mTORC1在肿瘤线粒体功能调控中的作用及其治疗靶点

0 Comments

Continuous proliferation of tumor cells requires constant adaptations of energy metabolism to rapidly fuel cell growth and division. This energetic adaptation often comprises deregulated glucose uptake and lactate production in the presence of oxygen, a process known as the “Warburg effect.” For many years it was thought that the Warburg effect was a result of mitochondrial damage, however, unlike this proposal tumor cell mitochondria maintain their functionality, and is essential for integrating a variety of signals and adapting the metabolic activity of the tumor cell. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of numerous cellular processes implicated in proliferation, metabolism, and cell growth. mTORC1 controls cellular metabolism mainly by regulating the translation and transcription of metabolic genes, such as peroxisome proliferator activated receptor γ coactivator-1 α (PGC-1α), sterol regulatory element-binding protein 1/2 (SREBP1/2), and hypoxia inducible factor-1 α (HIF-1α). Interestingly it has been shown that mTORC1 regulates mitochondrial metabolism, thus representing an important regulator in mitochondrial function. Here we present an overview on the role of mTORC1 in the regulation of mitochondrial functions in cancer, considering new evidences showing that mTORC1 regulates the translation of nucleus-encoded mitochondrial mRNAs that result in an increased ATP mitochondrial production. Moreover, we discuss the relationship between mTORC1 and glutaminolysis, as well as mitochondrial metabolites. In addition, mitochondrial fission processes regulated by mTORC1 and its impact on cancer are discussed. Finally, we also review the therapeutic efficacy of mTORC1 inhibitors in cancer treatments, considering its use in combination with other drugs, with particular focus on cellular metabolism inhibitors, that could help improve their anti neoplastic effect and eliminate cancer cells in patients.

肿瘤细胞的持续增殖需要能量代谢的不断适应,以快速燃料细胞的生长和分裂。这种能量适应通常包括在氧气存在下解除对葡萄糖的摄取和乳酸盐的产生,这个过程被称为“瓦伯格效应”多年来,人们认为瓦伯格效应是线粒体损伤的结果,然而,不同于这个提议,肿瘤细胞线粒体维持其功能,并对整合各种信号和适应肿瘤细胞的代谢活动至关重要。哺乳动物/机械靶雷帕霉素复合物1(mTORC1)是许多细胞过程的主要调节因子,涉及增殖、代谢和细胞生长。mTORC1主要通过调节过氧化物酶体增殖物激活受体 γ 辅活化子 -1α (pgc-1α)、固醇调节元件结合蛋白1/2(SREBP1/2)和缺氧诱导因子 -1α (hif-1α)等代谢基因的翻译和转录来控制细胞代谢。有趣的是,已经证明 mTORC1调节线粒体代谢,从而代表了线粒体功能的一个重要调节因子。本文综述了 mTORC1在肿瘤线粒体功能调节中的作用,同时考虑到新的证据表明 mTORC1调节细胞核编码的线粒体 mRNAs 的翻译,从而导致 ATP 线粒体产量的增加。此外,我们还讨论了 mTORC1与谷氨酰胺溶解以及线粒体代谢产物之间的关系。此外,还讨论了 mTORC1调节的线粒体分裂过程及其对癌症的影响。最后,我们也回顾了 mTORC1抑制剂在癌症治疗中的疗效,考虑到它与其他药物的联合使用,特别关注细胞代谢抑制剂,这可能有助于提高它们的抗肿瘤效果和消除患者的癌细胞。

Introduction

引言

Cellular metabolism involves a set of highly coordinated activities in which numerous enzymes collaborate to convert nutrients into building blocks toward generation of macromolecules, energy, and cellular biomass. In cancer, genetic, and epigenetic changes can disturb key enzymes or rewire oncogenic pathways, resulting in cell metabolism alterations (1). In 1924 Otto Warburg observed that tumor cells prefer aerobic glycolysis to generate ATP and lactate even in presence of oxygen, process known as the “Warburg effect” (2). For a long time it was believed that this preference for the Warburg effect was due to a failure in the mitochondrial function. Nevertheless, in recent years, there were significant progresses in our understanding of metabolic regulation in cancer and contrariwise, it was demonstrated that cancer cells have a functional mitochondrion. Furthermore, it was shown that oxidative phosphorylation (OXPHOS) is crucial for ATP production and tumor progression (3). However, mitochondria perform many functions beyond energetic production, including generation of redox molecules and the regulation of cell signaling, cell death, biosynthetic metabolism, and generation of reactive oxygen species (ROS) (4).

细胞新陈代谢涉及到一系列高度协调的活动,在这些活动中,许多酶协作将营养物质转化为生成大分子、能量和细胞生物量的积木。在癌症中,基因和表观遗传的改变可以扰乱关键酶或重新连接致癌途径,导致细胞代谢的改变。1924年,奥托 · 沃伯格观察到,即使在有氧环境中,肿瘤细胞也更喜欢有氧糖酵解来产生 ATP 和乳酸,这一过程被称为“沃伯格效应”(2)。长期以来,人们认为这种对沃伯格效应的偏爱是由于线粒体功能的失败。尽管如此,近年来我们对癌症代谢调控的研究取得了重大进展,而相反,癌细胞具有功能性的线粒体。此外,研究表明氧化磷酸化对于 ATP 的产生和肿瘤的进展至关重要。然而,除了能量产生之外,线粒体还有许多其他功能,包括产生氧化还原分子、调节细胞信号、细胞死亡、生物合成代谢以及产生活性氧类。

Mitochondrial ROS are the byproducts of metabolic processes during which electrons escape from the mitochondrial electron transport chain and then are captured by molecular oxygen to generate superoxide anions (O−2O2-) (5). Mitochondrial ROS exhibit both, a tumor promoting or tumor suppressing roles, depending on their levels and their oxidative potential. ROS are highly reactive species that produce oxidized proteins, lipids and nucleic acids, either behaving as damaging or as signaling species in cell metabolism. For instance, low levels of ROS have a pronounced proliferative effect but high levels induce tissue damage and consequently cell death (6). Despite the potential damaging roles of high ROS, cancer cells posses ROS-scavenging systems aimed to maintain ROS homeostasis, being the two major players Glutathione (GSH) and Thioredoxin (Txn) (7). Mitochondrial functions confer high levels of cellular plasticity, which permits a fast adaptation to challenging microenvironments conditions, such as hypoxia and nutrient deficiency, two very common consequences in tumors (8). On the other hand, accumulation of damaged mitochondria can be dangerous to cells; mitochondrial quality and quantity are processes severely monitored to ensure balance in cell physiology (9). Damaged or unwanted mitochondria can be selectively removed by mitophagy, a lysosome-dependent catabolic degradation process (10). Mitochondrial functions are matched by their morphological and structural changes, during the lifetime of a cell, the mitochondrial homeostasis network is constantly shaped by fission and fusion events (11).

线粒体活性氧是电子从线粒体电子传递链逃逸,然后被分子氧捕获产生超氧阴离子(o-2 O2 -)的代谢过程的副产品。线粒体活性氧具有促进或抑制肿瘤的双重作用,这取决于它们的水平和氧化能力。活性氧是一种高活性物质,可以产生氧化蛋白质、脂类和核酸,在细胞代谢中起破坏或信号物质的作用。例如,低水平的活性氧有明显的增殖作用,但高水平的活性氧会引起组织损伤,从而导致细胞死亡。尽管高活性氧具有潜在的破坏作用,但癌细胞具有旨在维持活性氧稳态的 ROS- 清除系统,是谷胱甘肽(GSH)和硫氧还蛋白(Txn)的两个主要参与者。线粒体功能赋予高水平的细胞可塑性,这使得细胞能够快速适应具有挑战性的微环境条件,如缺氧和营养缺乏,这是肿瘤中两个非常常见的后果(8)。另一方面,受损线粒体的积累可能对细胞有危险,线粒体的质量和数量都受到严格的监控,以确保细胞生理平衡。受损或不需要的线粒体可以通过噬菌体选择性地去除,这是一个依赖于溶酶体的分解代谢过程(10)。线粒体的功能与其形态和结构的变化相匹配,在一个细胞的生命周期中,线粒体内稳态网络不断地由分裂和融合事件形成(11)。

In the process of tumor initiation and progression, cancer cells are exposed to harsh condition such as hypoxia or nutrient depletion in the tumor microenvironment. To survive in this severe environment, cancer cells must sense, and respond to the status of nutrient availability in the extracellular environment. The cell has several nutrients sensors responsible for maintaining cell homeostasis with the extracellular environment, such as the mammalian/mechanistic target of rapamycin complex 1 (mTORC1) that drives ATP-consuming cellular processes (anabolic) necessaries for cellular proliferation and growth (12). Another important sensor is the serine/threonine kinase AMP-activated protein kinase (AMPK), which, as its name implies, senses cellular AMP levels and coordinate a metabolic switch from anabolism toward catabolism under energy deprivation conditions such as hypoxia and hypoglycemia (13). AMPK has a wide variety of cell targets, one of which is mTORC1. AMPK activation suppresses mTORC1 signaling, thus regulating energy metabolism by stimulating the activity of several transcriptional controllers of metabolic enzymes such as peroxisome proliferator activated receptor γ coactivator-1 α (PGC-1α), sterol regulatory element-binding protein 1/2 (SREBP1/2), and hypoxia inducible factor-1 α (HIF-1α) (14). Interestingly, has been shown that mTORC1 also regulates mitochondrial oxidative metabolism (1517). Moreover, mitochondrial oxidative metabolism is a very important mechanism for cancer development, acquired resistance against chemotherapy, and increased hypoxia tolerance in tumor microenvironment.

在肿瘤的发生发展过程中,肿瘤细胞暴露于恶性环境,如肿瘤微环境中缺氧或营养缺乏。为了在这种恶劣的环境中生存,癌细胞必须感觉到,并对细胞外环境中营养物质的可获得性作出反应。细胞有几个营养传感器负责维持细胞与细胞外环境的平衡,如哺乳动物/机械目标雷帕霉素复合物1(mTORC1) ,驱动 atp 消耗细胞过程(合成代谢)所需的细胞生长和生长。另一个重要的传感器是丝氨酸/苏氨酸激酶 AMP活化蛋白激酶(AMPK) ,正如它的名字所暗示的那样,它能感知细胞内的 AMP 水平,并协调代谢从合成代谢向分解代谢转换,在能量缺乏条件下,如缺氧和低血糖(13)。AMPK 具有多种细胞靶点,其中一种是 mTORC1。AMPK 激活抑制 mTORC1信号传导,从而通过刺激代谢酶的几个转录调控者如过氧化物酶体增殖物激活受体 γ 辅激活因子 -1α (pgc-1α)、固醇调节元件结合蛋白1/2(SREBP1/2)和缺氧诱导因子 -1α (hif-1α)(14)的活性来调节能量代谢。有趣的是,已经证明 mTORC1也调节线粒体唿吸作用(15-17)。此外,线粒体唿吸作用是一个非常重要的机制癌症的发展,获得性耐药化疗,并增加耐缺氧的肿瘤微环境。

In this review we explain the participation of mTORC1 in the regulation of mitochondrial ATP producing capacity and we discuss how this process affects tumor cells. On the other hand, the mitochondrial function is directly associated with mitochondrial morphology regulated through fusion and fission processes thus, we review the current knowledge about the relationship of mitochondrial morphology highlighting mTORC1 participation in cancer. On the other hand it is known that glutamine, the most abundant free amino acid in blood, is uptaked by tumor cells and converted into α-ketoglutarate (α-KG) that fuels the tricarboxilic acid (TCA) cycle and OXPHOS in tumor mitochondrial. Therefore, we discuss how glutamine and mTORC1 participate in tumor development. Additionally, it was shown that mutations in nuclear and mitochondrial DNA lead to deregulation of important metabolic enzymes promoting the accumulation of intermediary metabolites, known as oncometabolites which in turn support cancer development. In this review, we depict the role of mTORC1 in the regulation of oncometabolites, as well as the therapeutic efficacy of mTORC1 inhibitors in cancer treatment.

在这篇综述中,我们解释 mTORC1参与调节线粒体 ATP 产生能力,并讨论这一过程如何影响肿瘤细胞。另一方面,线粒体功能与线粒体形态的融合和分裂过程直接相关,因此,我们回顾了目前关于线粒体形态与 mTORC1参与癌症的关系的知识。另一方面,血液中最丰富的游离氨基酸谷氨酰胺被肿瘤细胞向上转化为 α- 酮戊二酸(α-kg) ,为肿瘤线粒体中的三羧酸(TCA)循环和 OXPHOS 提供动力。因此,我们讨论谷氨酰胺和 mTORC1如何参与肿瘤的发展。此外,研究表明,核和线粒体脱氧核糖核酸的突变会导致重要的代谢酶失调,促进中间代谢物的积累,这种代谢物被称为 oncometabolites,反过来又支持癌症的发展。本文综述了 mTORC1在肿瘤代谢调控中的作用,以及 mTORC1抑制剂在肿瘤治疗中的作用。

Structure and Functions of mTORC1

mTORC1的结构与功能

The protein serine threonine kinase TOR (target of rapamycin) was initially identified in Saccharomyces cerevisiae as a target of the macrolide fungicide rapamycin and later, the mammalian counterpart was identified and named mammalian/mechanistic target of rapamycin (mTOR), also known as FRAP (FKBP12-rapamycin-associated protein), RAFT (rapamycin and FKB12 target), and RAPT1 (rapamycin target 1) (1819). TOR is a large (~280 kDa) serine/threonine protein kinase that belongs to the family of phosphoinositide 3-kinase (PI3K)-related kinase (20). The mTOR protein interacts with other proteins and form two distinct multiprotein complexes: mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2), either one with a different sensitivities to rapamycin (21). mTORC1 is inhibited by rapamycin, while mTORC2 is resistant to acute rapamycin treatment, however in some types of cells (HeLa and PC3) this mTORC2 complex can be inhibited by longer rapamycin treatments (over 24 h) (22).

蛋白丝氨酸苏氨酸激酶 TOR (雷帕霉素靶蛋白)最初在酿酒酵母中被确定为大环内酯类杀菌剂雷帕霉素的靶蛋白,后来,这种哺乳动物被确定为雷帕霉素靶蛋白(mTOR) ,也称为 FRAP (fkbp12雷帕霉素相关蛋白)、 RAFT (雷帕霉素和 FKB12靶蛋白)和 RAPT1(雷帕霉素靶蛋白1)(18,19)。TOR 是一种大型(约280kda)丝氨酸/苏氨酸蛋白激酶,属于磷脂酰肌醇3- 激酶(PI3K)相关激酶家族(20)。mTOR 蛋白与其他蛋白质相互作用,形成两种不同的多蛋白复合物: mTORC1复合物和 mTORC2复合物。mTORC1受雷帕霉素抑制,mTORC2对雷帕霉素急性作用耐受,但在某些类型的细胞(HeLa 和 PC3)中,雷帕霉素处理时间延长(24小时以上)可抑制 mTORC2复合体。

mTORC1 is composed by the regulatory-associated protein of mTOR (Raptor), a scaffolding protein important for mTORC1 assembly, stability, substrate specificity and regulation (23), and by the proline-rich AKT substrate 40 kDa factor protein (PRAS40) (24), that associates with Raptor and inhibits mTORC1 activity. mTORC2 complex is composed by the rapamycin-insensitive companion (Rictor) (25), a component essential for both, complex formation, and their biological function, the mammalian stress-activated map kinase-interacting protein 1 (mSin1) an essential component required for complex formation and kinase activity (26), and by Protor 1 (Protein observed with Rictor 1), required to allow efficient regulation of mTORC2 targets (27). Both mTORC1 and mTORC2 are composed by mTOR protein, a mammalian lethal with sec13 protein 8 (mLST8, also known as GβL), DEP domain-containing mTOR interacting protein (DEPTOR), and Tel two interacting protein 1 (TTI1/TEL2) complex. mLST8 is associated with the catalytic domain of mTOR and may stabilize the kinase activation loop, DEPTOR on the contrary inhibits mTOR activity, TTI1/TEL2 is a mTOR interacting protein important for mTOR stability and assembly of the mTOR complex and maintain their activities (28) (Figure 1).

mTORC1是由 mTORC1的调节相关蛋白 mTOR (Raptor)和富含脯氨酸的 AKT 底物40kda 因子蛋白(PRAS40)(24)组成,mTORC1是一种对 mTORC1的组装、稳定性、底物特异性和调节起重要作用的支架蛋白。mTORC2复合物是由雷帕霉素不敏感伴侣(Rictor)(25)和 Protor 1(与 Rictor 1共同观察到的蛋白质)组成的,前者是复合物形成和生物功能的基本组成部分,哺乳动物应激激活的 map 激酶相互作用蛋白1(mSin1)是复合物形成和激酶活性的基本组成部分(26) ,后者是有效调控 mTORC2靶标(27)所必需的。mTORC1和 mTORC2都是由哺乳动物中与 sec13蛋白8(mLST8,又称 GβL)、 DEP 结构域的 mTOR 相互作用蛋白(detor)和 Tel 2相互作用蛋白1(TTI1/TEL2)复合物构成的。mLST8与 mTOR 的催化结构域有关,可以稳定激酶活化环,而 DEPTOR 则抑制 mTOR 活性,TTI1/TEL2是 mTOR 相互作用蛋白,对 mTOR 复合物的稳定和组装以及维持其活性至关重要(图1)。FIGURE 1 图1

Figure 1. Mechanistic target of rapamycin complex 1 (mTORC1) and regulation of cellular processes. mTORC1 is a complex with DEPTOR and PRAS40 as negative regulators and RAPTOR and mSLT8 as positive regulators. Rapamycin-FKBP12 inhibits the mTOR kinase by directly blocking substrates recruitment and by further restricting active-site access. mTORC1 regulates different cellular processes such as ribosomal biogenesis, mRNA translation, autophagy, lipid and nucleotide synthesis, and mitochondrial functions.

