Regulatory T Cell Apoptosis during Preeclampsia May Be Prevented by Gal-2.

There are several open questions to be answered regarding the pathophysiology of the development of preeclampsia (PE). Numerous factors are involved in its genesis, such as defective placentation, vascular impairment, and an altered immune response. The activation of the adaptive and innate immune system represents an immunologic, particularity during PE. Proinflammatory cytokines are predominantly produced, whereas immune regulatory and immune suppressive factors are diminished in PE. In the present study, we focused on the recruitment of regulatory T cells (Tregs) which are key players in processes mediating immune tolerance. To identify Tregs in the decidua, an immunohistochemical staining of FoxP3 of 32 PE and 34 control placentas was performed. A clearly reduced number of FoxP3-positive cells in the decidua of preeclamptic women could be shown in our analysis (p = 0.036). Furthermore, CCL22, a well-known Treg chemoattractant, was immunohistochemically evaluated. Interestingly, CCL22 expression was increased at the maternal-fetal interface in PE-affected pregnancies (psyncytiotrophoblast = 0.035, pdecidua = 0.004). Therefore, the hypothesis that Tregs undergo apoptosis at the materno-fetal interface during PE was generated, and verified by FoxP3/TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining. Galectin-2 (Gal-2), a member of the family of carbohydrate-binding proteins, which is known to be downregulated during PE, seems to play a pivotal role in T cell apoptosis. By performing a cell culture experiment with isolated Tregs, we could identify Gal-2 as a factor that seems to prevent the apoptosis of Tregs. Our findings point to a cascade of apoptosis of Tregs at the materno-fetal interface during PE. Gal-2 might be a potential therapeutic target in PE to regulate immune tolerance.

CCL22 as an independent prognostic factor in endometrial cancer patients.

OBJECTIVES: The chemokine CCL22 is recognized for recruiting immunosuppressive regulatory T-cells (Treg) that contribute to disease progression in various tumor entities helping them to evade the host immune response. Our study aims to identify the expressing cell types and to evaluate the prognostic significance of CCL22 secretion and its association with Treg invasion in endometrial cancer (EC), an immunogenic cancer. METHODS: Specimens from 275 patients with EC and 28 healthy controls were screened immunohistochemically for CCL22. Immunofluorescence double-staining for CCL22 and different immune cell markers was performed. In vitro regulation of CCL22-expression was examined in EC cell lines (Ishikawa+, RL95-2) and human PBMCs in coculture settings via qPCR and ELISA. RESULTS: Elevated CCL22 staining in tumor cells and CCL22-positive M1-macrophages in tumordistant areas were significantly associated with increased overall survival (OS). Conversely, high, secretory-appearing staining in the peritumoral and intratumoral stroma correlated with reduced OS. Although the analysis of the in vitro coculture model of epithelial tumor- and immune cells revealed PBMCs as the primary source of CCL22, we could confirm expression of the chemokine also in the EC epithelial cells. CONCLUSION: Our study suggests that CCL22 in EC is associated with OS, dependent on its location and the cell type producing it. Intracellular upregulation and extracellular secretion must be considered separately when investigating CCL22 expressing cell types in EC. These results may provide evidence for CCL22-mediated Treg recruitment in EC as a potential future therapeutic target.

Gal-2 Increases H3K4me3 and H3K9ac in Trophoblasts and Preeclampsia.

Preeclampsia (PE) is a severe pregnancy disorder with a pathophysiology not yet completely understood and without curative therapy. The histone modifications H3K4me3 and H3K9ac, as well as galectin-2 (Gal-2), are known to be decreased in PE. To gain a better understanding of the development of PE, the influence of Gal-2 on histone modification in trophoblasts and in syncytialisation was investigated. Immunohistochemical stains of 13 PE and 13 control placentas were correlated, followed by cell culture experiments. An analysis of H3K4me3 and H3K9ac was conducted, as well as cell fusion staining with E-cadherin and ß-catenin-both after incubation with Gal-2. The expression of H3K4me3 and H3K9ac correlated significantly with the expression of Gal-2. Furthermore, we detected an increase in H3K4me3 and H3K9ac after the addition of Gal-2 to BeWo/HVT cells. Moreover, there was increased fusion of HVT cells after incubation with Gal-2. Gal-2 is associated with the histone modifications H3K4me3 and H3K9ac in trophoblasts. Furthermore, syncytialisation increased after incubation with Gal-2. Therefore, we postulate that Gal-2 stimulates syncytialisation, possibly mediated by H3K4me3 and H3K9ac. Since Gal-2, as well as H3K4me3 and H3K9ac, are decreased in PE, the induction of Gal-2 might be a promising therapeutic target.

Presence of regulatory T-cells in endometrial cancer predicts poorer overall survival and promotes progression of tumor cells.

