Suppressor Protein Pdcd4 Research Articles

Human tumor suppressor protein Pdcd4 binds at the mRNA entry channel in the 40S small ribosomal subunit

Translation is regulated mainly in the initiation step, and its dysregulation is implicated in many human diseases. Several proteins have been found to regulate translational initiation, including Pdcd4 (programmed cell death gene 4). Pdcd4 is a tumor suppressor protein that prevents cell growth, invasion, and metastasis. It is downregulated in most tumor cells, while global translation in the cell is upregulated. To understand the mechanisms underlying translational control by Pdcd4, we used single-particle cryo-electron microscopy to determine the structure of human Pdcd4 bound to 40S small ribosomal subunit, including Pdcd4-40S and Pdcd4-40S-eIF4A-eIF3-eIF1 complexes. The structures reveal the binding site of Pdcd4 at the mRNA entry site in the 40S, where the C-terminal domain (CTD) interacts with eIF4A at the mRNA entry site, while the N-terminal domain (NTD) is inserted into the mRNA channel and decoding site. The structures, together with quantitative binding and in vitro translation assays, shed light on the critical role of the NTD for the recruitment of Pdcd4 to the ribosomal complex and suggest a model whereby Pdcd4 blocks the eIF4F-independent role of eIF4A during recruitment and scanning of the 5′ UTR of mRNA.

Abstract A019: eIF4A and PDCD4 expression in TNBC underlies sensitivity to natural product translation inhibitor

Abstract Despite recent advances in immunotherapy and adjuvant-therapy, resistance to cancer therapeutics is common and requires novel approaches. Moreover, highly aggressive triple-negative breast cancers lack effective targeted treatments. Deregulation of translation factors is associated with the increased proliferation of TNBC cells, heightened CSC activity, and poor patient outcomes. Thus targeting translation regulatory factors upregulated in TNBC is a promising approach to developing novel treatments. Pateamine A (PatA), a natural marine macrodiolide product, inhibits RNA cap-dependent translation by binding and sequestering the eukaryotic translation initiation factor eIF4A. eIF4A promotes the translation of polypurine-rich mRNA sequences, which are enriched within breast cancer oncogenes. Here we report the anticancer efficacy of novel PatA analogs we have synthesized against breast cancer cell lines. The PatA analogs exhibited selective cytotoxicity to breast and colorectal cancers compared to non-cancerous cells measured by Cell Titer-Blue viability studies. Novel analogs show greater antiproliferative potency against models of triple-negative than against human mammary epithelial cells (HMLE). To further elucidate eIF4A as a molecular target, we conducted analyses of patient tissue samples in the Cancer Genome Atlas (TCGA). Our analyses reveal the overexpression of eIF4A in tumor tissues compared to normal tissues and gene correlation studies reveal the upregulation of key co-expressed tumor drivers including beta-catenin, fibronectin, and epithelial-to-mesenchymal transition (EMT) markers such as SNAIL and SLUG. Further analyses show a downregulation of tumor suppressor protein PDCD4, an endogenous inhibitor of eIF4A, in cancer tissues. We have generated eIF4A shRNA knockdown and PDCD4 shRNA knockdown cell lines to uncover the eIF4A-PDCD4 regulatory axis across breast adenocarcinoma and colorectal adenocarcinoma cell types. We show that the loss of eIF4A attenuates the mesenchymal and stem-like phenotype of breast cancer stem cells revealed by phenotypic assays. Our results suggest that targeting translation initiator eIF4A is an effective approach against highly proliferative and metastatic breast cancer types. The presented findings underscore the potential of eIF4A as a promising molecular target and supports translational control as a viable approach against aggressive cancers. Citation Format: Alagu Subramanian, Haoran Xue, Kenneth G Hull, Daniel Romo, Joseph Taube. eIF4A and PDCD4 expression in TNBC underlies sensitivity to natural product translation inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A019.

Neuronal activity regulates the nuclear proteome to promote activity-dependent transcription.