图1。雷帕霉素复合物1(mTORC1)的机制靶点及其对细胞过程的调控。mTORC1是一个以 DEPTOR 和 PRAS40为负调节剂,RAPTOR 和 mSLT8为正调节剂的复合物。雷帕霉素 fkbp12通过直接阻断底物补充和进一步限制活性位点通路来抑制 mTOR 激酶。mTORC1调节不同的细胞过程,如核糖体的生成、 mRNA 的翻译、自噬、脂质和核苷酸的合成以及线粒体的功能。

mTORC1 is activated via growth factors stimulation [epidermal growth factor (EGF), insulin-like growth factor (IGF)], increase in amino acid levels such as leucin and arginine and cellular energy status (2931), promoting protein and lipid synthesis, as well as ribosome biogenesis impacting on cell proliferation and growth, autophagy and metabolic processes are also stimulated by mTORC1 action (32). Moreover, it was demonstrated that mTORC1 signaling is strongly implicated in the aging process of diverse organisms, including yeast, worms flies, and mammals (33).

mTORC1通过刺激生长因子[ EGF (表皮生长因子) ,胰岛素样生长因子(IGF)]激活,增加氨基酸水平,如亮氨酸和精氨酸和细胞能量状态(29-31) ,促进蛋白质和脂质合成,以及核糖体生物合成影响细胞增殖和生长,自噬和代谢过程也受到 mTORC1作用(32)的刺激。此外,还证明了 mTORC1信号与多种生物的衰老过程密切相关,包括酵母、蠕虫、苍蝇和哺乳动物(33)。

On the other hand, mTORC2 is activated by growth factors but unlike mTORC1 its activity is not regulated by amino acids. mTORC2 phosphorylates PKC-α, AKT/PKB (Ser473) and paxillin (focal adhesion-associated adaptor protein) (Tyr118), to regulate the activity of the small GTPases Rac and Rho, controlling cell survival and cytoskeletal organization and cell migration (34).

另一方面,mTORC2被生长因子激活,但不像 mTORC1,它的活性不受氨基酸的调节。mTORC2磷酸化 pkc-α、 AKT/PKB (Ser473)和 paxillin (collagen-associated adaptor protein)(Tyr118) ,调节小 GTPases Rac 和 Rho 的活性,控制细胞存活、细胞骨架组织和细胞迁移(34)。

Regulation of mTORC1 Signaling in Cancer

mTORC1信号通路在肿瘤中的调控作用

The mTORC1 is often deregulated in numerous cancer types, such as breast, cervical cancer, esophageal squamous cell carcinoma, lung and hepatic cancers (3539). mTORC1 is often activated by mutations in its upstream regulators. These include gain-of-function mutation of PI3K and loss-of-function mutation of the tumor suppressor PTEN (40). In a number of in vitro cell-lines and in vivo murine xenograft models, it has been demonstrated that aberrant mTORC1 contributes to tumor growth, angiogenesis, invasion and metastasis (41). Given its key role in the regulation of process associated with cell growth and metabolism in cancer, specifically with the mitochondrial functions, we focus on mTORC1.

mTORC1常常在许多类型的癌症中被解除,如乳腺癌、宫颈癌、食管鳞状细胞癌、肺癌和肝癌(35-39)。mTORC1通常被其上游调节子的突变所激活。这些突变包括 PI3K 功能增强突变和抑癌基因 PTEN (40)功能缺失突变。在许多体外细胞系和体内小鼠异种移植模型中,已证实异常 mTORC1参与肿瘤生长、血管生成、侵袭和转移(41)。鉴于其在调节癌细胞生长和代谢过程中的关键作用,特别是线粒体功能,我们重点研究 mTORC1。

It has been shown that mTORC1 is regulated by growth factors through the phosphoinositide 3-kinase/protein kinase B, also known as Akt (PI3K/PKB or Akt) pathway and by Ras/mitogen-activated protein kinase (MAPK) pathway (42). Binding of insulin or insulin-like growth factor (IGF) to their receptor lead to recruitment and phosphorylation of the insulin receptor substrate and subsequent recruitment of PI3K. PI3K phosphorylates the inositol ring of the membrane phospholipid, phosphatidylinositol-4,5-biphosphate (PI-4,5-P2) to generate phosphatidylinositol-3,4,5-trisphosphate (PIP3) at the cytoplasmic side of the cellular membrane (43). PIP3 recruits a subset of pleckstrin homology (PH) domain-containing proteins, such as the same protein kinase Akt and constitutively active phosphoinositide-dependent kinase 1 (PDK1) (4445). In turn PDK1 phosphorylates Akt in T308 (46), however the maximal activation of Akt requires its additional phosphorylation on S473 located at the carboxyl-terminus site, mediated by mTORC2 (47). Akt inhibits the tuberous sclerosis complex (TSC) that limit the mTORC1 signaling, TSC complex is composed by three subunits: TSC1 (Harmatin), TSC2 (Tuberin), and TBC1D7 (4849). Akt phosphorylate TSC2 on five residues (S939, S981, S1130, S1132, and T1462) leading to its inactivation (5051). The TSC complex is a negative regulator of the small GTPase Rheb (Ras homolog enriched in brain) (52), via stimulation of GTP hydrolysis. On the other hand Rheb-GTP is translocated to the lysosomal membrane, where directly interacts with the catalytic domain of mTOR promoting its activation (53). Once mTORC1 is activated, positively controls cell growth through stimulation of protein synthesis by induction the phosphorylation of its two main targets, the eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and the ribosomal protein S6 kinase (S6K). Raptor-mTOR binds to S6K and 4E-BP1 through their respective TOR signaling (TOS) motifs (5455) enhancing translation of proteins involved in the control of cell growth/size and cell cycle progression.

研究表明,mTORC1通过磷酸肌醇3- 激酶/蛋白激酶 b 途径(又称 Akt (PI3K/PKB 或 Akt))和 ras/MAPK 途径(42)受生长因子调节。胰岛素或胰岛素样生长因子与其受体结合导致胰岛素受体底物的补充和磷酸化,并随后补充 PI3K。PI3K 磷酸化膜磷脂的肌醇环,磷酸化磷脂酰肌醇 -4,5-二磷酸(PI-4,5-P2) ,在细胞膜胞质侧产生磷脂酰肌醇 -3,4,5-三磷酸(PIP3)。PIP3招募一个包含 pleckstrin 同源(PH)结构域蛋白的子集,如同样的蛋白激酶 Akt 和组成活性磷酸肌醇依赖性激酶1(PDK1)(44,45)。反过来 PDK1又使 T308(46)中的 Akt 磷酸化,但 Akt 的最大激活需要其位于羧基末端的 S473上的额外磷酸化作用,这是由 mTORC2(47)介导的。Akt 抑制了限制 mTORC1信号转导的结节性硬化症,TSC 复合物由三个亚基组成: TSC1(Harmatin) ,TSC2(Tuberin)和 TBC1D7(48,49)。5个残基(S939、 S981、 S1130、 S1132和 T1462)上的 Akt 磷酸化 TSC2,导致 TSC2失活(50、51)。TSC 复合物通过刺激 GTP 水解,是小 GTPase Rheb (脑内富集的 Ras 同系物)(52)的负性调节因子。另一方面,Rheb-GTP 位于溶酶体膜上,直接与 mTOR 的催化结构域相互作用,促进其活化(53)。一旦 mTORC1被激活,通过诱导其两个主要靶点—- 真核起始因子4 e 结合蛋白1(4E-bp1)和核糖体蛋白质 s 6激酶(S6K)的磷酸化,通过刺激蛋白质合成积极地控制细胞生长。Raptor-mTOR 通过各自的 TOR 信号(TOS)序列(54,55)与 S6K 和4E-BP1结合,促进涉及控制细胞生长/大小和细胞周期进程的蛋白质翻译。

The 4E-BPs are small (~15–20 kDa) proteins that interact with eukaryotic translation initiation factor 4E (eIF4E) inhibiting translation initiation, this being a very important regulation point in protein translation. Although there are three 4E-BPs known isoforms in mammals (4E-BP 1, 2, and 3), most studies had focus on 4E-BP1. mTORC1 phosphorylates 4E-BP1 in Thr37/Thr46, followed by Thr70, and finally Ser65 (56). Phosphorylation of Thr70 and Ser65 are part of the response to extracellular signals such as serum stimulation. Phosphorylation of all of these sites inhibits 4E-BPs’ binding to eIF4E. The 4E-BPs prevents the formation of the translation initiation complex (eIF4F) by competing with eIF4G for binding to the dorsal side of eIF4E and reduces cap-dependent translation initiation (57). On the other hand the ribosomal protein S6 kinase (rpS6) known as S6K was first identified in unfertilized Xenopus laevis eggs as a 90 kDa polypeptide, termed p90 or rpS6 kinase (RSK, also known as p90RSK) (58). Later a protein with a molecular weight of 65–70 kDa was purified from chicken embryos and 3T3 cells, and referred to as S6K (59). Mammalian cells express both S6K1 and S6K2 also known as S6Kα and S6Kβ, respectively, which are encoded by two different genes and share a very high level of overall sequence homology (60). S6K1 has cytosolic and nuclear isoforms (p70 S6K1 and p85 S6K1, respectively) (61), whereas both S6K2 isoforms (p54 S6K2 and p56 S6K2) are primarily nuclear. S6K was identified as the main kinase responsible for ribosomal protein S6 phosphorylation (60), S6K regulates the mRNA biogenesis, translation initiation, and elongation.

4E-BPs 是一种小型蛋白质,约15-20kda,与抑制翻译起始的真核起始因子4E 蛋白(eIF4E)相互作用,这是蛋白质翻译中一个非常重要的调控点。虽然在哺乳动物中有三种已知的4E-BP1(4E-BP 1、2和3) ,但大多数研究都集中在4E-BP1上。mTORC1在 Thr37/Thr46中磷酸化4E-BP1,其次是 Thr70,最后是 Ser65(56)。Thr70和 Ser65的磷酸化是细胞外信号(如血清刺激)反应的一部分。所有这些位点的磷酸化都会抑制4E-BPs 与 eIF4E 的结合。4E-BPs 通过与 eIF4G 竞争结合到 eIF4E 的背侧来阻止翻译起始复合物(eIF4F)的形成,并减少了依赖于翻译起始帽的翻译起始(57)。另一方面,在未受精的非洲爪蟾卵中首次发现核糖体蛋白质 S6激酶(rpS6) ,称为 p90或 rpS6激酶(RSK,也称为 p90RSK)(58)。随后从鸡胚和3T3细胞中分离纯化出一种分子量为65-70kda 的蛋白质,称为 S6K (59)。哺乳动物细胞同时表达 S6K1和 S6K2,又称 s6kα 和 s6kβ,它们由两个不同的基因编码,具有很高的全序列同源性(60)。S6K1具有胞质型和核型(分别为 p70 S6K1和 p85 S6K1)(61) ,而 S6K2亚型(p54 S6K2和 p56 S6K2)均为核型。S6K 被认为是负责核糖体蛋白质 S6磷酸化的主要激酶,S6K 调节 mRNA 的生物合成、翻译起始和延伸。

Mitochondria and Cancer

线粒体与癌症

In addition to genetic aberrations, tumor cells rewiring their metabolism, such as aerobic glycolysis, glutamine uptake, accumulation of intermediates of glycolysis, and upregulation of lipid and amino acid synthesis, and OXPHOS, enable support their high demands on nutrients for building blocks and energy production (62). In cancer development tumor cells reprogram their metabolism to guarantee survival and proliferation in an often nutrient-scare and stressful microenvironment. (40). Moreover, several findings demonstrate that mutations in oncogenes and /or tumor suppressor genes can mediate metabolic rewiring in cancer cells to support the high demands for building blocks and energy production (63). Tumor cells acquire a metabolic plasticity that allows alternate between aerobic glycolysis and OXPHOS in order to maintain malignant phenotypes, such as a chemotherapy resistance, tumor invasion, and metastasis, and mitochondria play a central role in this dynamic (64).Changes in mitochondrial respiration rates are accompanied by changes in mitochondrial mass, the rate of fission, fusion, mitochondria biogenesis and mitophagy as well as mtDNA copy number, transcription and translation (64). In recent years, several evidences have established the role of mTORC1 as a central regulatory node in such events, which coordinates energy consumption by the translation apparatus and ATP production in mitochondria (65) (Figure 2).

除了遗传异常外,肿瘤细胞还重组其代谢,例如有氧糖酵解、谷氨酰胺摄取、糖酵解中间体的积累、脂质和氨基酸合成的上调以及 OXPHOS,这些都使肿瘤细胞能够支持其对构建模块和能量生产所需的营养物质的高需求。在癌症的发展过程中,肿瘤细胞通过重新编程它们的新陈代谢来保证在营养稀缺和紧张的微环境中生存和增殖。(40).此外,一些研究结果表明,癌基因和/或肿瘤抑制基因的突变可以介导癌细胞的新陈代谢重组,以支持对积木和能量产生的高要求。肿瘤细胞获得了一种代谢可塑性,可以在有氧糖酵解和 OXPHOS 之间进行交替,以维持恶性表型,如化疗抵抗力、肿瘤侵袭和转移,而线粒体在这种动力中起着中心作用(64)。线粒体呼吸速率的变化伴随着线粒体质量、分裂、融合速率、线粒体生物发生和吞噬以及线粒体 dna 拷贝数、转录和翻译的变化(64)。近年来,一些证据已经证实 mTORC1在此类事件中起着中枢调控节点的作用,它通过翻译装置协调能量消耗和线粒体 ATP 生成(65)(图2)。FIGURE 2 图2

Figure 2. Mechanistic target of rapamycin complex 1 (mTORC1) as a regulator of mitochondrial functions. mTORC1 can be activated by growth factor, and can regulate the mitochondrial biogenesis, mitophagy, fission and fusion processes, glutaminolysis, and mitochondrial oncometabolites generation.

图2。雷帕霉素复合物1(mTORC1)作为线粒体功能调节剂的机制靶点。mTORC1可被生长因子激活,可调节线粒体的生物发生、吞噬、分裂融合、谷氨酰胺分解和线粒体共代谢产物的生成。

It has been demonstrated that the role of mitochondria in cancer can vary depending of input genetic, environmental, and tissue-of-origin between tumors (4). The mitochondrion, contains its own DNA (mDNA) which is replicated independently of the host genome, mDNA comprises a circular genome of 16, 569 base pairs and encodes 37 genes, including 13 subunits of the electron transport chain (ETC), 2 ribosomal RNAs and 22 tRNAs, the remaining mitochondrial proteins are encoded by the nuclear genome and are imported into the mitochondria (66). Higher mtDNA copy number and mitochondrial function may confer an invasive advantage to human colorectal cancer (67).

已经证明,线粒体在癌症中的作用取决于输入基因、环境和肿瘤之间的起源组织(4)。线粒体含有自己的 DNA (mDNA) ,它独立于宿主基因组而复制,mDNA 由16,569个碱基对组成的圆形基因组组成,编码37个基因,包括13个电子传递链(ETC)亚单位、2个核糖体 rna 和22个转录因子,其余的线粒体蛋白质由核基因组编码并输入线粒体(66)。较高的线粒体 dna 拷贝数和线粒体功能可能赋予人类大肠癌侵袭优势。

Respiratory chain protein complexes (complexes I-IV) are placed into the inner membrane of mitochondria together with adenosine triphosphate (ATP) synthase, protein import machinery and transport proteins regulating metabolites passage through the matrix. The generation of ATP in mitochondria is coupled to the oxidation of NADH and FADH2, and reduction of oxygen to water (68). Abnormalities in mitochondrial complex I activity increase the aggressiveness of human breast cancer cells (69). The complex I, II and IV have all been shown to be hyperactive in human breast cancer cells; compared to tumor stromal cells and normal epithelial ductal cells (70). Interestingly it was shown that COX7RP is overexpressed in breast and endometrial cancer cells and promotes in vitro and in vivo growth by stabilizing mitochondrial supercomplex assembly even in hypoxic states, and increases hypoxia tolerance (71). Recently it was shown that OXPHOS is regulated by fascin, an actin-bundling protein that promotes lung cancer metastatic colonization by augmenting metabolic stress resistance by remodeling mitochondrial actin filaments (72).

