Search

Recent studies have shown that autophagy plays an important role in gynecologic tumors, and ubiquitin modification of autophagy regulatory components is essential to regulate autophagic flux. In this study, we found that the ubiquitin-conjugating enzyme E2C (UBE2C) affects endometrial cancer cell apoptosis and proliferation by inhibiting autophagy. Electron microscopy observation of cell ultrastructure and experimental biochemical analysis showed that endometrial cancer cells with UBE2C expression knocked down display typical autophagic characteristics. Cells were cotreated with the autophagy pharmacologic inhibitors chloroquine and/or bafilomycin A1, and mRFP-GFP-LC3 assays were performed to monitor autophagic flux and determine whether UBE2C suppresses the autophagy program. Investigation of the corresponding mechanism by which UBE2C inhibits autophagy revealed that UBE2C induces K48-linked SIRT1 ubiquitination and promotes ubiquitination-dependent degradation of SIRT1, subsequently reducing H4K16 deacetylation levels and epigenetically inhibiting the expression of autophagy-related genes. The results of cell counting kit-8, Hoechst staining, and immunofluorescence assays further indicated that deletion of the autophagy-related gene BECN1 significantly attenuates UBE2C knockdown–induced cell apoptosis. Moreover, overexpression of UBE2C promoted tumor growth in the xenograft mice model. While, the introduction of rapamycin, an agonist of autophagy, successfully reversed tumor growth and apoptosis inhibition mediated by UBE2C overexpression in vitro and in vivo. Taken together, our results suggested that UBE2C-mediated ubiquitination and degradation of SIRT1 contribute to the malignant progression of endometrial cancer through epigenetic inhibition of autophagy. Implications: Our study highlights the tumorigenic role and regulatory mechanism of UBE2C in endometrial cancer; UBE2C inhibits endometrial cancer cell apoptosis through autophagy-related mechanisms and our findings provide new insights into the treatment of endometrial cancer.

Figure S8

Table S1. Primers for qPCR

Recent studies have shown that autophagy plays an important role in gynecologic tumors, and ubiquitin modification of autophagy regulatory components is essential to regulate autophagic flux. In this study, we found that the ubiquitin-conjugating enzyme E2C (UBE2C) affects endometrial cancer cell apoptosis and proliferation by inhibiting autophagy. Electron microscopy observation of cell ultrastructure and experimental biochemical analysis showed that endometrial cancer cells with UBE2C expression knocked down display typical autophagic characteristics. Cells were cotreated with the autophagy pharmacologic inhibitors chloroquine and/or bafilomycin A1, and mRFP-GFP-LC3 assays were performed to monitor autophagic flux and determine whether UBE2C suppresses the autophagy program. Investigation of the corresponding mechanism by which UBE2C inhibits autophagy revealed that UBE2C induces K48-linked SIRT1 ubiquitination and promotes ubiquitination-dependent degradation of SIRT1, subsequently reducing H4K16 deacetylation levels and epigenetically inhibiting the expression of autophagy-related genes. The results of cell counting kit-8, Hoechst staining, and immunofluorescence assays further indicated that deletion of the autophagy-related gene BECN1 significantly attenuates UBE2C knockdown–induced cell apoptosis. Moreover, overexpression of UBE2C promoted tumor growth in the xenograft mice model. While, the introduction of rapamycin, an agonist of autophagy, successfully reversed tumor growth and apoptosis inhibition mediated by UBE2C overexpression in vitro and in vivo. Taken together, our results suggested that UBE2C-mediated ubiquitination and degradation of SIRT1 contribute to the malignant progression of endometrial cancer through epigenetic inhibition of autophagy.Implications:Our study highlights the tumorigenic role and regulatory mechanism of UBE2C in endometrial cancer; UBE2C inhibits endometrial cancer cell apoptosis through autophagy-related mechanisms and our findings provide new insights into the treatment of endometrial cancer.

