Identification of translationally deregulated proteins during inflammation-associated tumorigenesis

The translation of mRNAs into proteins is an elaborate and highly regulated process. Translational regulation primarily takes place at the level of initiation. During initation the eukaryotic initiation factors (eIFs) form a complex that binds to the 5’end of the mRNA to scan for a start codon. Once recognized, the ribosome is recruited to the mRNA and protein synthesis starts. Initiation of translation can basically occur via two distinct mechanisms, i.e. cap-dependent and cap-independent that is mediated via internal ribosome entry sites (IRESs). The former is mediated by a 5’cap structure composed of a 7-methylguanylate which is added to every mRNA during transcription and recruits the initiation complex. IRES-dependent translation involves elements within the 5’untranslated region (UTR) of the mRNA that mostly bind IRES trans-acting factors (ITAFs) which associate either with the initiation complex or with the ribosome itself and consequently allow for internal initiation of translation. During tumorigenesis the demand for proteins is increased due to rapid cell growth, which consequently requires enhanced translation. Many factors that regulate translation are overexpressed in tumors. Moreover, signaling pathways that trigger translation or further hyperactivated by the surrounding tumor microenvironment. This environment is largely generated by infiltration of immune cells such as macrophages that secrete cytokines and other mediators to promote tumorigenesis. As the effects of inflammatory conditions on the translation of specific targets are only poorly characterized, my study aimed at identifying translationally deregulated targets during inflammation-associated tumorigenesis. For this purpose, I cocultured MCF7 breast tumor cells with conditioned medium of activated monocyte-derived U937 macrophages (CM). Polysome profiling and microarray analysis identified 42 targets to be regulated at the level of translation. The results were validated by quantitative PCR and one target - early growth response 2 (EGR2) - was chosen for in depth analysis of the mechanism leading to its enhanced translation. In order to identify upstream signaling molecules causing enhanced EGR2 protein synthesis the cytokine profile of CM was analyzed and the impact of several cytokines on EGR2 translation was examined. Preincubation of CM with neutralizing antibodies revealed that lowering interleukin 6 (IL-6) had only little effect, whereas depletion of IL 1β significantly reduced EGR2 translation. This finding was corroborated by the fact that treatment with recombinant IL-1β enhanced EGR2 translation to virtually the same extend as CM. Further experiments revealed that this effect was mediated via the p38-MAPK signaling cascade. Interestingly, I observed that the mTOR inhibitor rapamycin, which reduces cap-dependent translation, specifically stimulated EGR2 translation. This result argued for an IRES-dependent mechanism that might account for EGR2 translation. The use of bicistronic reporter assays verified this hypothesis. In line with the above mentioned results, CM, IL-1β and p38-MAPK induced EGR2-IRES activity. Since IRESs commonly require ITAFs to mediate translation initiation, the binding of proteins to the 5’UTR was analyzed using mass spectrometry. Among others, several previously described ITAFs, such as polypyrimidine tract-binding protein (PTB) and heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) were identified to directly bind to the EGR2-5’UTR. Furthermore, overexpression of hnRNP-A1 enhanced EGR2-IRES activity whereas a dominant negative form of hnRNP-A1 significantly decreased it, thus, showing its importance for EGR2 translation. In summary, my data provide evidence that EGR2 expression can be controlled by IRES-dependent translational regulation, which is responsive to an inflammatory environment. The identified mechanism may not be exclusive for one target but might be representative for gene expression regulation mechanisms during tumorigenesis. This is of special interest for the treatment of cancer patients and development of more specific therapies to reduce tumor outcome.Die Translation von mRNAs in Proteine ist ein komplexer Prozess, der aufgrund seines hohen Energieverbrauchs strikt kontrolliert wird. Die Regulation findet dabei primär auf Ebene der Translationsinitiation statt. Während der Initiation