I OncomiR: microRNA在癌症中的重要作用 | 生命奥秘
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I& OncomiR: microRNA在癌症中的重要作用
&&&&&& microRNA（miRNA）是长度在18～25个核苷酸左右的内源性非编码小分子RNA。miRNA在进化上高度保守，具有转录后基因调控功能。它由基因组DNA编码，在RNA聚合酶II的作用下被转录。这些小分子通过RNA诱导的沉默复合体（RNA-induced silencing complex, RISC）靶向到达mRNA，进而行使阻遏翻译或引导酶切的功能。最近有研究表明，miRNA具有多种生物学功能，如调节细胞发育、分化、增殖、凋亡等。有研究人员发现线虫体内的异时性基因（见文后小词典1）lin-4编码的小RNA与lin-14反义互补。据估计，脊椎动物基因组编码多达1000种不同的miRNA，研究人员推测，这些miRNA可能调控至少30%的基因的表达。尽管研究人员目前已在人体内发现超过530种miRNA，但仍需进一步研究以了解这些分子的确切的细胞学功能，及其在疾病发生中所扮演的角色。最近有研究报道，miRNA具有肿瘤抑制因子及癌基因的作用。那些在癌症发生发展中发挥作用的miRNA被称作oncogenic miRNA，即oncomiR。
microRNA与癌症&&&&&& oncomiR的失调与基因突变或表观遗传变异有关，这些变异包括缺失突变、扩增突变、点突变及DNA异常甲基化等。其表达情况与人类多种恶性肿瘤的发生、发展、诊断、预后相关（见表1）。&&&&&&
表1：microRNA在癌症中的作用
神经母细胞瘤
miR-15、miR-15a
白血病、垂体腺瘤
miR-16、miR-16-1
白血病、垂体腺瘤
miR-17-5p、miR-17-92
肺癌、淋巴瘤
肺癌、淋巴瘤
乳腺癌、胆管腺癌、头颈癌、白血病、宫颈癌
miR-29、miR-29b
白血病，胆管腺癌
结肠直肠癌
神经母细胞瘤
结肠直肠癌
白血病、胰腺癌
miR-125a、miR-125b
神经母细胞瘤、乳腺癌
胶质母细胞瘤
结肠直肠癌
结肠直肠癌
结肠癌、宫颈癌
乳腺癌、结肠直肠癌
乳腺癌、白血病、胰腺癌
miR-181、miR-181a、
miR-181b、miR-181c
白血病、胶质母细胞瘤、甲状腺癌
直肠结肠癌
神经母细胞瘤
miR-196a-2
胶质母细胞瘤、甲状腺癌
let-7、let-7a、 let-7a-1、
hsa- let-7a-2、let-7a-3
肺癌、结肠癌
&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&
microRNA在肿瘤诊断中的作用&&&&&& Lu等人采用串珠流式细胞miRNA表达谱分析技术对来自人恶性肿瘤组织的miRNA进行系统表达分析，其中包括结肠癌、肝癌、胰腺癌和胃癌。miRNA表达谱成功对低分化肿瘤进行了分类。研究揭示了miRNA表达水平在癌症诊断中的重要作用[1]。
&&&&&& Bottoni等人则采用芯片技术及RT-PCR分析了垂体腺瘤和正常垂体样本中的全部miRNA，结果发现miRNA表达水平可以区分垂体微腺瘤和垂体巨腺瘤，还有一些miRNA参与细胞增殖与凋亡的过程。miRNA可以作为有效的诊断标志，提高对垂体腺瘤的分类水平[2]。
&&&&&& Lee等人采用原位RT-PCR技术，经分析发现miR-221、miR-301和miR-376a的异常表达只在胰腺癌细胞中出现，而没有在良性胰腺肿瘤及正常间质和导管处出现。miRNA的异常表达可以作为胰腺肿瘤发生的研究线索，同时也可能为胰腺癌诊断提供新的生物学标志[3]。
microRNA在癌症预后判断中的作用&&&&&& Takamizawa等人报道指出，let-7 miRNA的表达往往在人肺癌中缺失，而let-7 miRNA表达的减少与术后生存期的减短显著相关。此外，let-7 miRNA在A549肺腺癌细胞株中的过表达可在体外抑制肺癌细胞的生长。他们的研究结果表明了let-7 miRNA表达水平的改变具有潜在临床和生物学作用[4]。
&&&&&& Yanaihara等人的研究可以区分肺癌组织和非恶性肿瘤组织的miRNA表达模式。前体miRNA hsa-miR-155的高表达和 hsa-let-7a-2的低表达与肺腺癌的低生存率相关。他们的研究表明，miRNA不仅可以作为肺癌的诊断学标志，还可以作为预后预测的标志[5]。
&&&&&& Roldo等人则发现，miR-21在胰腺肿瘤中的过表达与高Ki67增殖指数及出现肝转移高度相关。他们的研究结果表明，miRNA表达水平的改变与恶性肿瘤的进展状态相关[6]。此外，Bloomston等人将胰腺癌细胞中的miRNA的表达水平与正常胰腺和慢性胰腺炎组织细胞的miRNA进行比较，结果发现，胰腺癌细胞中miRNA表达水平与后两者不同，可以有效区分开来。miR-196a-2的高表达可以用于预测不良预后[7]。
&&&&&& 某些miRNA表达状态受到癌细胞中表观遗传学改变的控制，比如DNA甲基化和组蛋白修饰。采用染色质修饰药物激活肿瘤抑制因子miRNA可以调节靶向癌基因，这一策略也许能在不久的将来成为癌症的新型治疗方法。miRNA可以与基因组学、蛋白质组学中的生物学标志相结合，成为癌症诊断和判断预后的参考指标[8,9]。
&&&&&& 尽管每个miRNA可以调控成百个靶向基因，但在癌症研究中鉴别出miRNA的准确靶点仍然是一大难题。此外，由于干细胞可以分化发育成为多种类型的细胞，因而成为研究者们瞩目的焦点。已有研究指出miRNA通路在干细胞分化中起调控作用，而其机制是否可用于癌症的预防与治疗还需要进一步研究[10]。
&&&&&& miRNA在多种病理过程中的功能的发现，使得对疾病进行分子水平的诊断和预后预测成为可能，对于癌症尤其如此。
&&&&&& 2006年度的诺贝尔奖授予了Andrew Fire和Craig Mello。这两位科学家因为发现RNA干扰现象&&双链RNA诱导的基因沉默而获此殊荣。自从在一些生物体内发现相互作用的反义RNA具有调控功能以来，转录后的基因调节便跻身于主要基因调控机制的行列。miRNA通过碱基互补诱导RNA干扰路径，从而抑制靶向mRNA。数以百计的miRNA的发现使我们对于RNA干扰现象的生物医学意义有了全新的认识。研究者的目光开始集中于利用反义寡核苷酸抑制miRNA功能，或者采用小干扰RNA类技术研究miRNA。科学家们致力于揭开miRNA生物学的神秘面纱，挖掘其在疾病治疗方面的应用潜力。对患者进行的有关oncomiR的大型高通量研究，可以对癌症进行新的分类，并对患者预后做出更为准确的预测。联合基因组学、微RNA组学（miRomics）以及蛋白质组学的高通量靶向分析，有助于我们进一步发现miRNA的调节靶点。对oncomiR功能的进一步认识，将给癌症生物学领域带来革命性突破，并为生物医药研究提供新的视点与方法。
原文检索：
参考文献：Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, Golub TR. (2005).
MicroRNA expression profiles classify human cancers. Nature. 435, 834-838.
Bottoni A, Zatelli MC, Ferracin M, Tagliati F, Piccin D, Vignali C, Calin GA, Negrini M, Croce CM, Degli Uberti EC. (2007). Identification of differentially expressed microRNAs by microarray: a possible role for microRNA genes in pituitary adenomas. J Cell Physiol. 210, 370-377.
Lee EJ, Gusev Y, Jiang J, Nuovo GJ, Lerner MR, Frankel WL, Morgan DL, Postier RG, Brackett DJ, Schmittgen TD.( 2007). Expression profiling identifies microRNA signature in pancreatic cancer. Int J Cancer. 120, .
Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H, Harano T, Yatabe Y, Nagino M, Nimura Y, Mitsudomi T, Takahashi T. (2004). Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res. 64, .
Roldo C, Missiaglia E, Hagan JP, Falconi M, Capelli P, Bersani S, Calin GA, Volinia S, Liu CG, Scarpa A, Croce CM. (2006). MicroRNA expression abnormalities in pancreatic endocrine and acinar tumors are associated with distinctive pathologic features and clinical behavior. J Clin Oncol. 24, .
Yanaihara N, Caplen N, Bowman E, Seike M, Kumamoto K, Yi M, Stephens RM, Okamoto A, Yokota J, Tanaka T, Calin GA, Liu CG, Croce CM, Harris CC. (2006). Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell. 9, 189-198.
Bloomston M, Frankel WL, Petrocca F, Volinia S, Alder H, Hagan JP, Liu CG, Bhatt D, Taccioli C, Croce CM. (2007). MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA. 297, .
