cell文獻翻譯RNAi可對肝癌進行深入分析

1、cell文獻翻譯:RNAi可對肝癌進行深入分析 在癌細胞中發(fā)現的各種基因拷貝數的改變之中，那一種改變對癌癥表型最為關鍵？Zender等在本期Cell描述了鑒定與癌癥有關的新腫瘤抑制基因的基因組學綜合方法。 由于癌細胞基因組不穩(wěn)定性的增高，它們通常有復雜的核型，基因拷貝數有很大的增加或減少。盡管目前的基因組雜交比較(CGH)技術能夠在很大程度上解析基因拷貝數的增加或減少，但在這種研究中給出的基因組區(qū)域仍相當大，可能含有幾十個甚至幾百個基因。一個有待回答的關鍵問題是在這類區(qū)域中的眾多基因中，哪些是癌癥表型的“司機”，哪些是“乘客”，后者表達的改變不會產生癌癥。Zender等在本期Cell中發(fā)表的深

2、入研究描述了在肝癌的基因拷貝數丟失區(qū)域中鑒定癌癥啟動者的綜合方法，并用該方法鑒定了新的腫瘤抑制基因。 Zender等的研究始于建立98個人肝癌的高解析度CGH陣列圖。他們的工作集中在較小(<5 MB)的重復缺失上，因為這種大小的位點缺失很可能含有腫瘤抑制基因。他們總共鑒定了58個重復缺失，含有362個潛在抑制基因。為了研究其中哪些基因與肝癌的產生有關，Zender等使用了先前建立的小鼠肝癌模型，該模型包括因丟失腫瘤抑制基因p53和表達C-Myc致癌基因（這兩個過程頻繁出現在人肝癌中）而無限增殖的鼠類胚胎肝細胞。盡管這些肝細胞是無限增殖的，但它們并不形成腫瘤，只有在引入另外的致癌基因或腫瘤

3、抑制基因時，才被誘導產生腫瘤。為了找出在361個潛在人腫瘤抑制基因中哪些可以驅動致癌過程，Zender等首先鑒定了其中301個基因的小鼠直系同源基因并獲得了可傳遞短發(fā)夾RNA (shRNA)的反轉錄病毒載體，以便將這些基因敲除。他們在一個先導實驗中測試了與WNT信號傳導途徑的兩個組分有關的“正控制”shRNA，因為WNT途徑在肝癌中通常不受調節(jié)。用攻擊Axin或Apc的shRNA感染表達c-Myc和缺失p53的小鼠肝細胞導致小鼠迅速產生肝癌。其后的實驗表明，在用無活性的shRNA使有活性的shRNA的稀釋度為1：48時，仍可大量產生腫瘤，因此可篩選出少量有潛在致癌活性的shRNA。 當用上述測

4、定方法篩選作用潛在腫瘤抑制基因的shRNA庫時，可觀察到大量的誘導性癌變（13個被測試的shRNA庫中有7個可產生腫瘤），而隨機挑選的10個shRNA庫中沒有一個可引發(fā)腫瘤生長。這項結果清楚表明，在人腫瘤中發(fā)現的基因組缺失與腫瘤的產生有關。Zender等進一步將在小鼠腫瘤中的shRNA載體與在shRNA庫中的原有shRNA進行比較。在腫瘤中富集的特定shRNA可作為該shRNA可促進體內腫瘤生長的證據。用這種方法總共鑒定了36個shRNA，比在原始質粒庫中的數目至少增加了2.5倍，這使得進一步篩選其中哪些shRNA可促進體內腫瘤生長成為可能。由此篩選出6個基因：Pten, Xpo4, Ddx2

5、0, Gjd4, Fst15和Nrsn2，用多個不同的shRNA抑制這些基因可顯著增加無限增殖的肝細胞的癌變。這6個基因中只有脂磷酸酶PTEN曾被認為與人類癌癥有關。在篩選中得到最多富集的是為Exportin 4編碼的Xpo4基因。Exportin 4是細胞核轉運因子家族成員，可從細胞核中輸出Smad3 (TGF信號傳導中的組分)和Eif5a1及Eif5a2（兩個密切相關的翻譯起始因子）。Xpo4在肝細胞中的去除肯定會引起細胞核Smad3的增加，并伴隨有TGF靶基因的上調。對TGF的這種影響可以很好地解釋去除Xpo4后癌變的增加。另外，Zender等注意到，在得到研究的98個人肝癌中，有22個

6、肝癌中的一個為XPO4的底物EIF5A2編碼的基因被頻繁擴增。Eif5a2基因（不包括Eif5a1基因）的超量表達可啟動表達c-Myc并缺失p53的肝癌細胞轉化成癌細胞，這說明XPO4蛋白不僅作用Smad3，調節(jié)癌變，而且也作用Eif5a2蛋白。值得注意的是，當Zender等檢索乳腺癌中基因拷貝數變化的數據庫時，發(fā)現在30%以上的腫瘤中XPO4基因被刪除，這種刪除與腫瘤的低存活度有關。另外，為XPO4的底物EIF5A2編碼的基因存在于乳腺癌擴增子中。縱上所述，這項研究定義了一個由XPO4和EIF5A2組成的新癌變信號傳導途徑。 上述研究最令人驚訝的是鑒定了大量腫瘤抑制基因，但如果考慮到Zend

7、er等使用了有高度局限性的模型系統(tǒng)來尋找增加癌變的基因，并不是所有的敲除載體都可消除表型，這項工作的意義更為突出。其他的遺傳背景可能有更多的敲除載體，因而也可鑒定其他的基因。 上述研究鑒定了一個與跨越細胞核膜的蛋白質轉運有關的致癌信號傳導途徑，這是出乎意料的，但并非沒有先例。為核孔蛋白NUP98編碼的基因是癌癥轉移的重要標靶。NUP98的融合蛋白包括同源框轉錄因子、拓撲異構酶和RNA解旋酶。例如，在骨髓增生異常綜合癥、急性粒細胞白血病和慢性粒細胞白血病急變的病人中已發(fā)現有NUP98-HOXD13融合蛋白。 最近Firestein等使用類似的方法鑒定與結腸癌有關的新基因。這項研究第一次篩選了某些

8、基因，這些基因的抑制可抑制人結腸癌細胞的增殖和WNT傳導。將潛在致癌基因的名單與結腸癌基因拷貝數增加區(qū)域的基因名單進行比較，鑒定出了依賴于細胞周期蛋白的激酶8(CDK8)是新的結腸癌致癌基因。Firestein與Zender等人的工作指出了將功能喪失的遺傳篩選與人類癌癥中基因拷貝數改變的分析相結合所具有的巨大威力。特別值得關注的是，在Zender等詳細研究的6個基因中只有一個先前被認為與人類癌癥有關，這說明該類方法可不斷找出與癌癥有關的各種新的重要基因。這類基因因其在人類癌癥中的確切作用可成為重要的潛在藥物標靶。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,