Stefan Eugen Sonderegger

DeltaEF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells

Downregulation of E-cadherin is a crucial event for epithelial to mesenchymal transition (EMT) in embryonic development and cancer progression. Using the EpFosER mammary tumour model we show that during EMT, upregulation of the transcriptional regulator deltaEF1 coincided with transcriptional repression of E-cadherin. Ectopic expression of deltaEF1 in epithelial cells was sufficient to downregulate E-cadherin and to induce EMT. Analysis of E-cadherin promoter activity and chromatin immunoprecipitation identified deltaEF1 as direct transcriptional repressor of E-cadherin. In human cancer cells, transcript levels of deltaEF1 correlated directly with the extent of E-cadherin repression and loss of the epithelial phenotype. The protein was enriched in nuclei of human cancer cells and physically associated with the E-cadherin promoter. RNA interference-mediated downregulation of deltaEF1 in cancer cells was sufficient to derepress E-cadherin expression and restore cell to cell adhesion, suggesting that deltaEF1 is a key player in late stage carcinogenesis.

Wnt Signalling in Implantation, Decidualisation and Placental Differentiation – Review

The family of secreted Wingless ligands plays major roles in embryonic development, stem cell maintenance, differentiation and tissue homeostasis. Accumulating evidence suggests that the canonical Wnt pathway involving nuclear recruitment of β-catenin and activation of Wnt-dependent transcription factors is also critically involved in development and differentiation of the diverse reproductive tissues. Here, we summarise our present knowledge about expression, regulation and function of Wnt ligands and their frizzled receptors in murine and human endometrial and placental cell types. In mice, Wnt signalling promotes early trophoblast lineage development, blastocyst activation, implantation and chorion-allantois fusion. Moreover, different Wnt ligands play essential roles in the development of the murine uterine tract, in cycling endometrial cells and during decidualisation. In humans, estrogen-dependent endometrial cell proliferation, decidualisation, trophoblast attachment and invasion were shown to be controlled by the particular signalling pathway. Failures in Wnt signalling are associated with infertility, endometriosis, endometrial cancer and gestational diseases such as complete mole placentae and choriocarcinomas. However, our present knowledge is still scarce due to the complexity of the Wnt network involving numerous ligands, receptors and non-canonical pathways. Hence, much remains to be learned about the role of different Wnt signalling cascades in reproductive cell types and their changes under pathological conditions.

Wingless (Wnt)-3A Induces Trophoblast Migration and Matrix Metalloproteinase-2 Secretion through Canonical Wnt Signaling and Protein Kinase B/AKT Activation

Invasion of human trophoblasts is promoted through activation of wingless (Wnt) signaling, suggesting a role of the pathway in placental development and morphogenesis. However, details on the process such as involvement of canonical and/or noncanonical Wnt signaling cascades as well as their target genes are largely unknown. Hence, signal transduction via canonical Wnt signaling or phosphatidylinositide 3-kinase (PI3K)/AKT and their cross talk as well as trophoblast-specific protease expression were investigated in trophoblastic SGHPL-5 cells and primary extravillous trophoblasts purified from first-trimester placentas. Western blot analyses revealed that the recombinant Wnt ligand Wnt-3A increased phosphorylation of AKT and the downstream kinase glycogen synthase kinase (GSK)-3beta as well as accumulation of activated, nuclear beta-catenin. In accordance, luciferase expression of a canonical Wnt/TCF reporter and cell migration in first-trimester villous explant cultures and of SGHPL-5 cells were stimulated. Chemical inhibition of PI3K abolished Wnt-dependent phosphorylation of AKT and GSK-3beta and trophoblast motility but did not affect appearance of activated beta-catenin or Wnt/TCF reporter activity. In contrast, inhibition of the canonical pathway through soluble Dickkopf-1 did not influence AKT and GSK-3beta phosphorylation but reduced Wnt reporter activity, accumulation of active beta-catenin, and cell migration. Both inhibitors decreased Wnt-3A-induced secretion of pro- and active matrix metalloproteinase-2 from SGHPL-5 cells and pure EVT. The data suggest that Wnt-3A may activate canonical Wnt signaling and PI3K/AKT through distinct receptors. The two signaling cascades act independently in trophoblasts; however, both pathways promote Wnt-dependent migration and the release of matrix metalloproteinase-2, which has been identified as novel Wnt target in invasive trophoblasts.

Governing the invasive trophoblast: current aspects on intra- and extracellular regulation.

