Reproduction

Hypermethylation of the GRHL2 promoter region is associated with ovarian endometriosis

Abstract Abnormal gene expression caused by epigenetic changes, including DNA methylation, is associated with the development and progression of endometriosis. Grainyhead-like 2 gene (GRHL2), a suppressor of epithelial–mesenchymal transition, has been suggested to be associated with the occurrence, progression and poor survival of a variety of cancers. Although endometriosis is a benign disease, it has the biological behaviour of migration and invasion as malignant tumor. This study aims to determine whether the abnormal expression of the GRHL2 caused by aberrant methylation of its promoter is associated with the pathogenesis of ovarian endometriosis. Our results demonstrated that GRHL2 promoter region was significantly hypermethylated in the ectopic endometrium of patients with ovarian endometriosis compared with the normal endometrium of control patients. In contrast, the levels of GRHL2 mRNA and protein were significantly lower in the ectopic endometrium than in the control endometrium. Correlation analysis showed the methylation levels of GRHL2 were significantly negatively correlated with the mRNA expression of GRHL2. Moreover, the in vitro results suggested that the knockdown of GRHL2 could significantly increase the invasion and migration ability of EECs and may promote ZEB1 and vimentin expression while decreasing the expression of E-cadherin in EECs. Taken together, these results suggest that the low expression of GRHL2 caused by hypermethylation of the GRHL2 promoter is associated with ovarian endometriosis. The knockdown of GRHL2 may be involved in the occurrence of endometriosis by increasing EEC migration and invasion. This study provides more evidence for the hypothesis that endometriosis may be an epigenetic regulatory disorder.

Roads to the strategic targeting of ovarian cancer treatment

Abstract Although ovarian cancer mortality rates have slightly declined in the last 40 years, ovarian cancer continues to be the eighth cause of cancer death in women. Ovarian cancer is characterized by its high response to treatments but also by its high rate of recurrence. Although treatments are limited to cytoreductive surgery and platinum-based chemotherapy, other therapies using antiangiogenic agents and poly (ADP-ribose) polymerase inhibitors are being tested. Nevertheless, these therapeutic strategies have had poor results and new potential targets and approaches are thus needed. The present review focuses on the recent evidence on antiangiogenic strategies in ovarian cancer cells and on the mechanisms governed by Notch and β-catenin proteins. It also describes the concept of ‘vascular normalization’ by using the platelet-derived growth factor, PDGFB, molecule as a tool to regulate ovarian tumor angiogenesis and thus improve ovarian tumor treatment. It has been reported that alterations in the Notch system components and changes in the canonical Wnt/β-catenin signaling, the other pathway of our interest, are relevant to molecular events that contribute to ovarian cancer development. Thus, in this review, we consider these aspects of the ovarian tumor biology as potential new therapeutic strategies for the treatment of this disease.

Mitochondrial humanin peptide acts as a cytoprotective factor in granulosa cell survival

Abstract Humanin (HN) is a short peptide involved in many biological processes such as apoptosis, cell survival, inflammatory response, and reaction to stressors like oxidative stress, between others. In the ovary, a correct balance between pro- and anti-apoptotic factors is crucial for folliculogenesis. In the follicular atresia, survival or death of granulosa cells is a critical process. The goal of this study was to evaluate the action of HN on granulosa cell fate. To explore endogenous HN function in the ovary, we used a recombinant baculovirus (BV) encoding a short-hairpin RNA targeted to silence HN (shHN). HN downregulation modified ovarian histoarchitecture and increased apoptosis of granulosa cells. HN was also detected in a granulosa tumor cell line (KGN). Transduction of KGN cells with BV-shHN resulted in HN downregulation and increased apoptosis. On the other hand, treatment of KGN cells with exogenous HN increased cell viability and decreased apoptosis. In summary, these findings indicate that HN is a cytoprotective factor in granulosa cells of antral follicles, suggesting that this peptide would be involved in the regulation of folliculogenesis. Also, this peptide is a cytoprotective factor in KGN cells, and therefore, it could be involved in granulosa tumor cell behavior.

