cell文献翻译RNAi可对肝癌进行深入分析

1、cell文献翻译:RNAi可对肝癌进行深入分析 在癌细胞中发现的各种基因拷贝数的改变之中，那一种改变对癌症表型最为关键？Zender等在本期Cell描述了鉴定与癌症有关的新肿瘤抑制基因的基因组学综合方法。 由于癌细胞基因组不稳定性的增高，它们通常有复杂的核型，基因拷贝数有很大的增加或减少。尽管目前的基因组杂交比较(CGH)技术能够在很大程度上解析基因拷贝数的增加或减少，但在这种研究中给出的基因组区域仍相当大，可能含有几十个甚至几百个基因。一个有待回答的关键问题是在这类区域中的众多基因中，哪些是癌症表型的“司机”，哪些是“乘客”，后者表达的改变不会产生癌症。Zender等在本期Cell中发表的深

2、入研究描述了在肝癌的基因拷贝数丢失区域中鉴定癌症启动者的综合方法，并用该方法鉴定了新的肿瘤抑制基因。 Zender等的研究始于建立98个人肝癌的高解析度CGH阵列图。他们的工作集中在较小(<5 MB)的重复缺失上，因为这种大小的位点缺失很可能含有肿瘤抑制基因。他们总共鉴定了58个重复缺失，含有362个潜在抑制基因。为了研究其中哪些基因与肝癌的产生有关，Zender等使用了先前建立的小鼠肝癌模型，该模型包括因丢失肿瘤抑制基因p53和表达C-Myc致癌基因（这两个过程频繁出现在人肝癌中）而无限增殖的鼠类胚胎肝细胞。尽管这些肝细胞是无限增殖的，但它们并不形成肿瘤，只有在引入另外的致癌基因或肿瘤

3、抑制基因时，才被诱导产生肿瘤。为了找出在361个潜在人肿瘤抑制基因中哪些可以驱动致癌过程，Zender等首先鉴定了其中301个基因的小鼠直系同源基因并获得了可传递短发夹RNA (shRNA)的反转录病毒载体，以便将这些基因敲除。他们在一个先导实验中测试了与WNT信号传导途径的两个组分有关的“正控制”shRNA，因为WNT途径在肝癌中通常不受调节。用攻击Axin或Apc的shRNA感染表达c-Myc和缺失p53的小鼠肝细胞导致小鼠迅速产生肝癌。其后的实验表明，在用无活性的shRNA使有活性的shRNA的稀释度为1：48时，仍可大量产生肿瘤，因此可筛选出少量有潜在致癌活性的shRNA。 当用上述测

4、定方法筛选作用潜在肿瘤抑制基因的shRNA库时，可观察到大量的诱导性癌变（13个被测试的shRNA库中有7个可产生肿瘤），而随机挑选的10个shRNA库中没有一个可引发肿瘤生长。这项结果清楚表明，在人肿瘤中发现的基因组缺失与肿瘤的产生有关。Zender等进一步将在小鼠肿瘤中的shRNA载体与在shRNA库中的原有shRNA进行比较。在肿瘤中富集的特定shRNA可作为该shRNA可促进体内肿瘤生长的证据。用这种方法总共鉴定了36个shRNA，比在原始质粒库中的数目至少增加了2.5倍，这使得进一步筛选其中哪些shRNA可促进体内肿瘤生长成为可能。由此筛选出6个基因：Pten, Xpo4, Ddx2

5、0, Gjd4, Fst15和Nrsn2，用多个不同的shRNA抑制这些基因可显著增加无限增殖的肝细胞的癌变。这6个基因中只有脂磷酸酶PTEN曾被认为与人类癌症有关。在筛选中得到最多富集的是为Exportin 4编码的Xpo4基因。Exportin 4是细胞核转运因子家族成员，可从细胞核中输出Smad3 (TGF信号传导中的组分)和Eif5a1及Eif5a2（两个密切相关的翻译起始因子）。Xpo4在肝细胞中的去除肯定会引起细胞核Smad3的增加，并伴随有TGF靶基因的上调。对TGF的这种影响可以很好地解释去除Xpo4后癌变的增加。另外，Zender等注意到，在得到研究的98个人肝癌中，有22个