Rui Shi, Ziwei Wang, Jun Zhang, Zhicheng Yu, Lanfen An, Sitian Wei, Dilu Feng, Hongbo Wang Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, People’s Republic of ChinaCorrespondence: Dilu Feng; Hongbo WangDepartment of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, People’s Republic of ChinaTel +86 027 85350583Fax +86 278 572 6362Email fengdilu@126.com; hb_wang1969@sina.comPurpose: Endometrial cancer (EC) is a common gynaecologic malignancy with an increasing incidence rate and mortality in recent years. N6-methylandenosine (m6A)-related long noncoding RNA (lncRNA) plays a vital role in EC, emerging as one of the most abundant RNA modifications.Materials and Methods: The Cancer Genome Atlas (TCGA) database and UCSC Xena were used to download data related to EC. Survival and univariate and multifactorial prognostic analyses were performed for m6A-related lncRNAs. The expression levels of the three lncRNAs were verified using q-PCR. A nomogram was used to create a clinical tool to assess overall survival. To investigate the relationship between m6A-related lncRNA and EC, we downloaded differential genes related to EC from the TCGA database and mined three m6A-related lncRNAs, namely SCARNA9, TRAF3IP2-AS1, and AL133243.2. The data were categorized into high- and low-risk groups based on m6A-associated lncRNA.Results: Survival analysis revealed that the high-risk group had a lower survival rate. Survival analysis of three m6A-associated lncRNAs revealed that cases with high expression of SCARNA9 tended to have a poorer prognosis, whereas the opposite was true for TRAF3IP2-AS1, AL133243.2. Univariate and multifactorial prognostic analyses suggested statistical differences in patients’ age, FIGO stage, pathological grade, risk score, and prognosis of EC, which was confirmed by results of the separate prognostic factor analysis for the three lncRNAs. Risk status was validated as an independent prognostic indicator, and the prognostic nomogram combined patient age, pathological stage, and FIGO classification to assess 3– 5-year survival. Cases from high- and low-risk groups were analysed for the tumour microenvironment and immune cell scores, and stromal cell scores were found to be lower in the high-risk group. Correlations were analysed using different databases for immune cell classification.Conclusion: m6A-related lncRNAs may play a key role in the diagnosis and treatment of EC as targets of prognosis and the immune microenvironment.Keywords: N6-methylandenosine, long noncoding RNAs, endometrial cancer, tumour microenvironment

Zhicheng Yu,* Sitian Wei,* Jun Zhang, Rui Shi, Lanfen An, Dilu Feng, Hongbo Wang Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hongbo WangDepartment of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of ChinaTel +86 137-2022-8749Fax +86 027 857-26114Email hb_wang1969@sina.comObjective: Existing prognostic models for endometrial cancer are short of facility and effective validation. In this study, we aim to develop and validate a novel prognostic model for endometrial cancer based on clinical characteristics.Methods: The clinical data such as age, BMI (body mass index), FIGO stage, surgical approach, myometrial invasion, grade, lymph node metastasis, pathology and menopause status were collected for constructing and validating the prognostic model from The Cancer Genome Atlas (TCGA) and Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, respectively. COX regression and the least absolute shrinkage and selection operator (LASSO) COX were applied to identify the significant predictors of overall survival (OS) and construct the prognostic model. The discrimination, calibration, and clinical usefulness of the model were evaluated in both cohorts.Results: Three hundred and sixty-seven and 286 EC patients were collected for training and validation cohort, respectively. A clinical prognostic model integrating six clinical variables including age, BMI, FIGO stage, surgical approach, myometrial invasion and grade was established. K-M analysis shows a significant difference between the low- and high-risk groups. The area under the receiver operating characteristic curve (AUC-ROC) was 0.775 (95% CI, 0.708 to 0.843) and 0.870 (95% CI, 0.758 to 0.982) for the training and validation cohorts which indicating reliable discrimination. The calibration curve revealed excellent predictive accuracy and the Hosmer–Lemeshow test also verified this. Decision curve analysis (DCA) for the prognostic model indicated that it would add more benefits than either the detect-all-patients scheme or the detect-none scheme. In addition, our model has a superior AUC comparing with any single factor as predicting OS.Conclusion: Our predictive model offers a convenient and accurate tool for clinicians to estimate the prognosis of EC patients.Keywords: clinical characteristics, endometrial cancer, prognostic model, TCGA