bilden die eukaryotischen Initiationsfaktoren (eIFs) einen Komplex, der an das 5’Ende der mRNA bindet und die 5’untranslatierte Region (UTR) nach einem Startcodon scannt, woraufhin die ribosomalen Untereinheiten an die mRNA rekrutiert werden. Die Ribosomen vermitteln dann die eigentliche Proteinsynthese. Grundsätzlich können zwei verschiedene Arten der Initiation unterschieden werden – die Cap-abhängige sowie die Cap-unabhängige, wobei letztere über sogenannte internal ribosome entry sites (IRESs) vermittelt wird. Bei ersterer bindet der Initiationskomplex an die Cap-Struktur der mRNA, die aus einem N-terminalen 7 Methylguanylat besteht. Bei der IRES-vermittelten Initiation bindet der Initiationskomplex oder auch die kleine ribosomale Untereinheit direkt innerhalb der 5’UTR an die mRNA, allerdings in 3’-Distanz zur Cap-Struktur. Während der Tumorentwicklung kommt es aufgrund des verstärkten Zellwachstums zu einem gesteigerten Bedarf an Proteinen und somit zu erhöhter Translation. Viele Faktoren, die die Translation regulieren, werden in Tumoren überexprimiert oder sind überaktiv. Bei der Aktivierung der entsprechenden Signalkaskaden spielt das Tumormilieu eine zentrale Rolle. Dieses wird insbesondere von Zellen des Immunsystems wie z.B. Makrophagen beeinflusst. Makrophagen setzen dabei Mediatoren frei, welche das Tumorwachstum begünstigen. Während tumorigene Expressionsveränderungen auf Transkriptionsebene bereits detailliert untersucht wurden, gibt es nur wenig Information über Translationsveränderungen spezifischer Proteine. Deswegen war es das Ziel dieser Studie translationell (de-)regulierte Proteine in der entzündungsinduzierten Tumorigenese zu identifizieren. Dafür kokultivierte ich MCF7 Brustkrebszellen mit konditioniertem Medium von ausdifferenzierten U937 Makrophagen (CM). Die Translationsveränderung in den Tumorzellen wurde mit Hilfe von Polysomenfraktionierungen überprüft. Durch eine aufbauende Mikroarray Analyse wurden 42 mRNAs identifiziert, die translationell reguliert wurden. Die Ergebnisse des Mikroarrays wurden anschließend durch quantitative PCR validiert und der Regulationsmechanismus eines Targets – early growth response 2 (EGR2) – im Detail analysiert. Dafür untersuchte ich den Einfluss verschiedener im CM vorhandener Zytokine auf die EGR2-Translation mittels neutralisierender Antikörper. Es stellte sich heraus, dass die Abreicherung von Interleukin 6 (IL-6) die EGR2-Translationsinduktion durch CM nur minimal verringerte, wohingegen eine Depletion von IL-1β diese signifikant inhibierte. Dieser Befund wurde dadurch unterstützt, dass eine Behandlung mit rekombinantem IL-1β eine ähnlich starke Induktion der EGR2-Translation bewirkte wie CM. Anschließende Untersuchungen ergaben, dass dieser Effekt durch die p38-MAPK Signalkaskade vermittelt wurde. Desweiteren wurde beobachtet, dass der mTOR-Inhibitor Rapamycin, der die Cap-abhängige Translation hemmt, ebenfalls zu einer verstärkten EGR2-Translation führte. Dies ließ vermuten, dass ein IRES-vermittelter Mechanismus der Translation von EGR2 zu Grunde lag. Durch die Verwendung von bicistronischen Reporter-Vektoren wurde diese Hypothese bestätigt. Außerdem konnte ich beweisen, dass CM, IL 1β und p38-MAPK die EGR2-IRES-Aktivität in der gleichen Art beeinflussten wie bereits für die EGR2-Translation mittels Polysomenfraktionierung gezeigt. Da zelluläre IRES-Elemente oft durch sogenannte IRES trans-acting factors (ITAFs) induziert werden, wurden mittels Massenspektrometrie Proteine identifiziert, die an die 5’UTR von EGR2 binden. Unter anderem wurden die bereits bekannten ITAFs polypyrimidine tract-binding protein (PTB) und heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) gefunden. Abschließend konnte bewiesen werden, dass die Überexpression von hnRNP-A1 zu einer Erhöhung der EGR2-IRES-Aktivität führte, wohingegen eine dominant-negative Mutante von hnRNP-A1 diese signifikant inhibierte. Diese Ergebnisse ließen darauf schließen, dass hnRNP-A1 einen entscheidenden Einfluss auf die IRES-abhängige EGR2-Translation hat. Zusammenfassend konnte ich einen neuen Translationsregulationsmechanismus für EGR2 identifizieren, der durch ein entzündliches Tumormikroenvironment in Tumorzellen induziert wird. Dieser Mechanismus ist möglicherweise auch auf weitere translationell regulierte Targets übertragbar. Dies ist von besonderem Interesse, da es für eine optimale Behandlung von Tumorpatienten essentiell ist die zu Grunde liegenden Regulationsmechanismen zu verstehen

