The role of Pdcd4 in tumour suppression and protein translation

Programmed cell death 4 (Pdcd4), a tumour suppressor, is frequently down‐regulated in various types of cancer. Pdcd4 has been demonstrated to efficiently suppress tumour promotion, progression and proliferation. The biochemical function of Pdcd4 is a protein translation inhibitor. Although the fact that Pdcd4 inhibits protein translation has been known for more than a decade, the mechanism by which Pdcd4 controls tumorigenesis through translational regulation of its target genes is still not fully understood. Recent studies show that Pdcd4 inhibits translation of stress‐activated‐protein kinase interacting protein 1 to suppress tumour invasion, depicting a picture of how Pdcd4 inhibits tumorigenesis through translational inhibition. Thus, understanding the mechanism of how Pdcd4 attenuates tumorigenesis by translational control should provide a new strategy for combating cancer.     

Knockdown of Pdcd4 results in induction of proprotein convertase 1/3 and potent secretion of chromogranin A and secretogranin II in a neuroendocrine cell line

Background information. Pdcd4 (programmed cell death 4) is up‐regulated during apoptosis and seems to play an important role as a tumour suppressor. To gain further insights into its biological functions, we suppressed Pdcd4 expression in the neuroendocrine cell line Bon‐1 via siRNA (small interfering RNA) technology. Results. Using this cell line, we found that suppression of Pdcd4 resulted in an increased release of CgA (chromogranin A) and Sg II (secretogranin II), and was accompanied by an up‐regulation of intracellular PC1 (proprotein convertase 1/3). The enhanced secretion of CgA and Sg II seemed to be mediated by an activation of protein kinase Akt via PI3K (phosphoinositide 3‐kinase). In accordance with this, inhibition of PI3K activity and, thereby, reduced phosphorylation of Akt was shown to enhance Pdcd4 expression. Neither the PKC (protein kinase C) signal transduction cascade nor the MAPK (mitogen‐activated protein kinase) pathway seemed to play a role in the regulation of CgA and Sg II secretion by Pdcd4. Conclusions. CgA is considered to be a marker for neuroendocrine tumours, and up‐regulation of PC1 has been reported in various types of cancers. The repression of PC1 by Pdcd4 may represent a novel mechanism for the function of Pdcd4 as a tumour suppressor. Our results are of particular interest, as we observed that pioglitazone, an oral medication used in the treatment of Type 2 diabetes, decreased Pdcd4 levels, activated Akt, increased CgA and Sg II secretion and augmented PC1 protein in Bon‐1 cells. Enhanced PC1 levels, leading to improved processing of proinsulin and proglucagon, may contribute to the benefits of pioglitazone therapy. The in vivo relevance of our findings was highlighted by data indicating elevated CgA amounts in the sera of patients treated with pioglitazone. This is the first study connecting Pdcd4 levels, secretion behaviour of neuroendocrine cells and regulation of PI3K activity.     

The action of Pdcd4 may be cell type specific: evidence that reduction of dUTPase levels might contribute to its tumor suppressor activity in Bon-1 cells

Pdcd4 (programmed cell death protein 4) was identified as a gene up-regulated during apoptosis and, additionally, seems to have a function as a tumor suppressor. However, there are conflicting data concerning its role in programmed cell death and most results for its action as an inhibitor for neoplastic transformation are derived from experiments with epidermal cells. Therefore, we were interested to investigate if the action of Pdcd4 might be cell type specific. For that purpose we examined the expression of Pdcd4 and several other proteins in various tumor cell lines. We could not find any correlation of Pdcd4 levels and expression of proteins associated with cell cycle and/or apoptosis in different cell lines. Furthermore, we stably transfected two cell lines (Bon-1 and HCT116) to over-express Pdcd4 and analyzed protein expression. Although we found several regulated proteins none of these proteins were affected in both cell lines in the same manner. For instance, dUTPase expression was reduced in Bon-1 cells but not changed in HCT116 cells. This regulation might be important for the sensitivity of cells to anti-cancer drugs like inhibitors of thymidilate synthase. Therefore, we conclude that the function of Pdcd4 might be cell type specific. A role for Pdcd4 in apoptosis or as a tumor suppressor might be limited to certain cell types.     

