癌症治疗原则(ZT)

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攻击癌症标志（Attacking the Hallmarks of Cancer）
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      任何治疗或治愈癌症的目的或方法都必需基于对肿瘤细胞和正常细胞有不同的毒性，意思是指一些肿瘤细胞的独有特性并不是正常细胞所共有的，我们必需通过探索这些特性、并利用其来其来杀死癌细胞，此也就是所谓的合成致死。
6 K0 ?* V  r9 C, a- a4 ^7 J1 ?     原则上来说，可以通过诱导癌细胞进入凋亡、坏死、衰老或分化来治疗癌症。这些改变可以通过以下几个途径达到：破坏癌细胞的自主进程，干扰癌细胞之间的自分泌/旁分泌，阻断肿瘤细胞和周围间质组织或血管之间的异型信号。通过表达特异性的抗原来增强抗癌细胞的免疫监督被认为是一个很有潜力的方法，在特异性杀伤癌细胞中已经显示出了疗效(Muller and Scherle, 2006)。     1 O7 I4 W) S5 j* ~4 }. d8 L' m( `4 z6 X
        以抑制癌基因活性和恢复抑癌基因功能为目的的实验也显示出对癌细胞的高度删除作用。癌发生依赖癌基因的活化及抑癌基因的功能缺失，导致相关两个名词的产生，他们是“癌基因加法(OA，oncogene
9 e: q! e+ w5 m4 V/ Uaddiction)”和“抑癌基因高敏（tumor suppressor gene hypersensitivity）”，这些改变对于建立和维持肿瘤形成状态是必需的，但同时也会成为药物的合适靶点。确实，很多药效应已经通过抑制肿瘤蛋白的药理而体现出来。
- i! a( F; p) h0 a4 |      癌基因依赖现象的基础：在良好生长和生存的癌细胞中观察到癌基因诱导出强大的与促生存相反的以及调亡前的信号，而急性抑制癌基因功能使这个平衡倾向于细胞死亡。为了显示他们的表现形式，癌基因依赖细胞内本身不是致癌性的过程来延伸适应性。! ?# x9 n0 D/ A: a2 b) R
      另外，癌细胞也可显示一个依赖某些正常细胞的基因功能的增加，这些基因可能参与癌基因途径，但它们本身不是癌基因。 例如，在一癌形成途径内的许多基因的变异是不能直接促进肿瘤形成的，因为它们不能完全升高此途径的活性。但是许多这些基因活性的下降可能成为这些途径的限速因素，因此它们也显示了成为潜在药物靶点的效应所在。基于这个理论，癌细胞同时依赖于癌基因和非癌基因因素。为了描述癌细胞对非癌基因的功能依赖，我们已经称这个现象为“非癌基因依赖(NOA，non-oncogene
0 |0 Q) ], L: k  l0 Zaddiction)”。虽然与癌基因相似，非NOA基因在肿瘤发生、发展过程中也是需要的，可能起着维持作用。但NOA基因不经历像癌基因那样的基因变异或明显的基因组修饰改变。非癌基因依赖的概念强调了它们对癌形成网络的重要支持作用，强调了非癌基因作为癌治疗的可行干预措施。
& k7 \  \9 P4 K) X( C0 S     然而有时要把某些基因类型和途径完全归纳到OA或NOA范畴是很难的，因为他们有时可同时显示具有两种分类的特征，例如干扰素调节因子4(IRF4) (Iida et al., 1997) 在某些多发性骨髓瘤中是致癌因素，因为移位而过度表达；然而缺乏IRF4的转录或过表达也是骨髓瘤细胞生存的需要(Shaffer et al., 2008)。那么在后者中，它是否应该作为一个OA的例子呢？同样地，一种蛋白质可以由癌基因直接激活、并且是癌形成所必需的(但在癌中没有突变发生)，所以虽然它与癌发生存在明确的联系，但是否应该作为NOA的例子吗？2 K( O" @+ E5 d$ ?' L  w1 F: F1 f( g2 X
     两个例子都明确的提示，如果一个因素符合NAO的严格标准，那么他在肿瘤形成过程中不经历基因的变异。然而这些例子也常常违反我们想进行不同分类的初衷。但无论如何，虽然OA和NOA的界定是不完美的，但它们提供了一个考虑癌细胞弱点和癌治疗原理的有用框架。下面，我们将讨论癌基因与非癌基因依赖的例子，并描述现代工具怎样可以应用于鉴定这些基因类型，为将来可能的应用探索治疗。
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Attacking the Hallmarks of Cancer" z: W' ~! J! Z# P! X
     Any therapy with the stated goal to treat and possibly cure cancer must show differential toxicity toward tumor cells relative to normal cells. Implicit in this statement is that some unique properties of cancer cells not shared by normal cells, such as those depicted in Figure 1, must be exploited to the specifc detriment of cancer cells, i.e., the concept of synthetic lethality. In principle, cancer can be treated by inducing cancer cells to undergo apoptosis, necrosis, senescence, or differentiation. These changes can be brought about by disrupting cancer cell-autonomous processes, by interfering with autocrine/
0 f& e$ u8 B7 oparacrine signaling within tumors, or by blocking heterotypic signaling between tumor cells and the surrounding stromal tissue or blood vessels. Enhancing immune surveillance against cancer cells expressing novel antigens is also an attractive approach that has shown effcacy in specifcally killing cancer & `3 J4 Y8 C  X. ^2 T7 j5 o
cells (Muller and Scherle, 2006).Experiments aimed at either suppressing oncogene activity or restoring tumor suppressor function have revealed that 7 }" u& V! n6 V, q/ }; J
such intervention is highly deleterious to the cancer cell. The heightened state of dependency of cancer cells on oncogenes and the loss of tumor uppressors led to the terms “oncogene addiction” (OA) and “tumor suppressor gene hypersensitivity” (Weinstein, 2002; Weinstein and Joe, 2008). These lterations 0 a& K+ L( T  b9 L" c
are necessary for both the establishment and maintenance of the oncogenic state and therefore are logical drug targets. Indeed, much effort has been extended to pharmacologically inhibit oncoproteins. What is thought to underlie the phenomenon of oncogene addiction is the observation that oncogenes elicit strong, opposing prosurvival and proapoptotic signals in
8 Z) u( \9 w- c' Z/ O; j% vcancer cells that favor growth and survival, and the acute inhibition of oncogene function tilts this balance toward cell death (Downward, 2003; Sharma and Settleman, 2007).To bring about their phenotypic manifestations, oncogenes rely on extensive adaptations in cellular processes that are themselves not oncogenic. In addition, cancer cells may also display an increased dependence on the normal cellular functions of certain genes that act in oncogenic pathways but are not themselves classical oncogenes. For example, mutations in many genes in a given oncogenic pathway are unable : C2 {5 j9 L5 X+ }- B
to directly promote tumor formation because, despite being required for their pathway, they cannot increase the overall activity of the pathway because they are not rate-limiting. However, a reduction in the activity of many such genes can become rate-limiting to the pathway in question, and thus, they represent potential drug targets. By this rationale, cancer cells are addicted to both oncogenes and non-oncogenes. To describe this addiction of cancer cells to the functions of nononcogenes, we have termed this phenomenon “non oncogene addiction,” NOA (Solimini et al., 2007). Although NOA genes, l5 B, X8 \/ r4 W# A1 E: V2 V