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小词典：1 异时性基因（heterochronic gene）：&&&&&& 异时性基因在线虫发育过程中扮演着重要角色，它们的准时表达决定了胚胎中每一个细胞的特征，从而让胚胎发育成正常的成体线虫。
补充阅读：1 表观遗传学（epigenetics）：&&&&&& &表观遗传学&是研究在基因的核苷酸序列不发生改变的情况下，基因表达了可遗传的变化的一门遗传学分支学科。表观遗传的现象很多，已知的有DNA甲基化、基因组印记（genomic impriting）以及RNA编辑（RNA editing）等。
&&&&&& 表观遗传学是与遗传学（genetic）相对应的概念。遗传学是指基于基因序列改变所致的基因表达水平的变化，如基因突变、基因杂合丢失和微卫星不稳定等；而表观遗传学则是指基于非基因序列改变所致基因表达水平的变化，如DNA甲基化和染色质构象变化等；表观基因组学是在基因组水平上对表观遗传学改变的研究。所谓DNA甲基化是指在DNA甲基化转移酶的作用下，在基因组CpG二核苷酸的胞嘧啶5′碳位共价键结合一个甲基基团。正常情况下，人类基因组&垃圾&序列的CpG二核苷酸相对稀少，并且总是处于甲基化状态，与之相反，人类基因组中大小为100－1000 bp左右且富含CpG二核苷酸的CpG岛则总是处于未甲基化状态，并且与56％的人类基因组编码基因相关。人类基因组序列草图分析结果表明，人类基因组 CpG岛约为28890个，大部分染色体每1 Mb就有5－15个CpG岛，平均值为每Mb含10.5个CpG岛，CpG岛的数目与基因密度有良好的对应关系。由于DNA甲基化与人类发育和肿瘤疾病有密切关系，特别是CpG岛甲基化所致抑癌基因转录失活问题，DNA甲基化已经成为表观遗传学和表观基因组学的重要研究内容。
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肺干细胞和肺癌干细胞的研究进展
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肺干细胞和肺癌干细胞的研究进展
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一项新研究揭示了癌症干细胞调控机制
研究人员最近发现了一种干细胞信号传导通路，如果这条信号传导通路中断会引发肠肿癌。这项发现加深了人们对干细胞引起肿瘤机制的理解。特异性干细胞分子可作为靶标来抑制肠肿癌发生、发展和复发。
&&桑福德伯纳姆医学研究所的研究人员最近发现了一种干细胞信号传导通路，如果这条信号传导通路中断会引发肠肿癌。这项发现加深了人们对干细胞引起肿瘤机制的理解。特异性干细胞分子可作为靶标来抑制肠肿癌发生、发展和复发。这项研究成果发表于在线《Cell Reports》期刊上。
&&细胞程序性死亡和生存网络项目的负责人Moscat博士说：“越来越多的证据表明，癌症干细胞负责癌症的启动、发展、转移、复发和耐药性。我们的这项研究能帮助人们更好地理解调节肿瘤干细胞的信号级联放大，这对设计新的更有效地疗法具有重大意义。
&&我们已经发现蛋白激酶C-zeta(PKC-zeta)通常下调2个信号通路来抑制肿瘤干细胞活性，这2个信号通路分别是beta-连环蛋白通路和 Yap通路。我们先前的研究发现蛋白激酶C-zeta作为一种肿瘤抑制物维持肠道干细胞的平衡。现在这项研究揭示了这种机制是怎样起作用的。
&&肠道由一层上皮细胞覆盖，这些细胞3至5天更新一代。肠癌干细胞替代这些上皮细胞来调节并维持一种稳定状态。
&&Moscat博士说：“打乱干细胞库内的平衡早造成2方面影响，如减少肠道上皮细胞再生或者增大肿瘤干细胞的增殖。癌症是由控制细胞生长中枢机制的关键基因的突变累计造成的，干细胞是肠道内一类“永生”的细胞群，储存了很多的基因突变。所以，如果干细胞活性提高，同时如果出现肠道蛋白激酶C-zeta不足，那么肿瘤发生的风险就会增大很多，同时如果肿瘤一旦发生，肿瘤就会变得很有侵袭性。
&&研究人员通过使用转基因小鼠模型来研究肠癌，研究团队发现上述过程是由蛋白激酶C-zeta对2种关键的肿瘤促进物beta-连环蛋白和Yap直接磷酸化进行控制的。
&&Moscat博士说：“重要的是，我们确认了人体结肠癌样品中的蛋白激酶C-zeta、beta-连环蛋白和Yap，我们在人体和转基因小鼠模型中得到的结果有力的证明了Yap和beta-连环蛋白是蛋白激酶C-zeta作用的潜在靶标，基于此潜在靶标可开发出新的抗癌疗法。我们的研究结果为通过抑制导致肿瘤发生的途径来预防和治疗肠癌，还为急性或慢性损伤如化疗和放射后肠道再生提供了一种治疗策略。”
&&原文标题：New study sheds light on cancer stem cell regulation
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《Nature》：干细胞修复功能与癌症的联系
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这个帖子发布于13年零12天前，其中的信息可能已发生改变或有所发展。
Johns Hopkins的研究人员报道说越来越多的证据表明在修复组织损伤时出错的干细胞有可能解答长期发炎（因酒精或心痛导致的疼痛）引发某些癌症的原因。相关文章发表在日的Nature上。研究人员发现两种关键的化学信号分子Hedgehog和Wnt在修复损伤组织的干细胞中很活跃。最近，研究人员还在一些很难治愈的癌症中意外地发现了这些信号分子，这个发现支持了有关“一些癌症可能由不知何故变坏的正常干细胞引发”的观点。在过去的十年里，研究人员发现了所谓的癌症干细胞，但是一直不清楚这些癌症干细胞是否来自组织中的正常的干细胞或组织的成熟细胞。研究人员认为与慢性发炎有关的癌症是确定干细胞是否是肿瘤源头的较好的研究背景。慢性发炎能造成组织损伤，这种损伤能够引发一个修复过程，而这个修复过程则需要组织特异性的干细胞来完成修复过程。然而，发炎和损伤的再次出现将阻止这种修复的完成。理论上，那些帮助修复的干细胞可能会不断积累突变从而失去控制。研究人员在一些癌症如肺癌、脑瘤、胃癌等癌症中都发现了Hedgehog的活性。与它类似，Wnt的活性也与一些癌症如结肠癌、血癌、骨癌和肺癌联系在一起。在新的实验中，研究人员发现抑制培养的癌细胞和试验动物的Hedgehog和Wnt能够杀死癌细胞，因此这些途径有可能成为抗癌药物的潜在靶标。但研究人员提醒说Hedgehog和Wnt抑制剂有可能会影响到与这些信号有关的正常过程。Nature 432, 324 - 331 (18 November 2004); doi:10.1038/nature03100
Tissue repair and stem cell renewal in carcinogenesis PHILIP A. BEACHY1,4, SUNIL S. KARHADKAR1,2 & DAVID M. BERMAN2,3,4 1 Department of Molecular Biology and Genetics, The Howard Hughes Medical Institute, Baltimore, Maryland 21205, USA2 Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA3 Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA4 Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USApbeachy@jhmi.eduCancer is increasingly being viewed as a stem cell disease, both in its propagation by a minority of cells with stem-cell-like properties and in its possible derivation from normal tissue stem cells. But stem cell activity is tightly controlled, raising the question of how normal regulation might be subverted in carcinogenesis. The long-known association between cancer and chronic tissue injury, and the more recently appreciated roles of Hedgehog and Wnt signalling pathways in tissue regeneration, stem cell renewal and cancer growth together suggest that carcinogenesis proceeds by misappropriating homeostatic mechanisms that govern tissue repair and stem cell self-renewal.The tightly regulated growth of multicellular animals presents a striking contrast to single-celled organisms, which grow and divide in a manner limited only by nutrients available in the environment. The evolutionary compensation for loss of this exuberant style of growth comes in the form of organs that afford adaptability and efficiency