Evaluation of Rap1GAP and Epac1 Gene Expression in Endometriosis

Objective: Endometriosis is a female reproductive system disease in which endometrial tissue are found in other women organs. Various factors are effective in the development of endometriosis and due to the interaction of genetics and environmental factors, this disease is a multifactorial disease. MAPK/ERK and PI3K/Akt/mTOR pathways are activated by growth factors and steroid hormones and known as two important pathways involved in the processes of growth, proliferation and survival of endometriosis cells. Raps, monomeric GTPase of Ras family, are able to activate these pathways independently of Ras. The goal of our study was to evaluated the expression level of Rap1GAP and Epac1 gene, as two important RapGAPs (GTPase-activating proteins) and RapGEFs (guanine nucleotide exchange factors) respectively, in endometriosis tissues and normal endometrium tissues.Materials and Methods: In this study, 15 samples of women without signs of endometriosis were taken as control samples, 15 ectopic and 15 eutopic samples were taken from women with endometriosis using laparoscopic surgery. The expression of Epac1 and Rap1GAP genes was investigated by Real-time PCR technique and results were analysis by One-Way ANOVA test.Results: Epac1 upregulated significantly in ectopic tissues compared to eutopic and control tissues (Their P-value were <0.0001). Rap1GAP expression was lower in ectopic tissues compared to control samples (P-value was 0.003) and eutopic tissues (P-value was 0.001).Conclusion: Based on these results, it may be concluded that changes in the expression of the Rap1GAP and Epca1 genes may play role in the pathways involved in the pathogenesis, displacement, and migration of endometriosis cells.

Homing Peptide-Based Targeting of Tenascin-C and Fibronectin in Endometriosis

The current diagnostic and therapeutic strategies for endometriosis are limited. Although endometriosis is a benign condition, some of its traits, such as increased cell invasion, migration, tissue inflammation, and angiogenesis are similar to cancer. Here we explored the application of homing peptides for precision delivery of diagnostic and therapeutic compounds to endometriotic lesions. First, we audited a panel of peptide phages for the binding to the cultured immortalized endometriotic epithelial 12Z and eutopic stromal HESC cell lines. The bacteriophages displaying PL1 peptide that engages with angiogenic extracellular matrix overexpressed in solid tumors showed the strongest binding to both cell lines. The receptors of PL1 peptide, tenascin C domain C (TNC-C) and fibronectin Extra Domain-B (Fn-EDB), were expressed in both cells. Silver nanoparticles functionalized with synthetic PL1 peptide showed specific internalization in 12Z and HESC cells. Treatment with PL1-nanoparticles loaded with the potent antimitotic drug monomethyl auristatin E decreased the viability of endometriotic cells in 2D and 3D cultures. Finally, PL1-nanoparticless bound to the cryosections of clinical peritoneal endometriotic lesions in the areas positive for TNC-C and Fn-EDB immunoreactivities and not to sections of normal endometrium. Our findings suggest potential applications for PL1-guided nanoparticles in precision diagnosis and therapy of endometriosis.

Metalloproteinases in Endometrial Cancer—Are They Worth Measuring?

Endometrial cancer is one of the most common gynecological malignancies, yet the molecular mechanisms that lead to tumor development and progression are still not fully established. Matrix metalloproteinases (MMPs) are a group of enzymes that play an important role in carcinogenesis. They are proteases involved in the degradation of the extracellular matrix (ECM) that surrounds the tumor and the affected tissue allows cell detachment from the primary tumor causing local invasion and metastasis formation. Recent investigations demonstrate significantly increased metalloproteinase and metalloproteinase inhibitor levels in patients with endometrial cancer compared to those with normal endometrium. In this review, we aim to show their clinical significance and possible use in the diagnosis and treatment of patients with endometrial cancer. We have critically summarized and reviewed the research on the role of MMPs in endometrial cancer.

