综述节选译文
干细胞、肿瘤和肿瘤干细胞
干细胞生物学开创了一个新纪元。在造血系统中存在干细胞的尚不明确的证据已经为分离组织特异性干细胞和祖细胞带来了曙光,同时对它们特性和基因表达程序的描绘带来希望,以及为其应用于再生医学上带来前景。
干细胞最重要和最有实用价值的性质或许就是它的自我更新的性质。通过这个性质,我们看到干细胞和肿瘤细胞之间有着惊人的相似性,肿瘤可能来源于正常干细胞的变异,在干细胞和肿瘤细胞中有着相似的信号调节通路,肿瘤细胞中可能包含“肿瘤干细胞”,极少数这样的细胞却有着无限自我更新的能力,来促使肿瘤生成。
干细胞是一类具有持续的自我更新能力和分化为特定组织成体细胞的细胞。在大多数组织里,干细胞是非常少的,所以要研究干细胞的性质必须将干细胞进行分离纯化。过去假设每种组织都是来源于一种组织特异性的干细胞,尽管这似乎是合理的,然而仅仅在少数的组织中分离出了成体干细胞。比如,人们已经在小鼠和人身上分离出造血干细胞(HSCs),并且证实这些干细胞在血液的形成和免疫系统(造血淋巴系统)中起作用(图1)。也许将来能把来源于各种器官的干细胞用于治疗手段,而造血干细胞作为骨髓移植的重要成分已经广泛应用于治疗了。
最近的研究发现,骨髓和纯化的造血干细胞能够生成非造血系统组织,这表明比起过去的假设来,这些细胞可能有着更广泛的分化潜能。我们需要有确切的实验来明确这些来源于骨髓并且生成非造血系统组织的细胞究竟是造血干细胞还是其他的细胞群。如果进一步的研究能够支持造血干细胞的多向性的话,那么无疑会对造血干细胞发展潜力的认识打开新的局面,同时将会扩展它们的治疗应用。
由于造血干细胞的特点、分化潜能和临床应用已经在早期综述中报道了,在此我们讨论一下干细胞生物学能给肿瘤生物学带来新视野的证据。我们特别关注干细胞和肿瘤细胞三个方面的关系:首先,正常干细胞和肿瘤细胞在调节自我更新方面有着相似的机制;其次,肿瘤细胞来源于正常干细胞有着可能性;最后,肿瘤中可能存在“肿瘤干细胞”,极少数这样的细胞却有着无限增殖潜能,能够启动肿瘤的发生发展。在本文中我们着重关注造血系统,因为来源于这种组织的正常干细胞和肿瘤细胞都很容易标记。此外,造血系统的肿瘤(比如白血病)提供最佳证据证明了正常干细胞是转化突变的靶点,并且肿瘤细胞增殖是由肿瘤干细胞启动的。
造血干细胞的自我更新
干细胞生物学中最重要的问题之一就是要明确其自我更新的调节机制。自我更新对干细胞实现其功能来说是至关重要的,因为动物维系生命需要多种干细胞的持续更新。此外,尽管来源于不同器官的干细胞有着不同的发展潜能,但所有的干细胞都必须能够进行自我更新以及能够调节自我更新和分化之间的相对平衡。明确正常干细胞自我更新的调节机制对于理解肿瘤细胞增殖的调节机制同样是很重要的,因为肿瘤可以看作是一种自我更新失调的疾病。
在造血系统里,干细胞自我更新的能力存在异质性。在小鼠骨髓细胞里含有0.05%的多能干细胞,它们能够分化为三种不同的细胞群:长期自我更新的造血干细胞、短期自我更新的造血干细胞和无自我更新潜能的多能干细胞。其中长效的造血干细胞能够生成即时造血干细胞,最后生成多能干细胞。由于造血干细胞是在长期的自我更新到多能干细胞的过程中成熟起来的,所以它们逐渐丧失了自我更新的能力,但是分裂变得更加活跃。在小鼠体内,长效造血干细胞终生产生成熟的造血细胞,而即时造血干细胞和多能祖细胞在受过致死剂量辐射的小鼠体内只存活不到八周。
尽管人们已经确定了造血干细胞的表型和功能性质,它们是怎样调节自我更新能力的根本问题仍然不清楚。大多数情况下,造血干细胞在长期培养基中与生长因子的结合会诱导其强大的增殖能力,导致其不会分化。尽管人们改变过很多培养基的条件寻找保持造血干细胞的活性的原因,结果证明要确定培养基中是哪种生长因子导致了造血干细胞活性的显著增强仍然是很困难的。
调节干细胞自我更新和肿瘤发生的信号通路
由于正常干细胞和肿瘤细胞均有着自我更新的能力,似乎可以合理推测新生肿瘤细胞中有调控自我更新的成分,这些成分通常是在干细胞中表达。有证据表明许多跟肿瘤相关的经典通路同时也调控着正常干细胞的发展(图2),比如,原癌基因bcl-2表达的失调会引起细胞无法凋亡,从而会使体内造血干细胞数目剧增,这表明细胞死亡在调节造血干细胞的内环境稳定方面起着作用。
