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

Identification of erioflorin as a stabilizer of Pdcd4 by a HTS of natural products and discovery of its mechanism of action

The tumor suppressor programmed cell death 4 (Pdcd4) exerts its function by inhibiting protein translation initiation. Specifically, it displaces the scaffold protein eukaryotic initiation factor 4G (eIF4G) from its binding to the eukaryotic initiation factor 4A (eIF4A). Thereby, Pdcd4 inhibits the helicase activity of eIF4A, which is necessary for the unwinding of highly structured 5’ untranslated regions (UTRs) of messenger RNAs (mRNAs) often found in oncogenes like c-myc to make them accessible for the translation machinery and subsequent protein production. Overexpression of Pdcd4 inhibits tumorigenesis in vitro and in vivo and inversely, Pdcd4 knockout mice show enhanced tumor formation. In line, Pdcd4 is lost in various tumor types and proposed as prognostic factor in colon carcinomas. Unlike most other tumor suppressors that are rendered nonfunctional by mutations (e.g., p53), Pdcd4 loss is not attributable to mutational inactivation. It is regulated via translational repression by microRNAs and increased degradation of the protein under tumor promoting, inflammatory conditions and mitogens. Specifically, proteasomal degradation of Pdcd4 is controlled by p70 S6 Kinase (p70S6K)-mediated phosphorylation in its degron sequence (serines 67, 71 and 76). Stimulation of the PI3K-AKT-mTOR pathway by growth factors, hormones and cytokines initiates p70S6K activity. Phosphorylated Pdcd4 is subsequently recognized by the E3 ubiquitin ligase beta-transducin repeats-containing protein (β-TrCP) and marked with a polyubiquitin tail to be detected by the 26S proteasome for degradation. β-TrCP represents the substrate specific recognition subunit of the ubiquitin ligase complex responsible for protein-protein interaction with Pdcd4 as substrate for ubiquitin transfer and subsequent proteasomal disassembly. The first part of the present work aimed at identifying novel stabilizers of the tumor suppressor Pdcd4 in a high throughput screen (HTS). As assay design, a fragment of Pdcd4 from amino acid 39 to 91, containing the phosphorylation sensitive degron sequence, was fused to a luciferase reporter gene construct. Stable expression of this Pdcd4(39-91)luciferase (Pdcd4(39-91)luc) fusion protein in HEK 293 cells served as read-out for the Pdcd4 protein amount to be detected in a high throughput compatible cell-based assay. Loss of Pdcd4(39-91)luc was induced by treatment with 12-O- tetradecanoylphorbol-13-acetate (TPA), a phorbolester, which activates the PI3K signaling cascade leading to degradation of Pdcd4. The cut-off for hit definition was set at >50% activity in rescuing the Pdcd4(39-91)luc signal from TPA-induced degradation. Activity was calculated relative to the difference of DMSO- and TPA-treated cells (ΔDMSO-TPA = RLUDMSO-RLUTPA). Initial screening of a protein kinase inhibitor library (PKI) revealed hit substances expected to show Pdcd4 stabilizing activity by inhibition of kinases involved in Pdcd4 downregulation, e.g., the mTOR inhibitor rapamycin, the PI3K inhibitors wortmannin and LY294002 and the PKC inhibitors GF 109203X and Ro 31-8220. The Molecular Targets Laboratory (MTL) of the National Cancer Institute (NCI) in Frederick, USA, hosts one of the largest collections of crude natural product extracts as well as a big substance libraries from pure synthetic sources. Screening of over 15 000 pure compounds and over 135 000 natural product extracts identified 46 pure and 42 extract hits as Pdcd4 stabilizers. For nine synthetic and six natural product derived compounds (after bioassay-guided fractionation), dose-dependent activities for recovering the TPA-induced Pdcd4(39-91)luc loss defined IC50s in the low micromolar range. Most importantly, these compounds were confirmed to stabilize endogenous Pdcd4 protein levels from forced degradation as well. This result proved the assay design to be highly representative for endogenous cellular mechanisms regulating Pdcd4 protein stability. The next step was to stratify the hit substances according to their likely mechanism of action to be located either up- or downstream of the p70S6K-mediated phosphorylation of Pdcd4. Therefore, phosphorylation of S6, as proto-typical p70S6K