Demethylation of Secreted Frizzled-Related Protein2( SFRP2) Promoter Upregulates Wnt/β-Catenin Activity in Endometriosis

Wnt/β-catenin signalling contributes to the metastasis and invasion in the etiology and pathogenesis of endometriosis (EMS), but why the WNT pathway is dysregulated in EMS remains unclear. This study aimed to explore the effects of demethylation of SFRP2 promoter on the Wnt/β-catenin activity in EMS. Aberrantly methylated-differentially expressed genes were identified from GEO database microarray data. 5 ectopic endometrium and 5 normal endometrium were get, subsequently, ectopic endometrium epithelial cells (EEECs) and normal endometrium epithelial cells (NEECs) were isolated in vitro. MSP, BSP, luciferase reporter assay, Lentivirus infection of high expression of SFRP2 gene vector, low expression of DNMT1 gene vector, and 5-Aza stimulation, RT-PCR and western blot were performed in the tissues or cells. It was found that compared with the normal endometrium and NEECs, the RNA and protein expression levels of SFRP2 were significantly increased while the SFRP2 promoter was demethylated in ectopic endometrium and EEECs. The 5-Aza treatment significantly upregulated SFRP2 mRNA and protein levels in EEECs. Furthermore, after the knockdown of DNMT1 expression, the demethylation of the SFRP2 promoter and upregulation of SFRP2 mRNA and protein in EEECs were observed. Meanwhile, the expression of lentivirus carrying SFRP2 cDNA up-regulates the activity of Wnt signaling and the protein expression of β-catenin in EEECs. In summary, the increased SFRP2 expression-induced Wnt/β-catenin signaling due to the demethylation of the SFRP2 promoter plays an important role in the pathogenesis of EMS, suggesting that SFRP2 might be a therapeutic target for EMS treatment.

LC3 and NLRC5 Interaction Inhibits NLRC5-Mediated MHC Class I Antigen Presentation Pathway in Endometrial Cancer

Background: The major histocompatibility complex class I (MHC- I) transactivator, nucleotide binding oligomerization domain-like receptor family caspase recruitment domain containing 5 (NLRC5), serves as a target for immune evasion in many cancers, including endometrial cancer (EC). An inhibition of autophagy can contribute to immunotherapy by assisting the MHC-I-mediated antigen presentation in cancer. However, the underlying mechanism for autophagy-regulated MHC-I in EC remains unclear. Our study aimed to investigate the effect of autophagy on NLRC5 and MHC-I-mediated antigen presentation, and to identify the potential mechanisms underlying this process in EC.Methods: We examined the levels of autophagy and MHC-I genes by performing transmission electron microscopy (TEM), RNA-seq sequencing, western blotting, and qRT-PCR. The t-test, F-test, Kaplan-Meier analysis, and Pearson’s correlation analysis were used for statistical evaluations of tissue microarrays. Immunofluorescence staining, co-immunoprecipitation (CO-IP), and glutathione S-transferase (GST) pull-down assay were performed. HEC-1A, AN3CA, and Ishikawa EC cells were transfected designed, and the role of LC3 and NLRC5 in MHC-I-mediated antigen presentation in EC was further evaluated in a xenotransplantation model of HEC-1A cell line. Results: Autophagy was upregulated in EC endometrium as compared to that in normal endometrium. MHC I and NLRC5 expressions were lower in EC endometrium than in normal endometrium. Autophagy played a negative role in the MHC-I genes expression in vitro. Furthermore, a negative correlation was found between LC3 and NLRC5 levels, and LC3 interacted with NLRC5 to inhibit NLRC5-mediated MHC-I antigen presentation pathway in vitro and in vivo. Conclusion: An upregulation of LC3 in EC patients may contribute to tumor immune escape by restricting the NLRC5-mediated MHC-I antigen presentation pathway, suggesting that inhibiting LC3 and promoting NLRC5 may be a promising immunotherapy strategy in the management of EC.

Functional Interaction Between S100A1 and MDM2 May Modulate p53 Signaling in Normal and Malignant Endometrial Cells

