Therapeutic Target For Endometrial Cancer Research Articles

Atractylenolide II Suppresses Glycolysis and Induces Apoptosis by Blocking the PADI3-ERK Signaling Pathway in Endometrial Cancer Cells.

Atractylenolide II (AT-II), the major bioactive compound of Atractylodes macrocephala, exhibits anti-cancer activity against many types of tumors, but the roles and the potential mechanisms in endometrial cancer remain unclear. In the present study, AT-II treatment was found to significantly suppress RL95-2 and AN3CA cell proliferation and glycolysis, and induced their apoptosis by inactivating the ERK signaling pathway, accompanied by the changing expression of the glycolytic key enzymes and apoptotic-related proteins. Peptidyl arginine deiminase 3 (PADI3), as the candidate target gene of AT-II, was highly expressed in the endometrial cancer tissues and associated with a poor prognosis according to bioinformatics analysis. PADI3 knockdown inhibited proliferation and glycolysis in endometrial cancer cells and induced cell apoptosis. Furthermore, AT-II negatively regulated the expression of PADI3, and PADI3 overexpression reversed the effects of AT-II on endometrial cancer cells. Our findings suggested that the anti-cancer function of AT-II is associated with the suppression of glycolysis and induction of apoptosis by blocking the PADI3-ERK signaling pathway. Thus, AT-II represents a novel therapeutic target for endometrial cancer and targeting AT-II may serve as a potential strategy for the clinical therapy of endometrial cancer.

Targeting GRP75 with a Chlorpromazine Derivative Inhibits Endometrial Cancer Progression Through GRP75-IP3R-Ca2+-AMPK Axis.

Tumors often overexpress glucose-regulated proteins, and agents that interfere with the production or activity of these proteins may represent novel cancer treatments. The chlorpromazine derivative JX57 exhibits promising effects against endometrial cancer with minimal extrapyramidal side effects; however, its mechanisms of action are currently unknown. Here, glucose-regulated protein 75kD (GRP75) is identified as a direct target of JX57 using activity-based protein profiling and loss-of-function experiments. The findings show that GRP75 is necessary for the biological activity of JX57, as JX57 exhibits moderate anticancer properties in GRP75-deficient cancer cells, both in vitro and in vivo. High GRP75 expression is correlated with poor differentiation and poor survival in patients with endometrial cancer, whereas the knockdown of GRP75 can significantly suppress tumor growth. Mechanistically, the direct binding of JX57 to GRP75 impairs the structure of the mitochondria-associated endoplasmic reticulum membrane and disrupts the endoplasmic reticulum-mitochondrial calcium homeostasis, resulting in a mitochondrial energy crisis and AMP-activated protein kinase activation. Taken together, these findings highlight GRP75 as a potential prognostic biomarker and direct therapeutic target in endometrial cancer and suggest that the chlorpromazine derivative JX57 can potentially be a new therapeutic option for endometrial cancer.

EphA2- and HDAC-Targeted Combination Therapy in Endometrial Cancer.

Endometrial cancer is the most frequent malignant tumor of the female reproductive tract but lacks effective therapy. EphA2, a receptor tyrosine kinase, is overexpressed by various cancers including endometrial cancer and is associated with poor clinical outcomes. In preclinical models, EphA2-targeted drugs had modest efficacy. To discover potential synergistic partners for EphA2-targeted drugs, we performed a high-throughput drug screen and identified panobinostat, a histone deacetylase inhibitor, as a candidate. We hypothesized that combination therapy with an EphA2 inhibitor and panobinostat leads to synergistic cell death. Indeed, we found that the combination enhanced DNA damage, increased apoptosis, and decreased clonogenic survival in Ishikawa and Hec1A endometrial cancer cells and significantly reduced tumor burden in mouse models of endometrial carcinoma. Upon RNA sequencing, the combination was associated with downregulation of cell survival pathways, including senescence, cyclins, and cell cycle regulators. The Axl-PI3K-Akt-mTOR pathway was also decreased by combination therapy. Together, our results highlight EphA2 and histone deacetylase as promising therapeutic targets for endometrial cancer.

MiR-34b-5p suppresses the epithelial-mesenchymal transition and metastasis in endometrial cancer AN3CA cells by targeting ZEB1.