like oncogenes, are required for maintenance of the tumorigenic state, NOA genes do not undergo oncogenic mutations or functionally signifcant genomic alterations in tumors. The concept of non-oncogene addiction underscores the important contribution of these supporting networks to oncogenesis
; F7 r8 N* f8 l7 pand highlights the potential of non-oncogenes as points of intervention for cancer therapeutics.Whereas some gene classes and pathways fall neatly into the OA or NOA designations, others are more diffcult to categorize because they exhibit characteristics of both phenomena. For example, interferon regulatory factor 4 (IRF4) (Iida et al., 1997) is oncogenic and overexpressed due to translocations in some multiple myelomas. However, it is also required for the survival of myelomas lacking IRF4 translocations or overexpression
. A# w; T- w( F# R(Shaffer et al., 2008). Should it be considered as an example of OA in the latter cases? Also, should a protein that is directly activated by an oncogene and required for tumorigenesis—but is otherwise not mutated in cancer—be considered an example of NOA when it is so clearly linked to an oncogene? / e: F& L1 _" _) J! a
Both examples are clear if one adheres to a strict defnition of NOA stating that NOA genes do not undergo oncogenic mutations in tumors. However, these examples often run counter to our overall intuitive sense of the different categories. Regardless, although the OA and NOA designations are not perfect,
9 A1 [! D3 c1 C: d% z. Dthey provide a useful intellectual framework for thinking about cancer cell vulnerabilities and the principles of cancer therapies. Below, we will discuss examples of oncogene and nononcogene addiction and describe how modern tools are being applied to identify these classes of genes for possible therapeutic exploitation.
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marrowstem

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       治疗癌症有点像在黑暗中射击。有时奏效，有时没有准心，结果无法预测。原因则是我们目前对癌肿瘤如何产生和癌细胞为何增长失控还只是些粗略的想法，而癌肿瘤内部到底如何运行，绝大部分对我们至今仍然还是个迷。
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癌基因依赖和抑癌基因超敏反应
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       虽然在许多的癌中发现了存在大量的遗传和表观遗传的改变，但在一些肿瘤中却只有个别癌基因或抑癌基因的突变。“癌基因依赖”这个词的应用主要是用来阐述肿瘤的维持依赖于特定基因的持续活化 (Weinstein, 2002)。有多种癌基因的这种现象已经被确认：例如，经MYC癌基因诱导的鼠模型表现出MYC引起的皮肤乳头状瘤；另外，淋巴瘤、骨肉瘤也表现为可以在MYC撤退后出现逆转。相似的，对于HRAS或BCR-ABL癌基因的依赖，在黑色素瘤和白血病的鼠模型中也得到确认。
2 S4 @/ L$ P, S+ y5 K8 O    在带有KRAS基因变异的人类结直肠癌细胞株中，敲除KRAS癌基因将导致细胞表型的逆转和在裸鼠中形成肿瘤能力的消失。有许多癌基因被抑制后能导致癌细胞分化、阻滞或衰老发生，它们中的一些已成为治疗癌症的靶点：如蛋白酪氨酸癌基因BCR-ABL(伊马替尼/格列卫)、EGFR(吉非替尼/易瑞沙，厄罗替尼/特罗凯)、HER2(曲妥珠单抗/赫赛汀)已经在临床上被证明是成功的， 而对BRAF，MDM2T 和脂质激酶PI3K等基因作为抑制癌细胞的作用也正在研究中，但以RAS和MYC之类的非激酶类部基因作为靶点已经被证明是很难产生作用的。 # |& U( E% Z. g: B
      与癌基因的作用相反，肿瘤抑制基因表现为细胞生长的抑制作用，通过阻止异常规的生长与生存或基因组的不稳定性来导致。 通过缺失、灭活变异或表观沉默等引起的抑癌基因失能可最终导致肿瘤发生。而如果再引入抑癌基因到缺乏这个基因的肿瘤中会使肿瘤退缩，这个概今最近在鼠肿瘤模型中通过再激活P53而得到证实。 抑癌基因变异在药理学方面的研究远远落后于癌基因，因为通常难以用小分子物质去恢复或模拟一种已经变异或缺失的蛋白质的功能。在肿瘤抑制因子阴性的情况下调节原癌基因活性、针对原癌基因的靶向治疗将在缺乏这个抑制因子的肿瘤中产生有效的治疗作用。例如，丢失肿瘤抑制因子和脂酶PTEN(正常时具有抑制PI3K信号的作用)的肿瘤中似乎对PI3K抑制因子敏感。 相似地，丢失Rb,p16,p21或p27最终使细胞周期蛋白依赖激酶(CDK)(使细胞进入细胞周期)的活性上调。大体上来说，因为这些损伤引起的肿瘤可能对CDK抑制因子更敏感。这个预测是否准确只有在使用PI3K和CDK抑制剂的临床试验中才能得到明确。然而在那些丢失包括P53或ARF等抑癌因子的肿瘤中，没有明显的可作为靶向的信号通路及可代替的治疗策略可以考虑。 $ p) {" s$ v5 Q# h0 |
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Oncogene Addiction and Tumor Suppressor Gene : L" A9 D7 @, w% {2 {
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     Despite the multitude of genetic and epigenetic alterations found across cancers, a given tumor is likely to be driven by only a select few changes—those that result in the gain of an oncogene or the loss of a tumor ppressor. The phrase “oncogene addiction” was coined to describe the observation that tumor maintenance often depends upon the continued activity of certain oncogenes (Weinstein, 2002). This phenomenon has been demonstrated in vivo for several oncogenes. For example, mouse models using an inducible MYC oncogene have shown that MYC driven skin apillomas, lymphomas, and osteosarcomas can all be reversed upon MYC withdrawal . Similarly, addictions to the HRAS or BCR-ABL oncogenes have been demonstrated in mouse models of melanoma and leukemia, respectively (Chin et al., 1999; Huettner et al., 2000). In human colorectal cancer cells bearing a KRAS mutation, somatic knockout of the KRAS oncogene results in reversion of the transformed phenotype and abrogates the ability of these cells to form tumors in nude mice (Shirasawa et al., 1993).
