The macrophage migration inhibitory factor (MIF) is a potent pro-inflammatory cytokine and regulates the anti-inflammator

Background: The histone modification patterns in endometriosis have not been fully characterized. This gap in knowledge results in a poor understanding of the epigenetic mechanisms (and potential therapeutic targets) at play. We aimed to (1) assess global acetylation status of histone 3 (H3) and histone 4 (H4), (2) measure levels of H3 and H4 lysine (K) acetylation and methylation, and (3) to identify histone acetylation patterns in promoter regions of candidate genes in tissues from patients and controls. Methods: Global and K-specific acetylation/methylation levels of histones were measured in 24 lesions, 15 endometrium from patients, and 26 endometrium from controls. Chromatin immunoprecipitation (ChIP)-polymerase chain reaction was used to determine the histone acetylation status of the promoter regions of candidate genes in tissues. Results: The lesions were globally hypoacetylated at H3 (but not H4) compared to eutopic endometrium from controls. Lesions had significantly lower levels of H3K9ac and H4K16ac compared to eutopic endometrium from patients and controls. Tissues from patients were hypermethylated at H3K4, H3K9, and H3K27 compared to endometrium from controls. The ChIP analysis showed hypoacetylation of H3/H4 within promoter regions of candidate genes known to be downregulated in endometriosis (e.g., HOXA10, ESR1, CDH1, and p21 WAF1/Cip1 ) in lesions versus control endometrium. The stereoidogenic factor 1 (SF1) promoter region was enriched for acetylated H3 and H4 in lesions versus control tissues, correlating with its reported high expression in lesions. Conclusions: This study describes the histone code of lesions and endometrium from patients with endometriosis and provides support for a possible role of histone modification in modulation of gene expression in endometriosis.

Epigenetic mechanisms have been ascribed important roles in endometriosis. Covalent histone modifications at lysine residues have been shown to regulate gene expression and thus contribute to pathological states in many diseases. In endometriosis, histone deacetylase inhibition (HDACi) resulted in reactivation of E-cadherin, attenuation of invasion, decreased proliferation of endometriotic cells, and caused lesion regression in an animal model. This study was conducted to assess basal and hormoneregulated gene expression levels of HDAC1 and HDAC2 (HDAC1/2) in cell lines and protein expression levels in tissues. Basal and steroid hormone-regulated HDAC1/2 gene expression levels were determined by quantitative polymerase chain reaction in cell lines and tissues. Protein levels were measured by immunohistochemistry (IHC) in tissues on an endometriosis tissue microarray (TMA). Basal HDAC1/2 gene expression levels were significantly higher in endometriotic versus endometrial stromal cells, which was confirmed by Western blot analysis. Estradiol (E2) and progesterone (P4) significantly downregulated HDAC1 expression in endometrial epithelial cells. Levels of HDAC2 were upregulated by E2 and downregulated by E2 þ P4 in endometrial stromal cells. Hormone modulation of HDAC1/2 gene expression was lost in the endometriotic cell line. Immunohistochemistry showed that HDAC1/2 proteins were expressed in a substantial proportion of lesions and endometrium from patients, and their expression levels varied according to lesion localization. The highest proportion of strong HDAC1 immunostaining was seen in ovarian, skin, and gastrointestinal lesions, and of HDAC2 in skin lesions and endometrium from patients with endometriosis. These studies suggest that endometriosis etiology may be partially explained by epigenetic regulation of gene expression due to dysregulations in the expression of HDACs.

Epigenetic mechanisms may play an important role in the etiology of endometriosis. The modification of histones by methylation of lysine residues has been shown to regulate gene expression by changing chromatin structure. We have previously shown that endometriotic lesions had aberrant levels of histone acetylation (lower) and methylation (higher) than control tissues. We aimed to determine the levels of trimethylated histone 3 at lysine residue 27 (H3K27me3), a well-known repressive mark, by immunoassay of fresh tissues and immunohistochemistry (IHC) of an endometriosis-focused tissue microarray. Also, we aimed to determine levels of expression of enhancer of zeste homolog 2 (EZH2), the enzyme responsible for trimethylation of H3K27me3, in cell lines. Average levels of H3K27me3 measured by immunoassay were not significantly different in lesions compared to endometrium from patients and controls. However, there was a trend of higher levels of H3K27me3 in secretory versus proliferative endometrium. The results of IHC showed that lesions (ovarian, fallopian, and peritoneal) and secretory endometrium from controls have higher percentage of H3K27me3-positive nuclei than eutopic endometrium from patients. Endometriotic epithelial cells express high levels of EZH2, which is upregulated by progesterone. This study provides evidence in support of a role of H3K27me3 in the pathogenesis of endometriosis and for EZH2 as a potential therapeutic target for this disease, but more studies are necessary to understand the molecular mechanisms at play.

