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

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

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

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

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

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

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