Erioflorin stabilizes the tumor suppressor Pdcd4 by inhibiting its interaction with the E3-ligase β-TrCP1

Loss of the tumor suppressor Pdcd4 was reported for various tumor entities and proposed as a prognostic marker in tumorigenesis. We previously characterized decreased Pdcd4 protein stability in response to mitogenic stimuli, which resulted from p70S6K1-dependent protein phosphorylation, β-TrCP1-mediated ubiquitination, and proteasomal destruction. Following high-throughput screening of natural product extract libraries using a luciferase-based reporter assay to monitor phosphorylation-dependent proteasomal degradation of the tumor suppressor Pdcd4, we succeeded in showing that a crude extract from Eriophyllum lanatum stabilized Pdcd4 from TPA-induced degradation. Erioflorin was identified as the active component and inhibited not only degradation of the Pdcd4-luciferase-based reporter but also of endogenous Pdcd4 at low micromolar concentrations. Mechanistically, erioflorin interfered with the interaction between the E3-ubiquitin ligase β-TrCP1 and Pdcd4 in cell culture and in in vitro binding assays, consequently decreasing ubiquitination and degradation of Pdcd4. Interestingly, while erioflorin stabilized additional β-TrCP-targets (such as IκBα and β-catenin), it did not prevent the degradation of targets of other E3-ubiquitin ligases such as p21 (a Skp2-target) and HIF-1α (a pVHL-target), implying selectivity for β-TrCP. Moreover, erioflorin inhibited the tumor-associated activity of known Pdcd4- and IκBα-regulated αtranscription factors, that is, AP-1 and NF-κB, altered cell cycle progression and suppressed proliferation of various cancer cell lines. Our studies succeeded in identifying erioflorin as a novel Pdcd4 stabilizer that inhibits the interaction of Pdcd4 with the E3-ubiquitin ligase β-TrCP1. Inhibition of E3-ligase/target-protein interactions may offer the possibility to target degradation of specific proteins only as compared to general proteasome inhibition

Inflammatory conditions induce IRES-dependent translation of cyp24a1

Rapid alterations in protein expression are commonly regulated by adjusting translation. In addition to cap-dependent translation, which is e.g. induced by pro-proliferative signaling via the mammalian target of rapamycin (mTOR)-kinase, alternative modes of translation, such as internal ribosome entry site (IRES)-dependent translation, are often enhanced under stress conditions, even if cap-dependent translation is attenuated. Common stress stimuli comprise nutrient deprivation, hypoxia, but also inflammatory signals supplied by infiltrating immune cells. Yet, the impact of inflammatory microenvironments on translation in tumor cells still remains largely elusive. In the present study, we aimed at identifying translationally deregulated targets in tumor cells under inflammatory conditions. Using polysome profiling and microarray analysis, we identified cyp24a1 (1,25-dihydroxyvitamin D3 24-hydroxylase) to be translationally upregulated in breast tumor cells co-cultured with conditioned medium of activated monocyte-derived macrophages (CM). Using bicistronic reporter assays, we identified and validated an IRES within the 5′ untranslated region (5′UTR) of cyp24a1, which enhances translation of cyp24a1 upon CM treatment. Furthermore, IRES-dependent translation of cyp24a1 by CM was sensitive to phosphatidyl-inositol-3-kinase (PI3K) inhibition, while constitutive activation of Akt sufficed to induce its IRES activity. Our data provide evidence that cyp24a1 expression is translationally regulated via an IRES element, which is responsive to an inflammatory environment. Considering the negative feedback impact of cyp24a1 on the vitamin D responses, the identification of a novel, translational mechanism of cyp24a1 regulation might open new possibilities to overcome the current limitations of vitamin D as tumor therapeutic option

Oncogenic RAS enables DNA damage- and p53-dependent differentiation of acute myeloid leukemia cells in response to chemotherapy.

Acute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and are associated with beneficial response to cytarabine. Why AML-patients with oncogenic RAS benefit most from high-dose cytarabine post-remission therapy is not well understood. Here we used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. We show that oncogenic RAS synergizes with cytotoxic agents such as cytarabine in activation of DNA damage checkpoints, resulting in a p53-dependent genetic program that reduces clonogenicity and increases myeloid differentiation. Our data can explain the beneficial effects observed for AML patients with oncogenic RAS treated with higher dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells

Cyp24a1 contains an IRES element.

