The Role of Pdcd4 in Translation, Tumorigenesis and Tumor Progression

差异显示的信使核糖核酸分析确定Pdcd4(Cmarik等人，PNAS 1999)是一种新的转化抑制因子。Pdcd4基因的反义表达将耐转化(P-)转化为敏感(P+)细胞，pdcd4正义表达(Yang Et Al Oncogene 2001)将P+转化为P-细胞，从而建立了阻止肿瘤启动子诱导转化的因果关系。此外，pdcd4的表达抑制了转化的小鼠JB6细胞中的肿瘤表型(Yang Et Al Oncogene 2003)。一个令人惊讶的发现是，Pdcd4在人类癌细胞株中的表达可以预测对他莫昔芬和格尔达霉素的敏感性。此外，Pdcd4的表达实际上增加了对这些药物的敏感性(Jansen等人，Molec癌症治疗，2004)。对Pdcd4对已知促进肿瘤所需的分子事件的可能抑制作用的研究表明，Pdcd4的过表达抑制了转录因子AP-1的激活，但不能抑制NFkappa B或鸟氨酸脱羧酶的激活(Yang等人癌基因2001)。Pdcd4的AP-1抑制活性似乎可归因于阻断cJun和CFos的反式激活(Yang等人Oncogene 2003)。虽然Pdcd4蛋白的表达阻断了AP-1的激活，但Pdcd4并不直接与Jun或Fos蛋白相互作用。通过酵母双杂交和免疫共沉淀对Pdcd4结合伙伴的分析确定翻译起始因子RNA解旋酶eIF4A和支架eIF4G是主要的结合伙伴(Yang等人Molec Cell Biol 2003，MCB 2004)。Pdcd4需要与eIF4A结合，才能抑制eIF4A的RNA解旋酶活性，抑制翻译启动，并抑制肿瘤转化所需的AP-1依赖转录的激活。突变分析定义了与eIF4A结合和抑制翻译所需的两个螺旋MA-3结构域(Yang等人Molec Cell Biol 2004)。结合Pdcd4所需的eIF4A上的残基也已确定(Zakowicz RNA 2005)。与Alexander Wlowawer的实验室合作，已经解决了C-末端MA3结构域的晶体结构(LaRonde-LeBlanc，SanThanam等人MCB 2007)。对晶体结构的分析预测了Pdcd4通过与eIF4A结合伙伴eIF4G竞争抑制翻译起始的作用机制，这一预测已得到实验证实。Pdcd4在许多人类癌症中表达下调，对人类结肠癌分期和生存预后有诊断意义(Muduluru等人癌症，2007)。一种干预小鼠肺癌发生的基因疗法方法显示，给予Pdcd4可诱导小鼠肺组织细胞凋亡并抑制AP-1(Hwang等人的基因疗法2007和jin等人的Mol Ca Ther 2006)。Pdcd4过表达可抑制人类癌细胞的侵袭。该机制涉及靶向表达cJun N末端激酶上游的一种激酶，从而抑制AP-1依赖的转录(Yang等人MCB 2006)。最近对肿瘤抑制基因Pdcd4在癌变过程中下调的机制的研究表明，肿瘤启动子通过一种涉及Akt、S6Kinase和MEK/ERK的信号转导机制来诱导蛋白质的不稳定(SchmidJansen等人，癌症资源2008)。在纽约大学与Michele Pagano的合作下，Pdcd4已成为泛素连接酶BetaTRCP降解的目标(Dorrello等人，2006年)。此外，在与Heike Allgayer(Mannheim)和Yong Li(Louisville)的合作中，我们发现Pdcd4是microRNA miR-21的功能重要靶点(Asangani等人Oncogene 2008，Lu等人Oncogene 2008)。目前的研究主要集中在识别特定的转化相关的mRNAs，这些mRNAs的翻译被Pdcd4的表达抑制，并可能介导其肿瘤抑制活性。最近对区分对翻译抑制敏感的mRNAs的结构特征的研究揭示了一个令人惊讶的发现，即3‘UTR和microRNA结合位点是重要的(SanThanam等人，PLoS One，2009)。目前正在与Tobias Schmidd(歌德大学，法兰克福)、Bruce Shapiro、Stuart LeGrice、Nahum Sonenberg(McGill大学)和Curt Henrich(Blees等人，J BiolScreen 2010)合作开发针对翻译启动的药物发现工具。多聚体谱分析正在进行中，以确定当Pdcd4表达下调或上调时，进入或移出主动翻译部分的mRNAs。我们最近发现了Pdcd4的一个新的(间接)转录靶点，即赖氨酰氧化酶(SanThanam Et Al Oncogene 2010)。赖氨酰氧化酶在介导低氧反应和乳腺癌细胞侵袭中起重要作用。

Differential display of mRNA analysis identified Pdcd4 (Cmarik et al., PNAS 1999) as a novel suppressor of transformation. Antisense expression of the novel pdcd4 gene converts transformation resistant (P-) to sensitive (P+) cells and pdcd4 sense expression (Yang et al Oncogene 2001) converts P+ to P- cells, thus establishing a causal relationship to prevention of tumor promoter induced transformation. Furthermore pdcd4 expression suppresses tumor phenotype in transformed mouse JB6 cells (Yang et al Oncogene 2003). A surprising discovery is that Pdcd4 expression in human cancer cell lines is predictive for sensitivity to tamoxifen and geldanamycin. Moreover, expression of Pdcd4 actually confers sensitivity to these drugs (Jansen et al Molec Cancer Ther 2004). Examination of the possible inhibitory effect of Pdcd4 on molecular events known to be required for tumor promotion revealed that Pdcd4 over-expression inhibited the activation of transcription factor AP-1 but not of NFkappa B or of ornithine decarboxylase(Yang et al Oncogene 2001). The AP-1 inhibiting activity of Pdcd4 appears to be attributable to blocking the transactivation of cJun and cFos (Yang et al Oncogene 2003). Although expression of Pdcd4 protein blocks AP-1 activation, Pdcd4 does not interact directly with Jun or Fos proteins. Analysis of Pdcd4 binding partners by a yeast two-hybrid assay and co-immunoprecipitation