and γ subunits, even when the genes encoding those subunits lack genetic alterations. However, why it should only be the gene encoding β2, and not β1, that is amplified remains unclear.

当涉及到 ampk- 亚基时,PRKAB1和 PRKAB2基因在人类癌症中的表现存在显著差异,编码1和2(图6d,e)。虽然 PRKAB1的基因变化只在极少数癌症研究中发现,而且在基因类型上差异很大,但 PRKAB2基因经常在许多不同的癌症中被扩增(注意图6e 中的红条占多数) ,如果有的话,这表明了肿瘤促进剂的作用。由于亚基的 c 末端结构(- ctd)形成了异三聚体 AMPK 复合体的“核心”(见2.2) ,2的过度表达可能有助于稳定和增加亚基和亚基的表达,即使编码这些亚基的基因缺乏遗传改变。然而,为什么只有编码2而不是1的基因被扩增仍然不清楚。

Alterations in the genes encoding the three γ subunits tend to occur at a lower frequency than those encoding the α and β subunits, and are more mixed in genetic type (figure 6f–h). However, there were some interesting findings, such as the 41% of cases (albeit only five out of 12) in which the PRKAG1 gene was deleted in adenoid cystic breast cancer [183].

编码这三个亚单位的基因改变往往比编码这两个亚单位的基因改变发生的频率低,而且在遗传类型中混合得更多(图6f-h)。然而,也有一些有趣的发现,例如,在腺样囊性乳腺癌中,PRKAG1基因缺失的病例占41% (尽管只占12例中的5例)。

Looking at the genetic alterations occurring in the genes encoding LKB1 and AMPK subunits in human cancer, one striking observation is that all eight genes are frequently amplified in neuroendocrine prostate cancer (labelled NE prostate in figure 6). This is a subset of prostate cancer that has become resistant to anti-androgen treatment [184]. The significance of this intriguing observation remains unclear at present.

通过观察编码 LKB1和 AMPK 亚基的基因在人类癌症中的遗传变异,一个引人注目的发现是,所有8个基因在神经内分泌前列腺癌中都经常被扩增(图6中标记为 NE 前列腺)。这是前列腺癌的一个亚型,对抗雄激素治疗产生了耐药性[184]。这个有趣的发现的意义目前还不清楚。

7. Conclusion—is AMPK a tumour suppressor or a tumour promoter, or both?

7. 结论ー AMPK 是肿瘤抑制因子还是肿瘤促进因子,抑或两者兼有?

In this final section we will attempt to reconcile the apparently conflicting reports that AMPK can variously act to promote or suppress tumorigenesis. Our view is that AMPK can act either as a tumour suppressor or a tumour promoter, depending on the context. It can be argued that in all of the mouse studies where a tumour suppressor role was supported (e.g. in the Eµ-Myc model of B-cell lymphoma [163], the p53-null [166] and PTEN-null [167] models of T-cell lymphoma and the PTEN-null model of prostate cancer [170]), AMPK function had been knocked out prior to tumorigenesis. For example, in the Eµ-Myc model [185], loss of AMPK-α1 would have occurred during embryogenesis whereas, although over-expression of Myc in pre-B cells has certainly occurred by 35–50 days of age [186], lymphomas do not start to arise until 50 days and their median onset is ≈80 days [187]. Thus, events additional to Myc over-expression must occur before lymphomas are generated. The same applies to the PTEN knockout model of T-ALL, where the Lck promoter-driven knockout of PTEN and/or AMPK-α1 would have occurred by 30 days of age but lymphomas did not start to arise until later (figure 5).

在这最后一节,我们将试图调和明显矛盾的报告,AMPK 可以不同的行为,以促进或抑制肿瘤发生。我们的观点是,AMPK 可以作为肿瘤抑制或肿瘤启动子,这取决于上下文。可以认为,在所有支持肿瘤抑制因子作用的小鼠研究中(例如,在 b 细胞淋巴瘤的 e-myc 模型[163]、 t 细胞淋巴瘤的 p53-null [166]和 PTEN-null [167]模型以及前列腺癌的 PTEN-null 模型[170]) ,AMPK 功能在肿瘤发生之前就已被淘汰。例如,在 e-Myc 模型[185]中,ampk- 1的丢失可能发生在胚胎发育期间,然而,虽然 Myc 在前 b 细胞中的过度表达肯定发生在35-50天龄[186] ,但淋巴瘤直到50天才开始出现,它们的中位发病时间为≈80天[187]。因此,在生成淋巴瘤之前,除了 Myc 过度表达之外,还必须发生其他事件。同样的情况也适用于 T-ALL 的 PTEN 基因敲除模型,Lck 启动子驱动的 PTEN 和/或 ampk- 1基因敲除可能发生在30天龄之前,但淋巴瘤直到后来才开始出现(图5)。