The formation and plasticity of neuronal circuits relies on dynamic activity-dependent gene expression. Although recent work has revealed the identity of important transcriptional regulators and of genes that are transcribed and translated in response to activity, relatively little is known about the cell biological mechanisms by which activity alters the nuclear proteome of neurons to link neuronal stimulation to transcription. Using nucleus-specific proteomic mapping in silenced and stimulated neurons, we uncovered an understudied mechanism of nuclear proteome regulation: activity-dependent proteasome-mediated degradation. We found that the tumor suppressor protein PDCD4 undergoes rapid stimulus-induced degradation in the nucleus of neurons. We demonstrate that degradation of PDCD4 is required for normal activity-dependent transcription and that PDCD4 target genes include those encoding proteins critical for synapse formation, remodeling, and transmission. Our findings highlight the importance of the nuclear proteasome in regulating the activity-dependent nuclear proteome and point to a specific role for PDCD4 as a regulator of activity-dependent transcription in neurons.

Abstract 2265: Stat3 and ErbB-2 interaction in breast cancer metastasis

Abstract Metastasis is a complex multistep process, responsible for as much as 90% of cancer-related deaths, yet obtaining successful treatment for these patients remains an elusive challenge. It has long been recognized that the tyrosine kinase receptor ErbB-2 and the signal transducer and activator of transcription 3 (Stat3), two major players in the breast cancer (BC) scenario, are involved in BC metastatic dissemination through a mechanism where Stat3 acts as a downstream effector of ErbB-2 action. In addition, we and others have also disclosed the role of nuclear ErbB-2 (NErbB-2) in BC, whose presence we identified as a poor prognostic factor in MErbB-2-positive tumors. On the other hand, microRNAs are short non-coding endogenous RNAs with regulatory functions. In particular, high levels of microRNA-21 (miR-21), a well-known oncomiR, have been reported to actively promote invasion and metastasis in BC cell lines and tissues. Here, we describe a novel hierarchical interaction between Stat3, ErbB-2 and miR-21, underlying the metastatic phenotype of ErbB-2-positive BC. We disclosed that Stat3 acts as an upstream regulator of ErbB-2 expression and function. In a panel of cell lines corresponding to different BC subtypes we found that Stat3 induced ErbB-2 expression at the transcriptional level through its recruitment to response elements (called GAS) at the ErbB-2 promoter. Furthermore, we demonstrated that Stat3 co-opted NErbB-2 function, recruiting it as a coactivator, to assemble a transcriptional complex at the GAS sites of the miR-21 promoter, leading to miR-21 up-regulation. We showed that the increase in miR-21 levels resulted in the downregulation of the metastasis suppressor protein PDCD4, a well known miR-21 target. In order to assess the physiological relevance of our molecular findings, we developed an in vivo model of ErbB-2-overexpressing metastatic BC, in which Stat3 activation was inhibited by transfection with a Stat3 dominant negative variant (Stat3Y705F) or its expression was silenced by siRNAs. We demonstrated through reconstitution assays that ErbB-2 and miR-21 were necessary downstream mediators of Stat3-induced metastases development. Furthermore, we explored the clinical significance of our findings in a cohort of ErbB-2-positive primary invasive BC patients and demonstrated that Stat3 and ErbB-2 nuclear co-expression was associated with low PDCD4 expression levels, and that this correlated with the presence of nodal metastases. Our present results in experimental models and in the clinic shed light on the molecular mechanisms underlying BC metastasis and highlight targeting either Stat3 or NErbB-2 as novel therapeutic strategies for ErbB-2-positive BC patients. #: 15-A-2849-AACR Citation Format: Leandro Venturutti, Lucía V. Romero, Alejandro J. Urtreger, María F. Chervo, María F. Mercogliano, Rosalía I. Cordo Russo, Matías G. Pereyra, Gloria Inurrigarro, María C. Díaz Flaqué, Victoria Sunblad, Juan C. Roa, Pablo Guzmán, Elisa Bal de Kier-Joffe, Eduardo H. Charreau, Roxana Schillaci, Patricia V. Elizalde. Stat3 and ErbB-2 interaction in breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2265. doi:10.1158/1538-7445.AM2015-2265