through specialization for functions such as locomotion and reproduction, for sensing and responding to the environment, and for acquisition and use of nutrients. The assembly of such complex organs (pattern formation) requires mechanisms to establish intricate patterns of cell division and differentiation. Development of complex organs also takes longer than simple single-cell division, thus delaying the acquisition of reproductive maturity and exposing complex multicellular animals to a greater risk of tissue damage — whether from use, predation or exposure to a hostile environment.The evolution of mechanisms that increase the complexity of animal form thus is likely to be coupled to the evolution of mechanisms for the renewal and repair of complex organs (pattern maintenance). Given this link, it is perhaps not surprising that pattern formation and pattern maintenance share common mechanisms, such as regulation by Hedgehog (Hh) and Wnt signalling pathways. These pathways play central roles in directing embryonic pattern formation, but also function post-embryonically in stem cell renewal, tissue repair and regeneration. Moreover, when aberrantly activated, these pathways can have important roles in the initiation and growth of cancer.Here we focus on the relationship between the normal roles of Hh and Wnt pathways in pattern maintenance and on their pathological roles in the initiation and growth of malignant tumours. Using these pathways as central points of reference, we review recent developments in the area of cancer stem cells and the relationship of cancer stem cells
we concentrate in particular on stem cell renewal in the context of tissue repair as a common antecedent of cancer initiation.Cancer, stem cells and cancer stem cellsA long-standing idea in cancer biology is that tumours arise and grow as a result of the formation of cancer stem cells, which may constitute only a minority of the cells within a tumour but are nevertheless critical for its propagation. The concept of cancer stem cells1 dates back almost as far as the discovery of somatic stem cells in the haematopoietic system2, and was firmly established experimentally in acute myelogenous leukaemia (AML)3-5. In these studies, a minority of undifferentiated cells isolated from leukaemic patients proved to be the only cells capable of reconstituting tumours on transfer into NOD/SCID (non-obese diabetic/severe combined immunodeficient) the resulting tumours included a range of more differentiated cell types like those in the original leukaemia. In their cell-surface markers, in their multipotency and in their hierarchical self-renewal properties, these cancer stem cells resemble normal haematopoietic stem cells (HSCs), suggesting that the leukaemia stem cells either derive from HSCs or from more differentiated cells through acquisition of HSC properties.Stem cells are appealing candidates as the 'cell of origin' for cancer because of their pre-existing capacity for self-renewal and unlimited replication6, 7. In addition, stem cells are relatively long-lived in comparison to other cells within tissues. They therefore have more opportunity to accumulate the multiple additional mutations that may be required to increase the rate of cell proliferation and produce clinically significant cancers. The discovery of multipotent progenitor cells with the capacity for self-renewal (that is, stem cells) outside the haematopoietic system raises the possibility that cancer stem cells could arise from other tissue stem cells and initiate other cancer types, including solid cancers. Consistent with this possibility, a defined minority of cells within many human breast cancers are the only cells able to propagate the cancer in NOD/SCID mice, resulting in the reconstitution of tumours expressing the heterogeneous surface markers that were present in the original cancer8. In addition, cells with some of the properties of neural stem cells (NSCs), such as the ability to produce differentiated neurons and glia in vitro and in vivo and enhanced renewal activity, have recently been isolated from brain tumours and brain-tumour-derived cell lines9-11.The general validity of the cancer stem cell concept has not been proven, as the number of cancer types for which cancer stem cells have been identified is limited. This is, however, an important issue as successful therapy depends on targeting the cells