Pdcd4 is a novel transformation suppressor that inhibits tumor promoter-induced neoplastic transformation and the activation of AP-1-dependent transcription required for transformation. A yeast two-hybrid analysis revealed that Pdcd4 associates with the eukaryotic translation initiation factors eIF4AI and eIF4AII. Immunofluorescent confocal microscopy showed that Pdcd4 colocalizes with eIF4A in the cytoplasm. eIF4A is an ATP-dependent RNA helicase needed to unwind 5 mRNA secondary structure. Recombinant Pdcd4 specifically inhibited the helicase activity of eIF4A and eIF4F. In vivo translation assays showed that Pdcd4 inhibited cap-dependent but not internal ribosome entry site (IRES)-dependent translation. In contrast, Pdcd4 D418A , a mutant inactivated for binding to eIF4A, failed to inhibit cap-dependent or IRES-dependent translation or AP-1 transactivation. Recombinant Pdcd4 prevented eIF4A from binding to the C-terminal region of eIF4G (amino acids 1040 to 1560) but not to the middle region of eIF4G(amino acids 635 to 1039). In addition, both Pdcd4 and Pdcd4 D418A bound to the middle region of eIF4G. The mechanism by which Pdcd4 inhibits translation thus appears to involve inhibition of eIF4A helicase, interference with eIF4A association-dissociation from eIF4G, and inhibition of eIF4A binding to the C-terminal domain of eIF4G. Pdcd4 binding to eIF4A is linked to its transformation-suppressing activity, as Pdcd4-eIF4A binding and consequent inhibition of translation are required for Pdcd4 transrepression of AP-1.

Pdcd4 is a novel transformation suppressor that is highly expressed in promotion-resistant (P7) mouse epidermal JB6 cells but not in susceptible (P+) cells. Overexpression of pdcd4 cDNA in stably transfected P+ cells rendered cells resistant to tumor promoter-induced transformation, indicating that elevated expression of Pdcd4 protein is su cient to suppress neoplastic transformation. To determine whether Pdcd4 suppresses neoplastic transformation through inhibiting known transformation required events, we examined the possibility that pdcd4 inhibited the activation of AP-1 or NF-kB dependent transcription or of ornithine decarboxylase (ODC) activity. Activation of AP-1-dependent transcriptional activity was inhibited by pdcd4 expression in a concentration dependent manner. In contrast, Pdcd4 slightly increased NF-kB-dependent transcription and did not alter ODC enzymatic activity. Previous studies suggested that activation of AP-1 was required for P+ cell transformation as well as for tumor promotion in vivo. These results indicate that Pdcd4 functions as a transformation suppressor, possibly through inhibiting AP-1 activation in combination with other factors such as enhancing NF-kB activation. Pdcd4 may thus constitute a useful molecular target for cancer prevention. Oncogene (2001) 20, 669 ± 676.

BACKGROUND.Programmed cell death 4 (Pdcd4) inhibits malignant transformation, and initial studies of Pdcd4 suggested the regulation of Pdcd4 localization by protein kinase B (Akt). However, supporting patient tissue data are missing, and the diagnostic/prognostic potential of Pdcd4 rarely has been studied. The objectives of the current were 1) to determine Pdcd4 as a diagnostic marker in the adenoma‐carcinoma sequence, 2) to support phosphorylated Akt (pAkt)‐mediated Pdcd4 regulation in vivo, and 3) to obtain the first prognostic evidence of Pdcd4 in colorectal cancer.METHODS.Tumor samples and normal tissues from 71 patients with colorectal cancer who were followed prospectively (median follow‐up, 36 months) and 42 adenomas were analyzed for Pdcd4, Akt, and pAkt in immunohistochemical and Western blot analyses.RESULTS.A significant reduction in Pdcd4 was observed between normal mucosa and adenomas and between adenomas and tumor samples (P < .01 and P < .01, respectively). Normal mucosa demonstrated strong nuclear Pdcd4, which was reduced significantly in adenomas (P < .01) and almost was lost in tumors (P < .01). pAkt was correlated inversely with Pdcd4 and with the transition of Pdcd4 from nucleus to cytoplasm (P < .01). Kaplan‐Meier analysis (using the Mantel‐Cox log‐rank test) indicated a significant correlation between the loss of total and nuclear Pdcd4 in tumors and overall survival (P < .05 and P < .02, respectively) and disease‐specific survival (P < .01 and P < .01, respectively). In multivariate analysis, loss of total or nuclear Pdcd4 was an independent predictor of disease‐specific or overall survival.CONCLUSIONS.To the authors' knowledge, this is the first study to demonstrate an independent prognostic impact of Pdcd4 and its expression pattern in colorectal cancer. Data from this study support the regulation of Pdcd4 localization by pAkt in vivo. Pdcd4 immunohistochemistry may be useful as a supportive diagnostic tool for the transition between normal, adenoma, and tumor tissues. Cancer 2007. Published 2007 by the American Cancer Society.

Programmed cell death 4 (Pdcd4) is a novel repressor of in vitro transformation. Pdcd4 directly inhibits the helicase activity of eukaryotic translation initiation factor 4A, a component of the translation initiation complex. To ascertain whether Pdcd4 suppresses tumor development in vivo, we have generated transgenic mice that overexpress Pdcd4 in the epidermis (K14-Pdcd4). K14-regulated Pdcd4 expression caused a neonatal short-hair phenotype due to early catagen entry compared with matched wild-type siblings. In response to the 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) mouse skin carcinogenesis protocol, K14-Pdcd4 mice showed significant reductions in papilloma formation, carcinoma incidence, and papilloma-to-carcinoma conversion frequency compared with wild-type mice. The translational efficiency of an mRNA engineered to form a structured 5V untranslated region (UTR) was attenuated in primary keratinocytes when Pdcd4 was overexpressed. Pdcd4 inhibited by 46% TPA-induced activator protein-1 (AP-1)-dependent transcription, an event required for tumorigenesis. CDK4 and ornithine decarboxylase (ODC) are candidates for Pdcd4-regulated translation as their mRNAs contain 5Vstructured UTRs. In K14-Pdcd4 primary keratinocytes expressing activated Ha-Ras to mimic DMBA-initiated epidermis, ODC and CDK4 protein levels were decreased by 40% and 46%, respectively. Expression of a protein encoded by 5V unstructured mRNA showed no change. These results extend to an in vivo model the observations that Pdcd4 inhibits both translation initiation and AP-1 activation while decreasing benign tumor development and malignant progression. The K14-Pdcd4 mice seem to validate translation initiation as a novel target for cancer prevention. (Cancer Res 2005; 65(14): 6034-41)