Loss of programmed cell death 4 induces apoptosis by promoting the translation of procaspase-3 mRNA

The programmed cell death 4 (Pdcd4), a translation inhibitor, plays an essential role in tumor suppression, but its role in apoptosis remains unclear. Here we show that Pdcd4 is a critical suppressor of apoptosis by inhibiting the translation of procaspase-3 mRNA. Pdcd4 protein decreased more rapidly through microRNA-mediated translational repression following apoptotic stimuli than did the activation of procaspase-3, cleavage of poly(ADP)ribose polymerase (PARP) by active caspase-3, and nuclear fragmentation. Strikingly, the loss of Pdcd4 by the specific RNA interference increased procaspase-3 expression, leading to its activation and PARP cleavage even without apoptotic stimuli, and sensitized the cells to apoptosis. Thus, our findings provide insight into a novel mechanism for Pdcd4 as a regulator of apoptosis.     

Knock-down of Pdcd4 stimulates angiogenesis via up-regulation of angiopoietin-2

The tumor suppressor Pdcd4 is involved in multiple pathways. Considering its biological action conflicting data in the literature exist and, consequently, our own studies point to a cell type specific action of Pdcd4. In the present study, using several Pdcd4 knock down cell lines we succeeded to identify angiopoietin-2 (Ang-2) as a gene up-regulated on the mRNA and protein level. The subsequent enhanced peptide secretion forced wild type Bon-1 cells in a neoplastic direction demonstrated by increased proliferation and colony formation while cell adhesion was decreased. Most likely, the stimulation of Ang-2 is in part mediated by increased activation of AP-1 but different signal transduction pathways may also be involved since we found opposite activation of PI3K/Akt/mTOR and MAPK7ERK pathways (both known to regulate in Ang-2 expression). Ang-2 is a modulator of vascular remodeling. Therefore, we analyzed the effect of supernatants from Pdcd4 knock-down cell lines on endothelial cells. Again, we detected reduced cell adhesion and increased colony formation. Probably, the most impressive effect was described on tube formation in a model for angiogenesis. Tube length and junctions of endothelial cells treated with conditioned medium from Pdcd4 knock-down cells were considerably increased. Both, up-regulation of Ang-2 and down-regulation of Pdcd4 are described for many tumors. However, this is the first study showing a direct impact of Pdcd4 on Ang-2 levels and, thereby, angiogenesis. Our data suggest a completely new mechanism for Pdcd4 to act as a tumor suppressor rendering Pdcd4 an attractive target for new therapeutic strategies in cancer treatment.     

Programmed cell death protein 4 (pdcd4): A novel target for antineoplastic therapy?

Pdcd4 is a novel gene first identified as a differentially expressed protein during apoptosis. In the meantime not only the impact of Pdcd4 in programmed cell death but also an implication in transformation suppression by inhibition of protein translation is discussed. These features implicate a potential value of Pdcd4 as a molecular target in cancer therapy. This review summarizes the current knowledge about expression, structure and function of Pdcd4.     

Tumor suppressor protein Pdcd4 inhibits translation of p53 mRNA

The tumor suppressor protein Pdcd4 is thought to suppress translation of mRNAs containing structured 5'-UTRs by interacting with translation initiation factor eIF4A and inhibiting its helicase activity. However, natural target mRNAs regulated by Pdcd4 so far are mostly unknown. Here, we identified p53 mRNA as a translational target of Pdcd4. We found that Pdcd4 is associated with p53 mRNA and suppresses its translation. The inhibitory effect of Pdcd4 on the translation of p53 mRNA depends on the ability of Pdcd4 to interact with eIF4A and is mediated by the 5'-UTR of p53 mRNA, which is able to form a stable stem-loop structure. We show that treatment of cells with DNA-damaging agents decreases the expression of Pdcd4. This suggests that translational suppression by Pdcd4 plays a role in maintaining a low level of p53 in unstressed cells and that this suppression is abrogated due to low levels of Pdcd4 after DNA damage. Overall, our work demonstrates for the first time that Pdcd4 is directly involved in translational suppression of a natural mRNA with a 5'-structured UTR and provides novel insight into the translational control of p53 expression.     