呼吸链蛋白复合物(复合物 I-IV)与三磷酸腺苷(ATP)合成酶、蛋白质输入机制和调节代谢物通过基质传递的转运蛋白一起置于线粒体内膜。线粒体中 ATP 的产生与 NADH 和 FADH2的氧化以及氧气还原成水(68)相关。线粒体复合体 i 活性异常增加了人乳腺癌细胞的侵袭性(69)。复合物 i、 II 和 IV 在人类乳腺癌细胞中表现过度活跃,与肿瘤间质细胞和正常上皮导管细胞(70)相比。有趣的是,COX7RP 在乳腺和子宫内膜癌细胞中过度表达,在体内外通过稳定线粒体超复合体的组装来促进生长,即使在缺氧状态下,也能增强耐缺氧能力(71)。最近研究表明,OXPHOS 受 fascin 调节,fascin 是一种肌动蛋白捆绑蛋白,通过重塑线粒体肌动蛋白微丝增强代谢应激抵抗,促进肺癌转移定植。

The wide regulation of the mitochondria in cancer is of great importance and is a promising target in the development of cancer therapy (73), a number of therapeutic strategies have been based on targeting tumor mitochondrial proteins and their functions, such as metformin that has had currently a lot of impact on cancer therapy (74). Metformin induce the inhibition of OXPHOS due to reduced function of mitochondrial complex I underlies cellular and whole organism actions (75), this topic will be reviewed later in this review.

线粒体在肿瘤中的广泛调控具有重要意义,是肿瘤治疗发展中的一个有前途的靶点(73) ,许多治疗策略都是基于靶向肿瘤线粒体蛋白及其功能,如二甲双胍(74)。二甲双胍诱导 OXPHOS 的抑制,由于线粒体复合物 i 的功能下降,细胞和整个生物体的行动(75) ,这个主题将在本综述后面回顾。

mTORC1 and Mitochondrial Regulation by miRNAs in Cancer

肿瘤中 mTORC1与微 rna 对线粒体的调控

The expression of a large number of oncogenes and tumor suppressor genes is regulated by miRNAs, which altered expression, is currently though as a hallmark of cancer. miRNAs or microRNAs are small non-coding RNAs (21–25 nt), that regulate gene expression by targeting mRNAs for degradation or suppressing translation (76). In cancer, miRNAs are divided into two categories, oncogenic miRNAs and tumor suppressor miRNAs, which are up regulated and down regulated during tumorigenesis (77). According to its role as a master regulator of cell growth, mTORC1 activity is modulated by different extracellular signals and intracellular mechanisms, interestingly it has been shown that some miRNAs can also regulate the mTORC1 activity directly or through targeting upstream regulators such as PI3K/Akt pathway. For instance, miR-451 is upregulated in glioma compared with control brain tissue; furthermore decreased miR-451 expression was associated to a suppressed tumor cell proliferation via CAB39/AMPK/mTOR pathway in two glioma cell lines (78). Furthermore, over expression of miR-405 promoted caspase-3/-9 and Bax protein expression, and suppressed cyclin D1 protein expression and the PI3K/Akt/mTOR pathway inhibiting cell proliferation and promoting cell apoptosis in gastric cancer-derived cells (78). On the other hand evidence shown that mTORC1 regulates miRNAs biogenesis and given the broad function of miRNAs in cancer development, it is possible that a significant portion of mTORC1 function, may be through its ability to control miRNA biogenesis. It was shown that chronic treatment with rapamycin leads to significant alterations in miRNA profiles and these changes correlate with resistance to rapamycin. The miRNAs associated to rapamycin resistance were miR-370, miR-17-92 and its related miR-106a-92, and miR-106b-25 clusters, which have been shown to have oncogenic properties in several types of cancer (79). Ye and collaborators (2015) report that mTORC1 activation downregulates miRNA biogenesis through upregulation of Mdm2, which is a necessary and sufficient E3 ligase for ubiquitinylation of Drosha an essential RNase dedicated to processing pri-miRNA in response to the cellular environment (80). On the other hand it was shown that mTORC1 in TSC2 deficient cells, promotes the miRNA biogenesis through of GSK3β regulation. mTORC1 induces the activity of the microprocessor, a nuclear complex that includes the nuclease Drosha and its partner DGCR8, this complex cleaves the stem loop of pri-miRNA to form premiRNA via the nuclease activity of Drosha (81).

许多癌基因和肿瘤抑制基因的表达受微 rna 调控,微 rna 改变了表达,目前被认为是癌症的标志。miRNAs 或 microrna 是小型非编码 rna (21-25 nt) ,通过靶向 mRNAs 降解或抑制翻译来调节基因表达(76)。在癌症中,microrna 分为两类: 致癌性 mirna 和抑癌性 mirna,它们在肿瘤发生过程中表达上调和下调(77)。根据其作为细胞生长的主要调节因子的作用,mTORC1的活性受到不同的细胞外信号和细胞内机制的调节,有趣的是,一些微 rna 也可以直接或通过靶向上游调节因子如 PI3K/Akt 通路来调节 mTORC1的活性。例如,miR-451在神经胶质瘤中的表达比正常脑组织高,而且在两个神经胶质瘤细胞系(78)中,miR-451的表达下降与 CAB39/AMPK/mTOR 通路抑制肿瘤细胞增殖有关。miR-405的过度表达促进了 caspase-3/-9和 Bax 蛋白的表达,抑制了胃癌细胞株 cyclin D1蛋白的表达和 PI3K/Akt/mTOR 信号通路,抑制了细胞增殖,促进了细胞凋亡。另一方面,证据表明 mTORC1调节 microrna 的生物合成,并且考虑到 microrna 在癌症发展中的广泛作用,mTORC1的重要功能可能是通过其控制 miRNA 生物合成的能力。研究表明,长期雷帕霉素治疗会导致 miRNA 谱的显著改变,这些改变与雷帕霉素耐药性相关。与雷帕霉素耐药相关的 microrna 分别为 miR-370、 miR-17-92及其相关的 miR-106a-92和 miR-106b-25簇,这些分子在多种类型的癌症(79种)中都表现出致瘤性。Ye 和合作者(2015年)报告说,mTORC1的激活通过上调 Mdm2的表达来下调 miRNA 的生物合成,这是一个必要和充足的 E3连接酶,用于响应细胞环境处理 pri-miRNA (80)。另一方面,TSC2缺陷细胞中的 mTORC1通过 gsk3β 的调节促进 miRNA 的生成。mTORC1诱导微处理器的活动,这个微处理器是由核酸酶 Drosha 和它的伙伴 DGCR8组成的核复合体,这个复合体通过 Drosha (81)的核酸酶活性切断 pri-miRNA 的茎环形成 premiRNA。

On the other hand it was reported that several miRNAs targeting several mRNAs of nuclear-encoded mitochondrial proteins, integrating miRNAs into the landscape of translational regulation of mitochondrial functions such as TCA cycle, production of ROS and glutamine metabolism and mitochondrial fission process (82). miR-125a is frequently downregulated in several human cancer such as ovarian, non small-cell lung and gastric cancer and colorectal cancer (8385) Moreover low expression of miR-125a is associated with increased tumor diameter, high Ki67 expression and poor overall survival of patients with gastric carcinoma (86) Additionally miR-125a deficiency enhances agiogenic processes through metabolic reprogramming of endothelial cells (87). Interestingly it was demonstrated that miR-125a is decreased in pancreatic cancer cells (PANC-1), accompanied by an increase in the contents of mitofusin 2 (MFN2) an important regulator of mitochondrial fission. Interestingly reintroduction of miR-125a triggered mitochondrial fission via downregulation of MFN2. Excessive mitochondrial fission contributes to activation of mitochondria-dependent apoptosis and impairs cellular migration via induction of F-actin degradation (88).

另一方面,有报道指出,一些微小 rna 靶向几个核编码的线粒体蛋白质的微小 rna,将微小 rna 整合到线粒体功能的翻译调控中,如 TCA 循环、产生活性氧和谷氨酰胺代谢以及线粒体分裂过程(82)。miR-125a 在一些人类癌症如卵巢癌、非小细胞肺癌、胃癌和大肠癌(83-85)中常常下调。此外,miR-125a 的低表达与胃癌患者肿瘤直径增大、 Ki67表达增高和总体生存率低有关(86)此外,miR-125a 缺乏可通过内皮细胞的代谢重编程增强非增殖过程(87)。有趣的是,这表明 miR-125a 在胰腺癌细胞(PANC-1)中降低,同时伴随着线粒体融合蛋白2(MFN2)含量的增加,而线粒体融合蛋白2是线粒体分裂的重要调节因子。有趣的是,miR-125a 的重新引入通过降低 MFN2而触发了线粒体分裂。过多的线粒体分裂促进了线粒体依赖性凋亡的激活,并通过诱导 f- 肌动蛋白降解损害细胞迁移(88)。

miRNAs are encoded in the nuclear genome and exported to the cytosol where they perform most of their functions, however, the expression of miRNAs within the mitochondrion has been recently demonstrated, which can be either mitochondrial encoded or transcribed within the nucleus and subsequently localized to mitochondria, this miRNAs are termed as mitomiRs (89). MitomiRs are likely to contribute to some post-transcriptional regulation of gene expression related to the mitochondrial functions (90). Interestingly mitomiRs have been shown to play a very important role in chemotherapy resistance through the regulation of metabolic changes. For instance, it was demonstrated that mito-miR-2392 regulates the cisplatin resistance by reprogramming the balance between OXPHOS and glycolysis in tongue squamous cell carcinoma (TSCC) cells. Furthermore, in a retrospective analysis of TSCC patient tumor revealed a significant association of miR2392 and the expression of mitochondrial gene with chemosensitivity and overall survival (91).

miRNAs 编码在核基因组中,然后输出到细胞溶胶中,在那里它们发挥大部分功能,然而,最近证明线粒体内 miRNAs 的表达,可以是线粒体编码或转录在细胞核内,然后定位于线粒体,这种 miRNAs 称为 mitomir (89)。Mitomers 可能参与与线粒体功能相关的基因表达的转录后调控(90)。有趣的是,mitomiRs 通过调节代谢变化在化疗耐药性中发挥了非常重要的作用。例如,mito-miR-2392通过调节舌鳞癌细胞 OXPHOS 和糖酵解之间的平衡来调节顺铂耐药。此外,回顾性分析 TSCC 患者肿瘤发现 miR2392和线粒体基因表达与化疗敏感性和总生存率有显著关联(91)。

Although several cancer processes are regulated by miRNAs, there is a lacking of investigation aimed to determine the role of the mitomiRs and mTORC1 regulation either, in metabolic responses to therapy as well as mitochondrial functions, representing an open opportunity for future research.

虽然一些癌症过程是由微 rna 调节的,但是缺乏旨在确定 mitomir 和 mTORC1调节在治疗的代谢反应以及线粒体功能中的作用的研究,这为未来的研究提供了一个开放的机会。

mTORC1 Regulates Translation of Mitochondrial Proteins Encoded in the Nuclei

mTORC1调控细胞核线粒体蛋白质的翻译

Protein synthesis or mRNA translation, is the major energy-consuming process in the cell (9293). It is well-established that deregulation of mRNA translation is a prominent characteristic of cancer cells (94). Protein translation can be simplified into four stages: initiation, elongation, termination, and ribosomes recycling, however the critical regulation point occurs in the step of mRNA translation initiation, this step is regulated by several key signaling pathways, including PI3K/Akt/mTORC1 that in fact are over expressed in several neoplasms (95). The mitochondrial translation comprises the same four stages, although mitochondria have their own translation machinery with distinct mitochondrial ribosomes (mitoribosome), tRNAs and translation factors than the cytosolic counterparts. Yet the majority of the mitochondrial proteins, including all factors required for mtDNA maintenance and expression, and some components of the ETC complexes are encoded in the nuclear genome (96) and are translated in cytosolic ribosomes, and transported into mitochondria via peptides that function as import signals, this mitochondrial proteins are widely regulated via mTORC1 (97). Since mTORC1 regulates the cellular most energy consuming process, it is reasonable that mTORC1 responds to bioenergetics variation, a process controlled by mitochondria. Additionally, it was shown that mTORC1 regulates the capacity of the mitochondria to produce ATP as well as cell cycle progression in cancer cells (98).

蛋白质合成或 mRNA 翻译,是细胞中主要的能量消耗过程(92,93)。众所周知,mRNA 翻译的失调是癌细胞的一个显著特征。蛋白质翻译可以简化为起始、延伸、终止和核糖体循环四个阶段,然而关键调控点出现在 mRNA 翻译起始阶段,这一阶段受到几个关键信号通路的调控,其中包括 PI3K/Akt/mTORC1,它实际上在多个肿瘤中过度表达(95)。线粒体的翻译包括相同的四个阶段,尽管线粒体有自己的翻译机制,与细胞溶液相似的线粒体核糖体(mitoribosome)、 tRNAs 和翻译因子不同。然而,大多数线粒体蛋白质,包括线粒体 dna 维持和表达所需的所有因子,以及 ETC 复合物的一些组分,都编码在核基因组(96)中,在胞质核糖体中翻译,并通过作为输入信号的肽转运到线粒体中,这些线粒体蛋白质通过 mTORC1(97)被广泛调控。由于 mTORC1调节细胞最耗能的过程,因此 mTORC1对线粒体控制的生物能学变异作出反应是合理的。此外,研究表明 mTORC1调节线粒体产生 ATP 的能力以及癌细胞的细胞周期进程(98)。

Larsson et al. (99) evaluated the impact of different mTORC1 inhibitors in the global regulation of protein translation in MCF7 cells, interestingly, the authors found several mRNAs involved in mitochondrial functions (99). In another study, it was demonstrated that mTORC1 regulates the translation of the ATP synthase components, included ATP synthase subunit delta (ATP5D), and the transcription factor A, mitochondrial (TFAM), which promotes mitochondrial DNA replication and transcription through 4E-BPs, moreover, this was related with a higher mitochondrial activity (100). In conclusion, there is a feed-forward mechanism in the cells whereby translation of nucleus-encoded mitochondria-related mRNAs is regulated via mTORC1/4E-BP pathway to induce mitochondrial ATP production capacity and thus provide sufficient energy for protein synthesis (100). In support with this, using nano-cap analysis, which allows determination of transcription start sites on a genome-wide scale, a large number of non-TOP mRNAs were found to be mTOR sensitive (101). Among these non-TOP mRNAs, mRNAs with short 5′ UTRs were in fact mRNAs encoding for protein involved in mitochondrial functions, including components of the respiratory chain complexes (ATP50, ATP5D, UQCC2) (101).

Larsson 等(99)评估了不同 mTORC1抑制剂对 MCF7细胞蛋白质翻译全局调节的影响,有趣的是,作者发现几个 mRNAs 参与了线粒体功能(99)。在另一项研究中,证明 mTORC1调节 ATP 合成酶组分的翻译,包括 ATP 合成酶亚单位 delta (ATP5D)和转录因子 a 线粒体(TFAM) ,后者通过4E-BPs 促进线粒体脱氧核糖核酸的复制和转录,此外,这与较高的线粒体活性(100)有关。总之,在细胞中存在一种前馈机制,即通过 mTORC1/4E-BP 途径调节细胞核编码的线粒体相关 mRNAs 的翻译,以诱导线粒体 ATP 生产能力,从而为蛋白质合成提供足够的能量(100)。为了支持这一点,使用纳米帽分析,允许在全基因组范围内确定转录起始位点,大量非 top 的 mRNAs 被发现是 mTOR 敏感的(101)。在这些非 top 型 mRNAs 中,短5′ UTRs 的 mRNAs 实际上是编码参与线粒体功能的蛋白质的 mRNAs,包括呼吸链复合物(ATP50,ATP5D,UQCC2)(101)的组分。

This demonstrates that mTORC1 drives cell proliferation and neoplastic growth not only by inducing the translation of genes involved in cell growth but also by promoting the translation of mitochondrial proteins involved in cellular energy production, proteins implicated in mitochondrial DNA replication and mitochondrial repair, transcription, and translation.

这表明 mTORC1不仅通过诱导细胞生长相关基因的翻译,而且通过促进细胞能量产生所涉及的线粒体蛋白质的翻译,促进细胞增殖和线粒体修复、转录和翻译,从而推动细胞增殖和肿瘤生长。这些蛋白质涉及线粒体脱氧核糖核酸复制和线粒体。

Mitochondrial Localization of mTORC1: Regulation of the Mitochondrial Oxidative Metabolism

mTORC1的线粒体定位: 线粒体唿吸作用的调控

As described previously, mTORC1 regulates the translation of mitochondrial proteins encoded in the nucleus, however it is not the only function by which this important metabolic regulator acts. Interestingly, it has been shown that mTORC1 is found in mitochondrial fractions suggesting a regulatory ATP producing capacity.

如前所述,mTORC1调节细胞核中编码的线粒体蛋白质的翻译,然而它并不是这种重要的代谢调节因子发挥作用的唯一功能。有趣的是,有研究表明 mTORC1存在于线粒体组分中,这表明它具有调节性 ATP 生产能力。

Desai et al. (102) described the first association between mTOR and mitochondria through subcellular fractionation of human T cells. They identified that mTOR co-interact with purified mitochondria elements, and specifically mTOR is associated with the outer mitochondrial membrane. In addition, they demonstrated that when treating with mitochondrial inhibitors, the activity of mTORC1 was decreased (102). In support of these data, another study showed that mTOR-raptor complex is also present in the mitochondrial fraction of Jurkat T cells; this complex was tightly correlated with mitochondrial activity, specifically with high consumption of oxygen and mitochondrial membrane potential as well as with a higher capacity for ATP production. Moreover, disruption of the mTOR-raptor complexes with rapamycin or with RNAi resulted in a decreased mitochondrial metabolism (103).