( a  s9 g5 M6 ^, ]5 t) l3 w. `6 PThe subset of oncogenes whose inhibition can lead to tumor cell death, differentiation, arrest, or senescence is of great clinical interest as targets for cancer therapeutics (Table 1). This strategy has proven successful for the protein kinase oncogenes BCR-ABL (imatinib/Gleevec), EGFR (geitinib/Iressa,
2 D" }+ ^0 c9 V0 f* x% q6 G* g% |6 nerlotinib/Tarceva), and HER2 (trastuzumab/Herceptin) (Druker, 2002; Roberts and Der, 2007; Sharma et al., 2007), and efforts toward inhibition of BRAF, MDM2, and the lipid kinase PI3K are underway. Targeting non-kinase oncogenes such as RAS and MYC, however, has proven more dificult.In contrast to oncogenes, tumor suppressor genes act to provide the cellular restraints necessary to prevent aberrant growth and survival or genomic instability. Loss of tumor suppressor genes through deletion, inactivating mutation, or epigenetic silencing results in the removal of these restraints ( J. e# z" V) f! H7 m
leading to tumorigenesis. Reintroduction of a tumor suppressor gene into a tumor lacking that gene can result in tumor regression. This concept has been recently demonstrated by reactivation of p53 in mouse tumor models (Martins et al., 2006; Ventura et al., 2007; Xue et al., 2007). Pharmacological exploitation of tumor suppressor mutations, however, has lagged behind efforts aimed at oncogenes because it is often dificult to use a small molecule to either restore or mimic the function of a protein that is either mutated or
8 b/ b! v! G8 \1 }9 o8 _absent. In cases where a tumor suppressor negatively regulates the activity of a proto-oncogene, drugs targeting the corresponding proto-oncogene should prove eficacious in treating tumors lacking that tumor suppressor. For example, tumors that have lost the tumor suppressor and lipid phosphatase PTEN, which normally acts to constrain PI3K signaling, are likely to be sensitive to PI3K inhibitors. Similarly, loss of Rb, p16, p21, or p27 all result in upregulation of cyclin-dependent kinase (CDK) activity, which drives cell" Z9 ^: t4 q9 ]( C8 R6 h& Y- I4 J
cycle entry. In principle, tumors resulting from these lesions might be more sensitive to CDK inhibitors. Whether such predictions prove true will only become apparent from clinical trials employing PI3K and CDK inhibitors. In many other cases, however, such as those involving loss of the tumor
2 @: O) `) g3 M% x6 g2 C9 _% Hsuppressors p53 or ARF, there is no obvious positive signaling pathway to target, and alternative therapeutic strategies must therefore be considered. : X+ p5 j1 K$ `7 f0 V$ Y: u
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以非癌基因依赖为靶向的癌症治疗
5 e* q1 u4 r! }! B7 k" u1 Y8 H(Targeting Non-oncogene Addiction for Cancer Therapy)
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7 z( D8 e. k  W) s; ^        我们提出了非癌基因依赖性(NOA)这个概念是基于肿瘤形成状态依赖于大量基因及途径的活化的基础上，这些基因及途径中的大部份本身不是固有的癌基因(Solimini et al., 2007)。重要的是，这些基因和途径对于支持癌细胞的癌形成表现型来说是主要的但在正常细胞中不是同等需要的。+ ]/ c6 v$ U4 N% _/ F' ?  o7 u  \' [4 k
       单纯从遗传学的角度来说，当抑制造成根本的肿瘤基因型的综合致死，这些依赖可以提供大量的药物靶点。对酵母菌的基因相互作用的研究为这个概念提供了优先权。例如，当与某些其他变异成对出现后大部分基因的抑制增强了生长缺陷，一个在酵母中的研究证实每个基因平均存在相关6个基因的相互作用(Collins et al., 2007)。由于肿瘤可包含很多基因的改变，而每个改变提供了一个与第二个功能缺失的基因配对的机会，导致严重的和可能致命的生长或生存表型改变。，如果第二个基因是一种能抑制它的蛋白药物的靶点，那就意味着一种可能的癌治疗药物出现了。
8 t* F2 K! f& Z! s5 }; w8 M       非癌基因依赖途径为抗癌治疗提供了重要的靶点，在下面的部分我们将常见的NOA基因类型，如果可能的话会列出其特例。NOA基因可以分成两类，肿瘤内在的与肿瘤外在的基因途径。 肿瘤内在的NOA基因以细胞自主的形式支持肿瘤的发生和瘤细胞的形成状态，肿瘤外在NOA基因的功能是间质和血管细胞为肿瘤提供异型信号，这些辅助细胞的靶向治疗研究进展表明，与肿瘤细胞不同，它们在遗传学上更倾向于更稳定和更少形成耐药性。但是在某些情况下，肿瘤可能会通过发展而减少对辅助细胞的依赖。肿瘤形成状态的交替使用是指肿瘤细胞经历大量正常细胞没有经历的细胞压力，因而肿瘤细胞更依赖于它们生存的压力支持途径。 2 \. t; `2 K8 g
       一般来说，这种依赖于特定杀伤肿瘤细胞有两种方法可以利用：第一种方法，压力致敏，旨在减少压力支持途径的活性，结果肿瘤细胞不再能应付癌生成有状态的压力，所以不是停止增殖就是开始凋亡或坏死。第二种方法，压力过载，旨在加剧已经存在的癌生成压力，从而使肿瘤细胞中的压力支持途径挫败，导致生长停滞或细胞死亡。两种方法都是通过干扰促生存或抗生存信号平衡来损伤肿瘤细胞。NOA的特殊类型列在图2中并将在下文中讨论。
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    Targeting Non-oncogene Addiction for Cancer Therapy( Z% a, N. F, n3 q: B
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      We proposed the concept of non-oncogene addiction (NOA) based on the understanding that the tumorigenic state depends on the activities of a wide variety of genes and pathways, many of which are not inherently ncogenic themselves (Solimini et al., 2007). Importantly, these genes and pathways are essential to support the oncogenic phenotype of cancer cells but are not required to the same degree for the viability of normal cells. From a purely genetic point of view, these dependencies should provide an ample number of drug targets that when inhibited will constitute synthetic lethality with the underlying tumor genotype. Gene interaction studies in yeast have provided precedence for this notion. For example, most mutations exhibit enhanced growth defects when paired with certain other mutations, and