其他与肿瘤发生相关的信号通路像Notch、Shh和Wnt信号通路可能同时调节干细胞的自我更新。在体内外的培养基中用配体Jagged-1激活Notch通路可观察到原始祖细胞总数持续增加,表明Notch通路的激活可以促进造血干细胞的自我更新,或者至少维系其多向性。Shh信号通路同样跟自我更新的调节有关,因为在体外结合其他生长因子导致Shh通路被激活后,人们发现人类造血干细胞(CD34+Lin–CD38–)富集的细胞群自我更新能力会增强。Notch和Shh在造血干细胞自我更新中的作用非常有趣,因为这表示这些通路也调控其他组织中的干细胞的自我更新。
另一个极其有趣的在不同器官中同时调控自我更新和肿瘤发生的通路是Wnt信号通路(图2)。Wnt蛋白是一种胞间信号分子,在多种机制中调节生长,并且当其失调时会导致肿瘤。Wnt蛋白在骨髓的表达说明它们可能也影响造血干细胞。我们利用高度纯化的小鼠骨髓造血干细胞证明长期培养中β-catenin(一种能够激活Wnt信号通路下游的分子)的过表达会使由功能(能够在体外复原造血系统)和表型(Thy1.1loLin–/loSca1+c-kit+)确定的可移植造血干细胞得到扩增。此外,Wnt信号通路中的一个抑制因子Axin的异位表达会导致体外造血干细胞增殖阻滞和死亡增多,以及体内造血干细胞的修复减缓。另一项研究证明,从控制条件的上清中收集的可溶性Wnt蛋白能够影响来源于小鼠胚肝和人骨髓的造血干细胞的增殖。
对上皮细胞和小肠祖细胞的研究表明Wnt信号通路可能在调节其他组织中的干细胞或者祖细胞的自我更新方面起作用。经培养的有着更强增殖能力的人角质形成细胞,其β-catenin的表达水平比低增殖能力的角质形成细胞要高。此外,经逆转录病毒转导激活的β-catenin会引起上皮干细胞的自我更新增强和分化减弱。体外转基因小鼠的实验数据表明在上皮干细胞中Wnt信号通路的激活能够导致上皮癌的发生。另外,如果小鼠缺少了介导Wnt信号通路的分子之一TCF-4,会在胚胎发育时期就迅速耗尽小肠上皮隐窝处未分化的祖细胞,提示Wnt信号通路在维系小肠上皮干细胞的自我更新方面是必需的。
综合上述发现,提示Wnt信号通路除了在造血干细胞中以外还在多种不同的上皮细胞中促进干细胞的自我更新。Wnt信号通路影响干细胞的分子机制尚未明了。明确Wnt、Notch和Shh通路的相互作用是否调控干细胞和祖细胞的自我更新也是十分重要的。
原文:
Stem cells, cancer, and cancer stem cells
Stem cell biology has come of age. Unequivocal proof that stem cells exist in the haematopoietic system has given way to the prospective isolation of several tissue-specific stem and progenitor cells, the initial delineation of their properties and expressed genetic programmes, and the beginnings of their utility in regenerative medicine. Perhaps the most important and useful property of stem cells is that of self-renewal. Through this property, striking parallels can be found between stem cells and cancer cells: tumours may often originate from the transformation of normal stem cells, similar signalling pathways may regulate self-renewal in stem cells and cancer cells, and cancer
cells may include ‘cancer stem cells’ — rare cells with indefinite potential for self-renewal that drive tumorigenesis.