Citation Fitzgerald JS, Germeyer A, Huppertz B, Jeschke U, Knöfler M, Moser G, Scholz C, Sonderegger S, Toth B, Markert UR. Governing the invasive trophoblast: current aspects on intra‐ and extracellular regulation. Am J Reprod Immunol 2010

REVIEW ARTICLE: Governing the Invasive Trophoblast: Current Aspects on Intra- and Extracellular Regulation

Citation Fitzgerald JS, Germeyer A, Huppertz B, Jeschke U, Knöfler M, Moser G, Scholz C, Sonderegger S, Toth B, Markert UR. Governing the invasive trophoblast: current aspects on intra‐ and extracellular regulation. Am J Reprod Immunol 2010

Interleukin 11 regulates endometrial cancer cell adhesion and migration via STAT3

Endometrial carcinoma is the most common gynaecological malignancy. There is however a lack of curative therapies, especially for patients diagnosed with late stage, recurrent or aggressive disease, who have a poor prognosis. Interleukin (IL) 11 is a pleiotropic cytokine that has a role in a number of cancers including colon and breast cancer. IL11 was recently found to be upregulated in endometrial cancers, however the function of IL11 in endometrial cancer is not known. This study aimed to determine the effects of IL11 on endometrial cancer cell proliferation, adhesion and migration. Three endometrial cancer cell lines, Ishikawa, HEC-1A and AN3CA (derived from endometrial cancers grade I, II and III, respectively), were used to determine the effect of IL11 on endometrial cancer cell function. Cell proliferation and viability were assessed by BrdU and Wst-1 assays. Cell adhesion to the extracellular matrix proteins fibronectin, collagen I and IV, vitronectin and laminin was assessed. Modified boyden chambers were utilized to access IL11 action on migration and invasion, respectively. The specific effect of IL11 action on these processes was determined using a unique IL11 inhibitor. IL11 phosphorylated (p)-STAT3 protein abundance in all 3 cell lines but had no effect on pERK and pAKT abundance. Similarly, IL11 had no effect on cell proliferation and viability but increased adhesion of ANC3A cells to fibronectin while having no effect on the other extracellular matrix proteins. IL11 did not alter the adhesive properties of the Ishikawa and HEC-1A cells. In the AN3CA cells, IL11 treatment resulted in a 50% increase in migration and co-treatment with the specific IL11 inhibitor or a STAT3 inhibitor abolished the effect. This study shows a role for IL11 in endometrial cancer and suggests IL11 may be involved in endometrial cancer development and thus may be useful as a therapeutic target.

Expression, Regulation and Functional Characterization of Matrix Metalloproteinase-3 of Human Trophoblast

MMP-3 has been detected in human placenta and reduced expression of the enzyme was observed in invasive trophoblasts of patients with severe preeclampsia. However, detailed expression pattern, regulation and biological properties of the placental protease have not been elucidated so far. RT-PCR analyses, Western blotting and enzyme activity assays revealed that pro- and active form of MMP-3 were predominantly expressed in purified first trimester villous trophoblasts, in invasive cytotrophoblasts of differentiating explant cultures and in trophoblastic SGHPL-4 cells. Accordingly, immunofluorescene of first trimester placental tissues detected MMP-3 mainly in villous and extravillous cytotrophoblasts. IL-1beta, an inducer of MMP-3 in decidual cells, increased secretion and activity of the protease in trophoblast supernatants in a dose- and time-dependent manner. IL-1beta-stimulated production of the enzyme was suppressed in the presence of inhibitors of MAPK and AKT signalling. Similar to recombinant MMP-3, MMP-3 in supernatants of IL-1beta-stimulated decidual stromal or SGHPL-4 cells degraded IGFBP-1 in vitro resulting in the appearance of cleavage products at approximately 25, 22, 17, 14 and 11kD. However, cleavage assays using recombinant MMP-2 suggested that the gelatinase may contribute to IGFBP-1 degradation in trophoblast supernatants. Despite its effects on MMP-3 expression IL-1beta failed to significantly alter invasion of SGHPL-4 cells through Matrigel-coated transwells. In conclusion, the data suggest that invasive trophoblast cell models secrete bioactive MMP-3. Inducible expression of the protease involves MAPK and AKT signalling. In addition to the decidua, MMP-3 of trophoblasts may contribute to the regulation of the IGF system by degrading IGFBP-1.

Interleukin (IL)11 mediates protein secretion and modification in human extravillous trophoblasts