6、肝癌中的一个为XPO4的底物EIF5A2编码的基因被频繁扩增。Eif5a2基因（不包括Eif5a1基因）的超量表达可启动表达c-Myc并缺失p53的肝癌细胞转化成癌细胞，这说明XPO4蛋白不仅作用Smad3，调节癌变，而且也作用Eif5a2蛋白。值得注意的是，当Zender等检索乳腺癌中基因拷贝数变化的数据库时，发现在30%以上的肿瘤中XPO4基因被删除，这种删除与肿瘤的低存活度有关。另外，为XPO4的底物EIF5A2编码的基因存在于乳腺癌扩增子中。纵上所述，这项研究定义了一个由XPO4和EIF5A2组成的新癌变信号传导途径。 上述研究最令人惊讶的是鉴定了大量肿瘤抑制基因，但如果考虑到Zend

7、er等使用了有高度局限性的模型系统来寻找增加癌变的基因，并不是所有的敲除载体都可消除表型，这项工作的意义更为突出。其他的遗传背景可能有更多的敲除载体，因而也可鉴定其他的基因。 上述研究鉴定了一个与跨越细胞核膜的蛋白质转运有关的致癌信号传导途径，这是出乎意料的，但并非没有先例。为核孔蛋白NUP98编码的基因是癌症转移的重要标靶。NUP98的融合蛋白包括同源框转录因子、拓扑异构酶和RNA解旋酶。例如，在骨髓增生异常综合症、急性粒细胞白血病和慢性粒细胞白血病急变的病人中已发现有NUP98-HOXD13融合蛋白。 最近Firestein等使用类似的方法鉴定与结肠癌有关的新基因。这项研究第一次筛选了某些

8、基因，这些基因的抑制可抑制人结肠癌细胞的增殖和WNT传导。将潜在致癌基因的名单与结肠癌基因拷贝数增加区域的基因名单进行比较，鉴定出了依赖于细胞周期蛋白的激酶8(CDK8)是新的结肠癌致癌基因。Firestein与Zender等人的工作指出了将功能丧失的遗传筛选与人类癌症中基因拷贝数改变的分析相结合所具有的巨大威力。特别值得关注的是，在Zender等详细研究的6个基因中只有一个先前被认为与人类癌症有关，这说明该类方法可不断找出与癌症有关的各种新的重要基因。这类基因因其在人类癌症中的确切作用可成为重要的潜在药物标靶。原文地址:urlRNAi Delivers Insights into Liver

9、 CancerCell, Volume 135, Issue 5, Pages 793-795R. BernardsRNAi Delivers Insights into Liver Cancer René Bernards1, , 1Division of Molecular Carcinogenesis, Center for Biomedical Genetics, and Cancer Genomics Center, The Netherlands Cancer Institute, 1066 CX Amsterdam, The NetherlandsAvailable o

10、nline 27 November 2008. Refers to:An Oncogenomics-Based In Vivo RNAi Screen Identifies Tumor Suppressors in Liver CancerCell, Volume 135, Issue 5, 28 November 2008, Pages 852-864, Lars Zender, Wen Xue, Johannes Zuber, Camile P. Semighini, Alexander Krasnitz, Beicong Ma, Peggy Zender, Stefan Kub

11、icka, John M. Luk, Peter Schirmacher, W. Richard McCombie, Michael Wigler, James Hicks, Gregory J. Hannon, Scott Powers, Scott W. LowePDF (2398 K) | Supplementary ContentOf the myriad alterations in gene copy number found in cancer cells, which alterations are critical for the cancer phenotype? In t

12、his issue of Cell, Zender et al. (2008) describe an integrative genomics approach to identify new tumor suppressor genes involved in hepatocellular carcinoma.Article OutlineMain Text ReferencesMain TextBecause of heightened genomic instability, cancer cells often have a complex karyotype with many g

13、ains and losses in gene copy number. Although comparative genome hybridization (CGH) technologies now enable the mapping of copy number gain or loss at high resolution, the genomic regions pinpointed in such studies are still quite large and may contain tens or even hundreds of genes. A key question

14、 that often remains to be answered is which of the many genes in these regions are the “drivers” of the oncogenic phenotype and which are “passengers” whose altered expression does not contribute to the genesis of the cancer. In an elegant study in this issue, Zender et al. (2008) describe an integr