Genomic profiling of meiotic errors and early malignant transformation events in ovarian mature teratoma

Abstract Ovarian mature teratoma (OMT) exhibits copy-neutral loss of heterozygosity (CN-LOH) derived from meiotic errors. Studies have classified OMT into five types using these CN-LOH patterns. However, tumor purity issues have hindered accurate classification and molecular characterization. Moreover, the relationship between OMT genomic abnormalities and malignant transformation remains unclear. Here, we used laser microdissection to selectively collect 29 epithelial regions from 22 OMT cases and 1 carcinoma in situ (CIS) region from a squamous cell carcinoma case arising from OMT. We then conducted whole-exome sequencing. Copy number analysis enabled classification according to the established system. In cases with multi-regional sampling, the CN-LOH patterns were identical within the same tumor, while bilateral tumors showed distinct patterns, indicating that each tumor originated independently. Among the type II OMTs, which are believed to result from meiosis II error, 2 cases exhibited a chromosomal gain. Assessment of heterozygosity suggested that these cases had meiosis I error before meiosis II error. Somatic mutation analysis revealed an extremely low mutation burden, with 1 case harboring a PIK3CA mutation. In the CIS region, additional copy number alterations were present alongside the underlying CN-LOH pattern. TP53 mutations and loss of the wild-type allele were detected. The tumor mutation burden of CIS was 6.2 mutations per megabase. Mutational signature analysis showed enrichment of apolipoprotein B mRNA editing enzyme catalytic subunit-associated signatures. This study suggests novel perspectives on the mechanisms of OMT tumor development and malignant transformation.

ZNF146 regulates cell cycle progression via TFDP1 and DEPDC1B in ovarian cancer cells

In brief Aberration in cell cycle progression is one of the essential mechanisms underlying tumorigenesis, making regulators of cell cycle reasonable anti-cancer therapeutic targets. Here, we dissected the regulatory mechanism involving the novel axis ZNF146/TFDP1/DEPDC1B in the cell cycle in ovarian cancer. Abstract Ovarian cancer (OC) is the third most common kind of gynecological tumor, in addition to being the most lethal. Transcription factor Dp-1 (TFDP1) functions as a binding partner for E2F transcription factors, and its target genes include those involved in DNA synthesis, cell cycle, and apoptosis. However, the regulatory role of TFDP1 in OC remains incompletely understood. This study aimed to investigate the role and mechanism of TFDP1 in OC. TFDP1 was highly expressed in the ovarian epithelial tissues of OC patients, and the expression of TFDP1 in OC cells was higher than that in normal ovarian epithelial cells. Silencing of TFDP1 inhibited the biological activity of OC cells and hindered cell cycle entry. Zinc finger protein 146 (ZNF146) knockdown induced cell cycle arrest at the G0/G1 phase and tumor growth by blocking TFDP1 transcription, which was overturned by ectopic expression of TFDP1. TFDP1 stimulated DEP domain-containing protein 1B (DEPDC1B) expression through transcriptional activation. DEPDC1B increased the proportion of OC cells in the G2/M phase and potentiated tumor malignant progression in nude mice inhibited by sh-ZNF146. Taken together, these findings demonstrate that ZNF146 participates in TFDP1/DEPDC1B activation and plays a vital role in the cell cycle in OC.

Loss of PRICKLE1 leads to subfertility, aberrant extracellular matrix and abnormal myometrial architecture in mice

In brief PRICKLE1, a WNT/planar cell polarity (PCP) protein that is downregulated in uterine leiomyoma, plays an important role in myometrial tissue architecture and extracellular matrix (ECM) deposition. This paper shows that myometrial-specific ablation of the mouse Prickle1 gene results in a uterine leiomyoma phenotype. Abstract Uterine leiomyomas (ULs) are the most prevalent benign tumors of the female reproductive tract, originating from the myometrium and affecting over 75% of reproductive-age women. Symptoms of UL include pelvic pain, pressure, dysmenorrhea, menorrhagia, anemia and reproductive dysfunction. Currently, there is no effective long-term pharmacotherapy for UL, making them the leading cause of hysterectomies in the United States. The lack of treatment options is attributed to the absence of accurate animal models and a limited understanding of UL pathogenesis. Previous research has shown that the loss of repressor of element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) within the myometrium promotes UL pathogenesis. In addition, deletion of Rest in the mouse myometrium leads to a UL phenotype. PRICKLE1, also known as Rest-interacting LIM-domain protein (RILP), is required for nuclear localization of REST and Wnt/PCP signaling, making it a critical target for UL studies. In the context of PCP, smooth muscle cells in UL show abnormal organization, aberrant ECM structure and expression levels, potentially influenced by PRICKLE1 loss. The exact role of PRICKLE1 and Wnt/PCP in UL pathogenesis remains unclear. To explore PRICKLE1’s role in UL, we deleted Prickle1 using our myometrial-specific iCre. Our findings demonstrate that Prickle1 loss in the myometrium results in a UL phenotype characterized by altered collagen expression, excessive ECM deposition, aberrant smooth muscle cell organization, increased Esr1 and Pgr expression and dysregulated Wnt/PCP signaling. This novel mouse model serves as a valuable preclinical tool for understanding UL pathogenesis and developing future pharmacotherapies.