1 T# c" R1 W5 ?2 D* x! @one study in yeast identifed an average of six genetic interactions per gene (Collins et al., 2007). As a tumor contains many genetic alterations, each of these changes provides an opportunity to pair with the loss of function of a second gene to result in a severe and possibly lethal growth and survival phenotype. Furthermore, if this second gene is targeted with a drug that inhibits its protein, then a potential cancer therapy can result.NOA genes and pathways provide important targets for anti-tumor therapies. In the sections below, we will discuss general classes of NOA genes with specifc examples when available. NOA genes fall into two general categories, tumor intrinsic and tumor extrinsic. Whereas tumor-intrinsic NOA genes support the oncogenic state of the tumor cell in a cell-autonomous manner, tumorxtrinsic NOA genes function in stromal and vascular cells that provide heterotypic support for the tumor. An advantage of targeting these accessory cells is that, unlike tumor cells, they tend to be genetically more stable and therefore are less likely to evolve drug resistance. However, in cer-$ s4 l3 U5 H: ^- P& I
tain circumstances tumors may be able to evolve reduced dependency on these accessory cells.The trade-off for the tumorigenic state is that tumor cells experience numerous cellular stresses not experienced by normal cells and therefore tumor cells are more dependent on stress support pathways for their survival. In principle, there are two approaches to exploit this dependency to selectively kill tumor cells. The frst approach, stress sensitization, aims to diminish the activity of the stress support pathways such that the tumor cell can no longer cope with the stress of its oncogenic state and either ceases to proliferate or initiates apoptosis or necrosis. The second approach, stress overload, aims to exacerbate existing oncogenic stress in order to overwhelm the stress support pathways in the tumor cell, leading to growth arrest or cell death. Both approaches, therefore, disrupt the balance of pro- and antisurvival signaling to the detriment of tumor cells. Examples of specifc types of NOA are illustrated in Figure 2 and discussed below.
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内源性非癌基因依赖(Intrinsic Non-oncogene Addiction)
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! W+ M  c$ z5 {6 p  {        几乎在所有类型的肿瘤中，DNA自然损伤率和复制压力程度都被增强，癌基因的出现同样可以导致重要的DNA损伤 (Halazonetis et al., 2008)。DNA损伤压力可以通过压力增敏和压力过载用于治疗，细胞中存在着一套精细的DNA损伤反应途径，通过启动DNA修复来改善DNA损伤的效果 (Harper and Elledge, 2007)。一般来说，经历过自然DNA损伤的细胞对干扰这个压力途径的药物的敏感性会增强(图 2)。 : ~& a' O) O' T
       作为这个概念的支持，最近的研究表明DDR激酶ATM和Chk1的抑制剂有针对癌细胞的选择性毒性(Chen et al., 2006; Kennedy et al., 2007)。正常服务于抑制增殖和启动凋亡的途径将对癌细胞有保护作用的，这与直觉相背离，这个途径所提供的DNA修复和基因组稳定性使癌细胞避免持续的DNA损伤和复制压力所致的死亡。DDR（DNA损伤反应）途径压力过载同样表现出在癌症治疗中的有效性。
9 {! L: [% O6 Z8 [- A& w      虽然目前仍不清楚如IR和化疗之类的DNA损伤药物为什么是癌症的有效治疗。它们可能是压力过载的例子，癌细胞原已提高的DNA损伤水平和复制压力不能修复由这些药物强加的额外损伤。另一种解释是在肿瘤形成过程中，持续性的DNA损伤对拥有废除DDR途径部分的变异细胞选择，导致对DNA损伤失去正常的敏感性和反应。这些部份不健全的DDR可能对放疗和化疗引起的广泛的DNA损伤更敏感，没有正常的DDR途径是致命的。+ h. m  u7 ^5 T  p6 H  G! E
      在这个背景下，DNA损伤提供了一下类似于非癌基因依赖的肿瘤压力表现型。因为很多癌症对DNA损伤药物的敏感性，可能存在着基因，对这种基因的抑制将产生内源性DNA损伤并导致这种敏感性的增强。例如在携带有BRCA2变异的癌症中。这些肿瘤在同源重组介导DNA修复方面有缺陷，对DNA交联剂如DDP特别敏感(Sakai et al., 2008)。另外这类肿瘤依赖于其他类型的DNA修复，如碱基切除修复(结果对ADP核糖聚合酶抑制剂(PARP)敏感，这个酶可以促进单链断裂的修复) (Bryant et al., 2005; Farmer et al., 2005)。在正常细胞中，这种由PARP抑制剂产生的内源性DNA损伤由于同源重组介导DNA修复的功能代偿可以得到很好的耐受。确实，PARP1缺陷的鼠是广泛存在的，却没有观察到肿瘤易感性的增加 (Conde et al., 2001)。因而BRCA2变异细胞对PARP1功能的依赖是NOA在治疗研究方面的例子。这些肿瘤中可能存如PARP1的非癌基因依赖靶点可以开发用于肿瘤治疗。同样地，其他类型癌症的DNA敏感性可能也可以用这种方式进行利用，虽然内源性损伤是否优于添加的外源性损伤尚不确定。
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- I  s( ?/ @: \( ?' b7 sIntrinsic Non-oncogene Addiction
. N; K7 l+ `7 q, {DNA Damage and Replication Stress: u2 N$ P$ _" f; i# f: E
      In nearly all tumor cell types, the rate of spontaneous DNA damage and the degree of replication stress are enhanced. The presence of oncogenes can also elicit substantial DNA damage (Halazonetis et al., 2008). DNA damage stress can be exploited therapeutically through both stresss ensitization and stress overload. An elaborate DNA damage response (DDR)
7 c! _, |7 p5 a' o2 D6 jpathway exists in the cell to ameliorate the effects of DNA damage by promoting DNA repair. Mutations in genes of this pathway result in increased sensitivity to DNA damage (Harper and Elledge, 2007). In principle, cells experiencing spontaneous DNA damage should show enhanced sensitivity to agents that interfere with this stress response pathway (Figure 2). In sup-+ c/ M2 _5 }/ Z9 q
port of this notion, recent studies have shown that inhibitors of the DDR kinases ATM and Chk1 exhibit selective toxicity toward cancer cells (Chen et al., 2006; Kennedy et al., 2007). Although it seems counterintuitive that a pathway that normally serves to restrain proliferation and promote apoptosis would protect a cancer cell, the DNA repair and genomic stability afforded by this pathway could save a cancer cell from death caused by persistent DNA damage and replication stress.Stress overload of the DDR pathway should also show effcacy in cancer treatment. Although it is not clear why DNAdamaging agents, such as IR and chemotherapy, are effective cancer therapies, it is possible that these are examples of stress overload, where cancer cells with already elevated levels of DNA damage and replication stress cannot repair the additional damage inficted by these agents. An alternative explanation is that during tumorigenesis, the persistence of DNA damage selects for cells with mutations that abrogate part of the DDR pathway ; N' ?4 }8 x0 \" d& `9 y6 f! ?
