The tumor suppressor programmed cell death 4 (Pdcd4) is lost in various tumor tissues. Loss of Pdcd4 has been associated with increased tumorigenic potential and tumor progression. While various mechanisms of Pdcd4 regulation have been described, the effect of an inflammatory tumor microenvironment on Pdcd4 protein expression has not been characterized so far. In the present study, we aimed to elucidate the molecular mechanisms of Pdcd4 protein regulation in tumor cells under inflammatory conditions. 12-O-tetradecanoylphorbol 13-acetate-induced differentiation of human U937 monocytes increased the expression and secretion of inflammatory cytokines such as tumor necrosis factor α, interleukin (IL)-6 and IL-8. Exposure to conditioned medium (CM) of these activated macrophages markedly decreased Pdcd4 protein expression in various tumor cells. Similarly, indirect coculture with such activated U937 monocyte-derived macrophages resulted in the loss of Pdcd4 protein in tumor cells. Decreased Pdcd4 protein levels were attributable to enhanced proteasomal degradation, diminishing Pdcd4 protein half-life. Proteasomal degradation required activation of phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling. Since macrophage-CM sufficed to induce Pdcd4 degradation, Pdcd4 downregulation was determined to be an indirect unidirectional effect of the macrophages on the tumor cells. Pdcd4 protein expression was also attenuated in vivo in mouse colon tissue in response to dextran sodium sulfate-induced colitis. In summary, we characterized PI3K-mTOR-dependent proteasome-mediated Pdcd4 degradation in tumor cells in the inflammatory tumor microenvironment. Consequently, stabilization of Pdcd4 protein could provide a promising novel avenue for therapeutics targeting inflammation-associated tumors.

Ample evidence has shown key roles of inflammation in tumor promotion and carcinogenesis, and tumor-associated macrophages are known to promote tumor growth and dissemination. Programmed cell death 4 (Pdcd4) is a novel tumor suppressor, and although various studies have revealed that the functions and expression mechanisms of Pdcd4 in tumor promotion, those in regard to inflammation remain unclear. In the present study, we examined whether inflammatory stimuli regulate Pdcd4 expression. 12-O-tetradecanoylphorbol 13-acetate (TPA) suppressed expression of pdcd4 mRNA in human monocytic cell lines (U937, THP-1). Similarly, the bacterial endotoxin lipopolysaccharide (LPS) downregulated pdcd4 level in mouse RAW264.7 and peritoneal macrophages. Furthermore, conditioned medium from LPS-stimulated RAW264.7 macrophages suppressed pdcd4 mRNA in RAW264.7 macrophages, and findings obtained with recombinant tumor necrosis factor-α (TNF-α) and TNF-α-specific siRNA suggested that TNF-α partly mediates LPS-triggered Pdcd4 downregulation via an autocrine mechanism. Specific inhibitors of phosphoinositide-3-kinase (PI3K) and c-jun N-terminus kinase (JNK) restored LPS-abolished pdcd4 mRNA. Consistently, in MCF7 mammary carcinoma cells, conditioned medium from TPA-differentiated/activated U937 cells suppressed pdcd4 mRNA. Additionally, knockdown of pdcd4 in RAW264.7 macrophages using siRNA significantly enhanced LPS-induced TNF-α protein production, and interferon-γ, CC chemokine ligand (Ccl) 1, Ccl20, and interleukin-10 mRNA expression. These results suggest that Pdcd4 suppresses the induction of these inflammatory mediators. Taken together, loss of Pdcd4 in macrophages may be a critical step in establishing the inflammatory environment while that in tumor cells contributes to tumor progression.

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.

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.

Adjusting translation is crucial for cells to rapidly adapt to changing conditions. While pro-proliferative signaling via the PI3K-mTOR-pathway is known to induce cap-dependent translation, stress conditions, such as nutrient deprivation or hypoxia often activate alternative modes of translation, e.g., via internal ribosome entry sites (IRESs). As the effects of inflammatory conditions on translation are only poorly characterized, we aimed at identifying translationally deregulated targets in inflammatory settings. For this purpose, we cocultured breast tumor cells with conditioned medium of activated monocyte-derived macrophages (CM). Polysome profiling and microarray analysis identified early growth response-2 (egr2) to be regulated at the level of translation. Using bicistronic reporter assays, we found that egr2 contains an IRES within its 59 UTR, which facilitated enhanced translation upon CM treatment. We further provide evidence that the activity of egr2-IRES was induced by IL-1b and p38-MAPK signaling. In addition, we identified several potential IRES trans-acting factors (ITAFs) such as polypyrimidine tract binding protein (PTB) and hnRNP-A1 that directly bind to the egr2-59UTR. In summary, our data provide evidence that egr2 expression is translationally regulated via an IRES element, which is responsive to an inflammatory environment.