CRL3IBTK Regulates the Tumor Suppressor Pdcd4 through Ubiquitylation Coupled to Proteasomal Degradation

The human inhibitor of Bruton''s tyrosine kinase isoform α (IBtkα) is a BTB protein encoded by the IBTK gene, which maps to chromosomal locus 6q14.1, a mutational hot spot in lymphoproliferative disorders. Here, we demonstrate that IBtkα forms a CRL3^IBTK complex promoting its self-ubiquitylation. We identified the tumor suppressor Pdcd4 as IBtkα interactor and ubiquitylation substrate of CRL3^IBTK for proteasomal degradation. Serum-induced degradation of Pdcd4 required both IBtkα and Cul3, indicating that CRL3^IBTK regulated the Pdcd4 stability in serum signaling. By promoting Pdcd4 degradation, IBtkα counteracted the suppressive effect of Pdcd4 on translation of reporter luciferase mRNAs with stem-loop structured or unstructured 5′-UTR. IBtkα depletion by RNAi caused Pdcd4 accumulation and decreased the translation of Bcl-xL mRNA, a well known target of Pdcd4 repression. By characterizing CRL3^IBTK as a novel ubiquitin ligase, this study provides new insights into regulatory mechanisms of cellular pathways, such as the Pdcd4-dependent translation of mRNAs.     

Structure of the tandem MA-3 region of Pdcd4 protein and characterization of its interactions with eIF4A and eIF4G: molecular mechanisms of a tumor suppressor

One of the key regulatory points of translation initiation is recruitment of the 43S preinitation complex to the 5' mRNA cap by the eIF4F complex (eIF4A, eIF4E, and eIF4G). The tumor suppressor protein Pdcd4 has been shown to inhibit cap-dependent translation by interacting tightly with the RNA helicase eIF4A via its tandem MA-3 domains. The NMR studies reported here reveal a fairly extensive and well defined interface between the two MA-3 domains in solution, which appears to be stabilized by a network of interdomain salt bridges and hydrogen bonds, and reveals a unique orientation of the two domains. Characterization of the stoichiometry of the Pdcd4-eIF4A complex suggests that under physiological conditions Pdcd4 binds to a single molecule of eIF4A, which involves contacts with both Pdcd4 MA-3 domains. We also show that contacts mediated by a conserved acidic patch on the middle MA-3 domain of Pdcd4 are essential for forming a tight complex with eIF4A in vivo, whereas the equivalent region of the C-terminal MA-3 domain appears to have no role in complex formation in vivo. The formation of a 1:1 eIF4A-Pdcd4 complex in solution is consistent with the reported presence in vivo of only one molecule of eIF4A in the eIF4F complex. Pdcd4 has also been reported to interact directly with the middle region of eIF4G, however, we were unable to obtain any evidence for even a weak, transient direct interaction.     

An evolutionarily conserved interaction of tumor suppressor protein Pdcd4 with the poly(A)-binding protein contributes to translation suppression by Pdcd4

The tumor suppressor protein programmed cell death 4 (Pdcd4) has been implicated in the translational regulation of specific mRNAs, however, the identities of the natural Pdcd4 target mRNAs and the mechanisms by which Pdcd4 affects their translation are not well understood. Pdcd4 binds to the eukaryotic translation initiation factor eIF4A and inhibits its helicase activity, which has suggested that Pdcd4 suppresses translation initiation of mRNAs containing structured 5′-untranslated regions. Recent work has revealed a second inhibitory mechanism, which is eIF4A-independent and involves direct RNA-binding of Pdcd4 to the target mRNAs. We have now identified the poly(A)-binding protein (PABP) as a novel direct interaction partner of Pdcd4. The ability to interact with PABP is shared between human and Drosophila Pdcd4, indicating that it has been highly conserved during evolution. Mutants of Pdcd4 that have lost the ability to interact with PABP fail to stably associate with ribosomal complexes in sucrose density gradients and to suppress translation, as exemplified by c-myb mRNA. Overall, our work identifies PABP as a novel functionally relevant Pdcd4 interaction partner that contributes to the regulation of translation by Pdcd4.     

Up-regulation of PDCD4 in senescent human diploid fibroblasts

Programmed cell death 4 (PDCD4) has a common MI domain sharing with death associated protein 5 (DAP5) and a component of eukaryotic translation initiation factor (eIF4G) complex and it might also work as a tumor suppressor. We could find that the message and product of Pdcd4 gene were up-regulated in senescent human diploid fibroblasts. In yeast two hybrid analysis, the C-terminal region of PDCD4 interacted with ribosomal protein S13 (RPS13), ribosomal protein L5 (RPL5), and TI-227H. In in vitro binding assay, RPS13, a component of 40S ribosome was stably bound to PDCD4. We also found that PDCD4 was localized to polysome fractions. We could pull out eIF4G with GST-PDCD4, but eIF4E did not interact with PDCD4. From these results, we could assume that PDCD4 might regulate the eIF4G-dependent translation through direct interactions with eIF4G and RPS13 in senescent fibroblasts.     