within a tumour that drive cancer growth. However, as many cancers are heterogeneous both in their cell composition and in the relative abundance of cells capable of propagating tumour growth, it will not be surprising if cancer propagation turns out to be an exclusive property of defined subsets of cells within many particular cancer types. The key to developing effective future therapies thus seems to be the identification and characterization of these cancer stem cells, and the development of drugs that specifically target them.Another important issue for understanding the origins of cancer is the relationship between cancer stem cells and normal tissue stem cells. The best-studied cancer stem cells are those in AML, which have been isolated and individually marked before being serially transferred through several host animals3-5. In most respects examined so far, these cells resemble normal HSCs, consistent with a stem cell origin for the AML-cancer stem cell. The possibility cannot be excluded, however, that cancer stem cells might be derived from more committed progenitors by genetic or epigenetic changes that confer self-renewal ability12. Genetic or epigenetic changes that bestow and activate the ability to self-renew on a committed progenitor cell seem less likely to occur than changes that activate renewal in a stem cell — particularly as the window of cellular plasticity within which committed progenitors might acquire renewal ability is generally limited by progression towards irreversible differentiation and replicative quiescence. De-differentiation of committed progenitors back to stem cells under tightly controlled genetic conditions has nevertheless been reported in the Drosophila testis and ovary. For example, prematurely differentiated spermatogonia in the Drosophila testis can be induced to regenerate male germline stem cells on restoration of a critical signalling pathway, Jak-STAT (Janus kinase-signal transducers and activators of transcription)13. Moreover, cystocytes that have already begun to form oocytes within the ovary can be induced to de-differentiate to form productive female germline stem cells on ectopic activation of signalling by Dpp (Decapentaplegic), the Drosophila homologue of BMP4 (bone morphogenetic protein 4)14, a member of the TGF- (transforming growth Factor-) family.Of particular recent interest in the origin of cancer is the observation that transient Hh and Wnt pathway activities promote stem cell self-renewal in normal tissues, whereas continuous activation is associated with the initiation and growth of many types of human cancer. These pathways thus provide a potential link between the normal self-renewal of stem cells and the aberrantly regulated proliferation of cancer stem cells.Hh and Wnt signalling in stem cell maintenanceIn addition to their well-established roles in directing the patterning of embryonic tissues and structures (reviewed in refs 15–17), the Hh and Wnt pathways have more recently been implicated in the maintenance of stem or progenitor cells in a growing list of adult tissues that now include skin, blood, gut, prostate, muscle and the nervous system18-29 (Table 1). Evidence for a role of these pathways in stem cell maintenance functions comes from genetic interventions in vivo or the treatment of isolated stem cells in vitro (in the case of HSCs and NSCs), followed by assays for proliferation, engraftment and multilineage potential of presumptive stem cell populations. A general feature of the results of these studies is that Wnt and Hh pathway activities seem to increase presumptive stem cell number by stimulating stem cell proliferation. Thus, for example, loss of Hh signalling does not immediately obliterate hippocampal populations of neural stem cells, but affects their number by decreasing their proliferative capacity, both in vivo and in vitro19, 20. A similar effect of Hh pathway activity on numbers of somatic stem cells (follicle stem cells) has been noted in the Drosophila ovary18. Likewise, in vitro treatment of isolated HSCs with Wnt or Hh proteins increases their proliferative capacity and improves their ability to form colonies in vitro and to colonize NOD/SCID mice22, 27. Similarly, loss or inhibition of Wnt pathway activity in the intestine does not abrogate the initial development of normal epithelial architecture, but instead causes a progressive degradation