Desai 等人(102)描述了 mTOR 和线粒体之间通过人类 t 细胞亚细胞分离的第一个联系。他们发现 mTOR 与纯化的线粒体元素相互作用,特别是 mTOR 与线粒体外膜有关。此外,他们证明,当与线粒体抑制剂处理时,mTORC1的活性降低(102)。为了支持这些数据,另一项研究表明 mTOR-raptor 复合体也存在于 Jurkat t 细胞的线粒体部分; 这种复合体与线粒体活性紧密相关,特别是与高耗氧量和线粒体膜电位以及更高的 ATP 生产能力有关。此外,破坏 mTOR-raptor 复合物与雷帕霉素或与 rna 干扰导致减少线粒体代谢(103)。

The voltage-dependent anion channels (VDACs) are pore forming proteins found in the outer mitochondrial membrane of all eukaryotes, and are the binding sites for several cytosolic enzymes, including the isoforms of hexokinase and glycerol kinase, allowing a preferential access to mitochondrial ATP (104). This mitochondrial protein is often overexpressed in several cancers, and it has been shown that VDAC1 depletion leads to a rewiring of cancer cell metabolism in breast cancer, lung cancer and glioblastoma, resulting in cell growth arrest, and tumor growth inhibition (105). Ramanathan et al. (106) showed that leukemic cells treated with rapamycin, showed a decreased mitochondrial activity. Interestingly, they found that mTOR coimmunoprecipitates with the VDAC1 and with the anti-apoptotic protein B-cell lymphoma-extra-large (Bcl-xl). They also demonstrated that mTOR phosphorylates Bcl-xl in serine 62 and increases its activity. Since Bcl-xl is a key mediator of mitochondrial function and cellular apoptosis that has been shown to bind to VDAC1 and increase the substrate permeability, its suggested that mTOR could control mitochondrial metabolism in a Bcl-xl-VDAC1 dependent manner (106). On the other hand, it was demonstrated that under radiation stress, mTOR relocates to mitochondria in MCF7, HCT116, and U87 cells, where it interacts with hexokinase II, an enzyme that phosphorylates glucose during glycolysis switching bioenergetics from aerobic glycolysis to OXPHOS which is related to an increased tumor resistance to radiation treatment (107), this interaction was also observed in another study in neonatal rat ventricular myocytes under glucose starvation (108). In another study, it was demonstrated that mTOR/Akt pathway regulates the mitochondrial respiratory activities and the expression of complex I, II and IV of the electron transport chain trough 4E-BP1 (109). Furthermore, another study suggested that mTOR-raptor may acts as a metabolic checkpoint in G1 phase of cell cycle by regulating mitochondrial function (110).

电压依赖性阴离子通道(vdac)是在所有真核生物的线粒体外膜中发现的造孔蛋白,是几种胞浆酶的结合位点,包括己糖激酶和甘油激酶的同工酶,允许优先进入线粒体 ATP (104)。这种线粒体蛋白经常在几种癌症中过度表达,已经证明 VDAC1的缺失导致乳腺癌、肺癌和胶质母细胞瘤中癌细胞代谢的重新布线,导致细胞生长停滞和肿瘤生长抑制(105)。Ramanathan 等(106)表明,经雷帕霉素处理的白血病细胞线粒体活性下降。有趣的是,他们发现 mTOR 与 VDAC1和抗凋亡蛋白 b 细胞淋巴瘤-特大(Bcl-xl)共同免疫沉淀。他们还证明 mTOR 可以磷酸化 Bcl-xl 在丝氨酸62中的表达并增加其活性。Bcl-xl 是线粒体功能和细胞凋亡的关键调控因子,与 VDAC1结合并增加底物通透性,提示 mTOR 可能以 Bcl-xl-VDAC1依赖的方式控制线粒体代谢(106)。另一方面,在辐射应激下,mTOR 位于 MCF7、 HCT116和 U87细胞的线粒体上,与糖酵解过程中磷酸化葡萄糖的己糖激酶 II 相互作用,将有氧糖酵解的生物能量学转换为 OXPHOS,后者与肿瘤对辐射治疗的抵抗力增强有关(107) ,这种相互作用也在葡萄糖饥饿下的新生大鼠心室肌细胞中观察到(108)。在另一项研究中,证实 mTOR/Akt 通路调节电子传递链谷4E-BP1(109)的线粒体呼吸活动和复合体 i、 II、 IV 的表达。此外,另一项研究提示 mTOR-raptor 可能通过调节线粒体功能在细胞周期 G1期起到代谢检查点的作用(110)。

Triple-negative breast cancer cells possess special metabolic characteristics compared to estrogen receptor (ER) positive cells, manifested by high glucose uptake, increased lactate production, and low mitochondrial respiration which is correlated with attenuation of mTOR pathway and decreased expression of p70S6K. Re-expression of p70S6K reverses their glycolytic phenotype to OXPHOS state, while knockdown of p70S6K in ER positive cells leads to suppression of mitochondrial OXPHOS (111). It was demonstrated that global targeting of mTOR caused both anti-survival and pro-survival mitochondrial response, which were differentially exhibited in diverse cancer cells according to their intrinsic sensitivity to mTOR inhibition and hyperactive PI3K/AKT/mTOR activity status and/or growth factor-dependence (112).

与 ER 阳性细胞相比,三阴性乳腺癌细胞具有特殊的代谢特征,表现为葡萄糖摄取高、乳酸产量增加、线粒体呼吸低,这与 mTOR 通路衰减和 p70S6K 表达降低有关。p70S6K 的再表达使其糖酵解表型转变为 OXPHOS 状态,而 ER 阳性细胞中 p70S6K 的降低导致线粒体 OXPHOS (111)的抑制。结果表明,mTOR 的整体靶向作用可引起抗生存和促生存线粒体反应,不同癌细胞对 mTOR 抑制的内在敏感性、高活性 pi3k/akt/mTOR 活性状态和/或生长因子依赖性(112)表现出不同的线粒体反应。

mTORC1 and Mitochondrial Dynamic in Cancer

mTORC1与肿瘤线粒体动力学

The mitochondrial dynamic is a balance between fission and fusion processes (113). Mitochondria fusion is the union of two mitochondria resulting in one mitochondrion; organelle movement along cellular tracks that permit the encounter between two different mitochondria facilitating the fusion process (114). Fusion helps cells to mitigate stress by sharing multiple elements, which sustain mitochondrial biology as a form of complementation. Mitochondrial fusion involves two sequential steps: first, the outer membranes (OMs) of two mitochondria fuse; second, the inner membranes (IMs) fuse. OM fusion is mediated by mitofusin 1 (MFN1) (115) and MNF2 (116), which are dynamin-related GTPases at the OM (117). IM fusion is mediated by the dynamin-related protein optic atrophy 1 (OPA1) (118).

线粒体的动力学是裂变和融合过程之间的平衡(113)。线粒体融合是两个线粒体的结合,形成一个线粒体; 细胞器沿着细胞轨道运动,使两个不同的线粒体之间的相遇促进融合过程(114)。融合通过共享多种元素帮助细胞缓解压力,这些元素支持线粒体生物学作为一种互补形式。线粒体融合包括两个步骤: 第一,两个线粒体融合的外膜(OMs) ; 第二,内膜(IMs)融合。OM 融合是通过 mitofusin 1(MFN1)(115)和 MNF2(116)介导的,它们是 OM (117)上与动力相关的 GTPases。IM 融合是由动力相关蛋白质视神经萎缩1(OPA1)(118)介导的。

On the other hand, the mitochondrial fission is characterized by the division of one mitochondrion in two daughters, this process is required for segregation of damaged mitochondria for mitophagy, mtDNA replication, and mitochondria redistribution and motility during cell division (113). The fusion process requires the recruitment of dynamin-related protein 1 (DRP1) (119) from the cytosol to the mitochondrial OM. Assembly of DRP1 on the mitochondrial surface causes constraint of the mitochondria and leads to division of the organelle (120). In mammals exist four DRP1 receptors: mitochondrial fission 1 (FIS1) (121), mitochondrial fission factor (MFF) (122), Mitochondrial dynamics proteins of 49 kDa (MID49), and MID51 that are located on the mitochondrial OM (123).

另一方面,线粒体分裂线粒体分裂是一个线粒体在两个子细胞中分裂的拥有属性,这个过程是分离受损的线粒体用于吞噬,线粒体 dna 复制,以及细胞分裂过程中线粒体重新分布和运动所必需的。融合过程需要动力蛋白相关蛋白1(DRP1)(119)从胞浆到线粒体 OM 的补充。DRP1在线粒体表面的聚集使线粒体受限,导致细胞器分裂(120)。哺乳动物存在4种 DRP1受体: 线粒体分裂1(FIS1)(121)、线粒体分裂因子(MFF)(122)、线粒体动力蛋白49kda (MID49)和 MID51,它们分别位于线粒体 OM (123)上。

It has been established that the alteration of mitochondrial dynamics impact tumor development broadly. Alterations to the mitochondrial dynamic network also result in specific therapeutic susceptibilities, in particular, tumors with increased mitochondrial fragmentation or connectivity are hypersensitive to SMAC mimetics and induce apoptosis by blocking the action of inhibitor of apoptosis proteins (IAPs) (124). On the other hand, it was demonstrated that Drp1 expression was strongly increased in distant metastasis of hepatocellular carcinoma (HCC) compared to primary tumors. In contrast, Mfn1 showed an opposite trend (125). Moreover, in vitro experiments with HCC cells, demonstrated that mitochondrial fission significantly promoted the reprogramming of focal adhesion dynamics and lamellipodia formation mainly, by activating the CA2+/CaMKII/ERK/FAK pathway, which was associated with a greater capacity for migration and invasion (123125).

线粒体动力学的改变广泛影响着肿瘤的发生发展。线粒体动力学网络的改变也导致特定的治疗敏感性,特别是,肿瘤的线粒体断裂或连接增加是过敏性 SMAC 模型和诱导凋亡阻断凋亡抑制蛋白蛋白(IAPs)(124)。另一方面,研究表明,与原发性肿瘤相比,在肝癌的远处转移中,Drp1的表达强烈增加。相反,Mfn1表现出相反的趋势(125)。此外,对 HCC 细胞的体外实验表明,线粒体分裂主要通过激活 CA2 +/CaMKII/ERK/FAK 途径促进局灶粘附动力学和板状微球形成的重编程,该途径与较大的迁移和侵袭能力有关(123,125)。

A very important protein in mitochondrial fission is the mitochondrial fission process protein 1 (MTFP1), also called mitochondrial fission process 1,18 kDa (MTFP18), an integral pro-fission protein located at the mitochondrial inner membrane whose loss results in a hyperfused mitochondrial reticulum, whereas its overexpression produces mitochondrial fragmentation (126). As mentioned earlier, mTORC1 promotes the translation of mitochondrial proteins encoded in nuclei, interestingly, using a genome-wide polysome profiling and translatome, it was demonstrated the treatment with rapamycin, PP242 and metformin (mTORC1 inhibitors) suppressed the translation of MTFP1 (99). Morita and collaborators recently demonstrated that mTORC1 is a regulator of mitochondrial dynamics and cell survival via MTFP1 translation. Using mouse embryonic fibroblasts (MEFs) and human malignant melanoma cells treated with active-site mTOR inhibitor (asTORi), was demonstrate that mTORC1 stimulates the translation of MTFP1 mediated by 4E-BP, and therefore the mTOR inhibition induces the phosphorylation of the DRP1 at Ser 637, this phosphorylation prevents it translocation to mitochondria, conversely, the pro-fission phosphorylation site of DRP1 at Ser 616 was decreased in asTORi treated cells. This process was associated with a high mitochondrial elongation, branching, and circularization (127).

线粒体分裂中一个非常重要的蛋白质是线粒体分裂过程蛋白1(MTFP1) ,也称为线粒体分裂过程1,18kda (MTFP18) ,它是位于线粒体内膜的一个完整的裂变蛋白,其缺失导致线粒体网状组织的超灌注,而其过度表达则导致线粒体断裂(126)。正如前面提到的,mTORC1促进线粒体蛋白质在细胞核中的翻译,有趣的是,利用全基因组范围的多体分析和转录体,证明雷帕霉素、 PP242和二甲双胍(mTORC1抑制剂)抑制 MTFP1(99)的翻译。森田和合作者最近证明,mTORC1是线粒体动力学和细胞存活的调节因子,通过 MTFP1翻译。使用小鼠胚胎成纤维细胞(MEFs)和经活性位点 mTOR 抑制剂(asTORi)处理的人类黑色素瘤细胞,证明 mTORC1刺激4E-BP 介导的 MTFP1的翻译,因此 mTOR 抑制诱导了 DRP1在 ser637位点的磷酸化,这种磷酸化阻止了它向线粒体的转位,相反,DRP1在 ser616位点的裂变磷酸化位点在 astoria 处理的细胞中下降。这一过程与高线粒体延长,分支和循环有关(127)。

In support with these results it has been shown that cellular starvation inhibits mTORC1 pathway, interestingly, it was shown that the cells show a mitochondrial elongation phenotype under starvation (128129) similar to that observed in asTORi treatment. Combination between mTOR inhibitors and an increase of mitochondrial fission activates cell apoptosis, converting the mTOR inhibitors action of cytostatic to cytotoxic (127). In other study, it was shown that S6K1 contributes to mitochondrial dynamics, homeostasis and function, since MEFs-lacking S6K1 exhibited more fragmented mitochondria and a higher level of Drp1 with greater phosphorylation levels in Ser 616 (130). The depletion of S6K1 induced mitochondrial fission but not mitophagy. These changes in mitochondrial morphology alter its function disrupting the balance of OXPHOS, ATP production and changing cellular energy metabolism (130).

结果表明,细胞饥饿抑制 mTORC1通路,有趣的是,饥饿(128,129)使细胞呈现线粒体表型延长,与 asTORi 处理相似。联合使用 mTOR 抑制剂和增加线粒体分裂激活细胞凋亡,将 mTOR 抑制剂的细胞抑制作用转化为细胞毒作用(127)。在其他研究中,研究表明 S6K1参与线粒体动力学、稳态和功能,因为缺乏 S6K1的 mefs 表现出更多的线粒体碎片化,在 Ser 616(130)中,Drp1水平较高,磷酸化水平较高。6k1基因缺失引起的是线粒体分裂,而不是吞噬能力。线粒体形态的这些变化改变了它的功能,破坏了 OXPHOS 的平衡,ATP 的产生和细胞能量代谢的改变(130)。

Mitochondrial Biogenesis and Mitophagy: mTORC1 in Cancer

线粒体生物发生与肿瘤细胞吞噬: mTORC1

Mitochondrial mass is regulated by two opposite pathways, mitochondrial biogenesis and mitophagy, both processes emerging as dual regulators of tumorigenesis (4). Mitochondrial biogenesis is the growth and division of pre-existing mitochondria, whereas mitophagy is a form of autophagy that selectively degrades damaged mitochondria (131).

线粒体质量由两条相反的途径调节,即线粒体生物发生和食肉,这两个过程都是肿瘤发生的双重调节因子(4)。线粒体生物发生是原有线粒体的生长和分裂,而吞噬则是自噬的一种形式,它选择性地降解受损的线粒体(131)。

Mitochondrial biogenesis is widely regulated at transcriptional, translational and post-translational levels. Peroxisome proliferator-activated receptor γ co-activator 1α (PGC1-α) and related transcription co-activator are the master transcriptional regulators of mitochondrial biogenesis (132). PCG1-α binds to various transcription factors and nuclear receptors that recognize specific sequences in their target genes and promotes the mitochondrial biogenesis and oxidative phosphorylation in cancer cells and also promotes tumor metastasis (133) and drug resistance in colorectal cancer cells by regulating endoplasmic reticulum stress (134). The targets of PGC1-α include enzymes of energy metabolism as well as essential factors for the replication and transcription of mtDNA. PGC1-α is a transcription factor for mitochondrial genes, which action depends on its association with other transcription factors such as yin-yang (YY1), nuclear respiratory factor 1(NRF1) and 2 (NRF2), estrogen-related receptor α (ERRα) (132135). YY1 is a zinc finger protein and a member of the GLI-Kruppel family that can activate or inactivate gene expression depending on its interacting partners (136), YY1 is overexpressed in multiple cancer types and correlates with poor clinical outcomes (137138). However, other papers report that YY1 inhibits the cell growth in different tumor cell types in vitro, including human breast carcinoma cells and glioblastoma cells (139).