Tumor metastasis is a primary cause of recurrence and mortality in endometrial cancer. miR-34b-5p is abnormally expressed in various cancers and participates in tumor cell progression and metastasis. The objective of this study was to elucidate the biological functions and molecular mechanisms of miR-34b-5p in regulating the epithelial-mesenchymal transition (EMT) and metastasis in AN3CA endometrial cancer cells. The expression levels of miR-34b-5p and zinc finger E-box-binding homeobox 1 (ZEB1) in endometrial cancer cells were analyzed by qRT-PCR, and ZEB1 expression in endometrial cancer tissues was examined by immunohistochemistry. Proliferation, migration, and invasion of endometrial cancer AN3CA cells were evaluated using CCK8, scratch, and transwell assays, respectively. Bioinformatic analysis and dual-luciferase reporter gene assays were used to validate the targeting relationship between miR-34b-5p and ZEB1. Western blotting was performed to analyze the expression levels of ZEB1 and EMT-related proteins. miR-34b-5p was significantly downregulated in endometrial cancer AN3CA cells. Overexpression of miR-34b-5p significantly inhibited proliferation, invasion, migration, and the EMT of endometrial cancer AN3CA cells. ZEB1, which was identified as a direct target gene of miR-34b-5p, exhibited high expression in endometrial cancer cells and tissues. Additionally, ZEB1 upregulation partially reversed the inhibitory effects of miR-34b-5p on proliferation, migration, invasion, and the EMT of endometrial cancer AN3CA cells. miR-34b-5p suppresses the EMT and metastasis in endometrial cancer AN3CA cells by targeting ZEB1, indicating that the miR-34b-5p-ZEB1-EMT axis may be a therapeutic target for endometrial cancer.

Autophagy-related risk signature based on CDNK2A to facilitate survival prediction of patients with endometrial cancer.

Autophagy plays an important role in immunity and inflammation. The present study aimed to explore the prognostic significance of autophagy-related genes (ARGs) in endometrial cancer (EC) using bioinformatics. The list of ARGs was obtained from the Human Autophagy Database. The differentially expressed ARGs (DEARGs) between the EC and normal endometrial tissue samples were screened from The Cancer Genome Atlas database. Cox regression analysis was performed on the DEARGs to screen the prognostic ARGs and construct risk signatures for overall survival (OS) and progression-free survival (PFS). The hub ARGs were identified from a protein-protein interaction network, and CDKN2A was obtained from the intersection of prognostic ARGs and hub ARGs. The association of CDKN2A expression with clinical characteristics and immune infiltration were analyzed. Finally, the role of CDKN2A in autophagy was confirmed in EC cell lines. CDKN2A, PTK6 and DLC1 were used to establish risk signatures for predicting the survival of EC patients. Receiver operating characteristic curve analysis indicated that the risk signatures can accurately predict both OS and PFS. CDKN2A was the only hub prognostic ARG, and showed significant association with the age, survival status, grade, histological type, body mass index and FIGO (i.e. International Federation of Gynecology and Obstetrics) stage (p < 0.05). Furthermore, CDKN2A expression was also correlated with the infiltration of immune cells, indicating that CDKN2A might play a critical role in regulating the immune microenvironment and immune responses in EC. In addition, silencing of CDKN2A gene promoted autophagy in the HEC-1A cell line and upregulated the expression levels of autophagy-related proteins. CDKN2A is a prognostic factor and therapeutic target in EC, and is likely associated with the tumor immune landscape and autophagy.

Explore the alterations of downstream molecular pathways caused by ARID1A mutation/knockout in human endometrial cancer cells.

As one of the most common gynecologic malignancies, endometrial cancer (EC) is driven by multiple genetic alterations that may be targeted for treatments. AT-rich interaction domain 1A (ARID1A) gene mutations were reported as early events in endometrial carcinogenesis. To explore the alterations of downstream molecular pathways caused by ARID1A mutations and the associated therapeutic implications, we edited ARID1A gene in human endometrial cancer cell line Ishikawa using the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-Associated Proteins (CRISPR/Cas9) technology. We successfully constructed a stable Ishikawa cell line with a confirmed 10bp deletion on the ARID1A gene, which resulted in a code-shift mutation and gene knockout. Compared with unedited wild-type cells, ARID1A knockout (KO) led to reduced apoptosis, accelerated transformation from G0/G1 to S phase, and enhanced cell proliferation. ARID1A deficiency would reduce the protein levels of p21, caspase 7, and caspase 9 in Ishikawa endometrial cancer cells compared with the wild-type cells. In addition, ARID1A KO resulted in high levels of microsatellite instability (MSI-H). Moreover, transcriptomic analyses showed that ARID1A KO can lead to activated phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling. Furthermore, experimental analyses demonstrated that ARID1A KO cells had reduced expression of genetic instability-associated markers mutL homologue 1 (MLH1) and progesterone receptor B (PR) and increased p-Akt expression. These findings support further exploration of ARID1A as a therapeutic target for EC and provide insight into developing more effective treatments in EC, such as the combinatory use of immune checkpoint inhibitors.