Background S100A1 expression is deregulated in a variety of human malignancies, but its role in normal and malignant endometrial cells is unclear. Methods We used endometrial carcinoma (Em Ca) cell lines to evaluate the physical and functional interaction of S100A1 with p53 and its negative regulator, mouse double minute 2 (MDM2). We also evaluated the expression of S100A1, p53, and MDM2 in clinical samples consisting of 89 normal endometrial and 189 Em Ca tissues. Results S100A1 interacted with MDM2 but not p53 in Em Ca cell lines. Treatment of cells stably overexpressing S100A1 with Nutlin-3A, an inhibitor of the p53/MDM2 interaction, increased expression of p53-target genes including p21waf1 and BAX. S100A1 overexpression enhanced cellular migration, but also sensitized cells to the antiproliferative and proapoptotic effects of Adriamycin, a genotoxic agent; these phenotypes were abrogated when S100A1 was knocked down using shRNA. In clinical samples from normal endometrium, S100A1 expression was significantly higher in endometrial glandular cells of the middle/late secretory and menstrual stages when compared to cells in the proliferative phases; high S100A1 was also positively correlated with expression of MDM2 and p21waf1 and apoptotic status, and inversely correlated with Ki-67 scores. However, such correlations were absent in Em Ca tissues. Conclusion The interaction between S100A1 and MDM2 may modulate proliferation, susceptibility to apoptosis, and migration through alterations in p53 signaling in normal-but not malignant-endometrial cells.

Loss of ten-eleven translocation 1 (TET1) expression as a diagnostic and prognostic biomarker of endometrial carcinoma

Endometrial carcinoma (EC) is the most common gynecological cancer. However, there is currently no routinely used biomarker for differential diagnosis of malignant and premalignant endometrial lesions. Ten-eleven translocation (TET) proteins, especially TET1, were found to play a significant role in DNA demethylation, via conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). TET1, 5-mC, and 5-hmC expression profiles in endometrial carcinogenesis are currently unclear. We conducted a hospital-based retrospective review of the immunohistochemical expression of TET1, 5-mC, and 5-hmC in 181 endometrial samples. A “high” TET1 and 5-hmC expression score was observed in all cases of normal endometrium (100.0% and 100.0%, respectively) and in most samples of endometrial hyperplasia without atypia (90.9% and 78.8%, respectively) and atypical hyperplasia (90.6% and 93.8%, respectively), but a “high” score was found in only less than half of the EC samples (48.8% and 46.5%, respectively). The TET1 and 5-hmC expression scores were significantly higher in normal endometrium and premalignant endometrial lesions than in ECs (p < 0.001). A “high” 5-mC expression score was observed more frequently for ECs (81.4%) than for normal endometrium (40.0%), endometrial hyperplasia without atypia (51.5%), and atypical hyperplasia (53.1%) (p < 0.001). We also found that TET1 mRNA expression was lower in ECs compared to normal tissues (p = 0.0037). TET1 immunohistochemistry (IHC) scores were highly proportional to the TET1 mRNA levels and we summarize that the TET1 IHC scoring can be used for biomarker determinations. Most importantly, a higher TET1 score in EC cases was associated with a good overall survival (OS) rate, with a hazard ratio (HR) of 0.31 for death (95% confidence interval: 0.11–0.84). Our findings suggest that TET1, 5-mC, and 5-hmC expression is a potential histopathology biomarker for the differential diagnosis of malignant and premalignant endometrial lesions. TET1 is also a potential prognostic marker for EC.

Expression of Cell Adhesion Molecules in Endometrial Tissue and Endometrioid Adenocarcinomas

Endometrioid endometrial adenocarcinomas are the most common histological variant of malignant tumors in the uterine cavity. In turn, the features of expression by neoplastic cells of intercellular adhesion molecules are a reliable prognostic factor in the progression of malignant tumors. One of the important indicators of cancer progression is E-cadherin, which determines the strength of intercellular adhesion and the ability of cells to spread. Among other adhesion molecules, considerable attention has recently been paid to the molecules of cell adhesion of carcino-embryonic antigen 1 (MCA-REA1). Therefore, the purpose of the study was to study the expression of E-cadherin and MCA-REA1 in normal endometrium and endometrioid adenocarcinomas. Materials and methods. To achieve this purpose, we performed tissue studies of 10 samples of normal endometrium and 30 samples of endometrioid endometrial adenocarcinoma (8380/3). Morphological features of neoplastic tissue were studied by hematoxylin and eosin staining. Visualization of E-cadherin and MCA-REA1 receptors was determined using the appropriate antibodies and the UltraVision Quanto Detection System HRP DAB Chromogen (Thermo scientific, USA) in similar areas of the tumor on serial sections. Results and discussion. It has been shown that endometrial tissue demonstrates different expression of MCA-REA1 and E-cadherin receptors in the normal state and in endometrioid adenocarcinomas. This indicates the absence of any functional correlation between them. Expression of MCA-REA1 was detected on the apical surface of the luminal and glandular columnar epithelium. In contrast, the endometrioid endometrial carcinoma tissues showed the pronounced heterogeneous location of MCA-REA1 in tumor cells. Moreover, due to the tumor dedifferentiation, these proteins disappear from the cell surface. On the other hand, E-cadherin is normally localized in intercellular contacts and epithelial-mesenchymal junctions. During carcinoma dedifferentiation, the intensity of E-cadherin expression decreased, which was accompanied by an increase in nuclear polymorphism of cancer cells and focal separation of cells from the total tumor mass. Conclusion. The variability of the expression patterns of MCA-REA1 and E-cadherin in the dedifferentiation of endometrioid adenocarcinoma may be an indicator of neoplastic transformation and progression of the malignant process

