Epigenetic Dysregulation of Trophoblastic Gene Expression in Gestational Trophoblastic Disease

Gestational trophoblastic diseases (GTDs) have not been investigated for their epigenetic marks and consequent transcriptomic changes. Here, we analyzed genome-wide DNA methylation and transcriptome data to reveal the epigenetic basis of disease pathways that may lead to benign or malignant GTDs. RNA-Seq, mRNA microarray, and Human Methylation 450 BeadChip data from complete moles and choriocarcinoma cells were bioinformatically analyzed. Paraffin-embedded tissues from complete moles and control placentas were used for tissue microarray construction, DNMT3B immunostaining and immunoscoring. We found that DNA methylation increases with disease severity in GTDs. Differentially expressed genes are mainly upregulated in moles while predominantly downregulated in choriocarcinoma. DNA methylation principally influences the gene expression of villous trophoblast differentiation-related or predominantly placenta-expressed genes in moles and choriocarcinoma cells. Affected genes in these subsets shared focal adhesion and actin cytoskeleton pathways in moles and choriocarcinoma. In moles, cell cycle and differentiation regulatory pathways, essential for trophoblast/placental development, were enriched. In choriocarcinoma cells, hormone biosynthetic, extracellular matrix-related, hypoxic gene regulatory, and differentiation-related signaling pathways were enriched. In moles, we found slight upregulation of DNMT3B protein, a developmentally important de novo DNA methylase, which is strongly overexpressed in choriocarcinoma cells that may partly be responsible for the large DNA methylation differences. Our findings provide new insights into the shared and disparate molecular pathways of disease in GTDs and may help in designing new diagnostic and therapeutic tools.

Investigation of Human Endogenous Retrovirus-K (ERVK) Expression and Function in Normal Placentation and Preterm Pregnancy

Background: There is a growing body of evidence indicating the importance of endogenous retrovirus (ERV) derived proteins during early development and reproduction in mammals. Recently, a protein derived from the youngest ERV in humans, ERVK (HML2), was shown to be expressed during human placentation. Since a number of highly similar ERVK proviral loci exist across the human genome, locus-specific analysis of ERVK transcription and identification of the coding sequence expressed in the human placenta is difficult. Thus, despite its activity in early human development, the native expression and function of ERVK in the human placenta remains largely uncharacterized. Results: In this study, we comprehensively examined locus-specific ERVK transcription across several human placental tissues and cell types. Through a combination of RNA-seq and siRNA knock-down analyses, we identified the expression of a single ERVK locus, ERVK11q23.3, as (1) being significantly upregulated in preterm compared to term placenta, (2) predominantly expressed by mononuclear trophoblasts, (3) capable of encoding a truncated viral-like envelope protein, and (4) contributing to the expression cytokines involved in both antiviral and anti-inflammatory innate immune responses in human placental trophoblasts and BeWo choriocarcinoma cells, respectively. Conclusions: Collectively, the results of this study highlight the utility of studying locus-specific ERVK expression, provide a thorough characterization of locus-specific ERVK transcription from human placental tissues, and indicate that altered expression of placental ERVK11q23.3 influences interferon antiviral response, which may contribute to preterm birth and other pregnancy complications.

Syncytialization alters the extracellular matrix and barrier function of placental trophoblasts

The human placenta is of vital importance for proper nutrient and waste exchange, immune regulation, and overall fetal health and growth. Specifically, the extracellular matrix (ECM) of placental syncytiotrophoblasts, which extends outward from the placental chorionic villi into maternal blood, acts on a molecular level to regulate and maintain this barrier. Importantly, placental barrier dysfunction has been linked to diseases of pregnancy such as preeclampsia and intrauterine growth restriction. To help facilitate our understanding of the interface, and develop therapeutics to repair or prevent dysfunction of the placental barrier, in vitro models of the placental ECM would be of great value. In this study we aimed to characterize the ECM of an in vitro model of the placental barrier using syncytialized BeWo choriocarcinoma cells. Syncytialization caused a marked change in syndecans, integral proteoglycans of the ECM, which matched observations of in vivo placental ECM. Syndecan-1 expression increased greatly and predominated the other variants. Barrier function of the ECM, as measured by electrical impedance, increased significantly during and after syncytialization, while the ability of THP-1 monocytes to adhere to syncytialized BeWos was greatly reduced compared to non-syncytialized controls. Furthermore, ECIS measurements indicated that ECM degradation with MMP-9, but not heparanase, decreased barrier function. This decrease in ECIS-measured barrier function was not associated with any changes in THP-1 adherence to syncytialized BeWos treated with heparanase or MMP9. Thus, syncytialization of BeWos provides a physiologically accurate placental ECM with a barrier function matching that seen in vivo.