Endometrial cancer (EC) is commonly diagnosed cancer in women, and the prognosis of advanced types of EC is extremely poor. Kinesin family member 2C (KIF2C) has been reported as an oncogene in cancers. However, its pathophysiological roles and the correlation with tumor-infiltrating lymphocytes in EC remain unclear. The mRNA and protein levels of KIF2C in EC tissues were detected by qRT-PCR, Western blot (WB), and IHC. CCK8, Transwell, and colony formation assay were applied to assess the effects of KIF2C on cell proliferation, migration, and invasion. Cell apoptosis and cell cycle were analyzed by flow cytometry. The antitumor effect was further validated in the nude mouse xenograft cancer model and humanized mouse model. KIF2C expression was higher in EC. Knockdown of KIF2C prolonged the G1 phases and inhibited EC cell proliferation, migration, and invasion in vitro. Bioinformatics analysis indicated that KIF2C is negatively correlated with the infiltration level of CD8+ T cells but positively with the poor prognosis of EC patients. The apoptosis of CD8+ T cell was inhibited after the knockdown of KIF2C and was further inhibited when it is combined with anti-PD1. Conversely, compared to the knockdown of KIF2C expression alone, the combination of anti-PD1 further promoted the apoptosis of Ishikawa and RL95-2 cells. Moreover, the knockdown of KIF2C inhibited the expression of Ki-67 and the growth of tumors in the nude mouse xenograft cancer model. Our study found that the antitumor efficacy was further evaluated by the combination of anti-PD1 and KIF2C knockdown in a humanized mouse model. This study indicated that KIF2C is a novel prognostic biomarker that determines cancer progression and also a target for the therapy of EC and correlated with tumor immune cells infiltration in EC.

Kinesin family member 4A (KIF4A) belongs to the kinesin superfamily proteins, which are closely associated with mitophagy. Nonetheless, the role of KIF4A in endometrial cancer (EC) remains poorly characterized. The present study showed that KIF4A not only was upregulated but also predicted poor prognosis in patients with EC. KIF4A knockdown in EC cells resulted in attenuated proliferative capacity in vitro and in vivo. Transcriptome sequencing and gene function analysis revealed that KIF4A contributed to the maintenance of EC cells' genomic stability and that KIF4A knockdown induced the DNA damage response, cell cycle arrest, and apoptosis. Mechanistically, KIF4A interacted with TPX2 (a protein involved in DNA damage repair to cope with the replication pressure) to enhance its stability via inhibition of TPX2 ubiquitination and eventually ensured the genomic stability of EC cells during mitosis. Taken together, our results indicated that KIF4A functions as a tumor oncogene that facilitates EC progression via the maintenance of genomic stability. Therefore, targeting the KIF4A/TPX2 axis may provide new concepts and strategies for the treatment of patients with EC.

Endometrial cancer (EC) is one of the most common gynecologic malignancies with increasing morbidity. Cell-cell and cell-matrix interactions within the tumour microenvironment (TME) exert a powerful influence over the progression of EC. Therefore, a comprehensive exploration of heterogeneity and intratumoral crosstalk is essential to elucidate the mechanisms driving EC progression and develop novel therapeutic approaches.4 EC and 2 normal endometrium samples were applied for single-cell RNA sequencing (scRNA-seq) analysis. In addition, we also included the public database to explore the clinical benefits of the single cell analysis.9 types of cells were identified with specific expression of maker genes. Both the malignant epithelial cells and cells comprising the immune microenvironment displayed a high degree of intertumoral heterogeneity. Notably, the proliferation T cells also showed an exhausted feature. Moreover, the malignant cells may induce an immunosuppressive microenvironment through TNF-ICOS pair. Cancer-associated fibroblasts (CAFs) were divided into four subsets with distinct characteristics and they maintained frequent communications with malignant cells which facilitating the progression of EC. We also found that the existence of vascular CAF (vCAF) may indicate a worse prognosis for EC patients through integrating TCGA database.The TME of human EC remains highly heterogeneous. Out finding that malignant cells interact closely with immune cells and vCAFs identifies potential therapeutic targets.