Stem cells are defined as cells that have the ability to perpetuate themselves through selfrenewal and to generate mature cells of a particular tissue through differentiation. In most tissues, stem cells are rare. As a result, stem cells must be identified prospectively and purified carefully in order to study their properties. Although it seems reasonable to propose that each tissue arises from a tissue-specific stem cell, the rigorous identification and isolation of these somatic stem cells has been accomplished only in a few instances. For example, haematopoietic stem cells (HSCs) have been isolated from mice and humans, and have been shown to be responsible for the generation and regeneration of the blood-forming and immune (haematolymphoid) systems (Fig. 1). Stem cells from a variety of organs might have the potential to be used for therapy in the future, but HSCs — the vital elements in bone-marrow transplantation — have already been used extensively in therapeutic settings.
The recent discovery that bone marrow, as well as purified HSCs, can give rise to non-haematopoietic tissues suggests that these cells may have greater differentiation potential than was assumed previously. Definitive experiments are needed to determine whether the cells from the bone marrow that are capable of giving rise to different non-haematopoietic lineages are indeed HSCs or another population. If further studies support the idea of HSC plasticity, this will undoubtedly open new frontiers for understanding the developmental potential of HSCs, as well as expand their therapeutic application.
As the characteristics of HSCs, their differentiation potential and clinical applications have been covered in earlier reviews, here we discuss emerging evidence that stem cell biology could provide new insights into cancer biology. In particular, we focus on three aspects of the relationship between stem cells and tumour cells: first, the similarities in the mechanisms that regulate self-renewal of normal stem cells and cancer cells; second, the possibility that tumour cells might arise from normal stem cells; and third, the notion that tumours might contain ‘cancer stem cells’ — rare cells with indefinite proliferative potential that drive the formation and growth of tumours. Through much of this review we focus on the haematopoietic system because both normal stem cells and cancer cells from this tissue are well characterized. Moreover, cancers of the haematopoietic system (that is, leukaemias) provide the best evidence that normal stem cells are the targets of transforming mutations, and that cancer cell proliferation is driven by cancer stem cells.
Self-renewal of haematopoietic stem cells
One of the most important issues in stem cell biology is understanding the mechanisms that regulate self-renewal. Self-renewal is crucial to stem cell function, because it is required by many types of stem cells to persist for the lifetime of the animal. Moreover, whereas stem cells from different organs may vary in their developmental potential, all stem cells must self-renew and regulate the relative balance between self-renewal and differentiation. Understanding the regulation of normal stem cell self-renewal is also fundamental to understanding the regulation of cancer cell proliferation, because cancer can be considered to be a disease of unregulated self-renewal.
In the haematopoietic system, stem cells are heterogeneous with respect to their ability to self-renew. Multipotent progenitors constitute 0.05% of mouse bone-marrow cells, and can be divided into three different populations: longterm self-renewing HSCs, short-term self-renewing HSCs, and multipotent progenitors without detectable self-renewal potential. These populations form a lineage in which the long-term HSCs give rise to short-term HSCs, which in turn give rise to multipotent progenitors. As HSCs mature from the long-term self-renewing pool to multipotent progenitors, they progressively lose their potential to self-renew but become more mitotically active. Whereas long-term HSCs give rise to mature haematopoietic cells for the lifetime of the mouse, short-term HSCs and multipotent progenitors reconstitute lethally irradiated mice for less than eight weeks.
