Genes Differentially Expressed During Tumor Promotion an

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JB 6细胞中的肿瘤表型（Yang等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，发现翻译起始因子RNA解旋酶eIF 4A和支架eIF 4G是主要结合配偶体（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、抑制翻译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的实验室合作，已经解决了C-末端MA 3结构域的晶体结构（LaRonde-LeBlanc，Santhanam等人MCB，2006年出版）。晶体结构的分析预测了Pdcd 4抑制翻译起始的机制，这一预测已被实验证实。pdcd 4在许多人类癌症中表达下调。pdcd 4过表达抑制人癌细胞的侵袭。该机制涉及靶向表达cJun N-末端激酶上游的激酶，从而抑制AP-1依赖性转录（Yang等MCB 2006）。最近对肿瘤抑制因子Pdcd 4在癌发生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，提交）。与纽约大学的Michele Pagano合作，Pdcd 4已经成为泛素连接酶betaTRCP降解的靶点（Dorrello et al Science in press）。目前的研究主要集中在鉴定Pdcd 4表达抑制其翻译的特异性转化相关mRNA。目前正在与布鲁斯·夏皮罗、斯图尔特·勒格莱斯、纳胡姆·索嫩伯格（麦吉尔大学）和吉姆·麦克马洪合作开发靶向翻译起始的药物发现工具。

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.