This study was aimed to explore the expression and mechanism of the transcription factor YAP-TEAD in the Hippo signaling pathway under the regulation of non-coding Ribonucleic Acid (RNA) LINC00857 in the proliferation of ovarian cancer cells, so as to provide a scientific research basis for clinical diagnosis and treatment of ovarian cancer. In the study, the ovarian cancer cell lines (BT 549) were rolled into a control group (normal culture-defined as BT549/NC) and a response group (transfected with non-coding RNA LINC00857 cultured cells-defined as BT 549YAP cells). The expression and proliferation ability of the transcription factor YAP-TEAD in the two groups of cancer cells were analyzed and compared. The results showed that the YAP-TEAD expression rate was the highest in Bt549 cells; the YAP content grade (0.18) in BT 549-YAP cells was lower than BT 549/NC (0.2) after transfection (P< 0.05); and the apoptotic rate of the response group (80%) was higher than that of the control group (25%) after the intervention. With the extension of culture time, the expression of CCN1 mRNA decreased (P< 0.05), and CCN2 mRNA increased (P< 0.05). After 12, 24, 36, and 48 hours, the apoptosis rate of the reaction group at different time points was higher than that of the control group (P< 0.01). When YAP-TEAD was down-regulated, the in vitro proliferation ability of BT 549- YAP cells was weakened compared with BT 549/NC and parental cells. It was concluded that the noncoding RNA LINC00857 can target the transcription factor YAP-TEAD in the Hippo signaling pathway to decrease its expression, thus inhibiting the proliferation, migration, and invasion of cancer cells, and promoting cell apoptosis.

Ovarian cancer is one of the most common gynecological cancers with high morbidity and mortality, which seriously endangers women's health and quality of life. Long noncoding RNAs (lncRNAs) can regulate the progression of cancers, including ovarian cancer. LINC00857 (long intergenic non‐protein coding RNA 857) has been discovered to be a crucial factor in the regulation of cancer development. Nevertheless, the specific functions and mechanisms of LINC00857 in ovarian cancer remain unclear. The Hippo signaling pathway can involve in cancer progression. In our research, we aimed to investigate the correlation of LINC00857 and Hippo pathway. Quantitative real‐time polymerase chain reaction assay was utilized to test the expression of LINC00857 in ovarian cancer tissues and cells. Functional experiments revealed that LINC00857 silencing led to the inhibition on cell proliferation, migration, invasion, and glycolysis but accelerated cell apoptosis in ovarian cancer. Mechanism experiments, including RNA immunoprecipitation, RNA pull‐down, and luciferase reporter experiments demonstrated that LINC00857 could regulate YAP1 (Yes1 associated transcriptional regulator) by competitively binding to miR‐486‐5p in ovarian cancer. In a word, this study unveiled that LINC00857 regulates YAP1 by competitively binding to miR‐486‐5p and accelerates ovarian cancer progression., MiR‐486‐5p restrains cell progression and promotes cell apoptosis in ovarian cancer. Long intergenic non‐protein coding RNA 857 accelerates ovarian cancer progression and glycolysis via regulating Yes1 associated transcriptional regulator.

Background: Data regarding human papillomavirus (HPV) prevalence and genotype distribution are limited in Shannan City, Tibet Tibetan Autonomous Region, China. The purpose of this study is to provide reliable data for guiding women in Shannan City in cervical cancer screening and HPV vaccine innoculation. Methods: HPV testing was performed on women aged 16 to 109 years (mean age 44.03 ± 9.25 years) from Shannan City in 2019 and 2020, which was implemented technically by gynecological examination, vaginal discharge smear microscopy, cytology, and HPV detection. The overall prevalence, age-specific prevalence, and genotype distribution were analyzed. Results: A total of 48,126 women received HPV testing, of which 3,929 were detected human papillomavirus. The HPV-positive rate was 8.16% (3,929/48,126), and the highest prevalence was in the ≤ 25-year-old age group (12.68%). After the age of 25, the prevalence rate decreased rapidly, and then slowly increased from 7.49% in the 46-55 age group to 9.82% in the ≥ 66 age group, showing a “U-shaped” pattern. The positive prevalence of HPV 16 or 18-only was 1.43%, that of other HPV genotypes except HPV 16 or 18 was 6.39%, and mixed HPV infections including HPV 16 or 18 was 0.34%. Conclusions: The HPV infection rate in Shannan city is rather low, and the age-specific prevalence of HPV infection presents a “U” curve, suggesting the importance of screening among younger women and the necessity of detection among older women.