of epithelial structure. This effect is associated with the loss of proliferative activity in the crypts, where stem cells reside21, 26.In contrast to these effects of Hh and Wnt pathway activities on stem cell self-renewal, other signals within the stem cell niche seem to function more immediately in the maintenance of stem cell identity. For example, the Jak/STAT and TGF- signalling pathways seem to specify male and female germ cell identity in the Drosophila testis and ovary, genetic manipulation of these pathways rapidly and qualitatively alters cell phenotypes13, 14. Signals for stem cell maintenance might therefore be classified as signals with immediate effects on the maintenance of stem cell identity, and signals that regulate renewal divisions. Identity maintenance functions cannot be ruled out for Hh and Wnt pathway activities, but most evidence points to stem cell renewal as the main target in most tissues. A role of these pathways in normal stem cell renewal is consistent with their known role in the regulation of stem cell renewal genes such as nestin (encoding an intermediate filament protein) and Bmi-1 (encoding a component of the Polycomb transcriptional-silencing complexes) in tumours that depend on Hh or Wnt signalling (discussed in section 'Cancer and persistent states of repair' below).Hh and Wnt signalling in cancerThe roles of Hh and Wnt pathways in stem cell renewal are particularly interesting given the genetically implied connection between activity of these pathways and the initiation and growth of a substantial fraction of lethal cancers (Table 2). Familial mutations that facilitate Hh and Wnt pathway activation have been associated with increased incidence of specific brain, skin, skeletal muscle, liver and colon cancers in humans and mice, and of bladder cancer in mice. Additional studies in which pathway activities are antagonized by treatment with pharmacological agents, with antibodies that bind and block ligand action, or by overexpression of negatively acting pathway components further demonstrate an ongoing requirement for pathway activity in the growth of additional cancer types which include small-cell lung cancer and carcinomas of the oesophagus, stomach, pancreas, biliary tract and prostate. The range of organs from which Hh- and Wnt-pathway-dependent cancers originate is therefore similar to the range of organs in which these pathways have a role in stem cell renewal. In terms of medical significance, about one-third of total cancer deaths are caused by the cancer types in which current evidence implicates Hh or Wnt pathway activity in most cases30.The Hh pathway in cancerThe link between Hh pathway activity and cancer was initially established by the identification of heterozygous mutations affecting Patched (PTCH), a negatively acting component of the Hh receptor, as the cause of Gorlin's syndrome. This syndrome is associated with an increased incidence of basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma, and PTCH is also mutated in sporadic forms of these cancers, thus identifying PTCH as a tumour suppressor (reviewed in refs 6, 31). A similar range of tumours was also found to be associated with sporadic activating mutations affecting the positive receptor component and proto-oncogene Smoothened (SMO). In normal Hh pathway function, the transporter-like Ptch protein acts catalytically to restrain activation of the seven-transmembrane protein Smo. Ptch activity is blocked by binding of Hh, which liberates Smo for activation of transcriptional targets through the Gli family of latent transcriptional factors (see Fig. 1a). These features of Hh signalling are broadly conserved between Drosophila and mammals, as are common mechanisms for Hh protein processing and lipid modification and a dedicated mechanism for the release of lipid-modified Hh protein from producing cells. (reviewed in refs 32, 33). In addition the Gli proteins, like their Drosophila counterpart Ci, can be regulated by interactions with the Suppressor of fused (Su(fu)) protein and can exist in activating forms (primarily Gli and Gli2) as well as in proteolytically processed repressing forms (primarily Gli3). The human SU(FU) gene has also been implicated as a tumour suppressor, with mutations found in familial and sporadic medulloblastoma and in sporadic basal cell carcinoma (see Table 2). Figure 1 Hh and Wnt signalling pathways.