The (pro)renin receptor and soluble (pro)renin receptor in choriocarcinoma

This study aimed to determine if the (pro)renin receptor (ATP6AP2) changes the cellular profile of choriocarcinomas from cytotrophoblast cells to terminally syncytialised cells and ascertain whether this impacts on the invasive potential of choriocarcinoma cells. Additionally, we aimed to confirm that FURIN and/or site 1 protease (MBTPS1) cleave soluble ATP6AP2 (sATP6AP2) in BeWo choriocarcinoma cells and determine whether sATP6AP2 levels reflect the cellular profile of choriocarcinomas. BeWo choriocarcinoma cells were treated with ATP6AP2 siRNA, FURIN siRNA, DEC-RVKR-CMK (to inhibit FURIN activity) or PF 429242 (to inhibit MBTPS1 activity). Cells were also treated with forskolin, to induce syncytialisation, or vehicle and incubated for 48h before collection of cells and supernatants. Syncytialisation was assessed by measuring hCG secretion (by ELISA) and E-cadherin protein levels (by immunoblot and immunocytochemistry). Cellular invasion was measured using the xCELLigence real-time cell analysis system and secreted sATP6AP2 levels measured by ELISA. Forskolin successfully induced syncytialisation and significantly increased both BeWo choriocarcinoma cell invasion (P<0.0001) and sATP6AP2 levels (P=0.02). Treatment with ATP6AP2 siRNA significantly inhibited syncytialisation (decreased hCG secretion (P=0.005), the percent of nuclei in syncytia (P=0.05)), forskolin-induced invasion (P=0.046) and sATP6AP2 levels (P<0.0001). FURIN siRNA and DEC-RVKR-CMK significantly decreased sATP6AP2 levels (both P<0.0001). In conclusion, ATP6AP2 is important for syncytialisation of choriocarcinoma cells and thereby limits choriocarcinoma cell invasion. We postulate that sATP6AP2 could be used as a biomarker measuring the invasive potential of choriocarcinomas. Additionally, we confirmed that FURIN, not MBTPS1, cleaves sATP6AP2 in BeWo cells, but other proteases (inhibited by DEC-RVKR-CMK) may also be involved.

Chemical Induction of Trophoblast Hypoxia by Cobalt Chloride Leads to Increased Expression of DDIT3

Choriocarcinoma cells BeWo b30 are used to model human placental trophoblast hypoxia using cobalt (II) chloride and hydroxyquinoline derivative (HD) as chemical inducers of hypoxia-inducible factor (HIF). In this study, it was shown that both substances activate the hypoxic pathway and the epithelial–mesenchymal transition and inhibit the pathways of cell proliferation. However, CoCl2 caused activation of the apoptosis pathway, increased the activity of effector caspases 3 and 7, and increased the expression of the unfolded protein response target DDIT3. The mTORC1 pathway was activated upon exposition to CoCl2, while HD suppressed this pathway, as it happens during real trophoblast hypoxia. Thus, effect of CoCl2 on BeWo cells can be a model of severe hypoxia with activation of apoptosis, while HD mimics moderate hypoxia.

FURIN and placental syncytialisation: a cautionary tale

FURIN is a pro-protein convertase previously shown to be important for placental syncytialisation (Zhou et al. [1]), a process of cell fusion whereby placental cytotrophoblast cells fuse to form a multinucleated syncytium. This finding has been broadly accepted however, we have evidence suggesting the contrary. Spontaneously syncytialising term primary human trophoblast cells and BeWo choriocarcinoma cells were treated with either FURIN siRNA or negative control siRNA or the protease inhibitor, DEC-RVKR-CMK, or vehicle. Cells were then left to either spontaneously syncytialise (primary trophoblasts) or were induced to syncytialise with forskolin (BeWo). Effects on syncytialisation were measured by determining human chorionic gonadotrophin secretion and E-cadherin protein levels. We showed that FURIN is not important for syncytialisation in either cell type. However, in primary trophoblasts another protease also inhibited by DEC-RVKR-CMK, may be involved. Our results directly contrast with those published by Zhou et al. Zhou et al. however, used first trimester villous explants to study syncytialisation, and we used term primary trophoblasts. Therefore, we suggest that FURIN may be involved in syncytialisation of first trimester trophoblasts, but not term trophoblasts. What is more concerning is that our results using BeWo cells do not agree with their results, even though for the most part, we used the same experimental design. It is unclear why these experiments yielded different results, however we wanted to draw attention to simple differences in measuring syncytialisation or flaws in method reporting (including omission of cell line source and passage numbers, siRNA concentration and protein molecular weights) and choice of immunoblot loading controls, that could impact on experimental outcomes. Our study shows that careful reporting of methods by authors and thorough scrutiny by referees are vital. Furthermore, a universal benchmark for measuring syncytialisation is required so that various studies of syncytialisation can be validated.