Risk Factors for Typical Endometrial Polypoid Adenomyoma

Objective: Typical endometrial polypoid adenomyoma (TPA) is a rare type of lesion in the uterine cavity or cervical canal. Although the clinical and pathologic features of TPA have been described extensively, reports on its pathogenesis and treatment remain inconclusive. This study was conducted to investigate the risk factors for endometrial TPA and recurrence and to further define the pathogenesis and treatment.Study design: This was a retrospective analysis of 488 cases of TPA and 500 cases of normal endometrium. Age, menopausal status, body mass index (BMI), reproductive history, and method of surgery were retrospectively analyzed. Among TPA cases, 360 were treated by conservative surgery. Risk factors for TPA and recurrence were assessed.Results: There was a significant difference in age, menopausal status, BMI, gravidity, and parity between the two groups (P<0.05). Age >50 years, menopause, obesity, gravidity >3, and parity >2 were risk factors for TPA (P<0.05). The incidence rate of endometriosis and adenomyosis in the TPA group was significantly higher than that in the normal endometrium group (P<0.05). Follow-up data (22–77 months) were obtained for 360 patients, revealing gravidity >3, menopause, curettage, and polyp clamp as independent risk factors for the recurrence of TPA (P<0.05). Conclusions: In addition to high estrogen levels, endometrial injury was the main contributor to TPA pathogenesis. Hysteroscopic electrotomy was the preferential treatment for TPA to avoid recurrence, especially for women with risk factors. Increasing the depth of ablation may prevent the recurrence of TPA more efficiently.

Role of SIRT1 and Progesterone Resistance in Normal and Abnormal Endometrium

Context Progesterone resistance, a known pathologic condition associated with a reduced cellular response to progesterone and heightened estrogen responses, appears to have a normal physiologic role in mammalian reproduction. The molecular mechanism responsible for progesterone resistance in normal and abnormal endometrium remains unclear. Objective To examine the roles of Sirtuin-1 (SIRT1) in normal endometrium as well as endometrium associated with infertility and endometriosis, as an epigenetic modulator associated with progesterone resistance. Methods SIRT1 expression was examined by Western blot, RT-qPCR and immunohistochemistry in mouse uterus and human endometrium. Mice with uterine specific Sirt1 overexpression were developed to examine SIRT1’s role in endometrial function and endometriosis development. EX-527, a SIRT1 inhibitor, and SRT1720, a SIRT1 agonist, were also used to evaluate SIRT1 effect on endometriosis. Results In normal healthy women, endometrial SIRT1 is expressed only during menses. SIRT1 was dramatically overexpressed in the endometrium from women with endometriosis in both the epithelium and strom. In mice, SIRT1 is expressed at the time of implantation between day 4.5 and 5.5 of pregnancy. Overexpression of SIRT1 (Sirt1 over) in the mouse uterus leads to subfertility due to implantation failure and decidualization defects and progesterone resistance. SIRT1 overexpression in endometriotic lesion promotes worsening endometriosis development. EX-527 (SIRT1 inhibitor) significantly reduced the number of endometriotic lesions in the mouse endometriosis model. Conclusions SIRT1 expression and progesterone resistance appears to play -roles in normal endometrial functions. Aberrant SIRT1 expression contributes to progesterone resistance and may participate in the pathophysiology of endometriosis. SIRT1 is a novel and targetable protein for the diagnosis as well as treatment of endometriosis and the associated infertility seen in this disease.