Abstract 892: Vitamin D deficiency regulates TLR7 to promote hepatocellular cancer in TGF-β/Smad3 heterozygous mice

Abstract Hepatocellular carcinoma (HCC) is the fifth most common tumor and the third leading cause of cancer-related deaths. Loss of TGF-β signaling has been associated with development of HCC. Cancer preventive effects of Vitamin D (VD) have been implicated in multiple cancers, however a clear role for Vitamin D in specific high risk populations remains undefined for HCC. Therefore, we examined for a potential chemopreventive role of VD in HCC in the context of TGF-β inactivation. Methods: (1) We screened for somatic mutation of the TGF-β pathway and VD related genes in 202 HCCs from The Cancer Genome Atlas (TCGA). (2) Wild type, Sptbn1+/- and Smad3+/- mice were fed with low VD (200 IU VD/kg) or high VD diet (10,000 IU VD/kg) for 9 weeks. Liver tissues were subjected to microarray analyses and further evaluation by quantitative PCR. (3) Wild type, Sptbn1+/- and Smad3+/- mice were injected Diethylnitrosamine (DEN) and at 8 month, 26 mice receiving low VD (200 IU VD/kg) and 26 mice receiving high VD (10,000 IU VD/kg) chow. HCC development was assessed at 4 months after VD treatment. (4) Reverse Phase Protein Array (RPPA) was performed to analyze expression profiles of 164 proteins of mouse liver tumors. (5) Liver samples from patients with HCV cirrhosis receiving VD supplements were examined to evaluate expression of TGF-β, Wnt and VD pathway molecules by immunohistochemistry. Results: (1) We observed a high rate of somatic mutation in TGF-β and VD pathway related genes in the TCGA genomic analysis. (2) None of the VD treated mice developed HCC but high VD treatment increased TLR7 mRNA expression about 3-fold in liver from Smad3+/- mice compared with livers from WT mice. (3) Smad3+/- mice with low VD showed 3-fold larger HCC formation, compared to the high VD group (Smad3+/-) that did not develop significant tumors. However, correction of VD in the Chow after 10 months did not reverse HCC formation. (4) RPPA data revealed that the tumor suppressor protein PDCD4 was reduced in Smad3+/- mice with low VD treatment. However, oncoproteins such as β-catenin, Stat5A and Bcl2-XL were induced in the same sample. (5) Expression of β2SP and TβRII were higher in HCV cirrhosis patients receiving VD supplementation compared to non-treated group. Conclusions: Loss of TGF-β signaling pathway developed HCC and VD deficiency promotes tumor growth in the context of Smad3 disruption potentially through regulation of TLR7 expression. However, after 10 months restoring VD does not have any significant effect on altering tumors. Therefore VD could be a potential candidate for prevention in early identified HCC high risk individuals who has inactivation of TGF-β/Smad3 signaling. Citation Format: Ji-hyun Shin, Lior H. Katz, Nina M. Munoz, Andrea Cortes, Vivek Shukla, Sang-Bae Kim, Franklin Herlong, Keigo Machida, Hidekazu Tsukamoto, Kirti Shetty, Aiwu R. He, Lynt B. Johnson, Asif Rashid, Jian Chen, Ju-Seog Lee, Lopa Mishra. Vitamin D deficiency regulates TLR7 to promote hepatocellular cancer in TGF-β/Smad3 heterozygous mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 892. doi:10.1158/1538-7445.AM2015-892

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.