Background Activation of NLPR3 inflammasome is associated with the development and progression of some types of malignant tumors, but its role in endometrial cancer is unclear. This study aimed to investigate the expression and function of NLRP3 inflammasome in endometrial cancer. Materials and methods The expression levels of NLRP3, its inflammasome components and estrogen receptor β in endometrial cancer and paired non-tumor tissues were detected. The effects of NLPR3 silencing or overexpression on the proliferation, migration, and invasion of Ishikawa and HEC-1A cells were determined. The impact of NLPR3 silencing on the growth of implanted tumors was determined in vivo. The effects of estrogen on NLPR3 inflammasome activation and Ishikawa cell proliferation were determined. Results The upregulation of NLRP3, ASC, caspase-1, and IL-1β was associated with the progression of endometrial cancer and poor survival. NLPR3 silencing inhibited the proliferation, migration, and invasion of endometrial cancer cells while NLPR3 overexpression had opposite effects. NLPR3 silencing reduced IL-1β and caspase-1 expression and the growth of implanted endometrial tumors, accompanied by decreased pro-IL-1β maturation. Estrogen enhanced NLPR3, ERβ, pro-IL-1β, IL-1β expression, and endometrial cancer cell proliferation, which were mitigated by treatment with ERβ inhibitor but not ERα inhibitor. Conclusion Our results suggest that estrogen acts through ERβ to enhance the activation of NLPR3 inflammasome and promote the progression of endometrial cancer. NLPR3 inflammasome may be a new therapeutic target for endometrial cancer.

Progesterone, particularly medroxyprogesterone acetate (MPA) has been mainly used for young endometrial carcinoma (EC) patients with conservative treatment. However, its treatment benefits are limited by insensitivity or acquired resistance. In this study, we aim to investigate the effect of long non-coding RNA HOTAIR on progesterone sensitivity in endometrial cancer, as well as the underlying mechanisms. The expression of HOTAIR was detected by quantitative real-time PCR. The impact of MPA on the endometrial cancer cells was examined by MTT, colony formation, apoptosis-related protein detection and flow cytometry. Chromatin immunoprecipitation (ChIP) assay was performed to detect the regulatory mechanism between HOTAIR and progesterone receptor B (PRB). We further confirm the function of HOTAIR in vivo though xenograft tumor assay. We found that HOTAIR inversely correlated with PRB expression in endometrial carcinoma. Knockdown of HOTAIR promoted the MPA sensitivity by upregulating PRB, which can be largely reversed by PRB downregulation. Moreover, inhibiting LSD1, a HOTAIR-associated protein that removed activating H3K4me2 chromatin marks, induced PRB expression and promoted apoptosis induced by MPA. We further showed that silencing HOTAIR strengthened the H3K4me2 occupation on the promotor of PRB. Our findings provide compelling evidence that HOTAIR and LSD1 collaboratively repress PRB expression and thus mediate progesterone sensitivity in endometrial carcinoma cells. HOTAIR is a potential predictor for progesterone response in EC and down-regulated expression of HOTAIR might be an effective strategy for overcoming progesterone resistance.

Background: The outbreak of Coronavirus Disease 2019 (COVID-19) is threatening a surging number of populations worldwide, including women in breastfeeding period. Limited evidence is available concerning breastfeeding in women with COVID-19. Methods: Twenty-three pregnant women and puerperae were enrolled in the study. To evaluate the effect of breastfeeding on SARS-CoV-2 transmission, the presence of SARS-CoV-2, IgG and IgM in breast milk, maternal blood and infant blood were assessed. Feeding patterns were also recorded in follow-up. Results: No positive detection for SARS-CoV-2 of neonates was found. All breast milk samples were negative for the detection of SARS-CoV-2. The presence of IgM ofSARS-CoV-2 in breast milk was correlated with maternal blood. The results of IgG detection for SARS-CoV-2 were negative in all breast milk samples. All the infants were in healthy condition while six of them were fed with whole or partial breast milk. Eight infants received antibody test for SARS-CoV-2 in one month after birth and the results were all negative. Conclusion: Findings from this small number of cases suggest that there is currently no evidence for mother-to-child transmission via breast feeding in women with COVID-19 in the third trimester and puerperium.