Exploring the role of macrophages in the progression from atypical hyperplasia to endometrial carcinoma through single-cell transcriptomics and bulk transcriptomics analysis.

In this study, we aimed to identify key genes in endometrial cancer by conducting single-cell analysis of macrophages. We sourced clinical data from the TCGA database as well as supplementary datasets GSE201926 and GSE173682. Using bulk-seq data of atypical endometrial hyperplasia and endometrial cancer, we pinpointed key differentially expressed genes. Single-cell RNA sequencing was utilized for further gene expression analysis. Cluster analysis was conducted on TCGA tumor data, identifying two distinct subtypes. Statistical methods employed included LASSO regression for diagnostic modeling and various clustering algorithms for subtype identification. We found that subtype B was closely related to cellular metabolism. A diagnostic model was established using LASSO regression and was based on the genes CDH18, H19, PAGE2B, PXDN, and THRB. This model effectively differentiated the prognosis of cervical cancer. We also constructed a prognosis model and a column chart based on these key genes. Through CIBERSORT analysis, CDH18 and PAGE2B were found to be strongly associated with macrophage M0. We propose that these genes influence the transformation from atypical endometrial hyperplasia to endometrial cancer by affecting macrophage M0. In conclusion, these key genes may serve as therapeutic targets for endometrial cancer. A new endometrial cancer risk prognosis model and column chart have been constructed based on these genes, offering a reliable direction for future cervical cancer treatment.

Fumarate hydratase functions as a tumor suppressor in endometrial cancer by inactivating EGFR signaling.

Fumarase hydratase (FH) is an enzyme that catalyzes the reversible hydration and dehydration of fumarate to malate in the tricarboxylic acid cycle. The present study addressed the role of FH in endometrial cancer and clinically observed that the expression of FH was significantly lower in endometrial cancer tissues compared with normal endometrial tissues and, furthermore, that the decreased FH expression in endometrial cancer tissues was significantly associated with increased tumor size and lymph node metastasis. Further analysis in invitro study showed that cell proliferation, migration and invasion abilities were increased when the expression of FH in the endometrial cancer cells was knocked down, but, by contrast, overexpression of FH in endometrial cancer cells decreased cell proliferative, migratory and invasive abilities. Mechanistic studies showed that the expression of vimentin and twist, being two well-studied mesenchymal markers in endometrial cancer cells, were upregulated in fumarate hydratase-knockdowned cells. In addition, phosphokinase array analysis demonstrated that the expression of phospho-EGFR (Y1086), which promotes carcinogenesis in cancers, was increased in endometrial cancer cells when FH was knocked down. In conclusion, the present study suggested that FH is a tumor suppressor and inhibits endometrial cancer cell proliferation and metastasis by inactivation of EGFR. Further studies are required to clarify its role as a prognostic biomarker and therapeutic target for endometrial cancer.

Glutamine and amino acid metabolism as a prognostic signature and therapeutic target in endometrial cancer.