A novel mechanism for the control of translation of specific mRNAs by tumor suppressor protein Pdcd4: inhibition of translation elongation

The tumor suppressor gene Pdcd4 (programmed cell death gene 4) has drawn considerable attention because its downregulation is involved in the development of several types of cancer. Because Pdcd4 interacts with the translation initiation factor eIF4A and inhibits its helicase activity, Pdcd4 has been implicated in the translational suppression of cellular mRNAs containing structured 5'-untranslated regions. However, Pdcd4's role in translation regulation is still poorly understood, because only very few physiological Pdcd4 target mRNAs are known. By using a Pdcd4-deficient clone of the chicken B-cell line DT40, we have discovered that the mRNA of the A-myb proto-oncogene is a novel Pdcd4 target RNA whose translation is suppressed by Pdcd4. Interestingly, the inhibitory effect of Pdcd4 is independent of the Pdcd4-eIF4A interaction, but is dependent on an RNA-binding domain at the N terminus of Pdcd4 and on sequences located within the coding region of A-myb mRNA, indicating that Pdcd4 suppresses A-myb translation by a novel mechanism. Our data show that the Pdcd4 RNA-binding domain preferentially recognizes an RNA secondary structure element formed by the part of the A-myb coding region that mediates Pdcd4-dependent suppression. Previously, we have shown that Pdcd4 also suppresses the translation of the c-myb mRNA by a similar mechanism involving binding of Pdcd4 to RNA secondary structure formed by the c-myb coding region. Surprisingly, our data show that Pdcd4 exerts its inhibitory activity only when the target region of Pdcd4 in A-myb and c-myb mRNA is itself translated, consistent with a mechanism in which Pdcd4 suppresses translation by interfering with translation elongation. Taken together, our work reveals a novel mechanism by which Pdcd4 affects the translational of cellular RNAs. Furthermore, as c-myb and A-myb are members of the Myb proto-oncogene family whose deregulation has been implicated in tumorigenesis, inhibiting their translation might contribute to the tumor-suppressive activity of Pdcd4.

Abstract 3190: Inhibition of the translation of A-myb mRNA by tumor suppressor protein Pdcd4.

Abstract Pdcd4 (Programmed cell death protein 4) was first identified as a protein to be expressed during apoptosis. Later it was shown that Pdcd4 acts as a tumor suppressor protein and regulates the cellular processes on various levels, encompassing transcription and translation. Pdcd4 interacts with the translation initiation factor eIF4A and inhibits its helicase activity which is required to unwind secondary structures in the 5’UTRs of mRNAs. Previous studies on artificial mRNAs have revealed that it inhibits the translation of mRNAs with secondary structures in their 5’ UTRs. Recently, c-myb mRNA was identified as a natural target RNA of Pdcd4. A-myb is a member of the myb family which is expressed in certain hematopoietic cells, such as B-cells, but also in certain non-hematopoietic cells. We have observed that Pdcd4 inhibits the translation of A-myb mRNA in co-transfection experiments using Pdcd4 expression vectors as well as on the endogenous level, as demonstrated by the analysis of a Pdcd4 knock-out model in the Chicken B-cell line DT40. We found that a mutant of Pdcd4 which no longer interacts with eIF4 is still able to inbihit the translation of A-myb RNA, suggesting that Pdcd4 acts in an eIF4A independent manner and contradicting the current view of how Pdcd4 suppresses translation. Similar to the situation in the case of c-myb RNA we have localized the Pdcd4 responsive region in the coding region of A-myb mRNA. We therefore conclude that Pdcd4 suppresses the translation of A-myb RNA by a novel mechanism. Ribosome density profiling technique is widely used nowadays to explore the topography of the various stages of translation. Currently we aim to exploit the technique of ribosome density profiling to further understand how Pdcd4 affects the translation of A-myb RNA. Citation Format: Abhiruchi Biyanee, Karl-Heinz Klempnauer. Inhibition of the translation of A-myb mRNA by tumor suppressor protein Pdcd4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3190. doi:10.1158/1538-7445.AM2013-3190

Tumor suppressor protein Pdcd4 interacts with Daxx and modulates the stability of Daxx and the Hipk2-dependent phosphorylation of p53 at serine 46

