Interaction of transcription factor FoxO3 with histone acetyltransferase complex subunit TRRAP modulates gene expression and apoptosis.

Forkhead box O (FoxO) transcription factors are conserved proteins involved in the regulation of life span and age-related diseases, such as diabetes and cancer. Stress stimuli or growth factor deprivation promotes nuclear localization and activation of FoxO proteins, which-depending on the cellular context-can lead to cell cycle arrest or apoptosis. In endothelial cells (ECs), they further regulate angiogenesis and may promote inflammation and vessel destabilization implicating a role of FoxOs in vascular diseases. In several cancers, FoxOs exert a tumor-suppressive function by regulating proliferation and survival. We and others have previously shown that FoxOs can regulate these processes via two different mechanisms: by direct binding to forkhead-responsive elements at the promoter of target genes or by a poorly understood alternative process that does not require direct DNA binding and regulates key targets in primary human ECs. Here, we performed an interaction study in ECs to identify new nuclear FoxO3 interaction partners that might contribute to FoxO-dependent gene regulation. Mass spectrometry analysis of FoxO3-interacting proteins revealed transformation/transcription domain-associated protein (TRRAP), a member of multiple histone acetyltransferase complexes, as a novel binding partner of FoxO family proteins. We demonstrate that TRRAP is required to support FoxO3 transactivation and FoxO3-dependent G1 arrest and apoptosis in ECs via transcriptional activation of the cyclin-dependent kinase inhibitor p27kip1 and the proapoptotic B-cell lymphoma 2 family member, BIM. Moreover, FoxO-TRRAP interaction could explain FoxO-induced alternative gene regulation via TRRAP-dependent recruitment to target promoters lacking forkhead-responsive element sequences.

Efficient Suppression of NRAS-Driven Melanoma by Co-Inhibition of ERK1/2 and ERK5 MAPK Pathways.

Cutaneous melanoma is a highly malignant tumor typically driven by somatic mutation in the oncogenes BRAF or NRAS, leading to uncontrolled activation of the MEK/ERK MAPK pathway. Despite the availability of immunotherapy, MAPK pathway‒targeting regimens are still a valuable treatment option for BRAF-mutant melanoma. Unfortunately, patients with NRAS mutation do not benefit from such therapies owing to the lack of targetable BRAF mutations and a high degree of intrinsic and acquired resistance toward MEK inhibition. Here, we demonstrate that concomitant inhibition of ERK5 removes this constraint and effectively sensitizes NRAS-mutant melanoma cells for MAPK pathway‒targeting therapy. Using approved MEK inhibitors or a pharmacologic ERK inhibitor, we demonstrate that MAPK inhibition triggers a delayed activation of ERK5 through a PDGFR inhibitor-sensitive pathway in NRAS-mutant melanoma cells, resulting in sustained proliferation and survival. ERK5 phosphorylation also occurred naturally in NRAS-mutant melanoma cells and correlated with nuclear localization of its stem cell-associated effector KLF2. Importantly, MEK/ERK5 co-inhibition prevented long-term growth of human NRAS-mutant melanoma cells in vitro and effectively repressed tumor progression in a xenotransplant mouse model. Our findings suggest MEK/ERK5 cotargeting as a potential treatment option for NRAS-mutant melanoma, which currently is not amenable for targeted therapies.

Preoperative short-term parenteral administration of polyunsaturated fatty acids ameliorates intestinal inflammation and postoperative ileus in rodents.

PURPOSE: Abdominal surgery results in an inflammation of the intestinal muscularis externa (ME), subsequently leading to postoperative ileus (POI). Polyunsaturated fatty acids (PUFA) are known to modulate inflammation. The aim of this study was to analyze the effect of preoperative parenteral administration of marine (n-3) or soybean (n-6) PUFA lipid emulsions (PUFA-LE) on POI and tissue fatty acid profiles. METHODS: Rodents underwent intestinal manipulation (IM) after 5 days of parenteral administration of 10-mL/kg body weight saline, (n-3), or (n-6) PUFA-LE. Sham animals received saline treatment without IM. In rats, postoperative inflammation was quantified by ME neutrophil levels and NO production in organ culture, and ME function was determined by an in vitro contractility measurement. Additionally, in vivo gastrointestinal transit (GIT) was analyzed in mice. Lipopolysaccharide-induced IL-6 expression of rat bone marrow-derived mononuclear cells and ME was analyzed. Fatty acids were measured by gas chromatography in rat blood, bone marrow cells, and ME. RESULTS: The (n-3) PUFA-LE reduced neutrophil levels and NO production after IM and improved in vitro jejunal contractility and GIT time. The (n-6) PUFA-LE significantly reduced postoperative inflammation and tended to improve intestinal motility (P < 0.06). Interestingly, (n-6) PUFA-LE significantly reduced the levels of arachidonic acid in ME (-63%), while (n-3) PUFA-LE reduced arachidonic acid (-20%) and additionally raised EPA (+550%). CONCLUSION: Short-term preoperative parenteral administration of (n-3) or (n-6) PUFA-LE significantly alters tissue-specific fatty acid profiles. Preoperative parenteral PUFA-LE supplementation, preferably by marine (n-3) PUFA, ameliorates postoperative intestinal inflammation and dysmotility and could be a promising therapeutic option in POI prophylaxis.

Immunogenicity of HLA-A1-restricted peptides derived from S100A4 (metastasin 1) in melanoma patients.

S100A4 (metastasin 1) belongs to the S100 family of Ca(2+) binding proteins. While not present in most differentiated adult tissues, S100A4 is upregulated in the micromilieu of tumors. It is primarily expressed by tumor-associated macrophages, fibroblasts, and tumor endothelial cells. Due to its strong induction in tumors S100A4 is a promising target for cancer immunotherapy. By reverse immunology, using epitope prediction programs, we identified 3 HLA-A1-restricted peptide epitopes (S100A4 A1-1, A1-2, and A1-3) which are subject to human T cell responses as detected in peripheral blood of melanoma patients by means of IFN-gamma ELISPOT and cytotoxicity assays. In addition, IFN-gamma responses to S100A4 A1-2 can not only be induced by stimulation of T cells with peptide-loaded DC but also by stimulation with S100A4 protein-loaded DC, indicating that this epitope is indeed generated by processing of the endogenously expressed protein. In addition, S100A4 A1-2 reactive T cells demonstrate lysis of HLA-A1(+) fibroblasts in comparison to HLA-A1(-) fibroblasts. In summary, this HLA-A1-restricted peptide epitope is a candidate for immunotherapeutical approaches targeting S100A4-expressing cells in the tumor stroma.