Endometrial cancer (EC) is the most common female reproductive system cancer in developed countries with growing incidence and associated mortality, which may be due to the growing prevalence of obesity. Metabolism reprogramming including glucose, amino acid, and lipid remodeling is a hallmark of tumors. Glutamine metabolism has been reported to participate in tumor proliferation and development. This study aimed to develop a glutamine metabolism-related prognostic model for EC and explore potential targets for cancer treatment. Transcriptomic data and survival outcome of EC were retrieved from The Cancer Genome Atlas (TCGA). Differentially expressed genes related to glutamine metabolism were recognized and utilized to build a prognostic model by univariate and multivariate Cox regressions. The model was confirmed in the training, testing, and the entire cohort. A nomogram combing prognostic model and clinicopathologic features was established and tested. Moreover, we explored the effect of a key metabolic enzyme, PHGDH, on the biological behavior of EC cell lines and xenograft model. Five glutamine metabolism-related genes, including PHGDH, OTC, ASRGL1, ASNS, and NR1H4, were involved in prognostic model construction. Kaplan-Meier curve suggested that patients recognized as high risk underwent inferior outcomes. The receiver operating characteristic (ROC) curve showed the model was sufficient to predict survival. Enrichment analysis recognized DNA replication and repair dysfunction in high-risk patients whereas immune relevance analysis revealed low immune scores in the high-risk group. Finally, a nomogram integrating the prognostic model and clinical factors was created and verified. Further, knockdown of PHGDH showed cell growth inhibition, increasing apoptosis, and reduced migration. Promisingly, NCT-503, a PHGDH inhibitor, significantly repressed tumor growth in vivo (p = 0.0002). Our work established and validated a glutamine metabolism-related prognostic model that favorably evaluates the prognosis of EC patients. DNA replication and repair may be the crucial point that linked glutamine metabolism, amino acid metabolism, and EC progression. High-risk patients stratified by the model may not be sufficient for immune therapy. PHGDH might be a crucial target that links serine metabolism, glutamine metabolism as well as EC progression.

Novel insights into tumorigenesis and prognosis of endometrial cancer through systematic investigation and validation on mitophagy-related signature.

In-depth studies on the pathogenesis of endometrial cancer (EC) are critical because of the increasing global incidence of EC. Mitophagy, a mitochondrial quality control process, plays an important role in carcinogenesis and tumor progression. This study aimed to develop a novel mitophagy-based signature to predict the tumorigenesis and prognosis of EC. Data was downloaded from The Cancer Genome Atlas and Gene Expression Omnibus databases, and 29 mitophagy-related genes were downloaded from the Pathway Unification Database. EC patients were classified into two risk groups based on the two-key- gene signature, TOMM40 and MFN1, which were constructed using Cox regression analysis. A better prognosis was noted in the low-risk group. The model was validated for four aspects: clinical features, mutation status, clinical therapeutic response, and immune cell infiltration status. Moreover, according to the contribution to the risk model, TOMM40 was selected for further in vitro experiments. The silencing of TOMM40 inhibited mitochondrial degradation; suppressed cell proliferation; induced cell apoptosis and G1 phase cell cycle arrest; inhibited migration, invasion, and epithelial-mesenchymal transition; and suppressed cell stemness. In conclusion, the mitophagy-related risk score provides a novel perspective for survival and drug selection during the individual treatment of EC patients. TOMM40 serves as an oncogene in EC and promotes tumor progression via a mitophagy-related pathway. Thus, TOMM40 is a potential therapeutic target in EC.

A novel necroptosis-associated miRNA signature predicting prognosis of endometrial cancer and correlated with immune infiltration.

Necroptosis is a form of programmed cell death identified irrelevant to caspases, which plays an important role in the tumorigenesis and development of cancer. MicroRNAs (miRNAs) are important regulators of both necroptosis and cancer. Expression of sixteen necroptosis-associated miRNAs were analyzed in 546 endometrial cancer (EC) tissues and 33 paracancerous samples from the Cancer Genome Atlas (TCGA). Cox regression analysis was used to evaluate the correlations between miRNAs and overall survival. MiRNAs risk score (Mrs) and nomogram were established to assess the potential value of necroptosis-related miRNAs on prognosis. Expression of miRNA-148a-3p in endometrial cancer cells and endometrial epithelial cells was detected by quantitative real-time PCR (qRT-PCR). The targets genes of miR-148a-3p were predicted using miRDB, miRTarBase and TargetScan and the prognostic-related genes were screened. Immune infiltration analysis was conducted to explore the potential mechanism of these target genes. We identified fourteen differentially expressed miRNAs and selected seven miRNAs (miR-15a-5p, miR148a-3p, miR-7-5p, miR-141-3p, miR-200a-5p, miR-223-3p, miR-16-5p) for prognostic-model construction. The area under the curve (AUC) of receiver operating characteristic (ROC) curve for 1-, 2- and 5-year survival were 0.678, 0.652 and 0.656 respectively. Multivariate analysis revealed that the Mrs was an independent prognostic factor considering other risk factors (HR=1.928, 95% CI=1.072-3.467, P=0.028). Among these miRNAs, miRNA-148a-3p was up-regulated in cancer tissues and cells, and Kaplan-Meier analysis showed its significance in overall survival (OS). The target genes, DNAJB4 and PRNP, were associated with poor prognosis and correlated with tumor immune infiltration. Our study constructed a novel necroptosis-associated miRNAs model for prognosis prediction, and DNAJB4 and PRNP may be therapeutic targets for EC.