Mutational analysis of the DEAD-box RNA helicase eIF4AII characterizes its interaction with transformation suppressor Pdcd4 and eIF4GI

Eukaryotic initiation factor (eIF) 4A unwinds secondary and tertiary structures in the 5'-untranslated region of mRNA, permitting translation initiation. Programmed cell death 4 (Pdcd4) is a novel transformation suppressor and eIF4A-binding partner that inhibits eIF4A helicase activity and translation. To elucidate the regions of eIF4A that are functionally significant in binding to Pdcd4, we generated point mutations of eIF4A. Two-hybrid analysis revealed that five eIF4A mutants completely lost binding to Pdcd4 while four eIF4A mutants retained wild-type levels of binding. The residues that, when mutated, inactivated Pdcd4 binding specified ATP binding, ATP hydrolysis, or RNA binding. With the exception of the Q-motif mutant eIF4AP56L, the eIF4A mutants inactivated for Pdcd4 binding were inactivated for binding to eIF4G (GM, GC, or both) and for enhancing translation. Several eIF4A mutants showing wild-type level binding to Pdcd4 were also inactivated for binding to eIF4G and for enhancing translation. Thus, significant dissociation of eIF4A's Pdcd4- and eIF4G-binding regions appears to occur. Because three of the four eIF4A mutants that retained Pdcd4 binding also suppressed translation activity in a dominant-negative manner, the structure that defines the Pdcd4-binding domain of eIF4A may be necessary but is insufficient for translation. A structural homology model of eIF4A shows regions important for binding to Pdcd4 and/or eIF4G lying on the perimeters of the hinge area of eIF4A. A competition experiment revealed that Pdcd4 competes with C-terminal eIF4G for binding to eIF4A. In summary, the Pdcd4-binding domains on eIF4A impact both binding to eIF4G and translation initiation in cells.     

A novel function of the MA-3 domains in transformation and translation suppressor Pdcd4 is essential for its binding to eukaryotic translation initiation factor 4A

An alpha-helical MA-3 domain appears in several translation initiation factors, including human eukaryotic translation initiation factor 4G (eIF4G) and DAP-5/NAT1/p97, as well as in the tumor suppressor Pdcd4. The function of the MA-3 domain is, however, unknown. C-terminal eIF4G (eIG4Gc) contains an MA-3 domain that is located within the eIF4A-binding region, suggesting a role for eIF4A binding. Interestingly, C-terminal DAP-5/NAT1/p97 contains an MA-3 domain, but it does not bind to eIF4A. Mutation of amino acid residues conserved between Pdcd4 and eIF4Gc but not in DAP-5/NAT1/p97 to the amino acid residues found in the DAP-5/NAT1/p97 indicates that some of these amino acid residues within the MA-3 domain are critical for eIF4A-binding activity. Six Pdcd4 mutants (Pdcd4(E249K), Pdcd4(D253A), Pdcd4(D414K), Pdcd4(D418A), Pdcd4(E249K,D414K), and Pdcd4(D253A,D418A)) lost >90% eIF4A-binding activity. Mutation of the corresponding amino acid residues in the eIF4Gc also produced similar results, as seen for Pdcd4. These results demonstrate that the MA-3 domain is important for eIF4A binding and explain the ability of Pdcd4 or eIF4Gc but not DAP-5/NAT1/p97 to bind to eIF4A. Competition experiments indicate that Pdcd4 prevents ca. 60 to 70% of eIF4A binding to eIF4Gc at a Pdcd4/eIF4A ratio of 1:1, but mutants Pdcd4(D253A) and Pdcd4(D253A,D418A) do not. Translation of stem-loop structured mRNA is susceptible to inhibition by wild-type Pdcd4 but not by Pdcd4(D253A), Pdcd4(D418A), or Pdcd4(D235A,D418A). Together, these results indicate that not only binding to eIF4A but also prevention of eIF4A binding to the MA-3 domain of eIF4Gc contributes to the mechanism by which Pdcd4 inhibits translation.