The nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a lipid ligand-inducible transcription factor associated with macrophage polarization. However, its function in tumor-associated macrophages (TAMs) has not been investigated to date. Here, we report the PPARβ/δ-regulated transcriptome and cistrome for TAMs from ovarian carcinoma patients. Comparison with monocyte-derived macrophages shows that the vast majority of direct PPARβ/δ target genes are upregulated in TAMs and largely refractory to synthetic agonists, but repressible by inverse agonists. Besides genes with metabolic functions, these include cell type-selective genes associated with immune regulation and tumor progression, e.g., LRP5, CD300A, MAP3K8 and ANGPTL4. This deregulation is not due to increased expression of PPARβ/δ or its enhanced recruitment to target genes. Instead, lipidomic analysis of malignancy-associated ascites revealed high concentrations of polyunsaturated fatty acids, in particular linoleic acid, acting as potent PPARβ/δ agonists in macrophages. These fatty acid ligands accumulate in lipid droplets in TAMs, thereby providing a reservoir of PPARβ/δ ligands. These observations suggest that the deregulation of PPARβ/δ target genes by ligands of the tumor microenvironment contributes to the pro-tumorigenic polarization of ovarian carcinoma TAMs. This conclusion is supported by the association of high ANGPTL4 expression with a shorter relapse-free survival in serous ovarian carcinoma.

Background/Aims: Neuroendocrine neoplasms of the small intestine (SI-NENs) constitute 25-30% of all gastroenteropancreatic NEN. These tumors arise from enterochromaffin cells, and little is known about their microRNA (miRNA) expression. The purpose of this study was to characterize the expression of miRNAs in SI-NEN and to determine the potential of miRNAs as noninvasive blood-based biomarkers. Methods: miRNA was purified from 15 tumor and 7 control tissue samples, converted to cDNA, and applied to a miScript miRNA PCR. The small nucleolar RNA, SNORD95, was used as an endogenous control. Results: Microarray analysis revealed 7 miRNAs that showed a promising distinction between tumorous and healthy tissue. The miRNAs miR-7-5p and miR-96-5p were clearly upregulated in the tumor compared to the healthy tissue. In contrast, miRNAs miR-9-5p, miR-122-5p, miR-124-3p, miR-143-3p, and miR-144-3p showed a distinct downregulation in the tumor compared to the healthy tissue. These results were validated on a further 15 tumor samples, and the findings held true. As the miR-7-5p was significantly upregulated and revealed a low range across tumor samples, its presence was tested in the sera of 32 tumor patients and 25 healthy controls. Sera from all patients with SI-NENs had significantly higher levels of miR-7-5p than those from the 25 healthy controls (p = 0.0002), whereas there was no correlation with age, gender, or T-stage or UICC stage. Conclusion: The miRNA miR-7-5p may be a promising biomarker test for SI-NEN, which should be validated in a large-scale prospective study.

In the absence of ligands, the nuclear receptor PPARβ/δ recruits the NCOR and SMRT corepressors, which form complexes with HDAC3, to canonical target genes. Agonistic ligands cause dissociation of corepressors and enable enhanced transcription. Vice versa, synthetic inverse agonists augment corepressor recruitment and repression. Both basal repression of the target gene ANGPTL4 and reinforced repression elicited by inverse agonists are partially insensitive to HDAC inhibition. This raises the question how PPARβ/δ represses transcription mechanistically. We show that the PPARβ/δ inverse agonist PT-S264 impairs transcription initiation by decreasing recruitment of activating Mediator subunits, RNA polymerase II, and TFIIB, but not of TFIIA, to the ANGPTL4 promoter. Mass spectrometry identifies NCOR as the main PT-S264-dependent interactor of PPARβ/δ. Reconstitution of knockout cells with PPARβ/δ mutants deficient in basal repression results in diminished recruitment of NCOR, SMRT, and HDAC3 to PPAR target genes, while occupancy by RNA polymerase II is increased. PT-S264 restores binding of NCOR, SMRT, and HDAC3 to the mutants, resulting in reduced polymerase II occupancy. Our findings corroborate deacetylase-dependent and -independent repressive functions of HDAC3-containing complexes, which act in parallel to downregulate transcription.

In the absence of ligands, the nuclear receptor PPARβ/δ recruits the NCOR and SMRT corepressors, which form complexes with HDAC3, to canonical target genes. Agonistic ligands cause dissociation of corepressors and enable enhanced transcription. Vice versa, synthetic inverse agonists augment corepressor recruitment and repression. Both basal repression of the target gene ANGPTL4 and reinforced repression elicited by inverse agonists are partially insensitive to HDAC inhibition. This raises the question of how PPARβ/δ represses transcription mechanistically. We show that the PPARβ/δ inverse agonist PT-S264 impairs transcription initiation by decreasing recruitment of activating Mediator subunits, RNA polymerase II, and TFIIB, but not of TFIIA, to the ANGPTL4 promoter. Mass spectrometry identifies NCOR as the main PT-S264-dependent interactor of PPARβ/δ.