GINS2 regulates epithelial mesenchymal transformation and cell cycle in endometrial carcinoma by stimulating ERK/MAPK signaling

Endometrial cancer (EC) is one of the three main gynecological cancers. Identifying new therapeutic targets and further elucidating the molecular mechanisms of EC tumorigenesis have important implications for women’s health. The Go-Ichi-Ni-San (GINS) family, which includes four subunits (GINS1–4), has specific functions in DNA replication and cell cycle. The Cancer Genome Atlas (TCGA) data showed that GINS2 transcription level is upregulated in endometrial cancer tissue. However, the possible role of GINS2 in EC progression is still unknown. Herein, we explored the role of GINS2 in EC. We noticed that GINS2 was overexpressed in EC cells. GINS2 knockdown suppressed the proliferation of EC cells and induced cell cycle arrest. We further noticed that GINS2 knockdown restrained the Epithelial mesenchymal transformation (EMT) of EC cells. Mechanically, its downregulation suppressed the extracellular regulated protein kinase (ERK)/Microtubule-Associated Protein Kinase (MAPK) pathway, thereby suppressing EC progression. Thus, GINS2 has the potential to act as a therapeutic target for EC.

Calcium-Related Genes Predicting Outcomes and Serving as Therapeutic Targets in Endometrial Cancer.

Endometrial cancer (EC) is the most common gynecologic cancer with increasing incidence. The dysregulation of intracellular calcium plays a crucial role in cancer progression. However, the relationship between calcium-related genes and prognosis remains unclear. In this study, we aimed to establish a risk model based on calcium-related genes for prognosis prediction in patients with EC. The TCGA-total set was divided into a training set and a testing set (1:1). The four-gene prognostic signature (CACNA2D1, SLC8A1, TRPM4 and CCL2) was established and classified all EC patients into a low-risk or high-risk group. This model was validated in both the testing dataset and the total set. The EC patients with high RiskScores showed significantly shorter overall survival than those with low RiskScores, and this trend was consistent among most subgroups. Moreover, an enrichment analysis confirmed that calcium-related and estrogen-response signalings were significantly enriched in the high-risk group. The knockdown of CACNA2D1 by siRNA or its blocker, amlodipine (AM) inhibited cell proliferation and induced cycle arrest in vitro. The calcium channel blocker AM inhibited cell proliferation and induced cycle arrest in vitro. AM also showed marked tumor inhibition effects in vivo. In summary, the prognostic model constructed by four calcium-related genes can reliably predict the outcomes of EC patients, and a calcium channel blocker, AM, has significant potential for EC treatment.

Investigation of Transcriptome Patterns in Endometrial Cancers from Obese and Lean Women.

Endometrial cancer is the most common gynaecological malignancy in developed countries. One of the largest risk factors for endometrial cancer is obesity. The aim of this study was to determine whether there are differences in the transcriptome of endometrial cancers from obese vs. lean women. Here we investigate the transcriptome of endometrial cancer between obese and lean postmenopausal women using rRNA-depleted RNA-Seq data from endometrial cancer tissues and matched adjacent non-cancerous endometrial tissues. Differential expression analysis identified 12,484 genes (6370 up-regulated and 6114 down-regulated) in endometrial cancer tissues from obese women, and 6219 genes (3196 up-regulated and 3023 down-regulated) in endometrial cancer tissues from lean women (adjusted p-value < 0.1). A gene ontology enrichment analysis revealed that the top 1000 up-regulated genes (by adjusted p-value) were enriched for growth and proliferation pathways while the top 1000 down-regulated genes were enriched for cytoskeleton restructure networks in both obese and lean endometrial cancer tissues. In this study, we also show perturbations in the expression of protein coding genes (HIST1H2BL, HIST1H3F, HIST1H2BH, HIST1H1B, TTK, PTCHD1, ASPN, PRELP, and CDH13) and the lncRNA MBNL1-AS1 in endometrial cancer tissues. Overall, this study has identified gene expression changes that are similar and also unique to endometrial cancers from obese vs. lean women. Furthermore, some of these genes may serve as prognostic biomarkers or, possibly, therapeutic targets for endometrial cancer.