Visfatin increases the invasive potential of ovarian granulosa tumor spheroids by reprogramming glucose metabolism

In brief The role of visfatin in ovarian granulosa cell tumor (GCT) invasion and glucose metabolism reprogramming is largely unexplored. These studies imply that visfatin or its inhibitor is involved in regulating ovarian granuloma invasion by reprogramming glucose metabolism and may be a potential candidate for the diagnosis and treatment of ovarian GCT. Abstract Visfatin is an adipokine with nicotinamide phosphoribosyltransferase (NAMPT) activity, the concentration of which is higher in ascitic fluid than in serum, and is associated with ovarian cancer peritoneal dissemination. Potentially important effects of visfatin on glucose metabolism have been previously reported. However, the mechanism underlying the effects of visfatin on ovarian cancer cell invasion, and whether this involves altered glucose metabolism, has not been elucidated. Here, we tested the hypothesis that visfatin, which can reprogram cancer metabolism, promotes invasion by ovarian cancer spheroids. Visfatin increased glucose transporter (GLUT)1 expression and glucose uptake in adult granulosa cell tumor-derived spheroid cells (KGN) and also increased the activities of hexokinase 2 and lactate dehydrogenase. We showed a visfatin-induced increase in glycolysis in KGN cells. Moreover, visfatin increased the potential invasiveness of KGN spheroid cells by upregulating MMP2 (matrix metalloproteinase 2) and downregulating CLDN3 and CLDN4 (claudin 3 and 4) gene expression. Interestingly, an inhibitor of GLUT1 and lactate dehydrogenase (LDHA) abolished the stimulatory effect of visfatin on the potential invasiveness of KGN cells. More importantly, silencing expression of the NAMPT gene in KGN cells demonstrated its important effect on glycolysis and invasiveness in adult granulosa cell tumor cells (AGCTs). In summary, visfatin appears to increase AGCT invasiveness through effects on glucose metabolism and to be an important regulator of glucose metabolism in these cells.

Alterations in egg white-related genes expression in response to hormonal stimulation

Abstract The reproductive tract in avian females is sensitive to hormonal regulation. Exogenous estrogen induces immature oviduct development to improve egg production after molting. In this process, regressed female reproductive tract is regenerated in response to the secretion of estrogen. However, there is limited knowledge on the physiological mechanisms underlying the regulation of the avian female reproductive system. In our previous study, results from microarray analysis revealed that the expression of genes encoding egg white proteins is affected during molting. Herein, we artificially induced the molting period in chickens through a zinc-containing diet. Subsequently, changes in the expression of genes encoding egg white proteins were confirmed in the oviduct tissue. The levels of MUC5B, ORM1, RTBDN, and TENP mRNA were significantly high in the oviduct, and the genes were repressed in the regression phase, whereas these were expressed in the recrudescence phase, particularly in the luminal epithelium and glandular epithelium of the oviduct, during molting. Moreover, we observed that gene expression was induced in the magnum, the site for the secretion of egg white components. Next, differences in expression levels of the four genes in normal and cancerous ovaries were compared. Collectively, results suggest that the four selected genes are expressed in the female chicken reproductive tract in response to hormonal regulation, and egg white protein-encoding genes may serve as modulators of the reproductive system in hens.

Placenta-derived trophoblast extracellular vesicles contain unique miRNAs that inhibit ovarian cancer cell growth

Abstract Previous reports have indicated that placental trophoblast extracellular vesicles (EVs) possess unique properties that enable them significantly to inhibit the proliferation of ovarian cancer cells in vitro and slow ovarian tumour growth in an in vivo model, while EVs derived from monocytes did not. However, the mechanisms underlying the inhibitory effects of trophoblast EVs remain unclear. In this study, we characterized the microRNAs (miRNAs) uniquely present in placental trophoblast EVs but absent from THP-1 monocyte-derived EVs. Through bioinformatic analysis, we elucidated the potential involvement of these unique miRNAs in the negative regulation of proliferation pathways implicated in ovarian cancer. Functional assays demonstrated that placental trophoblast EVs inhibited ovarian cancer cell proliferation, and this effect was reversed upon blocking EV uptake, indicating the transfer of the contents of the EVs as a crucial mechanism modulating cancer cell viability. Using miRNA mimics, we also demonstrated that specific miRNAs from placental trophoblast EVs exhibited inhibitory effects on ovarian cancer cell proliferation, highlighting the potential of placental trophoblast EVs as therapeutic agents. These findings not only shed light on the molecular mechanisms underlying the therapeutic efficacy of placental trophoblast EVs but also provide valuable insights into the potential development of miRNA-based therapies for ovarian cancer, including the use of trophoblast EVs as a therapeutic for ovarian cancer.