Although the phenotypic and functional properties of HSCs have been extensively characterized, the fundamental question of how self-renewal is regulated remains unanswered. In most cases, combinations of growth factors that can induce potent proliferation cannot prevent the differentiation of HSCs in long-term cultures. Although progress has been made in identifying culture conditions that maintain HSC activity in culture, it has proved exceedingly difficult to identify combinations of defined growth factors that cause a significant expansion in culture in the number of progenitors with transplantable HSC activity.
Pathways regulating stem cell self-renewal and oncogenesis
Because normal stem cells and cancer cells share the ability to selfrenew, it seems reasonable to propose that newly arising cancer cells appropriate the machinery for self-renewing cell division that is normally expressed in stem cells. Evidence shows that many pathways that are classically associated with cancer may also regulate normal stem cell development (Fig. 2). For example, the prevention of apoptosis by enforced expression of the oncogene bcl-2 results in increased numbers of HSCs in vivo, suggesting that cell death has a role in regulating the homeostasis of HSCs.
Other signalling pathways associated with oncogenesis, such as the Notch, Sonic hedgehog (Shh) and Wnt signalling pathways, may also regulate stem cell self-renewal. Notch activation in HSCs in culture using the ligand Jagged-1 have consistently increased the amount of primitive progenitor activity that can be observed in vitro and in vivo, suggesting that Notch activation promotes HSC self-renewal, or at least the maintenance of multipotentiality. Shh signalling has also been implicated in the regulation of self-renewal by the finding that populations highly enriched for human HSCs (CD34+Lin–CD38–) exhibit increased self-renewal in response to Shh stimulation in vitro, albeit in combination with other growth factors. The involvement of Notch and Shh in the self-renewal of HSCs is especially interesting in light of studies that implicate these pathways in the regulation of self-renewal of stem cells from other tissues as well.
One particularly interesting pathway that has also been shown to regulate both self-renewal and oncogenesis in different organs is the Wnt signalling pathway (Fig. 2). Wnt proteins are intercellular signalling molecules that regulate development in several organisms and contribute to cancer when dysregulated. The expression of Wnt proteins in the bone marrow suggests that they may influence HSCs as well. Using highly purified mouse bone-marrow HSCs, we have shown that overexpression of activated b-catenin (a downstream activator of the Wnt signalling pathway) in long-term cultures of HSCs expands the pool of transplantable HSCs determined by both phenotype (Thy1.1loLin–/loSca1+c-kit+) and function (ability to reconstitute the haematopoietic system in vivo). Moreover, ectopic expression of Axin, an inhibitor of Wnt signalling, leads to inhibition of HSC proliferation, increased death of HSCs in vitro, and reduced reconstitution in vivo (T.R. et al., submitted). In separate studies, soluble Wnt proteins from conditioned supernatants have also been shown to influence the proliferation of haematopoietic progenitors from mouse fetal liver and human bone marrow.
Studies of epidermal and gut progenitors suggest that the Wnt signalling pathway may contribute to the regulation of stem cell/progenitor cell self-renewal in other tissues. Cultured human keratinocytes with higher proliferative potential have increased levels of b-catenin compared with keratinocytes with lower proliferative capacity. Moreover, retroviral transduction of activated b-catenin results in increased epidermal stem cell self-renewal and decreased differentiation. In vivo data from transgenic mice suggest that activation of the Wnt signalling pathway in epidermal stem cells leads to epithelial cancers. Furthermore, mice lacking TCF-4, one of the transcriptional mediators of the Wnt signalling pathway, quickly exhaust the undifferentiated progenitors in the crypts of the gut epithelium during fetal development, suggesting that this pathway is required for the maintenance or self-renewal of gut epithelial stem cells.
Cumulatively, the above findings suggest that Wnt signalling may promote stem cell self-renewal in a variety of different epithelia in addition to HSCs. The molecular mechanisms by which Wnt signaling influences stem cells remain to be elucidated. It will also be important to determine whether the Wnt, Notch and Shh pathways interact to regulate stem and progenitor cell self-renewal.
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