Hypomethylated gene RAC3 induces cell proliferation and invasion by increasing FASN expression in endometrial cancer.

Endometrial cancer (EC) is one of the most prevalent gynecological cancers with a 5-year survival rate of 20-60%. Feasible prognostic molecular biomarkers of EC are necessary for accurate prediction of EC prognosis. RAC3 is a member of the Rho GTPases. Public databases including Gene Expression Profiling Interactive Analysis (GEPIA2), Tumor Immune Estimation Resource (TIMER), LinkedOmics, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), TISIDB and cBioPortal were employed to analyze the differential expression, clinicopathologic characteristics, functional networks, immune cell infiltrates and genetic alteration of RAC3 in EC patients. RAC3 expression was elevated in EC patients analyzed by TIMER and GEPIA. Overexpression of RAC3 was obviously correlated with clinical stage, histological type, histological grade and DNA hypomethylation. Patients with high RAC3 expression displayed poor overall survival. Functional enrichment analysis showed that RAC3 was involved in translational initiation, DNA replication and mRNA processing. RAC3 expression was negatively associated with infiltrating levels of B cells, CD8+T cells, macrophages and dendritic cells in EC. Experiments in vitro showed that RAC3 was upregulated in EC tissues and cell lines, and RAC3 induced cell proliferation and invasion by increasing fatty acid synthase (FASN) expression. High expression of RAC3iscorrelated with poor prognosis and low infiltration of immune cells in EC. RAC3 promotes cell proliferation and invasion via FASN. These results demonstrate thatRAC3 functions as an EC oncogene and reveal its underlying mechanism in EC progression, suggesting that RAC3 may serve as a potential therapeutic target in EC.

EGFL6 is an important growth factor involved in endometrial cancer tumorigenesis (073)