target, was analyzed and uncovered two natural derived compounds to influence p70S6K activity. Four substances did not affect p70S6K phosphorylation activity and were therefore considered to stabilize Pdcd4 by acting downstream, i.e. on the β-TrCP-mediated proteasomal degradation. In the second part of this work, one of these compounds, namely the sesquiterpene lactone erioflorin, isolated by bioassay-guided fraction from the active extract of Eriophyllum lanatum, Asteraceae, was further characterized in detail with respect to its molecular mechanism of action. Erioflorin dose-dependently protected both Pdcd4(39-91)luc and endogenous Pdcd4 protein from TPA-induced degradation with IC50s of 1.28 and 2.64 μM, respectively. Pdcd4 stabilizing activity was maximal at 5 μM erioflorin. Up to this concentration, erioflorin was verified not to inhibit p70S6K activity. In addition, it was observed that erioflorin rescued Pdcd4(39-91)luc from both, wild type and constitutively active p70S6K-mediated downregulation. Only wild type p70S6K was inhibitable by the mTOR inhibitor rapamycin which served as an upstream acting control. To study the next section of Pdcd4 regulation, i.e. recognition by the E3 ubiquitin ligase β-TrCP, Pdcd4(39-91)luc and endogenous Pdcd4 were immunoprecipitated from whole cell extracts with the corresponding antibodies. In this key experiment, treatment with TPA increased overexpressed β-TrCP binding to both and this coimmunoprecipitation could be strongly reduced by erioflorin treatment. This result strongly pointed to an inhibitory mechanism of the β-TrCP specific binding to Pdcd4 by erioflorin. In addition, erioflorin disrupted the binding of in vitro transcribed/translated β-TrCP to Pdcd4 in an in vitro interaction assay to exclude nonspecific intracellular signals. Furthermore, polyubiquitination of Pdcd4 was decreased by erioflorin treatment as well. To clarify questions regarding specificity of erioflorin for the E3 ubiquitin ligase β-TrCP, stability of another important β-TrCP target was explored, i.e. the tumor suppressor inhibitor of kappa B alpha (IκBα). Indeed, the tumor necrosis factor alpha (TNFα)-mediated loss of IκBα could be prevented by erioflorin cotreatment. On the other hand, the E3 ubiquitin ligase von Hippel Lindau protein (pVHL) was left unaffected as its target hypoxia inducible factor 1 alpha (HIF-1α) could not be stabilized from oxygen-dependent degradation by erioflorin treatment. These results argued strongly for erioflorin being a specific inhibitor of β-TrCP-mediated protein degradation. Functional consequences of erioflorin treatment were investigated by observing its influence on the transcriptional activities of the transformation marker activator protein 1 (AP-1, an indirect downstream target of Pdcd4) and nuclear factor κB (NF-κB which is directly inhibited by IκBα). Indeed, erioflorin showed significant inhibition of AP-1 and NF-κB reporter constructs at 5 μM, a concentration for which an impact on cell viability was excluded. Finally to characterize the significance of erioflorin in a cell-based tumorigenesis assay, the highly invasive colon carcinoma cell line RKO was tested in a two dimensional migration assay. Erioflorin was discovered to significantly lower cell migration in a wound closure assay. In conclusion, development of a high throughput compatible cell-based reporter assay successfully identified novel substances from pure synthetic and natural product derived background as potent stabilizers of the tumor suppressor Pdcd4. In addition, this work aimed at elucidating the detailed mechanism of action of the sesquiterpene lactone erioflorin from Eriophyllum lanatum, Asteraceae. Erioflorin was discovered to inhibit the E3 ubiquitin ligase β-TrCP, thereby preventing protein degradation of tumor suppressors like Pdcd4 and IκBα. This may offer the possibility to more specifically target protein degradation and generate less adverse side effects by blocking a particular E3 ubiquitin ligase compared to general proteasome inhibition.Das Tumorsuppressorprotein Programmed cell death 4 (Pdcd4) inhibiert die Initiationsphase der Proteintranslation. Es wurde gezeigt, das seine Überexpression sowohl in vitro als auch in vivo zu einer reduzierten Tumorigenität führte, sowie verringerte Pdcd4 Level in menschlichen Tumorgeweben beobachtet wurden. Pdcd4 wird