The tumor suppressor protein Pdcd4 is a nuclear/cytoplasmic shuttling protein that has been implicated in the development of several types of human cancer. In the nucleus, Pdcd4 affects the transcription of specific genes by modulating the activity of several transcription factors. We have identified the Daxx protein as a novel interaction partner of Pdcd4. Daxx is a scaffold protein with roles in diverse processes, including transcriptional regulation, DNA-damage signaling, apoptosis and chromatin remodeling. We show that the interaction of both proteins is mediated by the N-terminal domain of Pdcd4 and the central part of Daxx, and that binding to Pdcd4 stimulates the degradation of Daxx, presumably by disrupting the interaction of Daxx with the de-ubiquitinylating enzyme Hausp. Daxx has previously been shown to serve as a scaffold for protein kinase Hipk2 and tumor suppressor protein p53 and to stimulate the phosphorylation of p53 at serine 46 (Ser-46) in response to genotoxic stress. We show that Pdcd4 also disrupts the Daxx–Hipk2 interaction and inhibits the phosphorylation of p53. We also show that ultraviolet irradiation decreases the expression of Pdcd4. Taken together, our results support a model in which Pdcd4 serves to suppress the phosphorylation of p53 in the absence of DNA damage, while the suppressive effect of Pdcd4 is abrogated after DNA damage owing to the decrease of Pdcd4. Overall, our data demonstrate that Pdcd4 is a novel modulator of Daxx function and provide evidence for a role of Pdcd4 in restraining p53 activity in unstressed cells.

Hyaluronan-CD44v3 Interaction with Oct4-Sox2-Nanog Promotes miR-302 Expression Leading to Self-renewal, Clonal Formation, and Cisplatin Resistance in Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma

Human head and neck squamous cell carcinoma (HNSCC) is a highly malignant cancer associated with major morbidity and mortality. In this study, we determined that human HNSCC-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by high levels of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. These tumor cells also express several stem cell markers (the transcription factors Oct4, Sox2, and Nanog) and display the hallmark CSC properties of self-renewal/clonal formation and the ability to generate heterogeneous cell populations. Importantly, hyaluronan (HA) stimulates the CD44v3 (an HA receptor) interaction with Oct4-Sox2-Nanog leading to both a complex formation and the nuclear translocation of three CSC transcription factors. Further analysis reveals that microRNA-302 (miR-302) is controlled by an upstream promoter containing Oct4-Sox2-Nanog-binding sites, whereas chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-302 expression by HA-CD44 is Oct4-Sox2-Nanog-dependent in HNSCC-specific CSCs. This process results in suppression of several epigenetic regulators (AOF1/AOF2 and DNMT1) and the up-regulation of several survival proteins (cIAP-1, cIAP-2, and XIAP) leading to self-renewal, clonal formation, and cisplatin resistance. These CSCs were transfected with a specific anti-miR-302 inhibitor to silence miR-302 expression and block its target functions. Our results demonstrate that the anti-miR-302 inhibitor not only enhances the expression of AOF1/AOF2 and DNMT1 but also abrogates the production of cIAP-1, cIAP-2, and XIAP and HA-CD44v3-mediated cancer stem cell functions. Taken together, these findings strongly support the contention that the HA-induced CD44v3 interaction with Oct4-Sox2-Nanog signaling plays a pivotal role in miR-302 production leading to AOF1/AOF2/DNMT1 down-regulation and survival of protein activation. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, clonal formation, and chemotherapy resistance in HA-CD44v3-activated head and neck cancer.

A tricyclic sesquiterpene from Eriophyllum lanatum stabilizes the tumor suppressor protein Pdcd4 by Inhibiting the E3-Ligase β-TrCP1

A tricyclic sesquiterpene from Eriophyllum lanatum stabilizes the tumor suppressor protein Pdcd4 by Inhibiting the E3-Ligase β-TrCP1

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.

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.