Objectives: Epidermal growth factor-like domain 6 (EGFL6) has been linked with obesity and implicated in the tumorigenesis of multiple tumor types. Endometrial cancer (EnCa) is most strongly linked with obesity; however, little is known about the role of EGFL6 in the tumorigenesis of EnCa. The objective of this study was to investigate the role of EGFL6 in EnCa tumorigenesis. Methods: Cell proliferation, tumor spheroid, transwell migration, and scratch assays were performed using three established human endometrial cancer cell lines (HEC-1-A, Ishikawa, and MFE-280). Cell lines were treated with recombinant EGFL6 (rEGFL6), and Western blot analysis was performed to examine EGFL6 signaling pathways. A lentivirus transduction system was used to overexpress EGFL6 in a low-EGFL6 expressing cell line (HEC-1-A). shRNA was used to knock down EGFL6 in two cell lines with intermediate (Ishikawa) and high (MFE-280) EGFL6 baseline expression. Tumor xenografts of overexpression and knockdown EGFL6 clones and their respective vector controls were injected in NSG mice. Tumor volume was monitored twice weekly, and tumor weights were obtained at the time of euthanasia. Comparisons between groups were made with the student’s t-test (two-tail, p<0.05). One-way ANOVA was used to analyze differences in mean tumor volume across groups. EGFL6 overexpressing mice were evaluated for obesity and uterine tumor growth. The Human Protein Atlas dataset was analyzed for the impact of EGFL6 expression on overall survival in EnCa. Results: EGFL6 overexpression and treatment with rEGFL6 of low and intermediate EGFL6-expressing cell lines resulted in significantly increased total spheroid number (p<0.05). Parental cell lines exposed to rEGFL6 and EGFL6-overexpressing clones demonstrated increased migration compared to unstimulated cells (p<0.05) and vector control (p=0.009), respectively. EGFL6 knockdown clones showed decreased total tumor spheroid number and migration compared to vector controls (p<0.05). Western blot analysis demonstrated an increase in phosphorylated MAPK at 7.5 minutes after treatment with rEGFL6 compared to no-treatment controls. Tumor xenograft studies showed that EGFL6 overexpression significantly increased tumor growth with EGFL6 overexpression (p<0.0001), whereas EGFL6 downregulation significantly decreased tumor growth when compared to controls (p<0.001). Transgenic EGFL6 overexpressing mice developed massive obesity, uterine hypertrophy, and in some cases, uterine tumors. Consistent with these results, we found that patients whose EnCa express high levels of EGFL6 had significantly worse overall survival than patients whose tumors express low levels of EGFL6 (67% vs 80%, p=0.004). Conclusions: Our findings indicate EGFL6 plays an important role in EnCA growth. Importantly, EGFL6 may represent a biological link between obesity and the development of EnCa. Finally, these results indicate that EGFL6 represents a potential therapeutic target in endometrial cancer. Objectives: Epidermal growth factor-like domain 6 (EGFL6) has been linked with obesity and implicated in the tumorigenesis of multiple tumor types. Endometrial cancer (EnCa) is most strongly linked with obesity; however, little is known about the role of EGFL6 in the tumorigenesis of EnCa. The objective of this study was to investigate the role of EGFL6 in EnCa tumorigenesis. Methods: Cell proliferation, tumor spheroid, transwell migration, and scratch assays were performed using three established human endometrial cancer cell lines (HEC-1-A, Ishikawa, and MFE-280). Cell lines were treated with recombinant EGFL6 (rEGFL6), and Western blot analysis was performed to examine EGFL6 signaling pathways. A lentivirus transduction system was used to overexpress EGFL6 in a low-EGFL6 expressing cell line (HEC-1-A). shRNA was used to knock down EGFL6 in two cell lines with intermediate (Ishikawa) and high (MFE-280) EGFL6 baseline expression. Tumor xenografts of overexpression and knockdown EGFL6 clones and their respective vector controls were injected in NSG mice. Tumor volume was monitored twice weekly, and tumor weights were obtained at the time of euthanasia. Comparisons between groups were made with the student’s t-test (two-tail, p<0.05). One-way ANOVA was used to analyze differences in mean tumor volume across groups. EGFL6 overexpressing mice were evaluated for obesity and uterine tumor growth. The Human Protein Atlas dataset was analyzed for the impact of EGFL6 expression on overall survival in EnCa. Results: EGFL6 overexpression and treatment with rEGFL6 of low and intermediate EGFL6-expressing cell lines resulted in significantly increased total spheroid number (p<0.05). Parental cell lines exposed to rEGFL6 and EGFL6-overexpressing clones demonstrated increased migration compared to unstimulated cells (p<0.05) and vector control (p=0.009), respectively. EGFL6 knockdown clones showed decreased total tumor spheroid number and migration compared to vector controls (p<0.05). Western blot analysis demonstrated an increase in phosphorylated MAPK at 7.5 minutes after treatment with rEGFL6 compared to no-treatment controls. Tumor xenograft studies showed that EGFL6 overexpression significantly increased tumor growth with EGFL6 overexpression (p<0.0001), whereas EGFL6 downregulation significantly decreased tumor growth when compared to controls (p<0.001). Transgenic EGFL6 overexpressing mice developed massive obesity, uterine hypertrophy, and in some cases, uterine tumors. Consistent with these results, we found that patients whose EnCa express high levels of EGFL6 had significantly worse overall survival than patients whose tumors express low levels of EGFL6 (67% vs 80%, p=0.004). Conclusions: Our findings indicate EGFL6 plays an important role in EnCA growth. Importantly, EGFL6 may represent a biological link between obesity and the development of EnCa. Finally, these results indicate that EGFL6 represents a potential therapeutic target in endometrial cancer.

Box C/D snoRNA SNORD89 influences the occurrence and development of endometrial cancer through 2’-O-methylation modification of Bim

The small nucleolar RNA (snoRNA) is a type of small non-coding RNA widely distributed in the nucleoli of eukaryotic cells, promoting cancer development. The aim of this study was to assess box C/D snoRNA 89 (SNORD89) dysregulations in endometrial cancer. According to the TCGA database as well as the International Federation of Gynecology and Obstetrics (FIGO), higher SNORD89 expression is found in endometrial cancer tissues. In addition, the SNORD89 expression level was higher in endometrial carcinoma with lymph node metastasis than in endometrial carcinoma without lymph node metastasis. By interacting with the conservative chaperone protein methylase fibrillarin (Fbl), SNORD89 inhibits the translation process of the Bim gene, leading to a decrease in Bim protein. Cancer-promoting effect of SNORD89 can be reversed by Fbl knockdown or Bim overexpressing. What’s more, ASO-mediated silencing of SNORD89 could inhibit endometrial cancer cell proliferation and migration ability. Taken together, SNORD89 can modify Bim through 2′-O-methylation and affect downstream signaling pathways to promote endometrial cancer occurrence and development. The role of methylation modification in the prevention and treatment of endometrial cancer provides a new understanding and SNORD89 may be a new diagnostic and therapeutic target for endometrial cancer.