and therefore cannot properly sense and respond to DNA damage. These cells with a partially defective DDR might therefore be more vulnerable to the extensive DNA damage resulting from radiation or chemotherapy that is lethal without a normal DDR pathway. In this context, DNA damage exploits a stress phenotype of tumors that is analogous to non-oncogene addition.+ }$ g) r: \) F1 J9 k  h3 U
Given the sensitivity of many cancers to DNA-damaging agents, there should exist genes whose inhibition will generate endogenous DNA damage to exacerbate this sensitivity. Exemplifying this phenomenon are cancers bearing BRCA2 mutations. These tumors are defective in homologous recombination-mediated DNA repair and are particularly sensitive to DNA crosslinkers such as cisplatin (Sakai et al., 2008). Furthermore, these tumors 1 B6 m+ [# X* n/ |$ n
rely heavily on other forms of DNA repair such as base-excision repair, resulting in sensitivity to inhibitors of poly-ADP-ribose polymerase (PARP1), an enzyme that facilitates repair of singlestranded breaks (Bryant et al., 2005; Farmer et al., 2005). In normal cells, the endogenous DNA damage generated by PARP inhibition is well-tolerated because of functional compensation from homologous recombination-mediated repair. Indeed, mice defcient in PARP1 are fertile and do not exhibit increased tumor " ^* {' e" I) n3 K1 C
susceptibi l ity (Conde et al., 2001). The addiction of BRCA2 mutant
( J5 z, S- m2 E3 x  L/ m) @cells to PARP1 function is therefore an example of NOA that is exploited therapeutically. There are likely to be additional nononcogene targets like PARP1 that can be exploited to treat these tumors. Likewise, the DNA damage sensitivity of other cancer types might also be exploited in this manner, although whether generating endogenous damage is superior to adding exogenous damage remains to be determined. # s( d7 m# p3 D* Y
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marrowstem

有丝分裂压力Mitotic Stress
8 [& l9 v, O& t- ~( a4 }      如上文所述，一系列肿瘤表现出染色体错误分离发生率增加，CIN表现型，这是因为它们包含有能使准确的有丝分裂发生改变的变异。癌细胞携带的有丝分裂改变，如非倍体或有丝分裂纺锤体调节机制的减弱可能表现出更大程度上对染色体正确分离途径压力支持的依赖性。它们因此对严重的基因组不稳定性更敏感，这些基因组不稳定性是由压力过载或有丝分裂机制压力致敏引起的 (Weaver and Cleveland, 2005)。
( M, n8 w/ h) y" |$ ~     纺锤体检查点可以为有丝分裂机制缺陷的细胞提供压力支持，在这种情况下抑制纺锤体检查点作为压力致敏的结果将导致死亡。相反，某些有CIN表型的肿瘤具有削弱纺锤体检查点的变异(Cahill et al., 1998)可能会对由于抑制执行有丝分裂的蛋白所致的压力过载特别敏感。一个压力过载的典型例子是微管稳定剂，泰素，它可以干扰纺锤体着丝点的正常粘着，并在乳腺癌和卵巢癌的治疗中显示出其有效性(Weaver and Cleveland, 2005)。有丝分裂激酶抑制剂如PLK1和Aurora-B同样可以启动压力过载，目前正在进行临床试验 (Carpinelli and Moll, 2008; Strebhardt and Ullrich, 2006)。非整倍体本身可能就是一个NOA靶点，因为酵母菌的基因筛选已经明确表现综合致死的变异与四倍体和二倍体相关(Storchová et al., 2006)。
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Mitotic Stress" H" R2 b! N+ E# l! q% U
As noted above, a variety of tumors display increased rates of chromosome mis-segregation, the CIN phenotype, because they contain mutations that alter the fdelity of mitosis. Cancer cells bearing mitotic alterations such as aneuploidy or weakened mitotic spindle regulatory machinery are likely to
" m# R7 t: d4 A, Iexhibit greater reliance on stress support pathways for proper chromosome segregation. They are therefore more sensitive to catastrophic genomic instability caused by stress overload or stress sensitization of the mitotic machinery (Weaver and Cleveland, 2005).The spindle checkpoint can provide stress support in cells that have defects in the mitotic machinery, and in such cases inhibiting the spindle checkpoint could lead to lethality as a
2 H- R8 R; k" h; ]7 t% _; fresult of stress sensitization. Conversely, some tumors with the CIN phenotype possess mutations that weaken the spindle checkpoint (Cahill et al., 1998) and might be especially sensitive to stress overload caused by the inhibition of proteins that carryout mitosis. A notable example of stress overload is the microtubule stabilizer taxol, which interferes with proper