hauptsächlich durch proteasomalen Abbau reguliert, da das Protein ein Erkennungsmotiv für die p70 S6 Kinase (p70S6K) enthält, das direkt von der Erkennungssequenz für die E3 Ubiquitin Ligase beta-transducin repeats-containing protein (β-TrCP) gefolgt wird. Erhöhte Level an Entzündungsmediatoren im Tumormikromilieu sowie Tumor-promovierende Agenzien (z.B. der Phorbolester Tetradocanoyl-Phorbolacetat = TPA) stimulieren die PI3K-AKT-mTOR-p70S6K Signalkaskade, was zur Phosphorylierung von Pdcd4 durch p70S6K an Serinresten des Degronmotivs und anschließender Polyubiquitinierung durch β-TrCP führt. Daraufhin wird Pdcd4 durch das 26S Proteasom abgebaut. Das Ziel meiner Arbeit war zunächst die Identifizierung von neuen Substanzen, die das Pdcd4 Protein stabilisieren, indem sie dessen proteasomalen Abbau verhindern. Hierzu entwickelte ich einen High Throughput Screen (HTS) kompatiblen, zellbasierten Assay, um am National Cancer Institute (NCI, Frederick, USA) die dort vorhandenen, umfangreichen Substanz- und Naturstoffextraktbibliotheken zu testen. Um dies zu realisieren wurde ein kurzer Abschnitt des Pdcd4 Proteins, der die phosphorylierungsabhängige Abbausequenz enthält, an Luziferase fusioniert und stabil in HEK 293 Zellen zur Expression gebracht (Pdcd4(39-91)luc). Der Abbau dieses Pdcd4(39-91)luc Fusionsproteins wurde durch Stimulation der oben genannten Signalkaskade mit TPA induziert. Im HTS von über 15 000 isolierten Molekülen und 135 000 Naturstoffextrakten konnte ich 46 reine Substanzen sowie 42 Extrakte identifizieren, die eine Pdcd4-stabilisierende Wirkung zeigten. Im zweiten Teil meiner Arbeit erforschte ich den Wirkmechanismus einer Pdcd4 stabilisierenden Hitsubstanz, des Sesquiterpenlactons Erioflorin aus Eriophyllum lanatum, Asteraceae im Detail. Es verhinderte Dosis-abhängig den TPA-induzierten Abbau des Pdcd4(39-91)luc Fusionsproteins und den des endogenen Pdcd4 Proteins. Als Wirkort von Erioflorin identifizierte ich den β-TrCP vermittelten Abbau von Pdcd4, da die durch TPA verstärkte Interaktion von β-TrCP mit Pdcd4, bzw. mit dem Luziferasefusionsprotein, durch gleichzeitige Inkubation mit Erioflorin signifikant vermindert werden konnte. Um die Spezifität von Erioflorin zu untermauern, prüfte und beobachtete ich, dass eine Inkubation mit Erioflorin ebenfalls zu einer Stabilisierung eines anderen prominenten β-TrCP Zielproteins, des Tumorsuppressors Inhibitorisches Protein kappa B alpha (IκBα), führte. Dieses Resultat unterstützte die Annahme, dass Erioflorin β-TrCP generell hemmt. Erioflorin führte jedoch nicht zu einem Anstieg des Hypoxie-induzierbaren Faktor 1 alpha (HIF-1α), der durch eine andere E3 Ubiquitin Ligase, dem von-Hippel-Lindau Protein (pVHL), herunterreguliert wird, sodass die gleichzeitige Hemmung anderer E3 Ubiquitin Ligasen ausgeschlossen werden konnte. Um die Konsequenz der Erioflorinbehandlung auf das tumorigene Geschehen zu untersuchen, analysierte ich den Einfluss von Erioflorin auf die transkriptionelle Aktivität der Transformationsmarker AP-1 und NF-κB, die bekannte Targets von Pdcd4 bzw. IκBα darstellen. Deren Aktivität wurde durch Eriofloringabe signifikant reduziert ohne einen Einfluss auf die Zellviabilität zu haben. Außerdem verringte Erioflorin das migratorische Potential der Kolonkarzinom Zelllinie RKO. Diese ersten in vitro Tumorigenese-Assays deuten daraufhin, dass Erioflorin eine potentiell Tumor-protektive Wirksamkeit aufweist. Zusammenfassend identifizierte die Durchführung eines HTS neuartige Substanzen, die zu einer Stabilisierung des Tumorsuppressorproteins Pdcd4 führten. Ein neu extrahierter Wirkstoff, der sekundäre Pflanzenmetabolit Erioflorin aus Eriophyllum lanatum, Asteraceae, wurde detailliert untersucht und die Inhibition der E3 Ubiquitin Ligase β-TrCP als der pharmakologische Wirkmechanismus von Erioflorin identifiziert. Dadurch konnten Tumorsuppressorproteine wie Pdcd4 und IκBα in einem tumorfördernden Milieu stabilisiert werden. Die spezifische Hemmung einer E3 Ubiquitin Ligase stellt ein neuartiges therapeutisches Target dar, das sich als Ergebnis meiner Arbeit durch einen ebenfalls neu identifizierten Wirkstoff als hemmbar erwies. Hier wirkende Arzneimittel können gegenüber einer generellen Blockade des Proteasoms, wie z.B. durch Velcade®, den Vorteil haben, dass es durch den exakteren Wirkort zu weniger unerwünschten Nebeneffekten komm