线粒体生物发生在转录、翻译和翻译后水平上受到广泛调控。过氧化物酶体增殖物活化受体 γ 辅助激活子1α (pgc1-α)和相关转录辅助激活子是线粒体生物发生的主要转录调节子(132)。Pcg1-α 与各种转录因子和核受体结合,这些转录因子和核受体识别其靶基因的特定序列,促进癌细胞的线粒体生物合成和氧化磷酸化,并通过调节内质网应激促进癌细胞的转移(133)和耐药性(134)。Pgc1-α 的作用靶点包括能量代谢酶以及线粒体 dna 复制和转录的关键因子。Pgc1-α 是线粒体基因的转录因子,其作用取决于与其他转录因子如阴阳(YY1)、核呼吸因子1(NRF1)和2(NRF2)、雌激素相关受体 α (errα)(132,135)的关联。YY1是一种锌指蛋白,是 GLI-Kruppel 家族的成员,根据其相互作用的伙伴可以激活或失活基因表达(136) ,y1在多种癌症类型中过度表达,并与不良的临床结果相关(137,138)。然而,其他文献报道 YY1在体外抑制不同肿瘤细胞类型的细胞生长,包括人乳腺癌细胞和胶质母细胞瘤细胞(139)。

Using skeletal muscle cells was showed that rapamycin decreased the expression of the PGC1-α, RREα, and NRF1 in correlation with decreased oxygen consumption. Moreover, it was identified that mTOR-raptor complex interacts with YY1, and in association with PCG1-α, regulates the mitochondrial gene expression (ATP5G1, Cox5A, cytochrome c, NDUF88, and UCP2) (140). In support with this results, it was demonstrated that mTOR induces the phosphorylation of YY1 (T30 and S356) consequently favoring the interaction with PGC1-α and increased mitochondrial morphology and bioenergetics state, in skeletal muscle (141). These results demonstrate that mTORC1 regulates mitochondrial biogenesis by promoting the transcription of mitochondrial genes. On the other hand, mTORC1 controls mitochondrial activity and biogenesis by selectively promoting translation of nucleus-encoded mitochondria related mRNAs via inhibition 4E-BPs. Moreover, the stimulation of the translation increases ATP production capacity, a required energy source for translation in MCF7 cells (100). In addition to stimulation of mitochondrial biogenesis by antagonizing 4E-BP1 dependent translation repression of mitochondria related mRNAs, mTORC1 inhibits mitochondrial degradation by suppressing autophagy (100).

用骨骼肌细胞研究表明,雷帕霉素可降低 pgc1-α、 rreα 和 NRF1的表达,与降低耗氧量有关。此外,还发现 mTOR-raptor 复合物与 YY1相互作用,并与 pcg1-α 共同调节线粒体基因表达(ATP5G1、 Cox5A、细胞色素 c、 NDUF88和 UCP2)(140)。结果表明,mTOR 诱导骨骼肌 y1(T30和 S356)磷酸化,从而有利于与 pgc1-α 的相互作用,提高了骨骼肌(141)的线粒体形态和生物能状态。这些结果表明 mTORC1通过促进线粒体基因的转录调控线粒体的生物发生。另一方面,mTORC1通过抑制4E-BPs 选择性地促进核编码的线粒体相关 mRNAs 的翻译,从而控制线粒体活性和生物发生。此外,该翻译的刺激增加了在 MCF7细胞(100)中翻译所需的能量来源 ATP 的生产能力。除了通过拮抗4E-BP1依赖的线粒体相关 mRNAs 的翻译抑制来刺激线粒体的生物发生外,mTORC1还通过抑制自噬(100)来抑制线粒体的降解。

PGC-1β is also an important mitochondrial biogenesis regulator, through regulation of the expression of NRF1 (142). It was shown that the levels of PGC-1β and mTOR correlated with overall mitochondrial activity in breast cancer samples. Moreover, the knockdown of endogenous PGC-1β, leads to a decreased expression of mTOR pathway related genes and induces apoptosis in MDA-MB-231 cells (143). Interestingly, it was demonstrated that the branched chain amino acid transaminase 1 (BCAT1) actives mTORC1 and in consequence promotes the mitochondrial biogenesis, ATP production and defense of oxidative stress (143). The inhibition of mTORC1 with rapamycin, neutralized the roles of BCAT1 in mitochondrial function and breast cancer cell growth (143). Recently, it was shown that rapamycin, enhanced the processes of apoptosis and initiation of autophagy in LKB1 deficient urothelial carcinoma of the bladder both in vitro and in vivo, which was associated with deregulated mitochondrial biogenesis and AMPK activation (144). These results are relevant because AMPK is an important regulator of mitochondrial biogenesis via PGC1-α (145), which also inhibits the mTORC1 pathway.

Pgc-1β 通过调控 NRF1(142)的表达,也是一个重要的线粒体生物发生调控因子。结果表明,乳腺癌组织中 pgc-1β 和 mTOR 水平与线粒体活性总体水平相关。此外,抑制内源性 pgc-1β,导致 mTOR 通路相关基因表达下降,诱导 MDA-MB-231细胞(143)凋亡。有趣的是,支链氨基酸转氨酶1(BCAT1)激活 mTORC1,从而促进氧化应激的线粒体生物发生、 ATP 的产生和防御。雷帕霉素对 mTORC1的抑制,中和了 BCAT1在线粒体功能和乳腺癌细胞生长中的作用(143)。近年来研究表明,雷帕霉素在体内外均能促进 LKB1基因缺陷型膀胱尿路上皮癌细胞凋亡和自噬的发生,这与线粒体生物合成失调和 AMPK 激活(144)有关。这些结果是相关的,因为 AMPK 是通过 pgc1-α (145)调节线粒体生物发生的重要调节因子,同时也抑制 mTORC1通路。

Mitophagic status was assessed in a panel of human cytoplasmic hybrid (cybrid) cell lines carrying a variety of pathogenic mtDNA mutations. It was found that both genetic and chemically induced loss of mitochondrial transmembrane potentially caused recruitment of the pro-mitophagic factor Parkin to mitochondria but it was insufficient to prompt mitophagy. They found that mitophagy could be induced following treatment with the mTORC1 inhibitor rapamycin (146).

在一组携带多种致病性线粒体 dna 突变的人细胞质杂交(cybrid)细胞系中评估了噬细胞状态。研究发现,遗传和化学诱导的线粒体跨膜损失可能引起线粒体中噬菌体因子 Parkin 的补充,但不足以促进噬菌体的产生。他们发现,mTORC1抑制剂雷帕霉素(146)可诱导吞噬细胞。

These findings suggest that, mTORC1 is an important regulator of mitochondrial biogenesis, by regulating the expression of important factors in the regulation of mitochondrial biogenesis, both at the transcriptional level and at the translation level (Figure 3).

这些发现表明,mTORC1是一个重要的调节线粒体生物发生,通过调节重要因素的表达调节线粒体生物发生,无论是在转录水平和翻译水平(图3)。FIGURE 3 图3

Figure 3. Mechanistic target of rapamycin complex 1 (mTORC1) and mitochondrial biogenesis. mTORC1 promotes mitochondrial biogenesis via upregulation of translation genes and moreover via transcriptional regulation of TFAM, ATP50, NRF1, NRF2 genes.

图3。雷帕霉素复合物1(mTORC1)的机制靶点与线粒体生物发生。mTORC1通过翻译基因的上调以及 TFAM、 ATP50、 NRF1、 NRF2基因的转录调控促进线粒体的生物合成。

Glutaminolysis and mTORC1 in Cancer

谷氨酰胺溶解与 mTORC1在肿瘤中的表达

Glutaminolysis is a set of reactions that occurs in mitochondrial matrix and cytosol in proliferating cells. In such reactions, the amino acid glutamine is degraded to glutamate, ammonium, aspartate and pyruvate, among others. Glutamine, glutamate as well as aspartate, are used for nucleic acid synthesis, other important function of glutamine is replenishing the TCA cycle intermediate α-KG.

谷氨酰胺溶解是发生在线粒体基质和增殖细胞的胞浆中的一系列反应。在这些反应中,氨基酸谷氨酰胺降解为谷氨酸、铵、天冬氨酸和丙酮酸等。谷氨酰胺、谷氨酸和天冬氨酸用于核酸合成,谷氨酰胺的其他重要功能是补充 TCA 循环中间体 α-kg。

It has been reported that glutamine is the amino acid most frequently found in plasma and muscle (147), glutamine concentration ranges from 450 to 800 μM in human plasma (148). Glutamine has been defined as a non-essential amino acid; nevertheless, evidence has showed that glutamine becomes essential in stressful conditions (149). As an example, when cells are under hypoxic stress, glutamine-derived α-KG is used to stimulate lipids synthesis (150). Carbon and nitrogen from the glutamine present in blood are used for biosynthesis and also for providing energy to the cell (151). Specifically, glutamine is the leading donor of nitrogen for purine and pyrimidine nucleotide synthesis, as well as a supplier for amino groups for non-essential amino acids synthesis, such as aspartate, alanine, glycine and serine, moreover, nitrogen from glutamine participates in nucleic acid and de novo protein synthesis (149152). Finally, the glutamine-derived carbon is source for fatty acid and amino acid synthesis as well (151).

据报道,血浆和肌肉中最常见的氨基酸是谷氨酰胺(147) ,血浆中谷氨酰胺浓度在450ー800μM 之间(148)。谷氨酰胺被定义为一种非必需氨基酸,然而,有证据表明,谷氨酰胺成为必需的应激条件(149)。例如,当细胞处于低氧应激状态时,谷氨酰胺衍生的 α-kg 被用来刺激脂质的合成(150)。存在于血液中的谷氨酰胺中的碳和氮用于生物合成,也用于向细胞(151)提供能量。具体而言,谷氨酰胺是嘌呤和嘧啶核苷酸合成的主要供体,也是合成天冬氨酸、丙氨酸、甘氨酸和丝氨酸等非必需氨基酸的供应者,此外,谷氨酰胺中的氮参与核酸和新蛋白质的合成(149,152)。最后,谷氨酰胺衍生的碳也是脂肪酸和氨基酸合成的来源。

Glutamine enters to the cells via SLC (solute carrier)-type transporters. Fourteen of these transporters are known for transporting glutamine to the plasma membrane which are classified into four families: SLC1, SLC6, SLC7, and SLC38 (153). Glutamine is metabolized within the mitochondrion via two deamination steps. The first one produces glutamate through an irreversible reaction catalyzed by glutaminase (GLS1 and GLS2 in mammals); in the following deamination reaction, α-KG is produced by the enzyme glutamate dehydrogenase (GDH) (154). The α-KG generated by glutaminolysis is a major anaplerotic source in the TCA cycle.

谷氨酰胺通过 SLC (溶质载体)型转运蛋白进入细胞。其中十四种转运蛋白可以将谷氨酰胺转运到质膜上,这些转运蛋白分为4类: SLC1、 SLC6、 SLC7和 SLC38(153)。谷氨酰胺在线粒体内通过两个脱氨基作用进行代谢。第一种是通过谷氨酰胺酶(GLS1和 GLS2)催化的不可逆反应产生谷氨酸,在随后的脱氨反应中,谷氨酸脱氢酶(GDH)(154)产生 α- 千克。谷氨酰胺溶解产生的 α- 千克是 TCA 循环中的主要回指源。

Importantly, it was demonstrated that glutamine could be useful for cancer cells to drive tumor growth due to is used for energy generation as well as for biomass accumulation being a source of carbon and nitrogen as mentioned before (152), moreover glutamine can be consumed by proliferating cells more rapidly than needed to satisfy nitrogen requirements (155). As a result of glutamine depletion, most cancer patient’s loss body weight due to muscle mass consumption provoking weakness, all these symptoms are known as cachexia (155156). It is important to notice that, when cancer cells are deprived of glutamine, undergo cell cycle arrest due to nitrogen deficiency since nitrogen is necessary for nucleotides synthesis (157). In 1978, Lawrence et al. observed that glutamine is the major energy source in HeLa cell line (158). Additionally, evidence supports that glutaminolysis provides metabolites, such as glutamate to promote tumor growth, as observed by Dornier et al. (159). The group investigated the participation of glutamine metabolism in invasive processes so that, they showed that mammary epithelial cells from normal tissue uptake glutamine, yet glutamate secretion was not observed. Extracellular glutamate is needed at low concentrations for mammary epithelial phenotype maintenance, but higher concentrations promote key characteristics of the invasive phenotype, moreover, in primary cultures of invasive breast cancer cells it was observed a high conversion glutamine to glutamate (159).

重要的是,已经证明谷氨酰胺可以用于癌细胞促进肿瘤生长,因为谷氨酰胺用于能量生成以及生物量积累,是前面提到的碳和氮的来源(152) ,此外,增殖细胞消耗谷氨酰胺的速度比满足氮需求的速度更快(155)。由于谷氨酰胺耗竭,大多数癌症患者的体重减轻由于肌肉大量消耗引起的虚弱,所有这些症状被称为恶病质(155,156)。重要的是要注意,当癌细胞被剥夺谷氨酰胺,进行细胞周期停滞由于氮缺乏症,因为氮是必要的核苷酸合成(157)。1978年,Lawrence 等人观察到谷氨酰胺是 HeLa 细胞系(158)的主要能量来源。此外,证据支持谷氨酰胺溶解提供代谢产物,如谷氨酸促进肿瘤生长,正如多尼尔等人(159)所观察到的。研究组观察了谷氨酰胺代谢在侵袭过程中的作用,发现乳腺上皮细胞从正常组织摄取谷氨酰胺,但未观察到谷氨酸分泌。低浓度的细胞外谷氨酸是乳腺上皮细胞表型维持所必需的,但较高浓度的谷氨酸促进侵袭性表型的关键特征,此外,在侵袭性乳腺癌细胞的原代培养中观察到谷氨酰胺转化为谷氨酸(159)的高转化率。

Autophagy and cell growth are found to be under control of mTORC1; those two cellular processes are regulated by glutaminolysis, so that mTOR activity is tightly controlled to prevent inappropriate cell growth (Figure 4). In fact, it has been found an upregulation of mTORC1 in several cancers and such activation is required for cell growth and protein synthesis. Further, glutamine metabolism is found disrupted in several cancer types, including papillary thyroid cancer where using cell lines was demonstrated that such cells are dependent on glutamine and glutaminolysis-associated proteins.

自噬和细胞生长受 mTORC1的控制,这两个细胞过程是由谷氨酰胺分解调节的,因此 mTOR 活性受到严格控制,以防止不适当的细胞生长(图4)。事实上,它已经被发现在一些癌症中 mTORC1的上调,这种激活是细胞生长和蛋白质合成所必需的。此外,在几种类型的癌症中发现谷氨酰胺代谢紊乱,包括乳头状甲状腺癌,其中使用细胞系被证明这种细胞依赖于谷氨酰胺和谷氨酰胺溶解相关蛋白。FIGURE 4 图4

Figure 4. Glutaminolysis and Mechanistic target of rapamycin complex 1 (mTORC1) The α-ketoglutarate (α-KG) produced by glutaminolysis is used for tricarboxilic acid (TCA) cycle intermediates replenish, a process known as anaplerosis. Once α-KG is exported from the mitochondria to the cytosol activates EGLNs, which in turn triggers mTORC1 activity promoting cell growth and inhibits autophagy.

图4。雷帕霉素复合物1(mTORC1)的谷氨酰胺解及其机理靶标谷氨酰胺解产生的 α- 酮戊二酸(α-kg)用于三羧酸(TCA)循环中间体的补充,这一过程称为回补。一旦 α-kg 从线粒体输出到细胞质中,它就会激活 EGLNs,进而触发 mTORC1活性,促进细胞生长,抑制自噬。

Through different experimental approaches, an aberrant overexpression of GLS was showed in cancer; moreover, pharmacological inhibition (by using inhibitors BPTES and CB-8939 that target both isoforms of GLS) and genetic knockdown of GLS repressed glutaminolysis and diminished mitochondrial respiration. Additionally, using tissues and cells from patients with papillary thyroid cancer, an altered overexpression of glutaminase was observed. When GLS was inhibited using a siRNA, mTORC1-signaling pathway was deactivated leading to an increase of autophagy and apoptosis (160).

通过不同的实验方法,在肿瘤组织中发现了 GLS 的异常过表达,而且药物抑制(使用针对 GLS 同型异构体的抑制剂 BPTES 和 CB-8939)和 GLS 的基因敲除抑制谷氨酰胺溶解和线粒体呼吸减弱。此外,使用乳头状甲状腺癌患者的组织和细胞,观察到谷氨酰胺酶过度表达的改变。当使用 siRNA 抑制 GLS 时,mtorc1信号通路失活,导致自噬和凋亡增加(160)。

It has been demonstrated that arginine and leucine prompt mTORC1 by activating RAS-related GTPase (RAG) complex; as a result, mTORC1 is recruited and triggers lysosome activity. Studies have demonstrated that glutamine positively regulates the mTORC1 pathway when promoting leucine uptake (161) and as well-boosting mTORC1 assembly as well as its localization into the lysosome; indeed, the presence of α-KG is considered to be enough to promote mTORC1 localization into the lysosome (162) (Figure 3).