PURPOSE: Endometrial cancer (EC) is one of the most common gynaecologic malignancies. Tumor infiltrating regulatory T-cells (Treg) have been reported to have a prognostic impact in many malignancies. Immunotherapeutic strategies are gaining interest for advanced and recurrent EC cases, where treatment options are rare. Our study was aimed at determining the value of Treg in EC progression. METHODS: EC specimens from 275 patients and 28 controls were screened immunohistochemically for the presence of Treg represented by FoxP3. Correlations with clinicopathological and survival parameters were performed. Functional assays were performed using EC cell lines Ishikawa + and RL95-2 after co-culturing with isolated CD4 + CD25 + CD127dim Treg. To assess the influence of EC on the composition of peripheral blood mononuclear cells (PBMC), flow cytometric analyses were performed. RESULTS: We found that an increased infiltration of Treg was associated with high grades and a reduced overall survival. Treg were almost absent in endometrium tissues from healthy control patients. Co-culture of tumor cells with CD4 + CD25 + CD127dim Treg led to functional changes: enhanced invasion, migration and viability indicated that increased levels of Treg in the tumor microenvironment may promote tumor growth. Furthermore, we found that the presence of EC cells led to phenotypic changes in PBMC, showing significantly increased levels of CD25 and FoxP3. CONCLUSION: Our results indicate that the presence of Treg in the EC tumor environment is associated with a poorer outcome. A remarkable impact of Treg on tumor cell behaviour and vice versa of tumor cells on PBMC subpopulations support this notion mechanistically. Our findings provide a basis for focusing on Treg as potential future therapeutic targets in EC.

Platelet-Activating Factor Acetylhydrolase Expression in BRCA1 Mutant Ovarian Cancer as a Protective Factor and Potential Negative Regulator of the Wnt Signaling Pathway.

Aberrantly activated Wnt/ß-catenin signaling pathway, as well as platelet-activating factor (PAF), contribute to cancer progression and metastasis of many cancer entities. Nonetheless, the role of the degradation enzyme named platelet-activating factor acetylhydrolase (PLA2G7/PAF-AH) in ovarian cancer etiology is still unclear. This study investigated the functional impact of platelet-activating factor acetylhydrolase on BRCA1 mutant ovarian cancer biology and its crosstalk with the Wnt signaling pathway. PAF-AH, pGSK3ß, and ß-catenin expressions were analyzed in 156 ovarian cancer specimens by immunohistochemistry. PAF-AH expression was investigated in ovarian cancer tissue, serum of BRCA1-mutated patients, and in vitro in four ovarian cancer cell lines. Functional assays were performed after PLA2G7 silencing. The association of PAF-AH and ß-catenin was examined by immunocytochemistry. In an established ovarian carcinoma collective, we identified PAF-AH as an independent positive prognostic factor for overall survival (median 59.9 vs. 27.4 months; p = 0.016). PAF-AH correlated strongly with the Wnt signaling proteins pGSK3ß (Y216; nuclear: cc = 0.494, p < 0.001; cytoplasmic: cc = 0.488, p < 0.001) and ß-catenin (nuclear: cc = 0.267, p = 0.001; cytoplasmic: cc = 0.291, p < 0.001). In particular, high levels of PAF-AH were found in tumor tissue and in the serum of BRCA1 mutation carriers. By in vitro expression analysis, a relevant gene and protein expression of PLA2G7/PAF-AH was detected exclusively in the BRCA1-negative ovarian cancer cell line UWB1.289 (p < 0.05). Functional assays showed enhanced viability, proliferation, and motility of UWB1.289 cells when PLA2G7/PAF-AH was downregulated, which underlines its protective character. Interestingly, by siRNA knockdown of PLA2G7/PAF-AH, the immunocytochemistry staining pattern of ß-catenin changed from a predominantly membranous expression to a nuclear one, suggesting a negative regulatory role of PAF-AH on the Wnt/ß-catenin pathway. Our data provide evidence that PAF-AH is a positive prognostic factor with functional impact, which seems particularly relevant in BRCA1 mutant ovarian cancer. For the first time, we show that its protective character may be mediated by a negative regulation of the Wnt/ß-catenin pathway. Further studies need to specify this effect. Potential use of PAF-AH as a biomarker for predicting the disease risk of BRCA1 mutation carriers and for the prognosis of patients with BRCA1-negative ovarian cancer should be explored.

Cisplatin resistance in non-small cell lung cancer cells is associated with an abrogation of cisplatin-induced G2/M cell cycle arrest.

The efficacy of cisplatin-based chemotherapy in cancer is limited by the occurrence of innate and acquired drug resistance. In order to better understand the mechanisms underlying acquired cisplatin resistance, we have compared the adenocarcinoma-derived non-small cell lung cancer (NSCLC) cell line A549 and its cisplatin-resistant sub-line A549rCDDP2000 with regard to cisplatin resistance mechanisms including cellular platinum accumulation, DNA-adduct formation, cell cycle alterations, apoptosis induction and activation of key players of DNA damage response. In A549rCDDP2000 cells, a cisplatin-induced G2/M cell cycle arrest was lacking and apoptosis was reduced compared to A549 cells, although equitoxic cisplatin concentrations resulted in comparable platinum-DNA adduct levels. These differences were accompanied by changes in the expression of proteins involved in DNA damage response. In A549 cells, cisplatin exposure led to a significantly higher expression of genes coding for proteins mediating G2/M arrest and apoptosis (mouse double minute 2 homolog (MDM2), xeroderma pigmentosum complementation group C (XPC), stress inducible protein (SIP) and p21) compared to resistant cells. This was underlined by significantly higher protein levels of phosphorylated Ataxia telangiectasia mutated (pAtm) and p53 in A549 cells compared to their respective untreated control. The results were compiled in a preliminary model of resistance-associated signaling alterations. In conclusion, these findings suggest that acquired resistance of NSCLC cells against cisplatin is the consequence of altered signaling leading to reduced G2/M cell cycle arrest and apoptosis.