BACKGROUND Human trophoblast invasion and differentiation are essential for a successful pregnancy outcome. Dysregulation of these processes can lead to placental pathologies such as pre-eclampsia. The molecular mechanisms; however, are poorly understood. Interleukin (IL)11--a cytokine that regulates endometrial epithelial cell adhesion, trophoblast motility and invasion during implantation--may be involved in some of these processes. METHODS AND RESULTS The effect of IL11 on protein expression was investigated in trophoblastic HTR8/SVneo cells and primary extravillous trophoblasts (EVTs) purified from first- trimester placentas. Two-dimension (2D)-differential in-gel electrophoresis analyses revealed that 731 spots were significantly differentially regulated by IL11 in HTR8/SVneo cells: seven spots were analyzed by liquid chromatography-tandem mass spectrometry and 14 unique proteins identified. Protein disulfide isomerase family A, member 3 (PDIA3; endoplasmic reticulum p57) and glucose-regulated protein 78 (GRP78) were further validated to be regulated by IL11 in HTR8/SVneo and primary EVT. One dimension western blot analysis confirmed that PDIA3 was down-regulated in EVT. 2D western blot analysis revealed that GRP78 was post-translationally modified following IL11 treatment. Moreover, IL11 stimulated the secretion of GRP78 in EVT. CONCLUSIONS Data suggest that IL11, possibly via signal transducers and activators of transcription 3 signaling pathway, regulates PDIA3 protein expression and modification/secretion of GRP78. This is the first study to identify PDIA3 and GRP78 as IL11 targets in invasive trophoblasts and identifies a possible mechanism by which IL11 regulates trophoblast function.

AKT Isoforms 1 and 3 Regulate Basal and Epidermal Growth Factor-Stimulated SGHPL-5 Trophoblast Cell Migration in Humans

ABSTRACT Protein kinase B/AKT is critically involved in murine placental development and migration of human placental trophoblasts into maternal uterine tissue. However, localization of the three AKT isoforms within human placenta and their roles in extravillous trophoblasts have not been elucidated. Therefore, we analyzed the expression pattern and function of AKT1, AKT2, and AKT3 in migratory human trophoblasts using SGHPL-5 cell pools stably expressing small-hairpin microRNA (shRNAmir) against AKT1, AKT2, or AKT3 as a model. Western blot analyses using isoform-specific antibodies revealed ubiquitous expression of AKT1, AKT2, and AKT3 in primary villous and extravillous trophoblasts and the trophoblastic cell lines JEG-3, HTR-8/SVneo, and SGHPL-5. Immunofluorescence of first-trimester placentae localized AKT2 and AKT3 to the cytoplasm and nucleus, respectively, in all subtypes of cytotrophoblasts, whereas AKT1 was detected in both cellular compartments. A similar distribution of AKT isoforms was detectable in SGHPL-5 cells. Gene silencing using shRNAmir decreased protein expression of AKT1, AKT2, and AKT3 to 16%, 8%, and 11%, respectively, in SGHPL-5 cells. Compared with shRNAmir controls, proliferation and camptothecin-induced apoptosis were not affected in the different AKT knockdown cells. However, basal and epidermal growth factor (EGF)-induced trophoblast migration was significantly reduced in AKT1 and AKT3 gene-silenced cells, whereas downregulation of AKT2 was not effective. Accordingly, a decrease in EGF-stimulated phosphorylation of AKT (Ser473 and Thr308) and its downstream target mTORC1 (Ser2448) was noticed in AKT1 and AKT3 shRNAmir cell pools. In summary, the results suggest that the AKT isoforms 1 and 3 promote basal as well as EGF-induced trophoblast migration.

The role of leukemia inhibitory factor in tubal ectopic pregnancy.

INTRODUCTION Ectopic pregnancy is unique to humans and a leading cause of maternal morbidity and mortality. The etiology remains unknown however factors regulating embryo implantation likely contribute. Leukemia inhibitory factor (LIF) has roles in extravillous trophoblast adhesion and invasion and is present in ectopic implantation sites. We hypothesised that LIF facilitates blastocyst adhesion/invasion in the Fallopian tube, contributing to ectopic pregnancy. METHODS We immunolocalised LIF receptor (R) in tubal ectopic pregnancy (N = 5). We used an oviduct cell line (OE-E6/E7) to model Fallopian tube epithelial cells and a trophoblast spheroid co-culture model (HTR-8/SVneo cell line formed spheroids) to model blastocyst attachment to the Fallopian tube. We examined LIF signaling pathways in OE-E6/E7 cells by Western blot. The effect of LIF and LIF inhibition (using a novel LIF inhibitor, PEGLA) on first-trimester placental outgrowth was determined. RESULTS LIFR localised to villous and extravillous trophoblast and Fallopian tube epithelium in ectopic pregnancy. LIF activated STAT3 but not the ERK pathway in OE-E6/E7 cells. LIF stimulated HTR-8/SVneo spheroid adhesion to OE-E6/E7 cells which was significantly reduced after PEGLA treatment. LIF promoted placental explants outgrowth, while co-treatment with PEGLA blocked outgrowth. DISCUSSION Our data suggests LIF facilitates the development of ectopic pregnancy by stimulating blastocyst adhesion and trophoblast outgrowth from placental explants. Ectopic pregnancy is usually diagnosed after 6 weeks of pregnancy, therefore PEGLA may be useful in targeting trophoblast growth/invasion. CONCLUSION LIF may contribute to the development of ectopic pregnancies and that pharmacologically targeting LIF-mediated trophoblast outgrowth may be useful as a treatment for ectopic pregnancy.