研究表明,精氨酸和亮氨酸通过激活 ras- 相关的 GTPase 复合体促进 mTORC1,因此,mTORC1被招募并触发一些活动。研究表明,谷氨酰胺在促进亮氨酸摄取(161)和促进 mTORC1组装以及定位于溶酶体中时,对 mTORC1通路起到了积极的调节作用; 实际上,α-kg 的存在足以促进 mTORC1定位于溶酶体中(162)(图3)。

The mentioned RAG-dependent regulation of mTOR could rely on glutamine, arginine and leucine transporter SLC38A9 (163165). Although the mechanism is not well-understood, it has been hypothesized that α-KG could be able to regulate RAGB activity as well as mTOR activation at a downstream glutamine metabolism level (151). On the other hand, Jewell and her group reported in 2015 that mTORC1 activation could be independent of the Rag GTPases and supported the fact that mTORC1 is differentially regulated by the amino acids leucine and glutamine. Using mouse embryonic fibroblasts RagA and RagB knockout cells, they demonstrated that leucine stimulates mTORC1 by Rag GTPase-dependent mechanism meanwhile glutamine stimulates mTORC1 through a mechanism that is carried out by Rag in an GTPase-independent mechanism in order to translocate mTORC1 to the lysosome (166).

这种 rag 依赖的 mTOR 调节可能依赖于谷氨酰胺、精氨酸和亮氨酸转运体 SLC38A9(163-165)。尽管其机制尚不清楚,但有假设认为 α-kg 可能在下游谷氨酰胺代谢水平(151)调节 RAGB 活性和 mTOR 激活。另一方面,Jewell 和她的团队在2015年报告 mTORC1的激活可能独立于 Rag GTPases,并支持 mTORC1受氨基酸亮氨酸和谷氨酰胺差异调节的事实。以小鼠胚胎成纤维细胞 RagA 和 RagB 基因敲除细胞为实验材料,证明亮氨酸通过 Rag gtpase 依赖性机制刺激 mTORC1,而谷氨酰胺通过 Rag 非 gtpase 依赖性机制刺激 mTORC1,从而将 mTORC1转位到溶酶体(166)。

In 2013, Csibi and collaborators reported that mTORC1 pathway regulates glutamine uptake and metabolism. The results showed that the mTORC1 pathway negatively controls SIRT4, an ADP-ribosyltransferase that is found in the mitochondria and inhibits glutamine dehydrogenase (GDH), through stimulation of proteasome-mediated degradation of cAMP-responsive element-binding (CREB) 2. In fact, it has also been reported that SIRT4 expression is decreased in several human cancers (167).

2013年,Csibi 和合作者报道 mTORC1通路调节谷氨酰胺的摄取和代谢。结果表明,mTORC1通路通过刺激蛋白酶体介导的 camp 反应元件结合蛋白(cAMP-responsive element-binding,CREB)2的降解,对线粒体内存在的 adp- 核糖基转移酶 SIRT4产生负性调控,抑制谷氨酰胺脱氢酶(glutamine dehydrogenase,GDH)活性。事实上,也有报道说 SIRT4在几种人类癌症中的表达下降(167)。

The same research group postulated a previous model in which they concluded that cells are addicted to glutamine as a result of mTORC1 activation (168). It was shown that α-KG could be exported to the cytosol by the mitochondrial carrier protein α-KG/malate named SLC25A11(154). At high glutaminolyc rate, cytosolic α-KG activates the enzymes that function as oxygen and nutrient of the cell sensors EGLNs (prolyl hydroxylase enzymes PHD) such enzymes are required for mTORC1 activation-dependent of amino acids in a HIF-1α independent manner to promote cell growth and anabolism and inhibit autophagy (154162).

同一个研究小组假设了一个先前的模型,在这个模型中他们得出结论,细胞对谷氨酰胺上瘾是 mTORC1激活的结果(168)。结果表明,α-kg 可以被命名为 SLC25A11(154)的线粒体载体蛋白 α-kg/苹果酸导入胞浆。在高谷氨酰胺速率下,胞浆 α ー kg 活化细胞传感器的氧和营养酶,这些酶是 mTORC1以 hif-1α 独立方式活化依赖的氨基酸所必需的,以促进细胞生长和合成,抑制自噬(154,162)。

An elevated glutaminolysis is related to the promotion of cancer progression at early stages by stimulating cell growth through the mTORC1 pathway and diminishing elimination of altered proteins and organelles by inhibiting autophagy (154162). In another case, glutamine dependence was evaluated in six different cell lines from squamous cell carcinoma and it was found that five out of six cell lines were glutamine-dependent, also, glutamine depletion, using GLS1- inhibitors BPTES and compound 968, decreased cell proliferation in those five cell lines, meanwhile inhibition of cell proliferation in QG56 glutamine-independent cell line was not reported as significant. Further, it was observed that the inhibition of glutaminolysis suppressed mTORC1 activity, by evaluating pS6 levels in the glutamine-dependent RERF-LC-AI cell line but the activity of mTORC1 was not affected in the QG56 glutamine-independent cell line. Finally, inhibition of glutaminolysis induced autophagy in RERF-LC-AI cell line (169). Furthermore, the activation of mTORC1 inhibits the family of enzymes that catalyze phosphorylation of phosphatidyl inositol, one of the main phospholipids present in the cell, specifically at the d-3 position of the inositol ring, to generate PtdIns (3)P complex I and unc-51 like autophagy activating kinase complex (ULK), both proteins participate in the initiation step of autophagy and mTORC1 activation limits the initiation steps of autophagy. On the other hand, glutaminolysis products GSH, NADPH, and α-KG limit ROS production to prevent autophagy induction (154).

通过 mTORC1途径刺激细胞生长,通过抑制细胞自噬减少改变的蛋白质和细胞器的消除(154,162) ,提高谷氨酰胺溶解与促进早期癌症进展有关。另一例,用6种不同的鳞状细胞癌细胞系进行谷氨酰胺依赖性评价,发现6种细胞系中有5种是谷氨酰胺依赖性细胞,同时,谷氨酰胺缺失,使用 GLS1- 抑制剂 BPTES 和化合物968,降低了这5种细胞系的细胞增殖,同时抑制 QG56谷氨酰胺非依赖性细胞系的增殖未见报道。此外,通过评价 pS6水平,我们观察到抑制谷氨酰胺溶解可抑制 mTORC1活性,但对 QG56谷氨酰胺非依赖性细胞系 mTORC1活性没有影响。最后,抑制谷氨酰胺溶解诱导的 RERF-LC-AI 细胞自噬(169)。此外,mTORC1的激活抑制磷脂酰肌醇磷酸化的酶家族产生 PtdIns (3) p 复合物 i 和类自噬激活激酶复合物(ULK) ,这两种蛋白都参与自噬的启动步骤,mTORC1的激活限制了自噬的启动步骤。另一方面,谷氨酰胺溶解产物谷胱甘肽、 NADPH 和 α- 千克限制活性氧的产生,以防止自噬诱导(154)。

It has been observed that a reactivation of mTORC1 by glutaminolysis is also required for lysosome regeneration and autophagy termination (154). In the specific case of autophagy, it has been reported that autophagy has a dual role in cancer, acting as tumor suppressor in some cases. For instance, metabolic stress causes the expression of p62, a sustained autophagy substrate protein, resulting in autophagy defects and an altered expression of NF-kB, finally promoting tumorigenesis, this information indicates that autophagy suppresses tumorigenesis by limiting p62 accumulation (151170). On the contrary, autophagy seems to support cancer cells survival by facilitating nutrients and suppressing stress pathways. For instance, expression of H-ras and K-ras oncogenes in immortal non-tumorigenic baby mouse kidney epithelial cells upregulated basal autophagy promoting tumor cell survival (151171). Another interesting relation between mTOR pathway and autophagy is the association to lifespan and aging; mainly because it has been observed that inhibition of mTOR could bring as a consequence delay of aging due to autophagy stimulation resulting in a mitophagy increase (172). In fact, it is well-documented that inhibition of key components of mTOR and its counterpart in invertebrates TOR pathways, results in an extension of life span in part by the influence of mTOR on the called “hallmarks of aging,” an interesting an extensive review about this subject is broadly reviewed in Papadopoli publication (173).

据观察,通过谷氨酰胺溶解重新激活 mTORC1也是溶酶体再生和自噬终止所必需的(154)。在自噬的特殊情况下,已经有报道说,自噬在癌症中具有双重作用,在某些情况下作为肿瘤抑制剂。例如,代谢应激引起 p62的表达,这是一种持续的自噬底物蛋白,导致自噬缺陷和 NF-kB 表达的改变,最终促进了肿瘤发生,这一信息表明,自噬通过限制 p62的积累来抑制肿瘤发生(151,170)。相反,自噬似乎通过促进营养物质和抑制应激通路来支持癌细胞的存活。例如,H-ras 和 K-ras 癌基因在永生非致瘤小鼠肾上皮细胞中的表达上调了基础自噬促进肿瘤细胞存活(151,171)。mTOR 途径与自噬之间的另一个有趣关系是与寿命和衰老的关系; 主要是因为已经观察到,mTOR 的抑制作用可以作为由于自噬刺激而导致的衰老延迟的结果,从而导致吞噬能力的增加(172)。事实上,已有大量文献证明,抑制 mTOR 及其在无脊椎动物 TOR 途径中的对应物的关键成分,部分是由于 mTOR 对所谓的“衰老特征”的影响而导致寿命的延长。关于这一主题的一篇有趣的广泛评论在 Papadopoli 出版物(173)中得到了广泛的回顾。

The regulation of both, mTORC1 and glutaminolysis suggests that mTORC1 and glutaminolysis act in both directions hence they are found to regulate each other for promoting cell growth and cancer progression; mTORC1 also induces glutaminolysis by activating c-MYC-GLS and because c-MYC is GLS and GLUD1 transcription factor, glutamine metabolism is favored; additionally, the glutaminolysis-mediated activation of mTORC1 participates in autophagy inhibition and the DNA double-strand breaks sensor serine/threonine protein kinase ATM which participates in cell cycle delay after DNA damage. The mTORC1 pathway suppresses ATM via S6K1/2 signaling pathways and by upregulating mircroRNA-18a and microRNA-421 that target ATM (154174). An increase in glutamine synthetase abolishes the production of α-KG from glutaminolysis, as a result, an inhibition of mTORC1 is observed as well as an enhancement of autophagy, which is imperative for cancer cell survival (154175). There is an increasing interest in inhibiting simultaneously both, glutaminolysis and autophagy in order to trigger a synergistic effect that may be useful for patient outcome improvement and also to diminish toxicity.

mTORC1通过激活 c-MYC-GLS 诱导谷氨酰胺溶解,因为 c-MYC 是 GLS 和 GLUD1转录因子,所以谷氨酰胺代谢受益; 此外,谷氨酰胺介导的 mTORC1激活参与自噬抑制,DNA 双链断裂传感器丝氨酸/蛋白质蛋白激酶参与 DNA 损伤后细胞周期延迟。mTORC1通路通过 S6K1/2信号通路抑制 ATM,并上调靶向 ATM (154,174)的 mircroRNA-18a 和 microrna 421。谷氨酰胺合成酶的增加消除了谷氨酰胺分解产生的 α- 千克,因此,可以观察到 mTORC1的抑制以及自噬的增强,这对于癌细胞的存活至关重要(154,175)。同时抑制谷氨酰胺溶解和自噬,以引发一种协同效应,这种效应可能有助于改善患者的结果,并减少毒性。

A very interesting publication of 2016 shows that autophagy could be a survival mechanism upon rapamycin treatment. Interestingly, in conditions of nutrient restrictions, mTORC1 is activated by glutaminolysis during nutritional restrictions and autophagy is inhibited, so then apoptosis is induced, via upregulation of p62 in U-2 OS cells (176).

2016年一篇非常有趣的文章表明,自噬可能是雷帕霉素治疗后的一种生存机制。有趣的是,在营养限制条件下,mTORC1在营养限制条件下通过谷氨酰胺分解被激活,自噬被抑制,通过 p62上调 U-2 OS 细胞(176)而诱导细胞凋亡。

Mitochondrial Oncometabolites and mTORC1

线粒体 Oncometabolites 与 mTORC1

Dominant mutations in mitochondrial enzymes led to identification of mitochondrial derived signaling molecules, called oncometabolites. The term of oncometabolites refers to intermediates of metabolism that abnormally accumulate in cancer cells upstream or downstream of metabolic defects, often through loss-of-function or gain-of function mutations, respectively, in genes encoding the corresponding enzymes (177). This oncometabolites are 2-hydroxyglutarate (2HG), succinate and fumarate which have been demonstrated to contribute to the development and progression of cancer (178). The oncometabolites are produced by mutations in the nuclear-encoded TCA enzymes, isocitrate dehydrogenase 1 and 2 (IDH1/2), succinate dehydrogenase (SDH), and fumarate hydratase (FH) (177). Chin and co-workers discovered that metabolite α-KG increases the lifespan of adult C. elegans by inhibiting the highly conserved ATP synthase and mTORC1, mimicking dietary restriction in longevity (179). Interestingly, it has been shown that mTORC1 promotes the generation of oncometabolites in addition it was also shown that these oncometabolites regulate mTORC1, as a feedback regulation.

线粒体酶的显性突变导致线粒体衍生信号分子的鉴定,称为 oncometabolites。Oncometabolites 一词是指代谢缺陷上游或下游癌细胞中异常积累的代谢中间体,通常分别通过编码相应酶的基因中的功能丧失或功能增强突变(177)。这种共代谢产物是2- 羟基戊二酸酯(2HG)、琥珀酸酯和富马酸酯,它们已被证明有助于癌症的发展和进展(178)。核编码的 TCA 酶、异柠檬酸脱氢酶1和2(IDH1/2)、琥珀酸脱氢酶(SDH)和延胡索酸酶(FH)(177)的突变产生了 oncometabolite。Chin 和他的同事发现,代谢产物 α-kg 通过抑制高度保守的 ATP 合成酶和 mTORC1,模仿饮食限制长寿(179) ,延长了成年秀丽隐杆线虫的寿命。有趣的是,研究表明 mTORC1促进了肿瘤代谢物的产生,而且这些肿瘤代谢物还调节 mTORC1,作为一种反馈调节。

2HG and mTORC1

2HG 和 mTORC1

Isocitrate dehydrogenases 1 and 2 (IDH1, and IDH2) are key TCA cycle enzymes that are nicotinamide adenine dinucleotide phosphate (NADP+) dependent. IDH1 and 2 catalyze the oxidative decarboxylation of isocitrate to α-KG with production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) (180). Mutations in IDH1 and IDH2 genes are mostly missense variants leading to a single amino-acid substitution of arginine residues at codon 132 in exon 4 of the IDH gene or codons 140 or 172 of the IDH2 gene. Mutant of IDH1 and IDH2 enzymes have a gain the function of catalyzing the reduction of α-KG to its (R)-enantiomer of 2-hydroxyglutarate (2HG), which accumulates to exceedingly high levels in patients with glioma, acute myeloid leukemia, esophageal squamous cell carcinoma (180183) thus, 2HG levels being used as a biomarker for IDH mutation in these cancers (184). 2HG is an oncometabolite impairing epigenetic and hypoxic regulation through its binding to α-KG-dependent dioxygenases.

异柠檬酸脱氢酶1和2(IDH1和 IDH2)是依赖于烟酰胺腺嘌呤二核苷酸磷酸的关键 TCA 循环酶。IDH1和2催化异柠檬酸氧化脱羧转化为 α- 千克,产生还原烟酰胺腺嘌呤二核苷酸磷酸(NADPH)(180)。IDH1和 IDH2基因的突变大多是错义突变,导致 IDH2基因第4外显子第132号密码子或第140号密码子或第172号密码子的精氨酸残基发生单个氨基酸取代。IDH1和 IDH2的突变体具有催化将 α-kg 还原为2- 羟基戊二酸(2HG)的(r)-对映体的功能,这些对映体在神经胶质瘤、急性骨髓性白血病、食管鳞状细胞癌(180-183)患者体内积累到极高水平,因此2HG 水平可作为这些癌症中 IDH 突变的生物标志物(184)。2HG 通过与 α ー kg- 依赖的双加氧酶结合,是一种表观和缺氧调控的共代谢损伤基因。

Recently, it was shown that 2HG induces angiogenic activity, because it induces the levels of secreted vascular endothelial growth factor (VEGF) in breast cancer cells, and finally enhance the endothelial cell proliferation and migration cell inducing MMP2 activity (185).

近年来研究表明,2HG 具有促进血管生成的作用,因为它能诱导乳腺癌细胞分泌血管内皮生长因子(VEGF) ,并最终促进内皮细胞增殖和迁移,从而诱导 MMP2活性(185)。

It was shown that both (R)-2HG and (S)-2HG bind and inhibit ATP synthase and mTOR signaling. Consistently, this inhibition is sufficient for growth arrest and tumor cell killing under conditions of glucose limitation in glioblastoma cells (186). Contrary to these results, it was demonstrated that mutations IDH1R132H or IDH2R172K in MEF and HeLa cells induce an increase in 2HG levels that stimulate both mTORC1 and mTORC2 signaling as highlighted by enhanced phosphorylation of p70S6K, pS6 and Rictor, and Akt, respectively. They also showed that 2HG inhibits the α-KG-dependent enzyme KDM4A and consequently, this affects the stability of DEPTOR a negative regulator of mTORC1 and mTORC2, leading to mTOR activation independently of the PI3K/Akt/TSC1-2 pathway (187).