Full legend High resolution image and legend (46k) Despite extensive similarities between Drosophila and mammalian pathways, however, significant differences may exist, particularly in the transductory machinery between Smo and Gli. Thus, although recent genetic and biochemical studies in Drosophila have demonstrated that pathway activation is transmitted through association of Smo with a complex of cytoplasmic proteins that includes Ci and a kinesin-like protein, Cos2, a functional mammalian homologue of Cos2 has not been identified (reviewed in ref. 32). Because of its role in maintenance of pathway quiescence in Drosophila, a functional mammalian Cos2 homologue would be of interest as a potential tumour suppressor. In addition, several apparent pathway components identified in mammals either have no counterparts or do not function in the Drosophila Hh pathway (see ref. 34). These include components such as RAB23 (ref. 35) or FKBP8 (ref. 34), which have unknown function, but are of interest as potential tumour suppressors because of their action downstream of Smo as negative regulators of pathway activity (see Fig. 1).Some tumours of the type associated with Gorlin's syndrome are not associated with known pathway-activating mutations, despite clear evidence for pathway activity36, 37. This suggests that activation of the Hh pathway may occur through mechanisms other than by mutation of pathway components, and raises the possibility that such mechanisms may also have a role in pathway activation in other cancers not typically associated with Gorlin's syndrome. Consistent with this possibility, recent studies using cyclopamine, a specific Hh pathway antagonist38-40, indeed have demonstrated an ongoing requirement for pathway activity in the growth of a series of lethal cancers arising in organs of endodermal origin, and not typically associated with Gorlin's syndrome. These cancers include small-cell lung cancer and carcinomas of the oesophagus, stomach, pancreas, biliary tract, and prostate41-43. Pathway activity in these cancers requires ligand activation, as demonstrated with the use of Hh-blocking antibodies, and contrary to ligand-independent activation arising in tumours associated with Gorlin's syndrome. Curiously, the limiting factor in pathway activation in these non-Gorlin's tumours seems not to be ligand expression, but rather the acquisition of responsiveness to ligand. Thus, whereas the Hh family members Shh (Sonic hedgehog) and Ihh (Indian hedgehog) are expressed in normal endodermal tissues, high-level activation of Hh pathway targets occurs only in cancer cells. In the prostate, the limiting factor for ligand responsiveness is SMO, which is not expressed in normal prostate tissue29. Furthermore, isolated prostate stem/progenitor cells acquire Hh responsiveness simply by introduction of Smo expression constructs, and these cells are oncogenically transformed upon pathway activation. The genetic or epigenetic changes that trigger Smo expression are not identified, although they may be linked to epithelial regeneration (see section 'Cancer and persistent states of repair' below).The Wnt pathway in cancerThe Wnt signalling pathway has also been implicated in several types of cancer, initially through overexpression of the Wnt-1 protein signal in murine mammary tumours as a consequence of nearby mouse mammary tumour virus (MMTV) insertion44, and subsequently through the substantially increased incidence of colorectal and other cancers in familial adenomatous polyposis, caused by mutations affecting the tumour suppressor APC (adenomatous polyposis coli) (reviewed in ref. 45). In the absence of Wnt signal, APC fosters the degradation of the oncogene -catenin and prevents its entry into the nucleus (Fig. 1b). Wnt stimulation, loss of APC protein function, or of its associated partner Axin, all lead to the stabilization of -catenin and to its increased concentration in the nucleus. -catenin can then act as a transcriptional co-activator by associating with the Tcf/LEF family of transcription factors. A complex of APC with Axin and other proteins targets -catenin for proteasomal degradation by scaffolding an association between -catenin and kinases whose activities lead to -catenin ubiquitinylation. This action is abrogated by the recruitment of the degradation complex to the membrane upon Wnt activation of a receptor complex that includes Frizzled (Fz), a relative of Smo, and LRP5/6. Any lesion causing -catenin accumulation through the disruption of a degradation complex component or by mimicking complex recruitment to the receptor would be expected to promote tumour formation. This pathway can also be activated by mutations of -catenin that render it resistant to degradation (for detailed reviews of Wnt signalling see refs 6, 17, 46).Hh and Wnt signalling pathways are similar in that both signals are lipidated (an important process that affects their activity and tissue distribution33), and they use several related or identical components. The fundamental logic of pathway activation is also similar, in both cases involving receptor recruitment of multicomponent complexes with key roles in cytoplasmic anchoring and proteolysis of key transcriptional effectors. It is also possible that, like Hh, the Wnt pathway is activated in a wider range of cancers than has been revealed by familial or sporadic mutations that produce ligand-independent pathway activation. The possibility of Wnt ligand dependent pathway activation in cancer is suggested by a recent study demonstrating that epi-genetic silencing of SFRPs (secreted frizzled-related proteins), which encode an extracellular ligand-binding pathway antagonist, may have a critical role in the early establishment of colorectal cancer47. A broader range of cancers requiring Wnt pathway activity for growth may also be revealed, as potent and specific Wnt pathway antagonists are identified and become broadly available48.Hh and Wnt pathways in regeneration and tissue repairIf cancer stem cells arise from tissue stem cells, and if Hh and Wnt pathway activities are critical for the renewal of at least some of these stem cell types, then continuous Hh and Wnt pathway activities may promote cancer growth by continuously recapitulating their roles in promoting normal stem cell renewal. But stem cell renewal must be tightly regulated (otherwise tumours