Objective To explore the possible mechanism of protein kinase CK2, which participates in estrogen recruitment of endothelial progenitor cells (EPCs), and its role in the angiogenesis of endometriosis lesions. Design Laboratory study. Setting University. Animal(s) BALB/c mice. Intervention(s) Exposure of human endometrial stromal cells (HESCs) to estrogen and CK2 inhibitor CX-4945 and endometrial stromal cells transfected with the protein kinase CK2 vector (HESC-CK2). Endometriosis models were induced by allogeneic mice transplantation of the endometrium into dorsal skinfold chambers. The mice received an IP injection of 50 mg/kg emodin per day or were treated with 100 μg/kg estrogen by SC injection once a week. Main Outcome Measure(S) The concentration of cytokines in cells was measured with ELISA. The migration of EPCs was examined using the scratch assay method and Transwell, a capillary tube-formation assay to determine EPC tube-forming capacity, and protein and mRNA expression with Western blot and polymerase chain reaction analyses, respectively. Result(s) Protein kinase CK2 participates in estrogen-mediated EPC homing to endometriotic lesions through stromal cells in a stromal cell–derived factor-1 (SDF-1)-CXCR4-dependent manner. Conditioned medium from endometrial stromal cells that were stably transfected with the protein kinase CK2 vector (HESC-CK2) or pretreated with estrogen significantly enhanced the migration and recruitment of EPCs. In contrast, conditioned medium from HESCs that were treated with CX-4945, a selective inhibitor of CK2, inhibited the mobility and viability of EPCs. Furthermore, CK2 overexpression significantly upregulated SDF-1 expression and secretion in endometrial stromal cells by activating the AKT/mTOR pathway. Moreover, treatment with the SDF-1 receptor CXCR4-specific inhibitor AMD3100 completely reversed the CK2-enhanced migration of EPCs. Conclusion(s) This study demonstrates that CK2 participates in estrogen-mediated EPC homing to endometriotic lesions through stromal cells in an SDF-1-CXCR4-dependent manner and may be a therapeutic target.

Epithelial-mesenchymal transition (EMT) is a major cause of endometrial cancer (EC) to initiate invasion and metastasis. S100A4, a calcium-binding protein, is implicated in multistage of tumorigenesis and tumor progression. The correlation between S100A4 and EMT in EC is still unclear. This study was aimed to clarify the role of S100A4 in EC and the relationship between S100A4 expression and EMT markers. S100A4, E-cadherin, and vimentin were detected in tissues of EC patients (n=50) by immunohistochemistry. The impact of S100A4 on EC cell proliferation, migration and invasion was investigated via RNA interference, and the correlation between S100A4 and EMT markers were also explored. The results showed that S100A4 was significantly increased in epithelial cells of EC compared with the normal endometrium (P

Studies have shown that calcium sensing receptor (CaSR) is involved in the progressions of several human cancers. However, the role of CaSR in endometrial cancer remains unknown. This study provides a preliminary analysis of the CaSR effect on endometrial cancer development. Ectopic CaSR expression by lentiviral transfection (CaSR-OV) in Ishikawa cells significantly increased intracellular calcium ([Ca2+]i) levels and cell apoptosis. E-cadherin and β-catenin expression and complex formation at the membrane were increased in CaSR-OV Ishikawa cells relative to control Ishikawa cells (vector). Furthermore, CaSR-OV Ishikawa cells showed a reduced invasive potential, which was attributed to E-cadherin/β-catenin complex formation. Moreover, a reduction in CaSR expression in endometrial cancer relative to normal specimens was evident by immunohistochemistry and was positively associated with E-cadherin, but not β-catenin, expression. Furthermore, VEGFR3 was significantly down-regulated in CaSR-OV Ishikawa cells. Additionally, an immunohistochemical analysis showed that VEGFR3 was significantly increased in endometrial cancer compared with the normal endometrium and was inversely correlated with CaSR expression. However, the CaSR knockdown produced the opposite effects. These findings suggest an inhibitory role for CaSR in endometrial cancer. Therefore, reduced CaSR expression may be a suitable explanation and valuable predictor for endometrial cancer progression.