结果表明,(r)-2 HG 和(s)-2 HG 结合并抑制 ATP 合酶和 mTOR 信号。一致地,这种抑制作用足以阻止胶质母细胞瘤细胞在葡萄糖限制条件下生长和杀死肿瘤细胞(186)。与这些结果相反,研究表明 MEF 和 HeLa 细胞中的 IDH1R132H 或 IDH2R172K 突变诱导了2HG 水平的增加,这些水平刺激 mTORC1和 mTORC2信号通路,这些信号通路分别被 p70S6K、 pS6和 Rictor 和 Akt 的磷酸化增强所突出。他们还发现,2HG 抑制了 α-kg 依赖性酶 KDM4A,从而影响了 detor 的稳定性—— mTORC1和 mTORC2的负调节因子,导致 mTOR 的激活独立于 PI3K/Akt/TSC1-2通路(187)。

In other study it was shown that rapamycin reduced 2-HG levels derived of lactate, in IDH1 mutant fibrosarcoma cell line (HT-1080 cells). Furthermore, they shown that rapamycin inhibit the growth in HT-1080 xenografts in vivo and 2HG production (188). In support with this, using two mutant cell lines for IDH and orthotopic mutant IDH tumor model, showed that the treatment with dual PI3K/mTOR inhibitor (XL765), induced a significant reduction in 2HG levels, and enhanced the survival (189).

另一项研究表明,雷帕霉素降低了 IDH1突变型纤维肉瘤细胞系(HT-1080细胞)乳酸衍生物2-HG 的水平。此外,他们还发现雷帕霉素在体内抑制了 HT-1080异种移植物的生长和2HG 的产生(188)。为了证明这一点,我们利用两个突变细胞系建立了 IDH 和原位突变的 IDH 肿瘤模型,结果表明,双重 PI3K/mTOR 抑制剂(XL765)治疗后,2HG 水平显著降低,生存率提高(189)。

Fumarate and mTORC1

富马酸酯和 mTORC1

In the TCA cycle the reversible hydration of fumarate to malate is catalyzed by FH. The oncogenic properties of FH loss have been mostly associated with a high intracellular accumulation of fumarate. This oncometabolite shares structural similarity with another TCA cycle intermediate α-ketoglutarate, also referred to as 2-oxoglutarate (2-OG). 2-OG is a cofactor for a family of enzymes called 2-OG-dependent dioxygenases that catalyze the hydroxylation of a wide range of targets (190). The enzymes that belong to this family are the prolyl hydroxylases and the Jumonji C containing family of histone lysine demethylases and TET (ten-eleven translocases) enzymes (190). It was shown that high levels of fumarate inhibit the HIF-1α prolyl hydroxylases, leading to HIF-1α stabilization (191). HIF-1α is inactivated in normoxia by prolyl hydroxylase enzymes (PHD 1-3) using oxygen as a substrate. HIF-1α hydroxilated is associated to E3 ubiquitin ligase Von Hippel Lindau protein (VHLp) for its degradation, whereas in hypoxia condition stabilization and nuclear translocation occur, leading to oncogenes activation (192). HIF-1α is a transcription factor for metabolic genes such as hexokinase (HK), lactate deshydrogenase (LDHA) and glucose transporter (GLUT1) promoting tumor glycolysis (193). In other study it was demonstrated that fumarate accumulation promotes HIF-1α mRNA and protein accumulation independent of the VHL pathway but through an NF-kB dependent mechanism. Fumarate promotes p65 phosphorylation and p65 accumulation at the HIF-1α promoter through non-canonical signaling via the upstream Tank biding kinase (TBK1) promoting cell invasion of renal cancer cells (194). In accordance with the role of the fumarate accumulation with cytotoxicity and oncogenic capacity, it was demonstrated that cells exposed to high levels of fumarate and succinate lead to extensive DNA fragmentation and altering the global DNA methylation patterns via DNA hypermethylation in human hepatocellular carcinoma (195).

在 TCA 循环中,FH 催化富马酸可逆水合制苹果酸。跳频丢失的致癌特性主要与富马酸在细胞内的高积累有关。这种共代谢结构相似性与另一种 TCA 循环中间体 α- 酮戊二酸酯共享,也称为2- 氧戊二酸酯(2-OG)。2-OG 是一系列被称为2-OG- 依赖的双加氧酶的辅助因子,它催化一系列目标(190)的羟基化。属于这个家族的酶是脯氨酰羟基酶和 Jumonji c 家族的组蛋白赖氨酸去甲基酶和 TET (10-11转位酶)酶(190)。结果表明,高水平的富马酸抑制 hif-1α 脯氨酰羟基酶,导致 hif-1α 稳定(191)。缺氧诱导因子 -1α 在正常条件下被 prolyl 羟化酶(PHD 1-3)以氧气为底物灭活。Hif-1α 的羟基化与 E3泛素连接酶的降解有关,而缺氧条件下出现稳定和核转位,导致致癌基因激活(192)。Hif-1α 是代谢基因如己糖激酶(HK)、乳酸脱氢酶(LDHA)和葡萄糖转运蛋白(GLUT1)促进肿瘤糖酵解(193)的转录因子。在其他研究中,延胡索酸的积累不依赖于 VHL 通路,而是通过 NF-kB 依赖的机制促进 hif-1α 的 mRNA 和蛋白质的积累。延胡索酸通过上游 Tank biding kinase (TBK1)促进肾癌细胞侵袭,通过非正规信号途径促进 hif-1α 启动子 p65磷酸化和 p65积累(194)。根据延胡索酸的积累对细胞毒性和致癌能力的作用,研究表明,细胞暴露于高水平的延胡索酸和琥珀酸会导致 DNA 大面积断裂,并通过 DNA 超甲基化改变全球 DNA 甲基化模式在人类肝细胞性肝癌中发生作用(195)。

Interestingly it was shown that mTORC1 upregulation leads to accumulation of fumarate, and contributes to tumor transformation. Using a mouse model harboring the kidney specific inactivation of TSC1 that developed progressive renal lesions that eventually resulted in cortical renal papillary carcinoma, it was shown that TSC1 inactivation results in the accumulation of fumarate due to mTOR-dependent downregulation of the FH. The re-expression of FH rescued renal epithelial transformation (196). In support with these results, using primary samples from clear cell renal cell carcinoma (ccRCC) a total of 15 of 23 cancer samples displayed increased positive staining for pS6 protein (~65%), confirming mTORC1 upregulation in a large proportion of ccRCC cases. Among the 23 samples analyzed, 16 samples showed downregulation of FH mRNA levels compared with relative healthy tissue (196).

有趣的是,mTORC1上调导致富马酸的积累,并有助于肿瘤转化。使用一个小鼠模型携带的 TSC1肾脏特异性失活,发展成肾脏进行性病变,最终导致肾皮质乳头状癌,结果显示 TSC1失活导致延胡索酸的积累,由于 mtor 依赖的 FH 下调。FH 在肾上皮细胞转化中的再表达(196)。支持这些结果的是,使用透明细胞肾细胞癌(ccRCC)的初级样本,23个癌症样本中共有15个样本显示 pS6蛋白阳性染色增加(约65%) ,证实在大部分 ccRCC 病例中 mTORC1上调。在所分析的23个样本中,有16个样本的 FH mRNA 水平与相对健康组织(196个)相比呈下调。

mTORC1 as a Therapeutic Target

mTORC1作为治疗靶点的研究

Chemotherapy and radiotherapy represent the leading option for cancer treatment and although responses are observed, relapses in several cancer types are common so then, effective therapeutic options for recurrent disease are lacking. There is a link among mTORC1 signaling upregulation and tumor growth, which establish that tumors could be responsive to mTORC1 inhibitors. The correlation between tumor growth and hyperactive mTORC1 signaling suggests that tumors may be sensitive to mTORC1 inhibitors. mTOR inhibitors are known primarily as cytostatic, so inhibiting cell growth could induce cell death when mTOR inhibitors are administrated alone or combined with different therapeutic drugs. Such inhibitors are a promising therapeutic strategy for treating several cancer types (197).

化疗和放射治疗是癌症治疗的主要选择,虽然观察到反应,但在几种癌症类型中复发是常见的,因此,对复发性疾病缺乏有效的治疗选择。mTORC1信号通路上调与肿瘤生长有关,这表明肿瘤对 mTORC1抑制剂有反应性。肿瘤生长与高活性 mTORC1信号转导的相关性提示肿瘤可能对 mTORC1抑制剂敏感。mTOR 抑制剂主要被认为是细胞抑制剂,因此当 mTOR 抑制剂单独或与不同的治疗药物联合使用时,抑制细胞生长可能导致细胞死亡。这种抑制剂是治疗几种癌症类型的有希望的治疗策略(197)。

Rapamycin is the first known allosteric mTORC1 inhibitor studied, however, its poor water solubility and chemical stability have led to implement instead the use of semi-synthetic rapamycin analogs (or rapalogs) that show improved pharmacokinetic properties, solubility and reduced immunosuppressive effects (159160). To date, three rapalogs are being tested in clinical trials, CCI-779 (temsirolimus), AP23573 or MK-8669 (ridaforolimus), and RAD001 (everolimus) (198). Temsirolimus is an ester derivative drug, approved for renal-cell carcinoma patients since 2007, and is administrated to patients via intravenous or orally. Ridaforolimus was designed to improve aqueous solubility and is administered orally. And finally, everolimus is a hydroxyethyl ether derivative that is administrated to patients via oral (199). In addition, the prototype rapamycin (sirolimus) is also being tested in kidney transplant recipients, for preventing the occurrence of secondary skin cancers, which are common in these patients (200).

雷帕霉素是已知的第一种变构型 mTORC1抑制剂,但由于其水溶性差和化学稳定性差,导致人们改用半合成雷帕霉素类似物(或雷帕霉素类似物) ,这些物质具有更好的药代动力学性质、溶解性和减少免疫抑制作用(159,160)。迄今为止,已有三种雷帕霉素进行了临床试验: CCI-779(temsirolimus)、 AP23573或 MK-8669(ridaforolimus)和 RAD001(everolimus)(198)。替西莫司是一种酯类衍生物药物,自2007年以来被批准用于肾细胞癌患者,通过静脉或口服给患者服用。雷达福莫司的目的是提高水溶性和口服给药。最后,依维莫司是一种羟乙基醚衍生物,通过口服给患者服用(199)。此外,雷帕霉素(西罗莫司)的原型也正在肾移植受者中进行测试,以防止继发性皮肤癌的发生,这种皮肤癌在这些患者中很常见(200)。

These drugs induce apoptosis inhibition by forming a complex with the intracellular immunophilin FKBP12 thus inhibiting the phosphorylation of the mTOR targets, S6K1 and 4E-BP1, as a result, the activation of cyclin-dependent kinases (CDK) is prevented, specifically, the expression of cyclin D1 is found to be decreased meanwhile p27 increases and consequently, cells arrested at G1/S phase die either by autophagy or apoptosis (197198).

这些药物通过与细胞内的亲免疫蛋白 FKBP12形成复合物,从而抑制 mTOR 靶点 S6K1和4E-BP1的磷酸化,从而阻止细胞周期蛋白依赖性激酶(CDK)的活化,特别是发现细胞周期蛋白 D1的表达下降,同时 p27的表达增加,导致阻滞于 G1/S 期的细胞死于自噬或凋亡(197,198)。

In the specific case of everolimus, it is known that this drug inhibits the aberrant activity of mTOR by inducing arrest at G1-phase and sensitizing endothelial and tumoral cells to cisplatin and radiotherapy effects through apoptosis enhancement (197). Such effect occurs due to everolimus ability to block p53-induced p21 expression (201). Everolimus has also been tested in cervical cancer cell lines with a remarkable ability to inactivate efficiently the HPV16 E7 oncoprotein inhibiting cell proliferation (202). The capacity of everolimus-based combinations to inhibit cell proliferation from several cancer types has been reported for breast cancer (203204), renal cell carcinoma (205206), and thyroid cancer (207) in clinical trials.

在依维莫司的特殊情况下,已知这种药物通过诱导 g1期阻滞和增加内皮细胞和肿瘤细胞对顺铂的敏感性以及通过增强细胞凋亡来抑制 mTOR 的异常活性(197)。这种效应是由于依维莫司能阻断 p53诱导的 p21表达(201)。依维莫司也在宫颈癌细胞系中进行了试验,该细胞系具有显著的抑制 HPV16 E7肿瘤蛋白增殖的能力(202)。据报道,基于依维莫司的联合药物能够抑制多种癌症类型的细胞增殖,用于乳腺癌(203,204)、肾细胞癌(205,206)和甲状腺癌(207)的临床试验。

In addition to everolimus and temsirolimus, three natural compounds that have been reported as mTOR inhibitors including curcumin, resveratrol and epigallocatechin gallate (EGCG)(208).These compounds proved to be able to induce cytotoxicity in the HeLa cell line when administrated along with radiation. Nevertheless, it is worth to notice that neither everolimus nor temsirolimus seem to be selective for all cancer cell lines as EGCG, resveratrol or curcumin (209). The pro-apoptotic effect of everolimus combined with paclitaxel has been successfully shown for HeLa and SiHa cell lines. In addition, it has been demonstrated that both compounds inhibit the PI3K/AKT/mTOR pathway (210).

除了依维莫司和坦西罗莫司,3种天然化合物已被报道为 mTOR 抑制剂,包括姜黄素、白藜芦醇和表没食子儿茶素没食子酸酯(EGCG)(208)。这些化合物被证明能够诱导细胞毒性的 HeLa 细胞系时,连同照射。然而,值得注意的是,无论是依维莫司还是坦西罗莫司似乎都不能选择所有的癌细胞系,如 EGCG、白藜芦醇或姜黄素(209)。依维莫司联合紫杉醇对 HeLa 和 SiHa 细胞株的促凋亡作用已被成功证实。此外,两种化合物均能抑制 PI3K/AKT/mTOR 通路(210)。

Recently, the combination of a daily everolimus dose administrated with standard chemotherapy for newly diagnosed patients with glioblastoma was evaluated in order to determine its efficacy. Even though everolimus has proved to be effective in several published data, it was evident that its efficacy in clinical trials is not as equal than in in vitro models. The administrated treatment was not efficient for improving clinical outcomes yet lead to increased toxicity. Moreover, it was suggested that one of the reasons for such lacking of efficacy could be the activation of the Akt pathway due to S6 feedback loop driven by mTORC2 so, it has been proposed that a dual inhibition of mTORC1 and mTORC2 could prevent such Akt activation (211).

最近,我们对新诊断的胶质母细胞瘤患者进行了每日依莫司联合标准化疗的评估,以确定其疗效。尽管依维莫司在几个已发表的数据中被证明是有效的,但它在临床试验中的有效性显然不如体外模型。管理治疗不能有效改善临床结果,但导致毒性增加。此外,mTORC1和 mTORC2的双重抑制可能是导致 Akt 通路激活的原因之一,因此提出 mTORC1和 mTORC2的双重抑制可以阻止这种 Akt 通路激活(211)。

An mTOR inhibitor derived from an active fraction of the ethyl acetate extract of Streptomyces sp OA293 was reported in 2018. Although it was fully corroborated that such extract lacks any known natural inhibitor of mTOR to date, the metabolite or metabolites present in such active fraction completely inhibited mTORC1 and controlled Akt activation by blocking mTORC2 phosphorylation at Ser2481. Also, this fraction suppressed the activation of 4E-BP1 and P70S6k in cervical cancer cell lines and, induced autophagy and Bax mediated apoptosis. Such extract may represent a better option for improving clinical outcomes in patients once its proved to perform as well as in cell lines (212).

2018年报道了一种 mTOR 抑制剂,该抑制剂来自链霉菌 OA293乙酸乙酯提取物的活性部分。虽然这种提取物迄今为止没有任何已知的 mTOR 天然抑制剂,但是这种活性部位中的代谢物或代谢产物完全抑制 mTORC1,并通过阻断 Ser2481位点的 mTORC2磷酸化来控制 Akt 的活化。此外,这一部分抑制了4E-BP1和 P70S6k 在宫颈癌细胞系的激活,并诱导了自噬和 Bax 介导的凋亡。这种提取物一旦被证明在细胞系中表现良好,可能是改善患者临床结果的更好选择(212)。

Other rapalogs have been evaluated in clinical trials showing discouraging results in some cases. In 2013, was reported the use of temsirolimus in a phase II study using a dose of 25 mg once a week 4 times. Of 38 patients with cervical cancer enrolled in the study, one of them experienced partial response and 19 had stable disease rendering the effectiveness of temsirolimus alone as questionable (213). According to previous reports performed with cervical cancer cell lines, it was suggested that using mTOR inhibitors could be more efficient when the inhibitors are administrated in combination with other drugs. Three years later, in 2016, Ferreira and colleagues evaluated the maximum-tolerated dose (MTD) of everolimus combined with cisplatin and pelvic radiotherapy, as well as safety and toxicity in 15 patients with advanced stage of cervical cancer in a phase I study. The results showed that although the acceptable dose of everolimus was 5 mg/day, all patients had at least 1 adverse event. Concerning its efficacy, from 12 patients evaluated, 11 showed a complete response, suggesting that 5 mg everolimus together with cisplatin and chemotherapy is a feasible therapy for cervical cancer treatment (207).