This study documents that expression of LOX is differentially regulated in endometriotic lesions and endometrium. A role for LOX in mediating proliferation, migration, and invasion of endometrial and endometriotic cells was observed, which may be implicated in the establishment and progression of endometriotic lesions.

High levels of inflammatory factors including chemokines have been reported in peritoneal fluid and blood of women with endometriosis. CXCL12 mediates its action by interaction with its specific receptor, CXCR4, reported to be elevated in human endometriosis lesions and in the rat model of endometriosis. Activation of the CXCR4-CXCL12 axis increases cell proliferation, migration, and invasion of cancer cells. To obtain insights into the CXCR4 expression profile in lesions and endometrium, as well as functionality of the CXCR4-CXCL12 axis in endometriosis, we analyzed the expression of CXCR4 in tissues on a human tissue array and studied CXCL12-mediated activation of proliferation, invasion, and migration in vitro. We observed differences in levels of nuclear CXCR4 expression among lesion types, being higher in ovarian lesions. Endometriotic cell lines (12Z) showed higher levels of CXCR4, proliferative and migratory potential, and AKT phosphorylation/kinase activity compared to untreated control cells (endometrial epithelial cells). CXCL12 and endometriotic stromal cell-enriched media increased proliferation of non-endometriotic epithelial cells. CXCL12 caused a significant increase in 12Z cell invasion but had no effect on migration; AMD3100, a CXCR4-specific inhibitor, significantly increased invasion of 12Z cells but decreased their migration. However, treatment with CXCL12 plus AMD3100 significantly decreased invasion and migration of 12Z cells. In conclusion, the CXCR4-CXCL12 axis is functional in endometriosis cells, but the expression of CXCR4 varies among lesions. CXCL12 promoted proliferation, migration, and invasion of endometriotic cells, while inducing AKT phosphorylation and activity, but pharmacologically blocking this axis in the absence of the ligand induced their invasiveness.

618 Background: The NIVOREN GETUG-AFU 26 study reported safety and efficacy of N in mccRCC pts in a “real world setting”. A translationnal research program was launched to characterize immune cell populations in the tumor by immunohistochemistry (IHC) and correlate them with outcome on N. Methods: All pts treated with N in the GETUG AFU 26 NIVOREN trial who consented for translational program and with available archived paraffin- embedded (FFPE) tumor tissue samples were eligible. Tumor were centrally reviewed. Using IHC we quantified main immune populations (B-cells, CD8 T-cells, macrophages), and immune checkpoints receptors (TIM-3, LAG-3, PD-1) at the invasive margin (IM) and at the core of the tumor (CT). We also identified AXL and PBRM1/BAP1 expression. Results: Overall 324 pts were included. Pts had similar baseline characteristics (IMDC Good, Intermediate, Poor in 18%, 60% and 22%, respectively) and comparable outcomes than overall trial population (PFS/OS = 4.5 / 25.4 months). PD-1 (IM) expression was associated with better PFS whereas AXL expression by tumor cells (TC) was associated with worse PFS (table). LAG-3 expression tend to be associated with worse OS. PBRM-1 loss (15%) was associated with better OS and PFS and with a higher density of CD8 T-cells (p = 0.001) and CD163-macrophages (p = 0.01) (CT) and a higher expression of LAG-3 (CT) (p = 0.01) and PD-1 (CT) (p = 0.02). BAP-1 loss was not associated with PFS (p = 0.6) nor OS (p = 0.9) in this cohort. Conclusions: We report the largest translational analysis supporting that PD-1 and AXL expression are associated with PFS in pts with mccRCC receiving N. We further confirm that PBRM-1 loss is a strong prognostic factor in this setting.[Table: see text]

Cubiu is a vegetable of Solanaceae family, native to the Amazon, which is widely distributed through Brazil, Peru, and Colombia. It is used in food, medicine, and cosmetics by native populations. Research has shown that cubiu extracts have antioxidant activities with great biological relevance. We performed a phytochemical screening to identify the main chemical groups that could confer antioxidant activity to this extract. Several tests and qualitative precipitation specific staining for major classes of secondary metabolites were used. Antioxidant capacity in vitro tests (DPPH and ABTS) were also used to assess the extract's ability to sequester free radicals of 70% hydroethanolic and aqueous extracts of cubiu flour. Alkaloids, organic acids, phenols, flavonoid glycosides, and coumarins were found in the hydroethanolic extract while the aqueous extract presented anthocyanins, gums, tannins and mucilage, amino groups, and volatile and fixed acids. For in vitro tests, the IC50 value obtained in the DPPH assay was 606.3 ± 3.5 μg/mL while that for the ABTS assay was 290.3 ± 10.7 µg/mL. Although cubiu extracts present chemical compounds directly related to antioxidant activity, our results show that it has a low antioxidant activity. Additional studies will be needed to isolate and characterize specific compounds to further assess antioxidant activity.