15、ative genomics approach to identify the true drivers of oncogenicity in regions of copy number loss in hepatocellular carcinoma, leading to the identification of new tumor suppressor genes. Zender et al. began by generating high-resolution array CGH maps for 98 human hepatocellular carcinomas (Figur

16、e 1). They concentrated on small (<5 MB) recurrent deletions because such focal deletions are most likely to contain tumor suppressor genes. In total, 58 recurrent deletions were identified, together harboring 362 candidate tumor suppressor genes. To ask which of these genes are relevant to liver

17、 carcinogenesis, the authors turned to a previously established mouse model for this disease, consisting of murine embryonic hepatocytes immortalized by loss of the tumor suppressor p53 and expression of the c-Myc oncogene (two events that frequently occur in human hepatocellular carcinoma). Althoug

18、h these hepatocytes are immortal, they do not form tumors in mice and can only be induced to become tumorigenic by introduction of additional oncogenes or tumor suppressor genes (Xue et al., 2008, Zender et al., 2005 L. Zender, W. Xue, C. Cordon-Cardo, G.J. Hannon, R. Lucito, S. Powers, P. Flemming,

19、 M.S. Spector and S.W. Lowe, Cold Spring Harb. Symp. Quant. Biol. 70 (2005), pp. 251261. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (12)Zender et al., 2005 and Zender et al., 2006). To ask which of the 362 candidate human tumor suppressor genes drive the oncogenic process, t

20、he authors first identified mouse orthologs for 301 of these genes and obtained retroviral vectors to deliver short hairpin RNAs (shRNAs) to knock down these genes. In a pilot experiment, the authors tested “positive control” shRNAs corresponding to two components of the WNT signaling pathway, as th

21、is pathway is frequently deregulated in liver cancer. Infection of the c-Myc-expressing p53-deficient mouse hepatocytes with shRNAs targeting either Axin or Apc yielded rapidly growing hepatocellular carcinomas in mice. Subsequent experiments of these active shRNAs in a background of inactive shRNAs

22、 showed that a dilution of 1:48 of active shRNAs still yielded significant numbers of tumors when injected in vivo, thus enabling the screening of small pools of candidate shRNAs for tumor-promoting activity.Full-size image (47K) High-quality image (394K)Figure 1. Identifying Liver Cancer Tumor

23、 Suppressor GenesHigh-resolution array comparative genome hybridization (CGH) of a panel of hepatocellular carcinomas allowed the identification of a number of genes in areas of recurrent copy number loss. Knockdown vectors delivering short hairpin RNAs (shRNAs) were generated against the murine ort

24、hologs of these candidate tumor suppressor genes. These vectors were then introduced into immortal but nontumorigenic murine hepatocytes and injected into mice. shRNAs capable of producing a tumorigenic phenotype are likely tumor suppressor genes in hepatocellular carcinoma.View Within ArticleWhen p

25、ools of shRNAs targeting the candidate tumor suppressor genes were screened in this assay, a striking induction of tumorigenicity (seven out of 13 pools tested yielded tumors) was seen, whereas none of the ten pools of randomly selected shRNAs caused tumor growth. This result strongly suggested that

26、 the genomic deletions found in the human tumors are relevant to the genesis of hepatocellular carcinoma. The representation of the shRNA vectors in the mouse tumors was then compared to their original representation in the pools. An enrichment of a specific shRNA in a tumor was taken as evidence th

27、at the shRNA conferred a growth advantage in vivo. With this approach, a total of 36 shRNAs were identified that were enriched at least 2.5-fold over their representation in the initial plasmid pools. A selection of these shRNAs was individually validated for enhanced tumor growth in vivo, yielding

28、a total of six genes whose suppression by multiple independent shRNAs markedly enhanced tumorigenicity of immortalized hepatocytes: Pten, Xpo4, Ddx20, Gjd4, Fstl5, and Nrsn2. Of these, only the lipid phosphatase PTEN has previously been implicated in human cancer.Most enriched in the screen was Xpo4

29、, encoding Exportin 4, a member of a family of nuclear transporters, known to export Smad3 (a component of TGF signaling) and Eif5a1 and Eif5a2 (two closely related translation initiation factors) from the nucleus. Indeed, knockdown of Xpo4 in hepatocytes leads to an increase in nuclear Smad3, and a