在临床试验中,对其他一些指标进行了评估,结果显示在某些情况下结果令人沮丧。2013年,有报道称在第二阶段研究中使用25毫克的替西莫司,每周一次,共4次。38名宫颈癌患者参与了这项研究,其中1名患者出现了部分缓解,19名患者有稳定的疾病,这使得单独使用替米罗莫司的有效性成为疑问(213)。根据以往对宫颈癌细胞系的研究,建议将 mTOR 抑制剂与其他药物联合使用可能更有效。三年后,也就是2016年,费雷拉和同事们在 i 期研究中评估了15例晚期宫颈癌患者使用依维莫司联合顺铂和盆腔放疗的最大耐受剂量(MTD) ,以及安全性和毒性。结果表明,尽管依维莫司的可接受剂量为每天5毫克,但所有患者至少发生了1次不良反应。关于其疗效,从12名患者评估,11显示完全反应,提示5mg 依维莫司联合顺铂和化疗是一个可行的治疗宫颈癌的治疗(207)。

Another promising combination using everolimus has been reported in cancer cell lines using metformin, a drug commonly used for diabetes treatment. Metformin induces the inhibition of OXPHOS due to reduced function of respiratory complex I and AMPK activation, which in turn promotes tumor growth reduction through mTOR inhibition, cell cycle arrest and activation of autophagy; therefore, a combination of both drugs could be more successful for cancer treatment. This synergistic effect was evaluated in breast cancer cell lines (MCF-7, MDA-MB-231, and T47D), cultured with a physiological concentration of glucose under hypoxic or normoxic conditions. The obtained results showed that everolimus and metformin cooperate to inhibit mTOR activity, tumor cell growth and colony formation, independently of glucose and O2concentrations (214). A year later, the synergic effect of metformin and rapamycin was evaluated in a pancreatic cancer cell line (SW1990) where a reduced cell proliferation was observed, moreover, cell viability was also reduced when cells were treated with both rapamycin and metformin, importantly, an evaluation of phosphorylated mTOR showed that only a combination of the two drugs was capable to suppress the mTOR pathway. Finally, using a xenograft tumor model, the capacity of metformin and rapamycin to inhibit tumor growth was confirmed (215).

另一种使用依维莫司的有希望的组合已经在使用二甲双胍的癌细胞系中被报道,二甲双胍是一种常用的治疗糖尿病的药物。二甲双胍通过降低呼吸复合物 i 和 AMPK 的激活功能而诱导 OXPHOS 的抑制,这反过来又通过抑制 mTOR、细胞周期阻滞和激活自噬来促进肿瘤生长的减少; 因此,两种药物联合治疗肿瘤可能更为成功。这种协同效应在乳腺癌细胞系(MCF-7,MDA-MB-231,和 T47D)中进行了评估,在低氧或常氧条件下培养的葡萄糖生理浓度。结果表明,不论葡萄糖和 O2浓度(214)如何,依维莫司和二甲双胍都能抑制 mTOR 活性、肿瘤细胞生长和集落形成。一年后,二甲双胍和雷帕霉素的协同作用在一个胰腺癌细胞系(SW1990)中进行了评估,观察到细胞增殖减少,此外,雷帕霉素和二甲双胍处理细胞也降低了细胞活力,重要的是,对磷酸化 mTOR 的评估表明,只有两种药物的联合能够抑制 mTOR 通路。最后,使用异种移植瘤模型,二甲双胍和雷帕霉素抑制肿瘤生长的能力得到证实(215)。

As mentioned before, the use of mTORC1 inhibitors in clinical trials has not been as successfully demonstrated as it has been in cancer cell lines. A possible explanation to this phenomenon could be that upon mTORC1 inhibition, PI3K-AKT cell signaling is stimulated and, consequently it may increase the survival of cancer cells (199). All this because rapamycin and its rapalogs selectively target only mTORC1 without affecting mTORC2, such selective inhibition could prompt feedback loops resulting in AKT activation at ser473 (216). However, it is important to highlight once more that there is plenty of information, which suggests that the use of such inhibitors in combination with other drugs could improve clinical outcome; what is more, inhibiting both mTORC1 and mTORC2 could improve the poor response of other inhibitors observed in clinical trials.

如前所述,mTORC1抑制剂在临床试验中的应用还没有在癌细胞系中得到成功的证明。对这一现象的一个可能的解释是,在 mTORC1抑制作用下,PI3K-AKT 细胞信号传导受到刺激,从而可能增加癌细胞的存活率(199)。这一切都是因为雷帕霉素及其衍生物只选择性靶向 mTORC1而不影响 mTORC2,这种选择性抑制可以提示反馈环,导致 AKT 在 ser473(216)处激活。然而,重要的是要再次强调,有大量的信息,这表明使用这种抑制剂与其他药物联合可以改善临床结果; 更重要的是,抑制 mTORC1和 mTORC2可以改善在临床试验中观察到的其他抑制剂的不良反应。

Besides mTORC1 rapalogs, there is another group of mTOR inhibitors known as ATP analogs; such drugs inhibit mTOR kinase activity trough competing with ATP in order to bind to the mTOR kinase domain. ATP donates the phosphate group by which mTOR phosphorylates its target proteins. The ATP analogs inhibit both mTORC1 and mTORC2, interestingly, and because of the resemblance of the kinase domains of mTOR and PI3Ks, this analogs are able to inhibit also PI3K (199).

除了 mTORC1 rapalogs,还有另一组 mTOR 抑制剂称为 ATP 类似物; 这类药物通过与 ATP 竞争来抑制 mTOR 激酶活性,从而结合 mTOR 激酶结构域。ATP 供给磷酸基团,mTOR 通过这个磷酸基团磷酸化其目标蛋白。ATP 类似物能同时抑制 mTORC1和 mTORC2,并且由于 mTOR 和 PI3Ks 的激酶结构域的相似性,该类似物也能抑制 PI3K (199)。

Inhibition of both PI3K and mTOR ought be effective in eliminating cancer cells. A recent publication tested a low-dose triple drug combination that inhibits the pathways PI3K, Akt and mTOR in seven cell lines derived from ovarian clear cell carcinoma (OCCC). The use of the drugs AZD8055, GDC0941, and selumetinib decreased cell proliferation and significantly reduced tumor growth in two OCCC patient-derived xenograft mice models. The results and lack of adverse effects in the mice show that the combination of these three drugs could validate future clinical tests (217).

抑制 PI3K 和 mTOR 应该能有效地清除肿瘤细胞。最近的一份出版物测试了一种低剂量三联药物组合,它可以抑制来自卵巢透明细胞癌(occ)的七个细胞系中的通路 PI3K、 Akt 和 mTOR。AZD8055、 GDC0941和 selumetinib 药物的使用,在两个 OCCC 病人来源的异种移植小鼠模型中,减少了细胞增殖,显著减少了肿瘤生长。结果和小鼠的不良反应表明,这三种药物的联合可以验证未来的临床试验(217)。

CC-223 is a competitive inhibitor of the mTOR that targets mTORC1 and mTORC2, preventing up regulation of Akt phosphorylation, a great advantage, if comparing to the rapalogs. In a phase I Dose-Escalation study, CC-22 was evaluated in twenty-eight patients with advanced cancer. Safety, tolerability, non-tolerated dosage, maximum tolerated dosage (MTD), and preliminary pharmacokinetic profile were evaluated; the reported adverse effects were hyperglycemia, rash, fatigue and mucositis, 45 mg/d was determined as the MTD and an inhibition of phosphorylation of mTORC1/mTORC2 pathway biomarkers present in blood was observed. Taken together these results suggest that CC-223 was tolerable, with manageable toxicities representing a promising antitumor activity compound (218).

CC-223是 mTORC1和 mTORC2靶向的 mTOR 竞争性抑制剂,防止 Akt 磷酸化的上调,如果比较猛禽,这是一个巨大的优势。在 i 期剂量升级研究中,对28例晚期癌症患者进行了 CC-22评估。评价安全性、耐受性、非耐受剂量、最大耐受剂量(MTD)和初步药代动力学曲线,报道的不良反应为高血糖、皮疹、疲劳和粘膜炎,测定 MTD 为45mg/d,观察血液中 mTORC1/mTORC2途径生物标志物的磷酸化抑制作用。综合这些结果表明,CC-223是可耐受的,具有可控的毒性,代表一种有希望的抗肿瘤活性化合物(218)。

Sapanisertib (TAK-228) is a potent and highly selective mTORC1/mTORC2 inhibitor that has been tested in non-hematological malignancies. In this study, sixty-one patients with advanced solid tumors were given daily or a weekly dose of TAK-228 alone or in combination with paclitaxcel. The results showed that just one patient that received TAK-228 plus paclitaxel showed a complete response, moreover, three patients that took TAK-228 plus paclitaxel and two patients with a daily dose of TAK-228 showed a partial response. Additionally, safety analyses showed that fatigue was the main adverse effect, followed bypruritus, lack of appetite and diarrhea, among others but any severe effect related to the treatment was reported. Contrary to everolimus and temsirolimus treatment, anemia and thrombocytopaenia were not reported as adverse effects by consuming TAK-228. Even though the authors emphasize a positive response to TAK-228 alone or in combination with paclitaxel, which could guarantee further investigations, it is only highlighted a positive response for some solid tumors (219).

Sapanisertib (TAK-228)是一种高选择性的 mTORC1/mTORC2抑制剂,已用于非血液肿瘤的治疗。在这项研究中,61名晚期实体肿瘤患者每天或每周单独或联合使用 TAK-228或 pacliaxcel。结果显示,只有一名患者接受 TAK-228联合紫杉醇治疗后出现完全缓解,此外,三名患者接受 TAK-228联合紫杉醇治疗,两名患者接受日剂量 TAK-228治疗后出现部分缓解。此外,安全性分析表明,疲劳是主要的不良反应,其次是瘙痒,缺乏食欲和腹泻等,但任何严重的影响相关的治疗报告。与依维莫司和坦西莫司治疗相反,服用 TAK-228并未报告贫血和血小板减少症为不良反应。尽管作者强调对 TAK-228单独或与紫杉醇联合应用的阳性反应,这可以保证进一步的研究,但只是强调对某些实体肿瘤的阳性反应(219)。

Recently, specific mTORC1/mTORC2 inhibitors, torin2, INK-128, and NVP-Bez235 (which also inhibits PI2K), were tested on LNT-229 human glioblastoma cells. INK-228 and NVP-Bez235 inhibited the phosphorylation of mTOR targets S6RP and NDRG1, and together with torin2 showed a better capacity of inhibiting mTOR pathway when compared to rapamycin due to a more effective inhibition of 4EBP phosphorylation. The main contribution of this paper was that they highlight the metabolic effects of partial mTOR pathway inhibition by rapamycin and rapalogs to economize resources when cells are exposed to nutrient deficiency and hypoxic conditions, which could promote survival of tumor cells hence, highlighting the use of dual mTORC1/mTORC2 inhibition because such inhibitors are able to target dividing cells more efficiently (220).

最近,特异性 mTORC1/mTORC2抑制剂 torin2、 INK-128和 NVP-Bez235(也能抑制 PI2K)在 LNT-229人胶质母细胞瘤细胞上进行了试验。与雷帕霉素相比,ink228和 NVP-Bez235对 mTOR 靶点 S6RP 和 NDRG1的磷酸化有明显的抑制作用,与 torin2相比,它们对 mTOR 通路的抑制作用更强。本论文的主要贡献在于突出了雷帕霉素和雷帕霉素抑制部分 mTORC1/mTORC2通路的代谢效应,以节省细胞在营养缺乏和低氧条件下的资源,从而促进肿瘤细胞的存活,突出了双 mTORC1/mTORC2抑制剂的应用,因为这类抑制剂能够更有效地分裂靶细胞(220)。

Another mTORC1/mTORC2 inhibitor, CC-223, was evaluated in a phase II study including 47 patients with non-pancreatic neuroendocrine tumors. Tolerability, preliminary efficacy and pharmacokinetic of CC-223 was evaluated in a daily dose. The results were consistent with those presented in cell lines; anti-tumor activity was assessed, and the data obtained indicated that the drug was safe for patients (221). Additionally, other mTORC1/mTORC2 known as vistusertib was evaluated in a phase II study for patients with relapsed or refractory diffuse large B cell lymphoma, in this specific case, the dual inhibitor vistusertib did not show any advantage over mTORC1 inhibitors in the group of patients evaluated (222).

另一种 mTORC1/mTORC2抑制剂 CC-223在 II 期研究中被评估,包括47例非胰腺神经内分泌肿瘤患者。以每日剂量评价 CC-223的耐受性、初步疗效和药代动力学。结果与细胞系中的结果一致,抗肿瘤活性得到了评估,所获得的数据表明该药物对患者是安全的(221)。此外,其他 mTORC1/mTORC2称为 vistusertib 在 II 期研究中用于复发或难治性弥漫性大 b 细胞淋巴瘤患者,在这一特定病例中,双重抑制剂 vistusertib 与 mTORC1抑制剂相比没有任何优势(222)。

The combination of mTOR inhibitors with other drugs or treatments is thought to be more effective than just one treatment alone. Recently, the oral administration of PQR309, a dual PI3K and mTORC1/mTORC2 inhibitor, was evaluated in a phase I trial of patients with advanced solid tumors. The patients presented several adverse effects as fatigue, rash and loss of appetite and partial response was reported (223).

联合使用 mTOR 抑制剂和其他药物或治疗被认为比单独使用一种治疗更有效。最近,PQR309—- 一种双重的 PI3K 和 mTORC1/mTORC2抑制剂—- 的口服给药在晚期实体肿瘤患者的 i 期临床试验中被评估。患者表现出几个不良反应,如疲劳,皮疹和食欲下降和部分反应报告(223)。

In sum, rapamicyn and rapalogs inhibit mTORC1 as demonstrated in several in vitro experiments (160), though incomplete mTOR signaling occurs due to these drugs incapacity of inhibit mTORC2 too, and in consequence, it has been suggested that cancer cells could survive because of Akt activation, for this reason and aiming to replicate the successful results observed in cell lines to patients, it is imperative to evaluate the synergic effect of mTOR inhibitors with other drugs or treatments that have shown promising results in patients and also lead the inhibition of mTOR signaling by drugs to perform a complete inhibition of mTORC1 and mTORC2 in order to guarantee clinical outcome.

总之,rapamicyn 和 rapalogs 可以抑制 mTORC1,虽然由于这些药物不能抑制 mTORC2而导致 mTOR 信号的不完全发生,因此有人认为肿瘤细胞可以通过 Akt 激活而存活,为了将在细胞系中观察到的成功结果复制给患者,必须评价 mTOR 抑制剂与其他药物或治疗的协同作用,这些药物或治疗在患者中显示出有希望的结果,也可以通过药物抑制 mTORC1和 mTORC2信号发生完全的抑制,以保证临床结果。

Concluding Remarks

结语

mTORC1 is widely described as an important regulator of cell growth, acting on the regulation of anabolic processes such as the synthesis of proteins, lipids, and the inhibition of autophagy. Importantly, mTORC1 is also involved in the regulation of mitochondrial metabolism and mitochondrial functions. In tumor exists a continuous two-way communication between mitochondria and the nucelus that orchestrates production of the mitochondrial encoded proteins and the nuclear-encoded mitochondria proteins to meet the cells continually changing energy and biosynthetic requirements. mTORC1 plays the major role in the regulation of the mitochondrial protein translation, moreover mTOR is an important regulator of mitochondrial turnover by regulating mitochondrial fusion and fission processes mainly deregulated in cancer and that are associated with chemotherapy resistance.

mTORC1被广泛地描述为细胞生长的重要调节因子,作用于合成蛋白质、脂质和抑制自噬等合成过程。重要的是,mTORC1还参与了线粒体代谢和功能的调节。在肿瘤中,线粒体与核心之间存在着连续的双向通讯,协调线粒体编码的蛋白质和核编码的线粒体蛋白质的产生,以满足细胞不断变化的能量和生物合成的需要。mTORC1在线粒体蛋白质翻译的调节中起着重要作用,而且 mTOR 是线粒体转换的重要调节因子,主要通过调节肿瘤中线粒体融合和分裂过程,并与化疗耐药有关。

However, it is necessary to intensify research to clarify the participation of mTORC1 in the regulation of these mitochondrial functions and their impact on the aggressiveness of tumors. The fact that mitochondria promotes metabolic plasticity associated with resistance to therapy and the existence of several drug able regulators, proposes this events as promising therapeutic targets in cancer. In addition to the regulatory actions performed by mTOR in mitochondrial functions it represents an opportunity to deeply study for therapy, developing treatment plans with synergy, mainly using mTOR inhibitors, and mitochondrial inhibitors. In this manner, the use of metformin is an attractive therapeutic option with probed efficacy in clinical trials.

然而,有必要加强研究以澄清 mTORC1参与调节这些线粒体功能及其对肿瘤侵袭性的影响。事实上,线粒体促进代谢的可塑性与治疗的抵抗和存在一些药物可调节,提出这一事件作为有希望的治疗目标的癌症。除了 mTOR 在线粒体功能方面的调节作用之外,它还提供了一个深入研究治疗的机会,以协同方式制定治疗计划,主要使用 mTOR 抑制剂和线粒体抑制剂。在这种方式下,使用二甲双胍是一个有吸引力的治疗选择探讨疗效在临床试验。

发表评论

邮箱地址不会被公开。 必填项已用*标注