might arise), raising the critical question of how and under what circumstances normal regulation can be circumvented in cancer.Some insight into the regulation of stem cell renewal activity may be gained from a consideration of the role of Hh and Wnt pathways in regenerative responses (Table 3). Wnt pathway activation in the radially symmetric coelenterate Hydra is closely associated with the growth and patterning of new individuals. This may result either from normal asexual budding or from experimental manipulations, such as cell dissociation and re-aggregation49. Hydra tissue thus seems to exist in a constant state of growth and replacement. Amphibia, particularly urodeles (newts and salamanders), are also capable of mounting impressive regenerative responses to limb amputation or to extirpation of certain organs. The typical sequence of events involves de-differentiation of cells near the site of injury, followed by extensive proliferation and patterning of the regenerating tissues. In the cases of urodele limb and lens regeneration, Hh family members are expressed in the de-differentiated cells following injury, and regener-ation can be blocked by treatment with cyclopamine50-52. Fin regeneration in fish also entails expression of Hh genes and targets, and is disrupted by cyclopamine inhibition53, 54.The regenerating structures in these examples encompass several tissue types that are arranged in this respect pathway roles resemble those in embryonic pattern formation. But Hh and Wnt pathway activity also have a role in regenerative responses that are restricted to single tissue types within organs. For example, transient Hh pathway activity is required for androgen-triggered regeneration of prostate epithelium in male mouse castrates29, and Wnt pathway activities similarly are required for muscle regeneration in response to cardiotoxin-induced muscle injury23. Increased Hh pathway activity in Ptch+/- mice also contributes to an increase in photoreceptor-cell progenitor number and retinal repair in a model of retinal degeneration. Furthermore, mammary progenitors are enriched in mice with Wnt pathway activation caused by increased Wnt ligand levels or by a -catenin altered to increase its stability55. In addition to these demonstrations of functional Hh and Wnt pathway activity in tissue repair, correlative data suggest a possible role for Wnt pathway activity in response to biliary tract56, liver57 and kidney tubule injury58, and for Hh pathway activity in repair of bone fractures59, bile duct56 and airway injury41.Cancer and persistent states of repairWe have reviewed evidence highlighting the role of Hh and Wnt pathway activity in cancer growth on the one hand, and in stem cell renewal and tissue regeneration on the other. But is there a link between tissue repair and cancer? A connection is strongly suggested by the known association between chronic tissue injury and cancer60, 61, including cancers associated with Hh and/or Wnt pathway activity. Increased cancer risks are associated with exposure to toxins, such as alcohol, cigarette smoke and organic chemicals62-64, with chronic infection of Helicobacter pylori and other pathogens65, and with inflammatory conditions, such as sclerosing cholangitis and inflammatory bowel disease66, 67 — all of which entail chronic tissue damage. As discussed above, acute injury is accompanied by the expansion of stem cell pools and by the transient activation of the Hh and Wnt signalling pathways41. Under conditions of chronic injury, pathway activation and presumed expansion of stem cell pools would persist so long as repeated injury prevents full regeneration. This state of continuous pathway and progenitor-cell activation resembles the continuous pathway activity and cell division seen in cancer.These observations suggest that cancer growth may represent the continuous operation of an unregulated state of tissue repair and that continuous Hh/Wnt pathway activities in carcinogenesis may represent a deviation from the return to quiescence that normally follows regeneration (Fig. 2A, a, b). The simplest model for the emergence of this state is that genetic or epigenetic events prevent the return to quiescence of an activated stem or progenitor cell on completion of regeneration, thus initiating a tumour by trapping the cell in an activated state of continuous renewal. Consistent with this model, the Bmi-1 gene required for HSC renewal is also required for the propagation of leukaemias in transfer experiments68, 69. The expression of Bmi-1 and nestin, which are both associated with stem cell renewal68-70, is dependent on Hh pathway activity in Hh-dependent tumours29, 37, 41, 71. Of course, multiple genetic or epigenetic changes might be required to trap the activated stem cell initially, and numerous other events may contribute to rapid proliferation or to other aspects of the phenotype. Conversion of an activated stem cell into a clinically threatening cancer stem cell may involve changes that lock the cell in an active state of renewal and allow the cell to acquire independence from niche signals that are normally required to maintain stem cell identity. Figure 2 Model for carcinogenesis resulting from persistence of a state of injury repair.
Full legend High resolution image and legend (34k) The observed increase in cancer incidence associated with chronic injury strongly supports this model of cancer as a continuous state of repair. If, as hypothesized, the oncogenic event results in trapping activated stem cells in a continual state of renewal, then any condition that increases the pool size of activated stem cells should increase the probability of an oncogenic event by making the cellular substrates for such an event more numerous. The effect of repeated injury over time would be exactly this — to increase the pool size of stem cells in an active state of renewal (Fig. 3). Tissues that normally undergo rapid renewal might also be expected to experience increased cancer incidence, as a high turnover rate might require a sizeable pool of activated stem cells. Indeed, organs such as the skin, the lungs, and the gastrointestinal tract, which are continuously exposed to environmental insults, and consequently in a constant state of renewal, are the tissues of origin for a high proportion of cancers. Figure 3 Increased cancer risk during chronic injury.