Association of Tumor Suppressor Protein Pdcd4 With Ribosomes Is Mediated by Protein-Protein and Protein-RNA Interactions

The Pdcd4 (programmed cell death gene 4) gene has been implicated as a novel tumor suppressor gene in the development of several types of human cancer. The Pdcd4 protein is believed to act as a translation suppressor of mRNAs containing structured 5' UTRs. Pdcd4 contains 2 copies of so-called MA3 domains that mediate tight interactions with the translation initiation factor eIF4A, resulting in the inhibition of the eIF4A helicase activity. The N-terminal part of Pdcd4, which has been less well characterized, binds RNA in vitro, but as yet, it has not been clear whether RNA binding by Pdcd4 plays a role in vivo. Here, the authors have identified 2 highly conserved clusters of basic amino acid residues that are essential for the RNA binding activity of Pdcd4. They also show that a substantial fraction of Pdcd4 is present, together with small ribosomal subunits, in translation preinitiation complexes. Using mutants that disrupt RNA binding or the Pdcd4-eIF4A interaction, they demonstrate that the ribosomal association of Pdcd4 is dependent on its RNA binding activity as well as on its ability to interact with eIF4A. Their work provides the first direct evidence for an essential role of the Pdcd4 RNA binding activity in vivo and suggests that RNA binding is required for recruiting Pdcd4 to the translation machinery.

15N, 13C and 1H Backbone Resonance Assignments for the Complete Tandem MA-3 Region of the Tumour Suppressor Protein Pdcd4

15N, 13C and 1H Backbone Resonance Assignments for the Complete Tandem MA-3 Region of the Tumour Suppressor Protein Pdcd4

Resonance assignment and secondary structure of the middle MA-3 domain and complete tandem MA-3 region of the tumour suppressor protein Pdcd4

Pdcd4 (Programmed Cell Death Protein 4) is a novel eukaryotic tumour suppressor protein, which is involved in the regulation of both transcription and translation (reviewed in Lankat-Buttgereit and Göke 2009). The protein contains two interacting MA-3 domains (MA-3M and MA-3C), which are linked by a short semi-flexible linker region (Waters et al. 2007; Suzuki et al. 2008). The MA-3 domains are involved in mediating specific protein–protein interactions with functional partners such as eIF4A (Yang et al. 2003 ). Here we report essentially complete backbone and side chain 15N, 13C and 1H assignments for a construct composed of the middle MA-3 domain and subsequent linker region (MA-3M) and backbone assignments for the entire tandem MA-3 region of Pdcd4 (Pdcd4 MA-3M-C). Analysis of the backbone chemical shift data obtained indicates that Pdcd4 MA-3M contains eight helical regions corresponding to over 74% of the protein backbone and that Pdcd4 MA-3M-C contains fifteen helical regions (72%). Comparison of the position of these helical regions with those observed in the crystal structures suggests that the solution and crystal structures of both proteins are very similar.

Hyaluronan-CD44 Interaction with Protein Kinase Cϵ Promotes Oncogenic Signaling by the Stem Cell Marker Nanog and the Production of MicroRNA-21, Leading to Down-regulation of the Tumor Suppressor Protein PDCD4, Anti-apoptosis, and Chemotherapy Resistance in Breast Tumor Cells

Multidrug resistance and disease relapse is a challenging clinical problem in the treatment of breast cancer. In this study, we investigated the hyaluronan (HA)-induced interaction between CD44 (a primary HA receptor) and protein kinase Cepsilon (PKCepsilon), which regulates a number of human breast tumor cell functions. Our results indicate that HA binding to CD44 promotes PKCepsilon activation, which, in turn, increases the phosphorylation of the stem cell marker, Nanog, in the breast tumor cell line MCF-7. Phosphorylated Nanog is then translocated from the cytosol to the nucleus and becomes associated with RNase III DROSHA and the RNA helicase p68. This process leads to microRNA-21 (miR-21) production and a tumor suppressor protein (e.g. PDCD4 (program cell death 4)) reduction. All of these events contribute to up-regulation of inhibitors of apoptosis proteins (IAPs) and MDR1 (multidrug-resistant protein), resulting in anti-apoptosis and chemotherapy resistance. Transfection of MCF-7 cells with PKCepsilon or Nanog-specific small interfering RNAs effectively blocks HA-mediated PKCepsilon-Nanog signaling events, abrogates miR-21 production, and increases PDCD4 expression/eIF4A binding. Subsequently, this PKCepsilon-Nanog signaling inhibition causes IAP/MDR1 down-regulation, apoptosis, and chemosensitivity. To further evaluate the role of miR-21 in oncogenesis and chemoresistance, MCF-7 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and inhibit its target functions. Our results indicate that anti-miR-21 inhibitor not only enhances PDCD4 expression/eIF4A binding but also blocks HA-CD44-mediated tumor cell behaviors. Thus, this newly discovered HA-CD44 signaling pathway should provide important drug targets for sensitizing tumor cell apoptosis and overcoming chemotherapy resistance in breast cancer cells.