1 ~% B2 h3 _  I4 |: R+ Jspindle-kinetochore attachment and has shown effcacy in the treatment of breast and ovarian cancers (Weaver and Cleveland, 2005). Inhibitors of mitotic kinases such as PLK1 and Aurora-B that also promote stress overload are currently undergoing clinical trials (Carpinelli and Moll, 2008; Strebhardt , h5 n% B4 G& O% k# W6 c
and Ullrich, 2006). Aneuploidy itself might be a target of NOA as genetic screens in yeast have identifed mutations that show synthetic lethality with tetraploid verses diploid cells (Storchová et al., 2006). # g% R! }- s7 Y) ~! @
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marrowstem

蛋白质毒性压力Proteotoxic Stress
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       由于肿瘤中出现极度非整倍体、拷贝数的变异和转录改变，蛋白质毒性压力常常出现在癌细胞中。这些改变导致不同蛋白质复合物的蛋白亚单位的剂量平衡发生改变，而且通常给维持蛋白质组正常平衡的伴侣途径添加压力。热休克途径在促进蛋白折叠中起着重要途径并且在很多肿瘤中被活化(Whitesell and Lindquist, 2005)，因而是一个潜在的NOA靶点(图2)。对酵母菌的研究表明单一的染色体中额外的拷贝足以激活热休克反应 (Torres et al., 2007)。药理学和遗传学证据支持这个概念，用蛋白质毒性压力致敏的肿瘤可以强烈地抑制肿瘤形成。例如，HSP90伴侣的作用下可以折叠成新合成蛋白也可以再折叠成错误折叠蛋白 (Whitesell and Lindquist, 2005)。HSP90的伴侣活性需要它的ATP酶活性，它可以被抗癌药物格尔德霉素所抑制。虽然格尔德霉素的抗肿瘤活性归因于关键的HSP90受质蛋白（包括细胞增殖如CDK4和HER2）的不稳定性，它仍有可能致敏肿瘤细胞从而提高广泛的蛋白质毒性压力而实现其效果。NOA另外的基因学支持来自对HSF1敲除的小鼠研究 (Dai et al., 2007)。HSF1是主要的转录因子，其主要作用是对过度的非折叠蛋白反应激活热休克蛋白，包括HSP90。HSF1的丢失显著地减少由P53或Ras变异引起的肿瘤生成(Dai et al., 2007)。由于HSP90和HSF1都不是癌基因，它们成为NOA的代表例子。非折叠蛋白普遍由泛素－蛋白酶途径代谢，所以，蛋白质毒性压力反应途径的第二个关键组分是蛋白酶体。遗传学试验表明额外的染色体的出现足以提高酵母菌对蛋白酶体抑制剂的敏感性(Torres et al., 2007)。Bortezomib (Velcade)是一个有希望的抗癌新药，它在治疗多发性骨髓瘤方面显示于其有效性，作用机理是抑制蛋白酶体的蛋白酶功能 (Richardson et al., 2006)。由于蛋白酶体抑制作用可能使很多蛋白稳定，通常是这样认为的，Velcade的主要抗肿瘤生成活性是稳定蛋白质的特定亚基，但是同样可能的是它的效应来自于增强蛋白质毒性压力导致压力过载，从而成为一个主要的NOA范例。
& z5 g7 |- y* \- s, {        压力致敏和压力过载是同一事物的两个方面。大体而言，增强蛋白质解折叠的治疗是导致癌细胞的压力支持网络过载，而不是由内源性蛋白质毒性压力致敏癌细胞将达到相同结果。另一方面，在正常细胞由于它们的基础蛋白质毒性压力低而对这种治疗的敏感性差。一个简便的诱导蛋白质解聚的方法是提高温度A convenient method of 。确实，高温广泛用于肿瘤治疗研究，如结肠癌、乳腺癌、睾丸癌、前列腺癌和肝癌中的应用已经成为临床试验的课题，通常与化疗联合 (Fiorentini and Szasz, 2006)。通过高温诱导蛋白质解聚可以提供这种方法有效的原理。这个解释的强大支持同样来自于酵母菌，研究表明只要一条额外的染色体导致对加温敏感，与非整倍体细胞对温度普遍敏感相符合 (Torres et al., 2007)。 % z7 S/ @( u' G+ B2 i: T
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Proteotoxic Stress
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0 |$ {& ^2 u! p( N$ D    Proteotoxic stress is a frequent occurrence in cancer cells as a result of the extreme aneuploidy, copy-number variation, and transcriptional alterations present in tumors. These changes alter the dosage balance of protein subunits in different protein complexes and consequently place increased stress on chaperone pathways to maintain normal homeostasis of the proteome. The heat shock pathway plays a major role in promoting protein folding and is activated in many tumors (Whitesell and Lindquist, 2005) and is therefore a potential NOA target (Figure 2). Studies in yeast have shown * A8 a6 }' {4 f7 A5 K  z# x) ?
that an extra copy of a single chromosome is suffcient to activate the heat shock response (Torres et al., 2007). Pharmacological and genetic evidence supports the notion that sensitizing tumor cells toward proteotoxic stress can strongly suppress tumorigenesis. For example, the chaperone HSP90
6 g5 A1 L4 N) ]8 xacts to fold newly synthesized proteins and refold mis-folded proteins (Whitesell and Lindquist, 2005). The chaperone activity of HSP90 requires its ATPase activity, which can be inhibited by the anticancer drug geldanamycin. Although the antitumor activity of geldanamycin has been attributed to the ( v' n. y+ \3 H4 }5 f; U% d
destabilization of key HSP90 client proteins involved in cell proliferation such as CDK4 and HER2, it is also possible that the sensitization of tumor cells to elevated general proteotoxic stress leads to its effcacy.  T+ \* Z5 e- C9 c' _9 w9 y! n
     Additional genetic support for NOA comes from studies of HSF1 knockout mice (Dai et al., 2007). HSF1 is the major transcription factor responsible for activating the expression of heat shock proteins, including HSP90, in response to excess unfolded proteins. Loss of HSF1 markedly reduces tumorigenesis driven by either p53 or Ras mutations (Dai et al., 2007).