S100 calcium binding protein A4 (S100A4) is a well‑established tumor metastasis mediator in various malignancies, including endometrial cancer (EC). However, the regulatory mechanism underlying S100A4 expression remains elusive. In the present study, by analyzing public datasets and clinical samples, we found that estrogen‑related receptor γ (ERRγ) was upregulated and positively correlated with S100A4 transcription in EC. ERRγ knockdown inhibited S100A4 expression and promoted the expression of its downstream target E‑cadherin, and vice versa. Mechanistic studies indicated that ERRγ enhanced the promoter activity of S100A4 to facilitate its transcription. In addition, knockdown of ERRγ suppressed migration and invasion of EC cells in vitro, while ectopic ERRγ expression promoted migration and invasion of EC cells in vitro and tumor growth in vivo. Importantly, restoration of S100A4 expression prevented EC cells from undergoing ERRγ‑mediated changes in these biological features. In addition, synchronous changes in S100A4 and ERRγ expression were observed after incubation with estrogen. Overall, ERRγ may exert oncogenic activity mainly associated with aggressiveness of EC by activating S100A4 transcription and thus may be a novel therapeutic target in EC.

Summary In endometriosis research, endometriosis-like lesions are usually induced in rodents by transplantation of isolated endometrial tissue fragments to ectopic sites. In the present study, we investigated whether this approach is affected by the cellular composition of the grafts. For this purpose, endometrial tissue fragments covered with luminal epithelium (LE+) and without luminal epithelium (LE-) were transplanted from transgenic green fluorescent protein-positive (GFP+) donor mice into the dorsal skinfold chamber of GFP- wild-type recipient animals to analyze their vascularization, growth and morphology by means of repetitive intravital fluorescence microscopy, histology and immunohistochemistry during a 14-day observation period. LE- fragments developed into typical endometriosis-like lesions with cyst-like dilated endometrial glands and a well vascularized endometrial stroma. In contrast, LE+ fragments exhibited a polypoid morphology and a significantly reduced blood perfusion after engraftment, because the luminal epithelium prevented the vascular interconnection with the microvasculature of the surrounding host tissue. This was associated with a markedly decreased growth rate of LE+ lesions when compared to LE- lesions. Besides, we found that many GFP+ microvessels grew outside the LE- lesions and developed interconnections to the host microvasculature, indicating that inosculation is an important mechanism in the vascularization process of endometriosis-like lesions. Our findings demonstrate that the luminal epithelium crucially affects the vascularization, growth and morphology of endometriosis-like lesions. Therefore, it is of major importance to standardize the cellular composition of endometrial grafts in order to increase the validity and reliability of pre-clinical rodent studies in endometriosis research.

Endometriosis is a frequent gynecological disease, which is crucially dependent on the process of angiogenesis. However, the underlying regulatory mechanisms of blood vessel development are still poorly understood. CK2 is a pleiotropic protein kinase, which is implicated in the regulation of various cellular processes including angiogenesis. Herein we studied for the first time the function of protein kinase CK2 in angiogenesis of endometriotic lesions. For this purpose, we analyzed the anti-angiogenic activity of the CK2 inhibitor quinalizarin in a rat aortic ring assay and its effect on the expression of individual CK2 subunits and on kinase activity in endometrial tissue. Moreover, endometriotic lesions were induced in dorsal skinfold chambers of quinalizarin- and vehicle-treated C57BL/6 mice to study their vascularization and morphology by means of repetitive intravital fluorescence microscopy and histology. Our results demonstrate that quinalizarin dose-dependently inhibits vascular sprouting. In addition, treatment of endometrial tissue with quinalizarin reduces CK2 activity without affecting the expression of the three CK2 subunits α, α' and β. In the dorsal skinfold chamber model of endometriosis, quinalizarin inhibits the vascularization of endometriotic lesions, which exhibit a significantly decreased vascularized area and functional capillary density when compared to those of vehicle-treated controls. This is associated with a reduced lesion size and histological fraction of endometrial glands. These findings indicate that CK2 is a regulator of angiogenesis in endometriotic lesions. Accordingly, inhibition of CK2 represents a novel option in the development of anti-angiogenic strategies for the treatment of endometriosis.