On the other hand, in those mouse models of cancer where AMPK appeared to be acting as a tumour promoter, it can be argued that the knockout of AMPK usually occurred either simultaneous with, or even after, tumorigenesis had been initiated. For example, in the study of T-ALL by Kishton et al. [173] (§5.2), transformation was generated in vitro by forced expression of an oncogenic mutant of Notch1, and the T-ALL cells were then transferred to irradiated recipient mice and disease allowed to become established prior to AMPK being knocked out by treatment with tamoxifen. It is particularly instructive to compare this model with our own more recently published model of T-ALL [167], where AMPK-α1 had been specifically knocked out in T-cell progenitors prior to lymphomas starting to occur, in which basal AMPK was clearly protecting against development of lymphomas, and in which activation of AMPK using phenformin provided further protection.

另一方面,在那些腺苷酸活化蛋白激酶似乎充当肿瘤启动子的癌症小鼠模型中,可以说腺苷酸活化蛋白激酶的基因敲除通常发生在肿瘤发生的同时,甚至在发生之后。例如,Kishton 等人对 T-ALL 的研究[173](5.2)中,转化是通过迫使 Notch1致癌突变体在体外表达而产生的,然后将 T-ALL 细胞转移到受照射的小鼠体内,在 AMPK 被三苯氧胺击倒之前,疾病得以建立。将这个模型与我们最近发表的 T-ALL [167]模型进行比较尤其具有指导意义,在这个模型中 AMPK-1在淋巴瘤发生之前已经在 t 细胞祖细胞中被特异性敲除,在这个模型中,基础 AMPK 显然对淋巴瘤的发展具有保护作用,并且使用苯乙双胍激活 AMPK 提供了进一步的保护。

Coming to other mouse studies that support a tumour promoter role for AMPK, in the autochthonous model of non-small cell lung cancer [178], knockout of AMPK would have occurred simultaneously with expression of mutant K-Ras and loss of p53, which may have been sufficient to trigger tumorigenesis on their own. The only study that supported a tumour promoter role but where AMPK had been knocked out prior to disease onset was the model of AML by Saito et al. [174]. However in that case (as in the study of T-ALL by Kishton et al. [173]) transformation had been achieved by enforced expression of oncogenes in vitro in haematopoietic progenitor cells, and the real test of the role of AMPK was in the survival and/or proliferation of the leukaemia cells in vivo in irradiated recipient mice. It can be argued that these two studies, by carrying out transformation in vitro, may have been less likely to detect a tumour suppressor role of AMPK.

在其他支持 AMPK 启动子作用的小鼠研究中,在非小细胞肺癌的原生模型中,AMPK 基因的敲除可能与突变的 K-Ras 基因的表达和 p53基因的丢失同时发生,这可能足以触发肿瘤发生。唯一支持肿瘤促进剂的作用,但 AMPK 在疾病发作前已被淘汰的研究是斋藤等人的 AML 模型[174]。然而,在这种情况下(如 Kishton 等人对 T-ALL 的研究[173]) ,转化是通过在造血祖细胞中体外强制表达癌基因实现的,而 AMPK 的真正作用是在辐射受体小鼠体内白血病细胞的存活和/或增殖中发挥作用。可以认为,这两项研究,通过进行转化在体外,可能已经不太可能检测到肿瘤抑制作用的 AMPK。