& q3 o  Z1 K( |6 U! L( i6 K- a& `$ xGiven that neither HSP90 nor HSF1 have been shown to be oncogenes, they represent examples of NOA.Unfolded proteins are generally turned over by the ubiquitin-proteasome pathway. Thus, a second key component of * G5 g5 [: L% R0 S9 d
the proteotoxic stress response pathway is the proteasome. Genetic experiments have shown that the presence of an extra chromosome is suffcient to increase the sensitivity of yeast cells to proteasome inhibitors (Torres et al., 2007). One promising new anticancer drug bortezomib (Velcade), which has shown effcacy in treating multiple myeloma, acts to inhibit the protease function of the proteasome (Richardson et al., 2006). . H0 U% q! Z5 e: Q
As proteasome inhibition is likely to stabilize many different proteins, it has been traditionally thought that the stabilization of a particular subset of proteins might be responsible for the antitumorigenic activity of Velcade. However, it is equally likely that its effcacy results from enhanced proteotoxic stress leading to stress overload and represents a prime example of NOA.* ^7 y; ?$ o( O# c5 B! d7 C9 ?
Stress sensitization and stress overload are two sides of the same coin. In principle, instead of sensitizing cancer cells toward endogenous proteotoxic stress, therapies that enhance protein unfolding should overload the stress support network in cancer cells, achieving the same result. Normal cells, on the other hand, should be less sensitive to such therapies due to their low baseline proteotoxic stress. A convenient method of inducing protein unfolding is temperature elevation. Indeed, hyperthermia is being extensively investigated as a tumor treatment for colon, breast, testicular, prostate, and liver cancers and has been the subject of ongoing clinical trials, often in ( h/ S( P- b1 m' e1 b- T4 e- ?
combination with chemotherapy (Fiorentini and Szasz, 2006). The induction of protein unfolding by hyperthermia could provide a rationale for the effcacy of this approach. Strong support for this explanation again comes from yeast where it was shown that the presence of just one extra chromosome results in enhanced temperature sensitivity, consistent with a general sensitivity to temperature for aneuploid cells (Torres et al., ( ]& ~: h1 W' @6 S6 U: h# z& T
2007).
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marrowstem

代谢压力Metabolic Stress1 \- i0 D8 k' b' A8 c

& _, G4 A5 A  ?) |      肿瘤细胞表现代谢性质的改变，其原因有二：细胞的内在特性和肿瘤微环境。因为以上原因，肿瘤细胞对葡萄糖的摄取增加而且即使是在有氧的状态下仍是优先通过糖酵解代谢葡萄糖。虽然Warburg效应的原因仍不清楚，人们认为这个适应使肿瘤细胞把资源转变到生物合成中，通过线粒体氧化磷酸化减少活性氧（ROS）的生成，应付出现在肿瘤周围的氧波动(DeBerardinis et al., 2008; Kroemer and Pouyssegur, 2008)。
: g, L% U4 S+ {* b* Y8 V: z: G; D        由于与癌基因驱动的持续增殖不相容，抑制肿瘤细胞中的糖酵解/生物合成途径将导致压力过载。确实，抑制ATP柠檬裂解酶（从柠檬酸合成乙酰辅酶A），乳酸脱氢酶A（在糖酵解的最后步骤中将丙酮酸转变成乳酸的酶）的RNAi敲除，以及对乙酰辅酶A羧化酶和脂肪酸合成酶的抑制剂（控制乙酰辅酶A转变成丙二酸单酰辅酶A再转变成,软脂酸的酶）将导致肿瘤细胞生长明显变慢(DeBerardinis et al., 2008; Kroemer and Pouyssegur, 2008). 最近的研究表明，在肿瘤细胞中通常表达的可变剪切物丙酮酸激酶（M2）发生轻微的改变而成为可变剪切物（M1），这将对肿瘤生成状态产生阴性的影响MRecently, 假设是通过将丙酮酸分流到三羧酸循环中(Christofk et al., 2008)。因而以组成NOA的关键代谢酶为靶点将能有效地减弱肿瘤细胞的增殖。
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+ J$ X7 e6 Y1 w' \1 r( pMetabolic Stress% q8 [4 o# v8 D/ o& ~) W
       Tumor cells exhibit altered metabolic behavior due to both cell-intrinsic properties and the tumor microenvironment. As noted above, tumor cells have increased glucose uptake and preferentially metabolize glucose through glycolysis even in the presence of oxygen. Although the reason for the Warburg effect is unclear, it has been suggested that such adaptation allows   j- V1 Y6 K- \4 w! o
the tumor cell to divert resources toward biosynthesis, reduce the generation of ROS through mitochondrial oxidative phosphorylation, and cope with fuctuating oxygen availability in the tumor vicinity (DeBerardinis et al., 2008; Kroemer and Pouyssegur, 2008). Inhibition of the ycolytic/biosynthetic pathways in a tumor cell will lead to stress overload due to incompatibility with the persistent proliferative drive from oncogenes (Vander Heiden et al., 2001). Indeed, inhibition of ATP citrate lyase
  n: @% E7 ?: P0 ?" n( G, O(which synthesizes acetyl-CoA from citrate), RNAi knockdown of lactate dehydrogenase A (the enzyme that converts pyruvate to lactate in the last step of glycolysis), and inhibitors against acetyl-CoA carboxylase and fatty acid synthase (which control the conversion of acetyl-CoA to manonyl-CoA to palmitate) all lead to substantial attenuation of tumor cell growth (DeBerar-
8 ^: P) h7 h1 o) Rdinis et al., 2008; Kroemer and Pouyssegur, 2008). Recently, it has been shown that the mere switching of pyruvate kinase from one splice variant (M2) commonly expressed in tumor cells to another splice variant (M1) can negatively impact the tumorigenic state, presumably by shunting pyruvate to the TCA cycle (Christofk et al., 2008). Thus targeting key metabolic
8 p5 Q6 S7 H/ Yenzymes that constitute NOA could effectively attenuate tumor cell proliferation. * H8 X* W! H2 R$ G5 r! ~

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marrowstem

氧化压力Oxidative Stress