<p>(A) Sequence of the human cyp24a1-5′UTR. (B) Schematic representation of the bicistronic control (phpRF) and cyp24a1-5′UTR-containing (phpR-cyp-F) luciferase constructs used for reporter assays. (C) Bicistronic reporter plasmids phpRF (white bars) and phpR-cyp-F (black bars) were transfected into MCF7 cells. 24 h after transfection <i>renilla</i> and <i>firefly</i> luciferase activities were measured and data are presented as means ± SEM relative to phpRF (n≥3, ** p<0.01). (D) RNA isolated from cells transfected with phpRF or phpR-cyp-F was DNAse treated and reverse transcribed. <i>Upper panel</i>: PCR was performed with specific primers to amplify full length RL or R-cyp-L mRNAs. PCR products were visualized <i>via</i> agarose gel electrophoresis and ethidium bromide staining. Data are representative for at least 3 independent experiments. <i>Lower panel</i>: RT-qPCR analysis of the amount of <i>firefly</i> mRNA normalized to <i>renilla</i> mRNA. Data are presented as means ± SEM (n≥3). (E) <i>In vitro-</i>transcribed mRNAs of the control (hpRF, white bars) or the cyp24a1-5′UTR-containing vector (hpR-cyp-F, black bars) were transfected into MCF7 cells. 24 h after transfection <i>renilla</i> and <i>firefly</i> luciferase activities were measured. Luciferase activities are given relative to hpRF and data are presented as means ± SEM (n≥3, ** p<0.01).</p

CM induces cyp24a1 translation.

<p>MCF7 cells were treated with Ctr or CM for 4(A) and cyp24a1 (B) was analyzed in single fractions using RT-qPCR. The distribution of the respective mRNAs across the individual gradients was determined relative to the total RNA extracted from the gradients. Results from a representative experiment are given in A and B. (C+D) Changes of gapdh (C) and cyp24a1 (D) mRNA distribution induced by CM were normalized to Ctr. (E) cyp24a1 distribution (from D) was normalized to gapdh distribution (from C). Distribution changes are presented as means ± SEM (n≥3, * p<0.05, ** p<0.01, *** p<0.001).</p

Polysome profile of MCF7 cells.

<p>Representative profile of MCF7 lysates at 254 nm as determined during polysomal fractionation (<i>upper panel</i>). Equal aliquots of RNA isolated from single fractions were analyzed using denaturing agarose gel electrophoresis to verify 28S and 18S rRNA content as indicators for ribosome distribution (<i>lower panel</i>).</p

Cyp24a1 translation is initiated in part cap-independently.

<p>MCF7 cells were treated with rapamycin [100 nM] for 4 h and subjected to polysomal fractionation. RNA from single fractions was isolated and gapdh (A) and cyp24a1 (B) mRNA distribution changes were analyzed separately as described before. Data are presented as means ± SEM (n≥3).</p

CM induces cyp24a1 IRES activity in an Akt-dependent manner.

<p>(A) MCF7 cells were transfected with phpR-cyp-F. 48 h after transfection cells were treated for 4 h with Ctr, CM, or CM supplemented with LY294002 [10 µM] or SB203580 [10 µM]. IRES activity was calculated as ratio of <i>firefly</i> to <i>renilla</i> luciferase activities and is given relative to Ctr. Data are presented as means ± SEM (n≥3, * p<0.05). (B) <i>(upper panel)</i> HEK293 cells overexpressing HA-tagged myr Akt were transfected with phpR-cyp-F. 48 h after transfection IRES activity was calculated as ratio of <i>firefly</i> to <i>renilla</i> luciferase activities and is given relative to control vector transfected cells. Data are presented as means ± SEM (n≥3, * p<0.05). <i>(lower panel)</i> HEK293 cells stably overexpressing HA-tagged myr Akt were serum starved for 48 h. Protein expression and S6-phosphorylation was determined by Western analysis. (C) MCF7 cells were treated for 4 h with CM or CM in combination with LY294002 [10 µM] followed by polysomal fractionation. Changes in cyp24a1 mRNA distribution were analyzed as described before. Data of pooled polysomal fractions (7–10) are presented as means ± SEM (n≥3, * p<0.05).</p