The Role of Pdcd4 in Translation, Tumorigenesis and Tumor Progression

mRNA分析的差异显示鉴定了Pdcd 4（Cmarik等人，PNAS 1999）作为一种新的转化抑制剂。新pdcd 4基因的反义表达将转化抗性（P-）细胞转化为敏感性（P+）细胞，而pdcd 4正义表达（Yang等Oncogene 2001）将P+细胞转化为P-细胞，从而建立了与预防肿瘤启动子诱导的转化的因果关系。此外，pdcd 4表达抑制转化的小鼠JB 6细胞中的肿瘤表型（Yang等人Oncogene 2003）。令人惊讶的发现是，人癌细胞系中的Pdcd 4表达预测对他莫昔芬和格尔德霉素的敏感性。此外，Pdcd 4的表达实际上赋予了对这些药物的敏感性（Jansen et al Molec Cancer Ther 2004）。Pdcd 4对已知肿瘤促进所需的分子事件的可能抑制作用的检查显示，Pdcd 4过表达抑制转录因子AP-1的活化，但不抑制NF κ B或鸟氨酸脱羧酶的活化（Yang et al Oncogene 2001）。Pdcd 4的AP-1抑制活性似乎可归因于阻断cJun和cFos的反式激活（Yang et al Oncogene 2003）。虽然Pdcd 4蛋白的表达阻断AP-1活化，但Pdcd 4不直接与Jun或Fos蛋白相互作用。通过酵母双杂交试验和免疫共沉淀分析Pdcd 4结合配偶体，鉴定翻译起始因子RNA解旋酶eIF 4A和支架eIF 4G为主要结合配偶体（Yang et al Molec Cell Biol 2003，MCB 2004）。Pdcd 4与eIF 4A的结合是Pdcd 4抑制eIF 4A的RNA解旋酶活性、抑制翻译起始和抑制肿瘤转化所需的AP-1依赖性转录激活所必需的。突变分析定义了结合eIF 4A和抑制翻译所需的两个螺旋MA-3结构域（Yang等Molec Cell Biol 2004）。还对结合Pdcd 4所需的eIF 4A上的残基进行了表征（Zakowicz RNA 2005）。与亚历山大沃达维尔实验室合作，已解决了C-末端MA 3结构域的晶体结构（LaRonde-LeBlanc，Santhanam et al MCB 2007）。晶体结构的分析预测了Pdcd 4通过与eIF 4A结合伴侣eIF 4G竞争抑制翻译起始而起作用的机制，该预测已被实验证实。Pdcd 4表达在许多人类癌症中下调，是人类结肠癌分期的诊断和存活的预后（Muduluru et al Cancer 2007）。干预小鼠肺癌发生的基因治疗方法已经揭示，肺吸入施用Pdcd 4在小鼠肺中诱导细胞凋亡并抑制AP-1（Hwang等Gene Ther 2007和Jin等Mol Ca Ther 2006）以及抑制异种移植肿瘤生长（Kim YK，Kwon JT，Choi JY，Jiang HL，Arote R，Jere D，Je YH，Cho MH，Cho CS，Cancer Gene Ther. 2010年）。pdcd 4过表达抑制人癌细胞的侵袭。该机制涉及cJun N-末端激酶上游激酶的靶向表达，从而抑制AP-1依赖性转录（Yang et al MCB 2006）。最近对肿瘤抑制因子Pdcd 4在致癌过程中下调的机制的研究揭示了肿瘤启动子通过涉及经由Akt、S6激酶和MEK/ERK的信号传导的机制诱导蛋白质的不稳定化（Schmid，Jansen等人，Cancer Res 2008）。与纽约大学的Michele Pagano合作，Pdcd 4已成为泛素连接酶betaTRCP降解的靶标（Dorrello et al Science 2006）。此外，与Heike Allgayer（曼海姆）和Yong Li（Louisville）合作，我们发现Pdcd 4是microRNA miR-21的功能重要靶标（Asangani et al Oncogene 2008，Lu et al Oncogene 2008）。目前的研究主要集中在识别特定的转化相关的mRNA，其翻译被Pdcd 4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mRNA的结构特征的研究揭示了令人惊讶的发现，即3 'UTR和microRNA结合位点是重要的（Santhanam等PLoS ONE 2009）。 目前正在与Tobias Schmid（Goethe Univ，法兰克福）、布鲁斯夏皮罗（Bruce Shapiro）、Nahum Sonenberg（麦吉尔Univ）和Curt Henrich（Blees et al，J Biomol Screen 2010）合作产生靶向翻译起始的药物发现工具。正在进行多核糖体分析，以识别当Pdcd 4表达下调或上调时转移到或移出活跃翻译部分的mRNA。我们最近发现了Pdcd 4的新的（间接）转录靶标，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。最近的研究（Gaur et al Neuro-Oncology 2011）已经将Pdcd 4作为肿瘤抑制因子的观察结果扩展到胶质母细胞瘤（GBM）细胞。Pdcd 4是GBM细胞中致癌微小RNA miR 21的主要靶标，其显示几乎不可检测的Pdcd 4。 Pdcd 4在GBM细胞中的稳定表达抑制异种移植物生长。

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 lung inhalation 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)as well as inhibiting xenograft tumor growth (Kim YK, Kwon JT, Choi JY, Jiang HL, Arote R, Jere D, Je YH, Cho MH, Cho CS, Cancer Gene Ther. 2010). 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, 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. Recent studies (Gaur et al Neuro-Oncology 2011) have extended the observation of Pdcd4 as a tumor suppressor to glioblastoma (GBM)cells. Pdcd4 is a major target of oncogenic micro-RNA miR21 in GBM cells which show almost undetectable Pdcd4. Stable expression of Pdcd4 in GBM cells suppresses xenograft growth.