Overall we propose that, when loss of AMPK occurs prior to initiation of tumorigenesis in vivo, this would remove the restraints on the mTORC1 pathway and unleash other biosynthesis processes and the cell cycle, thus transforming the cells into a metabolic and proliferative state that is primed for tumour formation. Under these circumstances, AMPK acts as a tumour suppressor, and AMPK activators may provide additional protection against tumorigenesis, such as the effect of phenformin in T-ALL [167]. These results suggest that AMPK activators might one day find a place in providing protection against cancer, perhaps in individuals who are at high risk of developing the disease. If biguanides are used, it might also make sense to use phenformin which, being more membrane permeable than metformin even in the absence of a transporter, is much more likely to activate AMPK in the tumour progenitor cells. Although phenformin was withdrawn for treatment of type 2 diabetes because of the risk of life-threatening lactic acidosis, the risk of this complication was actually quite low (≈64 cases per 100 000 patient-years [188]), and might be more acceptable in the context of cancer rather than diabetes. Alternatively, some of the other AMPK activators discussed in §3 might perhaps be developed for this purpose.

总的来说,我们认为,当 AMPK 的缺失发生在体内肿瘤发生之前,这将消除对 mTORC1通路的束缚,并释放其他生物合成过程和细胞周期,从而将细胞转化为一种新陈代谢和增殖状态,为肿瘤的形成做好准备。在这些情况下,AMPK 作为肿瘤抑制剂,AMPK 激活剂可能提供额外的保护,对肿瘤发生,如苯乙双胍的影响 T-ALL [167]。这些结果表明,腺苷酸活化蛋白激酶有朝一日可能在预防癌症方面找到一席之地,也许在那些患癌症风险较高的个体身上。如果使用双胍类药物,也可以使用苯乙双胍,即使在没有转运蛋白的情况下,苯乙双胍比二甲双胍具有更高的膜通透性,更有可能激活肿瘤祖细胞中的 AMPK。虽然苯乙双胍由于存在危及生命的乳酸酸中毒风险而停止用于2型糖尿病的治疗,但这种并发症的风险实际上相当低(≈每10万病人-年龄64例[188]) ,而且可能更适用于癌症,而不是糖尿病。另外,在3中讨论的其他一些 AMPK 激活剂也许可以用于这个目的。

We also propose that, once the cancer cells have started to grow in vivo, AMPK switches from being a tumour suppressor to a tumour promoter (like the transformation of the benevolent Dr Jekyll into the malevolent Dr Hyde in Stevenson’s novel!). Under these circumstances, the role of AMPK is to protect the cell in which it is expressed, irrespective of whether that cell is a cancer cell or a normal cell. By protecting cancer cells against stresses such as shortage of oxygen or nutrients, or oxidative or genotoxic stress, AMPK would enhance their survival and thus, in the long term, promote growth of tumours. Under these circumstances, AMPK is acting as a tumour promoter, which suggests that AMPK inhibitors might be efficacious in treatment of cancer. They may be particularly effective: (i) in cases where the PRKAA1 or PRKAB2 genes are amplified, causing AMPK over-expression; (ii) when given in combination with genotoxic treatments such as etoposide or radiotherapy, thus reducing the viability of tumour cells during such therapies. At present we do not have any well-characterized and specific inhibitors of AMPK (see §3.4), but future work can be directed at correcting that deficiency.

我们还认为,一旦癌细胞开始在体内生长,AMPK 就从一个肿瘤抑制基因转变为一个肿瘤促进基因(就像斯蒂文森小说中善良的杰基尔博士转变为恶毒的海德博士一样).在这些情况下,AMPK 的作用是保护其表达的细胞,不管该细胞是癌细胞还是正常细胞。通过保护癌细胞免受氧气或营养缺乏、氧化或基因毒性应激等压力,AMPK 可以提高癌细胞的存活率,从长远来看,促进肿瘤的生长。在这些情况下,AMPK 是作为一个肿瘤促进剂,这表明 AMPK 抑制剂可能是有效的治疗癌症。它们可能特别有效: (i)在 PRKAA1或 PRKAB2基因被放大,导致 AMPK 过度表达的情况下; (ii)与依托泊苷或放疗等基因毒性治疗结合使用时,从而降低肿瘤细胞在此类治疗中的活力。目前我们还没有任何特异性的 AMPK 抑制剂(见3.4) ,但是未来的工作可以直接纠正这一缺陷。



Recent studies in our laboratory involving live animals were approved by the Ethics Review Committee of the University of Dundee in accordance with the UK Animal (Scientific Procedures) Act 1986.

根据1986年《英国动物(科学程序)法》 ,我们实验室最近对活体动物的研究得到了英国邓迪大学道德审查委员会的批准。


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