identified the translation initiation factors RNA helicase eIF4A and scaffold eIF4G as major binding partners (Yang et al Molec Cell Biol 2003, MCB 2004). Binding of Pdcd4 to eIF4A is required for Pdcd4 to inhibit the RNA helicase activity of eIF4A, to inhibit translation initiation, and to inhibit the activation of AP-1 dependent transcription required for neoplastic transformation. Mutational analysis defines two helical MA-3 domains as required for binding to eIF4A and for inhibiting translation (Yang et al Molec Cell Biol 2004). Residues on eIF4A required for binding Pdcd4 have also been characterized (Zakowicz RNA 2005). In collaboration with the laboratory of Alexander Wlodawer, the crystal structure of the C-terminal MA3 domain has been solved (LaRonde-LeBlanc, Santhanam et al MCB 2007). Analysis of the crystal structure predicts a mechanism by which Pdcd4 acts by competing with eIF4A binding partner eIF4G to inhibit translation initiation, a prediction that has been experimentally confirmed. Pdcd4 expression is downregulated in a number of human cancers, is diagnostic for human colon cancer staging and prognostic for survival (Muduluru et al Cancer 2007). A gene therapy approach to intervention in mouse lung carcinogenesis has revealed that administration of Pdcd4 induces apoptosis and inhibits AP-1 in mouse lung (Hwang et al Gene Ther 2007 and Jin et al Mol Ca Ther 2006). Pdcd4 overexpression inhibits invasion by human cancer cells. The mechanism involves targeting expression of a kinase upstream of cJun N-terminal kinase to consequently inhibit AP-1 dependent transcription (Yang et al MCB 2006). Recent investigation of the mechanism by which tumor suppressor Pdcd4 is down regulated during carcinogenesis revealed tumor promoter induced destabilization of the protein by a mechanism involving signaling through Akt, S6kinase and MEK/ERK (Schmid, Jansen et al, Cancer Res 2008). In collaboration with Michele Pagano at NYU, Pdcd4 has emerged as a target for degradation by the ubiquitin ligase betaTRCP (Dorrello et al Science 2006). Moreover, in collaboration with Heike Allgayer (Mannheim) and Yong Li (Louisville) we found that Pdcd4 is a functionally significant target of microRNA miR-21 (Asangani et al Oncogene 2008, Lu et al Oncogene 2008. Current research is focused on identifying specific transformation relevant mRNAs whose translation is inhibited by Pdcd4 expression and may mediate its tumor suppressor activity. A recent inquiry into the structural features that distinguish mRNAs that are sensitive to translation inhibition revealed the surprising finding that the 3'UTR and microRNA binding sites are important (Santhanam et al PLoS ONE 2009). Tools for drug discovery targeting translation initiation are currently being generated in collaboration with Tobias Schmid (Goethe Univ, Frankfurt), Bruce Shapiro, Stuart LeGrice, Nahum Sonenberg (McGill Univ) and Curt Henrich(Blees et al, J Biomol Screen 2010). Polysome profiling is underway to identify mRNAs that shift into or out of the actively translating fraction when Pdcd4 expression is down- or up-regulated. We have recently discovered a new (indirect) transcriptional target of Pdcd4, namely the enzyme lysyl oxidase (Santhanam et al Oncogene 2010). Lysyl oxidase is important in mediating hypoxic response and breast cancer cell invasion.