Abstract
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.
Highlights
Programmed cell death 4 (Pdcd4) is a novel tumor suppressor that inhibits translation rather than transcription
Our studies succeeded in identifying erioflorin as a novel Pdcd4 stabilizer that inhibits the interaction of Pdcd4 with the E3-ubiquitin ligase b-TrCP1
Degradation We have previously shown that TPA induces the phosphorylation-dependent proteasomal degradation of Pdcd4 [5] and further introduced a luciferase-based assay to identify compounds stabilizing Pdcd4 from TPA-induced degradation [23,28,29]
Summary
Programmed cell death 4 (Pdcd4) is a novel tumor suppressor that inhibits translation rather than transcription. Activation of p70S6K1 in response to mitogens such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) results in phosphorylation of Pdcd, followed by binding of b-TrCP, polyubiquitination and subsequent proteasomal degradation [5,11]. Overactivation of the PI3K-Akt-mTORp70S6K axis is common in many tumor types [13] Interference with this signaling cascade is widely used for current tumor therapeutic regimens, e.g. mTOR inhibitors are in clinical use for the treatment of renal cell carcinomas and mantle-cell lymphomas [14]. Attenuating the interaction of b-TrCP with its target-proteins could be a promising approach for the development of proteasomal degradation targeting drugs for tumor therapies. Stabilization of the b-TrCP-target Pdcd provides an attractive tool for the identification of novel b-TrCP-inhibitors, which might be further developed for use in anti-tumor therapies. We set out to identify novel stabilizers of the tumor suppressor Pdcd, which interfere with b-TrCPmediated degradation of Pdcd
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