Genes Differentially Expressed During Tumor Promotion an

mRNA的差异显示分析发现Pdcd4 （Cmarik等人，PNAS 1999）是一种新的转化抑制因子。新型pdcd4基因的反义表达将转化抗性（P-）细胞转化为敏感（P+）细胞，pdcd4的义表达（Yang et al Oncogene 2001）将P+细胞转化为P-细胞，从而与预防肿瘤启动子诱导的转化建立了因果关系。此外，pdcd4的表达在转化的JB6细胞中抑制肿瘤表型（Yang et al Oncogene 2003）。一个令人惊讶的发现是，人类癌细胞系中Pdcd4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的表达实际上赋予了对这些药物的敏感性（Jansen et al . Molec Cancer Ther 2004）。对pdcd4对已知促进肿瘤所需的分子事件可能的抑制作用的研究表明，pdcd4的表达抑制转录因子AP-1的激活，但不抑制NFkappa B或鸟氨酸脱羧酶的激活（Yang et al Oncogene 2001）。Pdcd4的AP-1抑制活性似乎可归因于阻断cJun和cFos的转激活（Yang et al Oncogene 2003）。虽然Pdcd4蛋白的表达阻断AP-1的激活，但Pdcd4并不直接与Jun或Fos蛋白相互作用。通过酵母双杂交实验和共免疫沉淀分析Pdcd4结合伙伴发现翻译起始因子RNA解旋酶eIF4A和支架eIF4G是主要的结合伙伴（Yang等Molec Cell Biol 2003， MCB 2004）。Pdcd4与eIF4A结合才能抑制eIF4A的RNA解旋酶，抑制翻译，抑制肿瘤转化所需的AP-1依赖性转录的激活。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 et al Molec Cell Biol 2004）。eIF4A结合Pdcd4所需的残基也已被鉴定（Zakowicz RNA 2005）。与Alexander Wlodawer实验室合作，已经解决了c端MA3结构域的晶体结构（LaRonde-LeBlanc, Santhanam et al MCB In press 2006）。晶体结构的分析预测了Pdcd4抑制翻译起始的机制，这一预测已被实验证实。在许多人类癌症中，Pdcd4表达下调。Pdcd4过表达抑制人类癌细胞的侵袭。其机制涉及靶向cJun n端激酶上游的激酶表达，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Pdcd4在癌变过程中下调机制的研究表明，肿瘤启动子通过Akt、S6kinase和MEK/ERK信号通路诱导该蛋白失稳（Schmid， Jansen等，提交）。在与纽约大学的Michele Pagano的合作中，Pdcd4已成为泛素连接酶β - rcp降解的靶标（Dorrello等科学出版）。目前的研究主要集中在鉴定受Pdcd4表达抑制的特异性转化相关mrna。针对翻译起始的药物发现工具目前正在与Bruce Shapiro， Stuart LeGrice, Nahum Sonenberg（麦吉尔大学）和Jim McMahon合作。

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 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 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 revealed 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 eIF4A's RNA helicase, to inhibit translation, 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 in press 2006). Analysis of the crystal structure predicts a mechanism by which Pdcd4 acts to inhibit translation initiation, a prediction that has been experimentally confirmed. Pdcd4 expression is downregulated in a number of human cancers. 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, submitted). In collaboration with Michele Pagano at NYU, Pdcd4 has emerged as a target for degradation by the ubiquitin ligase betaTRCP (Dorrello et al Science in press). Current research is focused on identifying specific transformation relevant mRNAs whose translation is inhibited by Pdcd4 expression. Tools for drug discovery targeting translation initiation are currently being generated in collaboration with Bruce Shapiro, Stuart LeGrice, Nahum Sonenberg (McGill Univ) and Jim McMahon.