Nucleoside transporter-guided cytarabine-conjugated liposomes for intracellular methotrexate delivery and cooperative choriocarcinoma therapy

Gestational trophoblastic tumors seriously endanger child productive needs and the health of women in childbearing age. Nanodrug-based therapy mediated by transporters provides a novel strategy for the treatment of trophoblastic tumors. Focusing on the overexpression of human equilibrative nucleoside transporter 1 (ENT1) on the membrane of choriocarcinoma cells (JEG-3), cytarabine (Cy, a substrate of ENT1)-grafted liposomes (Cy-Lipo) were introduced for the targeted delivery of methotrexate (Cy-Lipo@MTX) for choriocarcinoma therapy in this study. ENT1 has a high affinity for Cy-Lipo and can mediate the endocytosis of the designed nanovehicles into JEG-3 cells. The ENT1 protein maintains its transportation function through circulation and regeneration during endocytosis. Therefore, Cy-Lipo-based formulations showed high tumor accumulation and retention in biodistribution studies. More importantly, the designed DSPE-PEG2k-Cy conjugation exhibited a synergistic therapeutic effect on choriocarcinoma. Finally, Cy-Lipo@MTX exerted an extremely powerful anti-choriocarcinoma effect with fewer side effects. This study suggests that the overexpressed ENT1 on choriocarcinoma cells holds great potential as a high-efficiency target for the rational design of active targeting nanotherapeutics. Graphic abstract

Nucleoside Transporter-guided Cytarabine Conjugated Liposomes for Intracellular Methotrexate Delivery and Cooperative Choriocarcinoma Therapy

The gestational trophoblastic tumor seriously endangers child productive needs and the health of women in childbearing age. Nanodrug-based therapy mediated by transporters provides novel strategy for the treatment of trophoblastic tumors. Focus on the overexpressed human equilibrative nucleoside transporter 1 (ENT1) on the membrane of choriocarcinoma cells (JEG-3), the cytarabine (Cy, a substrate of ENT1) grafted liposome (Cy-Lipo) was introduced for targeted delivery of methotrexate (Cy-Lipo@MTX) for choriocarcinoma therapy in this study. The ENT1 has high affinity for Cy-Lipo and can mediate the endocytosis of the designed nanovehicles into JEG-3 cells. The ENT1 protein maintains its transporting function through circulation and regeneration during endocytosis. Therefore, Cy-Lipo-based formulations achieved high tumor accumulation and retention in pharmacokinetic and distribution studies. More importantly, the designed Cy-lipid conjugation exhibited a synergistic therapeutic effect on choriocarcinoma. Finally, Cy-Lipo@MTX exerts an extremely powerful anti-choriocarcinoma effect with fewer side effects. This study suggests that the overexpressed ENT1 on choriocarcinoma cells holds a great potential to be a high-efficiency target for the rational design of active targeting nanotherapeutics.

α-Solanine Inhibits Proliferation, Invasion, and Migration, and Induces Apoptosis in Human Choriocarcinoma JEG-3 Cells In Vitro and In Vivo

α-Solanine, a bioactive compound mainly found in potato, exhibits anti-cancer activity towards multiple cancer cells. However, its effects on human choriocarcinoma have not been evaluated. In the present study, we investigated the effect of α-solanine on cell proliferation and apoptosis in human choriocarcinoma in vitro and in vivo. The results showed that α-solanine, at concentrations of 30 μM or below, did not affect the cell viability of the choriocarcinoma cell line JEG-3. However, colony formation was significantly decreased and cell apoptosis was increased in response to 30 μM α-solanine. In addition, α-solanine (30 μM) reduced the migration and invasion abilities of JEG-3 cells, which was associated with a downregulation of matrix metalloproteinases (MMP)-2/9. The in vivo findings provided further evidence of the inhibition of α-solanine on choriocarcinoma tumor growth. α-Solanine suppressed the xenograft tumor growth of JEG-3 cells, resulting in smaller tumor volumes and lower tumor weights. Apoptosis was promoted in xenograft tumors of α-solanine-treated mice. Moreover, α-solanine downregulated proliferative cellular nuclear antigen (PCNA) and Bcl-2 levels and promoted the expression of Bax. Collectively, α-solanine inhibits the growth, migration, and invasion of human JEG-3 choriocarcinoma cells, which may be associated with the induction of apoptosis.