There has been emergence of evidence suggesting that specific variants of the vascular endothelia growth factor (VEGF) family, based on their ability to regulate angiogenesis, would be pivotal in the pathogenesis of endometriosis. This study was aimed at determining whether high levels of VEGF-A could be found in the serum and peritoneal fluid (PF) of patients with endometriosis. VEGF-A levels were measured by enzyme-linked immunosorbent assay (ELISA) in serum and PF from 46 patients with surgically confirmed endometriosis, and 40 controls with no clinical evidence of the disease or detectable endometriotic lesions at the time of surgical examination. The results showed the mean VEGF-A levels were significantly higher in the serum and PF of patients with endometriosis than in the controls. The VEGF-A levels in the serum and PF of patients with severe endometriosis (stages III–IV) were significantly higher than in those with minimal endometriosis (P

Arctigenin, a functional ingredient of several traditional Chinese herbs, has been reported to have potential antitumor activity. However, its mechanisms of action are still not well elucidated. Because the establishment and metastatic spread of tumors is crucially dependent on angiogenesis, here we investigated whether arctigenin inhibits tumor growth by disturbing blood vessel formation. For this purpose, human dermal microvascular endothelial cells were exposed to different arctigenin doses to study their viability, proliferation, protein expression, migration, and tube formation compared with vehicle-treated controls. In addition, arctigenin action on vascular sprouting was analyzed in an aortic ring assay. Furthermore, we studied direct arctigenin effects on CT26.WT colon carcinoma cells. Spheroids of these tumor cells were transplanted into the dorsal skinfold chamber of arctigenin-treated and vehicle-treated BALB/c mice for the in-vivo analysis of tumor vascularization and growth by intravital fluorescence microscopy, histology, and immunohistochemistry. We found that noncytotoxic doses of arctigenin dose dependently reduced the proliferation of human dermal microvascular endothelial cells without affecting their migratory and tube-forming capacity. Arctigenin treatment also resulted in a decreased cellular expression of phosphorylated serine/threonine protein kinase AKT, vascular endothelial growth factor receptor 2, and proliferating cell nuclear antigen and inhibited vascular sprouting from aortic rings. In addition, proliferation, but not secretion of vascular endothelial growth factor, was decreased in arctigenin-treated tumor cells. Finally, arctigenin suppressed the vascularization and growth of engrafting CT26.WT tumors in the dorsal skinfold chamber model. Taken together, these results show for the first time an antiangiogenic action of arctigenin, which may contribute considerably toward its antitumor activity.

Cancer-associated fibroblasts (CAFs) are thought to play an essential role in cancer initiation and development. However, little research has been done to evaluate the role of CAFs in epithelial ovarian cancer (EOC) development. To address this issue, ninety-one specimens were immunostained with α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) antibodies to quantify CAFs, and antibodies D2-40 and CD34 to evaluate the lymphatic vessel density (LVD) and microvessel density (MVD) of the lesions. We found there were no α-SMA or FAP positive fibroblasts in normal ovary tissues. More CAFs were found in EOC than in borderline tumors and benign tumors (P

Objective To study the effect of combretastatin A4 phosphate (CA4P) on the vascularization of endometriotic lesions. Design Intravital microscopic, histologic, and immunohistochemical study. Setting University institute. Animal(s) BALB/c mice. Intervention(s) Murine endometriotic lesions were induced by syngeneic transplantation of endometrium into dorsal skinfold chambers. After 6 days, the mice received an intraperitoneal injection of 80 mg/kg CA4P or vehicle. Main Outcome Measure(s) Vascularization of the lesions and the surrounding tissue was analyzed by intravital fluorescence microscopy over 8 days. Lesion morphology, vessel maturation, viability, and proliferation of endometrial glands and stroma were assessed by histology and immunohistochemistry. Result(s) All lesions were initially well vascularized, containing immature and mature microvessels. Injection of CA4P induced a selective vessel collapse in the lesions without affecting the surrounding microvasculature. This resulted in a decreased functional capillary density and blood perfusion of CA4P-treated lesions after 2 hours when compared with controls. However, the vascularization of the lesions progressively normalized, and their numbers of proliferating and apoptotic cells did not differ from those of controls. Conclusion(s) This study demonstrates a selective vascular disrupting effect of CA4P on endometriotic lesions, indicating that vascular disrupting agents may be suitable for endometriosis therapy.