30、 concomitant upregulation of bona fide TGF target genes. This effect on TGF signaling appears to be sufficient explanation for the observed enhancement of tumorigenicity due to Xpo4 knockdown. Nevertheless, the authors did notice that one of the genes encoding the other substrate of XPO4, EIF5A2, is

31、 often amplified in human cancer, including in 22 of the 98 human hepatocelluar carcinomas studied. Indeed, overexpression of Eif5a2, but not Eif5a1, triggered oncogenic transformation of the c-Myc-expressing p53-deficient hepatocytes, suggesting that Xpo4 not only targets Smad3 to modulate tumorige

32、nicity but also targets Eif5a2. Remarkably, when the authors searched a database of gene copy number alterations in breast cancer, they found that XPO4 is deleted in over 30% of tumors and that this deletion is associated with poor survival. Moreover, the gene encoding the XPO4 substrate EIF5A2 was

33、found to be present in a breast cancer amplicon. Together, this study therefore defines a new oncogenic signaling pathway consisting of XPO4 and EIF5A2.Perhaps the most surprising aspect of this study is the large number of tumor suppressor genes that were identified. This is especially striking if

34、one considers that the authors used a highly constrained model system to search for genes that enhance tumorigenicity and not all knockdown vectors yield sufficient knockdown to produce a phenotype. It is possible that in another genetic background with more knockdown vectors, yet other genes could

35、be identified.The identification of an oncogenic signaling pathway involved in protein transport across the nuclear membrane is unexpected, but not without precedent. The gene encoding the nucleoporin NUP98 is a recurring target of translocations in cancer. Among the fusion partners of NUP98 are hom

36、eobox transcription factors, a topoisomerase, and an RNA helicase. For instance, NUP98-HOXD13 fusions have been identified in patients with myelodysplastic syndrome, acute myelogenous leukemia, and chronic myeloid leukemia blast crisis (Moore et al., 2007).Recently, Firestein et al. (2008) used a re

37、lated approach to identify new relevant genes for colon cancer. In that study, the authors first screened for genes whose suppression could inhibit both proliferation and WNT signaling in human colon cancer cells. The list of candidate genes was then compared to a list of genes that are located in r

38、egions of copy number gain in colon cancer, allowing the identification of cyclin-dependent kinase 8 (CDK8) as a new colon cancer oncogene (Firestein et al., 2008). The efforts of Firestein et al. and Zender et al. highlight the power of combining loss-of-function genetic screens with analysis of co

39、py number alterations found in human cancer. That only one of the six genes studied in detail by Zender et al. has previously been implicated in human cancer warrants particular emphasis. This suggests that approaches like these can still yield a treasure trove of new genes relevant to cancer. Such

40、genes, by virtue of their in vivo validation and established role in human cancer, have great value as potential drug targets.ReferencesFirestein et al., 2008 R. Firestein, A.J. Bass, S.Y. Kim, I.F. Dunn, S.J. Silver, I. Guney, E. Freed, A.H. Ligon, N. Vena and S. Ogino et al., Nature 455 (2008), pp

41、. 547551. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (46)Moore et al., 2007 M.A. Moore, K.Y. Chung, M. Plasilova, J.J. Schuringa, J.H. Shieh, P. Zhou and G. Morrone, Ann. N Y Acad. Sci. 1106 (2007), pp. 114142. Full Text via CrossRef | View Record in Scopus | Cited By in Sco

42、pus (12)Xue et al., 2008 W. Xue, A. Krasnitz, R. Lucito, R. Sordella, L. Vanaelst, C. Cordon-Cardo, S. Singer, F. Kuehnel, M. Wigler and S. Powers et al., Genes Dev. 22 (2008), pp. 14391444. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (30)Zender et al., 2005 L. Zender, W. Xue, C. Cordon-Cardo, G.J. Hannon, R. Lucito, S. Powers, P. Flemming, M.S. Spector and S.W. Lowe, Cold Spring Harb. Symp. Quant. Biol. 70 (2005), pp. 251261. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (12)Zender et al., 2006 L. Zender, M.S. Spector,