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        肿瘤细胞的细胞内活性氧水平通常是增高的 (Szatrowski and Nathan, 1991)。多种因素与活性氧的生成有关。肿瘤微环境的乏氧再灌注导致活性氧的产生，这将依次启动线粒体损伤和进一步的活性氧生成的恶性循环(Gogvadze et al., 2008)。另外，如Ras的癌基因能剌激ROS产生，这个机制牵涉到癌基因诱导衰老的根本性原因 (Lee et al., 1999)。
- P" f+ R- z# h0 r     活性氧导致DNA、蛋白质、脂质和其他细胞组分的氧化损伤，从而形成明显的细胞压力（图1）。肿瘤细胞通过糖酵解和下调线粒体功能部分缓解这个压力(Gogvadze et al., 2008)。增加活性氧产生的药物可望导致癌细胞的压力过载。 作为这个主张的证据，二氯乙酸二异丙胺（肝乐）（抑制丙酮酸脱氢酶激酶PDK，剌激线粒体氧化磷酸化和活性氧的产生）选择性地剌激癌细胞凋亡而对正常细胞没有这个作用(Bonnet et al., 2007) 。同理，以抑制谷胺酸半胱氨酸连接酶（一种细胞内谷胱甘肽合成的限速酶）来减少细胞内的活性氧缓冲能力能显著提高癌细胞对放射线的敏感性(Diehn et al., 2009)。 , |$ k* s9 [* C% t; P% m

2 m8 S, M9 O1 e  P, M" ?Oxidative Stress3 n) K+ }2 o4 H9 y
       Tumor cells often show increased levels of intracellular ROS (Szatrowski and Nathan, 1991). Multiple causes contribute to ROS generation. Hypoxia-reperfusion in the tumor microenvironment can lead to ROS production, which in turn can initiate a viscous cycle of mitochondrial damage and further ROS generation (Gogvadze et al., 2008). In addition, oncogenes such as Ras can stimulate ROS production, a mechanism that has been implicated as an underlying cause of oncogene-induced senescence (Lee et al., 1999). ROS cause oxidative damage to DNA, proteins, lipids, and other cellular mponents and therefore pose a signifcant cellular stress (Figure 1). Cancer cells partially alleviate this stress by engaging glycolysis and downregulating mitochondrial function (Gogvadze et al., 2008). Agents that enhance ROS production, therefore, are expected to cause stress overload in cancer cells. In support of this notion, it has been shown that dichloroacetate, which inhibits pyruvate dehydrogenase kinase (PDK) and therefore stimulates mitochondrial oxidative phosphorylation and ROS production, can selectively elicit apoptosis in cancer cells but not in normal cells (Bonnet et al., 2007). Similarly, reducing the cellular ROS buffering capacity through the inhibition of glutamate-cysteine ligase (a rate-limiting enzyme in cellular glutathione synthesis) can markedly increase the radiosensitivity of cancer cells (Diehn et al., 2009).
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marrowstem

乏氧与营养压力Hypoxia and Nutrient Stress
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7 t$ z1 _, v$ m3 @1 T      实体瘤由于不良而不正常的肿瘤脉管系统(见下述)，通常存在着欠佳的氧和营养供应。这个压力潜在需要不断增长的血管生成。通常多数肿瘤是通过上调转录因子，乏氧诱导因子HIF-1(Pouyssegur et al., 2006)。HIF-1启动的协同转录程序可以同时剌激脉管芽殖[通过诱导VEGF-A和血管生成蛋白-2(angiopoietin-2)]和启动糖酵解(通过诱导葡萄糖载体1，己糖激酶，乳酸脱氢酶和丙酮酸脱氢酶激酶1)。
8 P; d- [3 v) U1 W       而且，HIF-1保护肿瘤细胞避免酸中毒和通过诱导碳酸酐酶来排出乳酸，单羧酸转运蛋白 MCT4,和Na+/H+交换器NHE1。针对HIF-1或它的效应器途径的靶向药物将使肿瘤细胞对乏氧压力增敏。确实，抗血管生成治疗已经在大量临床情况下显示其有效性(见下述)。抑制单羧酸转运蛋白，将导致胞内乳酸堆积和酸中毒，可能也是一个选择性杀伤依赖糖酵解乏氧肿瘤细胞的有效途径(Pouyssegur et al., 2006)。由于肿瘤细胞脱离了与脉管系统的亲密关系，它们存在着营养压力并且为了生存能进行自体吞噬。虽然损伤的自体吞噬通过启动基因组不稳定性和细胞因子释放而对肿瘤生成有贡献(Mathew et al., 2007)，在营养或生长因子限制的情况下，自体吞噬对凋亡抗拒的肿瘤细胞的生存是一种威胁。对于这种细胞，抑制自体吞噬将使它们对代谢压力敏感并启动坏死。在这个观点的支持下，自体吞噬抑制剂氯喹表现出与DNA损伤协同作用诱导鼠肿瘤细胞致死(Jin and White, 2007)。 0 R) @5 e/ {( {

$ A3 t9 y: i0 Y& vHypoxia and Nutrient Stress; ]/ d/ k! D& V7 `& h
      Solid tumors, due to poor and abnormal tumor vasculature (see below), often suffer from suboptimal oxygen and nutrient supplies. This stress underlies the requirement for augmented angiogenesis (Figure 1). Consequently many tumors upregulate the transcription factor hypoxia-inducible factor HIF-1 (Pouyssegur et al., 2006). HIF-1 initiates a coordinated transcriptional program to both stimulate vessel sprouting (through the induction of VEGF-A and angiopoietin-2) and promote glycolysis (through the induction of glucose transporter 1, hexokinase, lactate dehydrogenase, and pyruvate dehydrogenase kinase 1). Furthermore, HIF-1 protects tumor cells from acidosis and promotes the extrusion of lactate by inducing carbonate anhydrases, monocarboxylate transporter MCT4, and the Na+/H+ exchanger NHE1. Drugs targeting either HIF-1 or its effector
' M( \) z; V1 ~' [. N, d; Kpathways will therefore sensitize tumor cells toward hypoxic stress. Indeed, anti-angiogenic therapies have been effective in various clinical settings (see below). Inhibiting the monocarboxylate transporter, which would result in intracellular lactate accumulation and acidosis, might also be a viable approach to selectively kill hypoxic tumor cells addicted to glycolysis - Q( U+ `. ?% `9 u% q: S% U) Q
(Pouyssegur et al., 2006).As tumor cells grow away from the vasculature system, they experience nutrient stress and can resort to autophagy for sur-$ g! ~1 k! E9 m* i  x
vival. Although impaired autophagy could contribute to oncogenesis by promoting genomic instability and cytokine release (Mathew et al., 2007), autophagy is critical for the survival of apoptosis-resistant tumor cells during nutrient or growth factor restriction. Inhibiting autophagy in such cells would sensitize them toward metabolic stress and promote necrotic death. In
! A& _" \% r4 I  A, `support of this notion, the autophagy inhibitor chloroquine has been shown to synergize with DNA damage to induce tumor cell death in mice (Jin and White, 2007). * a& M8 Q2 F* o9 ^; S4 \' I$ F
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