Diaryl disulfides as novel stabilizers of tumor suppressor Pdcd4

The translation inhibitor and tumor suppressor Pdcd4 was reported to be lost in various tumors and put forward as prognostic marker in tumorigenesis. Decreased Pdcd4 protein stability due to PI3K-mTOR-p70S6K1 dependent phosphorylation of Pdcd4 followed by β-TrCP1-mediated ubiquitination, and proteasomal destruction of the protein was characterized as a major mechanism contributing to the loss of Pdcd4 expression in tumors. In an attempt to identify stabilizers of Pdcd4, we used a luciferase-based high-throughput compatible cellular assay to monitor phosphorylation-dependent proteasomal degradation of Pdcd4 in response to mitogen stimulation. Following a screen of approximately 2000 compounds, we identified 1,2-bis(4-chlorophenyl)disulfide as a novel Pdcd4 stabilizer. To determine an initial structure-activity relationship, we used 3 additional compounds, synthesized according to previous reports, and 2 commercially available compounds for further testing, in which either the linker between the aryls was modified (compounds 2–4) or the chlorine residues were replaced by groups with different electronic properties (compounds 5 and 6). We observed that those compounds with alterations in the sulfide linker completely lost the Pdcd4 stabilizing potential. In contrast, modifications in the chlorine residues showed only minor effects on the Pdcd4 stabilizing activity. A reporter with a mutated phospho-degron verified the specificity of the compounds for stabilizing the Pdcd4 reporter. Interestingly, the active diaryl disulfides inhibited proliferation and viability at concentrations where they stabilized Pdcd4, suggesting that Pdcd4 stabilization might contribute to the anti-proliferative properties. Finally, computational modelling indicated that the flexibility of the disulfide linker might be necessary to exert the biological functions of the compounds, as the inactive compound appeared to be energetically more restricted

Tricyclic guanidine alkaloids from the marine sponge Acanthella cavernosa that stabilize the tumor suppressor PDCD4

A cell-based high-throughput screen that assessed the cellular stability of a tumor suppressor protein PDCD4 (Programmed cell death 4) was used to identify a new guanidine-containing marine alkaloid mirabilin K (3), as well as the known compounds mirabilin G (1) and netamine M (2). The structures of these tricyclic guanidine alkaloids were established from extensive spectroscopic analyses. Compounds 1 and 2 inhibited cellular degradation of PDCD4 with EC50 values of 1.8 μg/mL and 2.8 μg/mL, respectively. Mirabilin G (1) and netamine M (2) are the first marine natural products reported to stabilize PDCD4 under tumor promoting conditions

Cell cycle.

<p>HEK293 cells stably expressing Pdcd4_(39–91)luc were incubated for 24 h with DMSO (blue) or increasing concentrations of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b> (3 μM (violet), 10 μM (yellow), and 30 μM (red)). Cell cycle analysis was performed after permeabilization using 7-AAD. Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).</p

Conformational analysis of compounds 1, 2 and 4.

<p>(A) A systematic approach was chosen to explore the conformational space around the three rotable bonds. (B-D) Global minimum energy conformations (<i>left panel</i>) and energy plots (<i>right panel</i>) of compounds <b>1</b> (B), <b>2</b> (C), and <b>4</b> (D).</p

Pdcd4 stabilizing activity.

<p>HEK293 cells stably expressing the Pdcd4 stability reporter Pdcd4_(39–91)luc were incubated for 8 h with TPA [10 nM] in combination with increasing concentrations (0.1–30 μM) of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b>. Pdcd4 stabilizing activity was determined relative to Δ(RLU_DMSO–RLU_TPA). Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).</p

Chemical structures.

<p>Structures of compounds <b>1</b> (1,2-bis(4-chlorophenyl)disulfide), <b>2</b> (4-chloro-<i>N</i>-(4-chlorophenyl)benzamide), <b>3</b> ((<i>E</i>)-1,2-bis(4-chlorophenyl)diazene), <b>4</b> (1,2-bis(4-chlorophenyl)hydrazine), <b>5</b> (1,2-bis(4-methoxyphenyl)disulfide), and <b>6</b> (1,2-bis(4-nitrophenyl)disulfide).</p