Full legend High resolution image and legend (30k) The nature of the oncogenic events that may trap stem cells in an active state of renewal is not always clear. As noted above, Hh pathway activation in tissues that give rise to non-Gorlin's tumours seem not to be limited by ligand availability, but by the responsiveness to ligand. In normal prostate, the limiting factor in pathway responsiveness is SMO expression, and SMO upregulation is uniformly noted in all metastatic prostate cancer29. Upregulation of Smo also occurs in mice in response to injury of other endodermal tissues (P.A.B., S.S.K. and D.M.B., unpublished data), and has been dramatically demonstrated to occur locally at the site of bone injury72. The acquisition of pathway responsiveness through SMO upregulation is therefore a common feature of both injury respon cancer cells in this respect closely resemble cells in injured tissues. The identity and source of the signal that induces Smo expression in injured tissues may therefore lend insight into the targets of oncogenic processes that lead to SMO expression and Hh ligand responsiveness.Tissue repair, invasion and metastasisMost cancer deaths are caused by the growth of tumours at distant metastatic sites rather than at the site of origin. Metastasis requires the capacity to detach from the original tumour mass, to migrate through several types of tissue and to colonize a permissive ectopic site. Current evidence suggests that there may be associations between the activities of Hh and Wnt pathways and metastasis, at least in some types of cancer. In colorectal and pancreatic adeno-carcinomas, Wnt and Hh pathway activities, respectively, have been linked to all stages of these diseases — from pre-invasive neoplasia to locally growing and metastatic lesions42, 43, 73.In contrast, high-level Hh pathway activity in prostate cancer is associated exclusively with metastatic tumours29, and cell lines with and without the ability to metastasize can be interconverted by modulation of Hh pathway activity. Hh signalling specifically promotes collagen-matrix invasion and the expression of genes associated with a transition from epithelial to mesenchymal character (epithelial–mesenchymal transition, EMT). These genes include Snail, a helix-loop-helix transcriptional repressor74. EMT and the expression of Snail or its homologue Slug is also associated with aggressive behaviour, including metastasis, of other cancers74, 75, and has been linked to activity of the Wnt pathway in colorectal cancer76, 77.How does the promotion of invasiveness seen in tumours relate to the physiological roles of Hh or Wnt? Migration through tissues is a normal feature of neural crest, germ cell and haematopoietic stem cell development, and is also observed during acute epithelial injury repair in adults, in a process called epithelial restitution78. During restitution, epithelial cells adjacent to a focally denuding injury detach from each other, assume an elongated shape and rapidly migrate (often within 2–4 hours) to the injured area, where they invade remnants of the injured tissue and reconstitute epithelial continuity. This behaviour has so far been observed in differentiated cells, but as convenient markers for tracking stem cells in such experiments are lacking, the possibility remains that stem cells also become motile and invasive during injury repair. The acquisition of such an ability to invade and move through tissues might represent part of a programme of stem cell activation for optimal completion of epithelial repair. Hh and Wnt pathway activities are thus linked to the activation of stem cells in injury repair, and this reparative state is associated with cell behaviour that is recapitulated in metastasis. It seems plausible therefore that the trapping of stem cells in a state of repair might predispose them not only to tumour formation in general, but to the formation of aggressive tumours in particular.Perspectives and implicationsIf cancer stem cells responsible for driving the growth of cancer types associated with Hh and Wnt pathway activation indeed come from stem cells trapped in a state of active renewal by pathway activities, then a logical therapeutic approach for these cancers would be to impose a state of pathway blockade (see introduction in this issue by Sawyers, page 294). Potential problems associated with such approaches might include cognitive or affective disturbances, as both Hh and Wnt pathways are active in the mature brain. In addition, the roles of pathway activities in normal stem cell renewal suggest that pathway blockade might cause a complete or partial loss of stem cell pools. Latent symptoms caused by such a loss might include an increased susceptibility to degenerative disorders, and appear only after passage of a significant fraction of lifespan. On the other hand, Hh or Wnt pathway activities might be restricted solely to the stimulation of stem cell self-renewal and not affect signals required for the maintenance of stem cell identity. In this case, endogenous stem cells may remain quiescent during pathway blockade but regain renewal capacity once therapy is completed and the blockade lifted. Stem cell niches might also persist and permit the regeneration of stem cells that are temporarily lost during a period of pathway blockade. Consistent with such a possibility, the well-characterized germline stem cell niches in the Drosophila ovary and testis have been reported to persist and supply continuous niche signals for a significant fraction of the adult lifespan, even after they are emptied of stem cells13, 79.The potential success of such therapeutic approaches is suggested by the achievement of growth inhibition or regression, complete in some cases with systemic treatment by the Hh pathway antagonist cyclopamine in murine models of several Hh-pathway-associated tumour types29, 37, 42, 43, 80. In addition, a recent report demonstrates growth inhibition of spontaneous medulloblastomas arising in Ptch+/-p53-/- mice on systemic treatment with a synthetic Hh pathway antagonist81. Cancer growth in these tumour types apparently requires an active state of renewal, without which cancer stem cells are depleted by differentiation or apoptosis. The lack of toxic effects in mice during periods of systemic cyclopamine treatment extending as long as seven weeks and during follow-up observation periods of nearly half a year also augurs well for this approach. More recently, cyclopamine-induced regression of human basal-cell carcinomas was reported82, suggesting the potential effectiveness of Hh pathway blockade in humans. As cyclopamine application in these human cases was topical, cognitive or affective disturbances that might be caused by systemic pathway blockade cannot be ruled out. Such effects, if they materialize, might be reduced or eliminated by the development of pathway-blocking agents that do not cross the blood/brain barrier. The feasibility of such an approach is suggested by the identification of several structurally distinct classes of Hh pathway antagonists40, 83. Some recent success has also been reported in the identification of Wnt pathway antagonists48, suggesting that the therapeutic effects of blocking this pathway may be tested in the near future.Systemic pathway blockade in humans may require consideration of other factors. For example, the inability of prostate epithelium or muscle to regenerate under conditions of Hh or Wnt pathway blockade may be typical of many tissues, and the loss of stem cell renewal divisions could result in increased sensitivity to injury or other transient demands being placed on stem cell pools. It may therefore be important for patients to avoid even mild sources of trauma while undergoing pathway blockade therapy. This consideration also raises doubts about combining any form of cytotoxic chemotherapy with pathway blockade, as indiscriminate injury imposed by such therapy might affect some of the tissues containing stem cells whose renewal depends on pathway activities. 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