Locked Nucleic Acid In situ Hybridization Analysis of miR-21 Expression during Colorectal Cancer Development

To better understand microRNA miR-21 function in carcinogenesis, we analyzed miR-21 expression patterns in different stages of colorectal cancer development using in situ hybridization (ISH). Locked nucleic acid (LNA)/DNA probes and a biotin-free tyramide signal amplification system were used in ISH analyses of miRNA expression. Conditions for specific detection of miR-21 were determined using human cell lines and miR-21-expressing lentiviral vectors. Expression was determined in 39 surgically excised colorectal tumors and 34 endoscopically resected colorectal polyps. In the surgical samples, miR-21 expression was much higher in colorectal cancers than in normal mucosa. Strong miR-21 expression was also observed in cancer-associated stromal fibroblasts, suggesting miR-21 induction by cancer-secreted cytokines. Protein expression of PDCD4, a miR-21 target, was inversely correlated with miR-21 expression, confirming that miR-21 is indeed a negative regulator of PDCD4 in vivo. In the endoscopic samples, miR-21 expression was very high in malignant adenocarcinomas but was not elevated in nontumorigenic polyps. Precancerous adenomas also frequently showed miR-21 up-regulation. Using the LNA-ISH system for miRNA detection, miR-21 was detectable in precancerous adenomas. The frequency and extent of miR-21 expression increased during the transition from precancerous colorectal adenoma to advanced carcinoma. Expression patterns of miR-21 RNA and its target, tumor suppressor protein PDCD4, were mutually exclusive. This pattern may have clinical application as a biomarker for colorectal cancer development and might be emphasized by self-reinforcing regulatory systems integrated with the miR-21 gene, which has been previously shown in cell culture.

Structure of the C-terminal MA-3 domain of the tumour suppressor protein Pdcd4 and characterization of its interaction with eIF4A

Programmed cell death protein 4 (Pdcd4) is a novel tumour suppressor protein, which is involved in the control of eukaryotic transcription and translation. The regulation of translation involves specific interactions with eukaryotic initiation factor (eIF)4A and eIF4G, which are mediated via the two tandem MA-3 domains. We have determined the structure of the C-terminal MA-3 domain of Pdcd4 (Pdcd4 MA-3(C)), characterized its interaction with eIF4A and compared the features of nuclear magnetic resonance (NMR) spectra obtained from the single domain and tandem MA-3 region. Pdcd4 MA-3(C) is composed of three layers of helix-turn-helix hairpins capped by a single helix and shows close structural homology to the atypical HEAT repeats found in many eIFs. The sequence conservation and NMR data strongly suggest that the tandem MA-3 region is composed of two equivalent domains connected by a somewhat flexible linker. Pdcd4 MA-3(C) was found to interact with the N-terminal domain of eIF4A through a conserved surface region encompassing the loop connecting alpha5 and alpha6 and the turn linking alpha3 and alpha4. This site is strongly conserved in other MA-3 domains known to interact with eIF4A, including the preceding domain of Pdcd4, suggesting a common mode of binding.

NMR Assignment and Secondary Structure Determination of the C-terminal MA-3 Domain of the Tumour Suppressor Protein Pdcd4

NMR Assignment and Secondary Structure Determination of the C-terminal MA-3 Domain of the Tumour Suppressor Protein Pdcd4