Role of the prorenin receptor in endometrial cancer cell growth.

Endometrial cancer is the most diagnosed gynecological malignancy. Despite numerous scientific advances, the incidence and mortality rate of endometrial cancer continues to rise. Emerging evidence suggests a putative role of the (pro)renin receptor ((P)RR), in the ontogenesis of endometrial cancer. The (P)RR is implicated in breast cancer and pancreatic carcinoma pathophysiology by virtue of its role in proliferation, angiogenesis, fibrosis, migration and invasion. Thus, we aimed to investigate the functional role of the (P)RR in human endometrial cancer.We employed an siRNA-mediated knockdown approach to abrogate (P)RR expression in the endometrial epithelial cell lines; Ishikawa, AN3CA and HEC-1-A and examined cellular proliferation and viability. We also carried out a sophisticated proteomic screen to explore potential pathways via which the (P)RR is acting in endometrial cancer physiology.These data confirmed that the (P)RR is critical for endometrial cancer development, contributing to both its proliferative capacity and in the maintenance of cell viability. This is likely mediated through proteins such as MGA, SLC4A7, SLC7A11 or DHRS2, which were reduced following (P)RR knockdown.These putative protein interactions/pathways, which rely on the presence of the (P)RR, are likely to contribute to endometrial cancer progression and could therefore, represent several novel therapeutic targets for endometrial cancer.

Mitochondrial protein LETM1 and its-mediated CTMP are potential therapeutic targets for endometrial cancer.

Leucine zipper/EF hand-containing transmembrane-1 (LETM1) is an important mitochondrial protein, while its function in endometrial cancer remains unknown. This study aimed to explore the function of LETM1 in endometrial cancer and reveal the underlying mechanisms involving carboxy-terminal modulator protein (CTMP). Immunohistochemistry was performed to detect the expression of LETM1 and CTMP in normal, atypical hyperplastic and endometrial cancer endometrial tissues. LETM1 and CTMP were silenced in two endometrial cancer cell lines (ISK and KLE), which were verified by western blot. Cell viability, colony number, migration and invasion were detected by cell counting kit-8, colony formation, wound healing and trans-well assays, respectively. A xenograft mouse model was established to determine the antitumor potential of LETM1/CTMP silencing in vivo . In addition, CTMP was overexpressed to evaluate its regulatory relationship with LETM1 in endometrial cancer cells. The expression of LETM1 and CTMP proteins were higher in endometrial cancer tissues than atypical hyperplastic tissues and were higher in atypical hyperplastic tissues than normal tissues. LETM1 and CTMP were also upregulated in ISK and KLE cells. Silencing of LETM1 or CTMP could decrease the viability, colony number, migration and invasion of endometrial cancer cells and the weight and volume of tumor xenografts. In addition, CTMP was downregulated by LETM1 silencing in KLE cells, and its overexpression enhanced the malignant characteristics of si-LETM1-transfected KLE cells. Silencing of LETM1 inhibits the malignant progression of endometrial cancer through downregulating CTMP.

CircESRP1 enhances metastasis and epithelial\u2013mesenchymal transition in endometrial cancer via the miR-874-3p/CPEB4 axis

BackgroundMetastasis is critical for endometrial cancer (EC) progression and prognosis. Accumulating evidence suggests that circular RNAs (circRNAs) can operate as independent functional entities. However, the functional regulatory mechanisms of circRNAs in EC remain unclear.MethodsThe levels of circESRP1, miR-874-3p, and CPEB4 mRNA in EC tissues and cells were determined by qRT-PCR. Sanger sequencing, PCR with divergent primers, an actinomycin D assay, and RNase R treatment were applied to verify the circular properties. Fluorescence in situ hybridization (FISH) and nuclear-cytoplasmic fractionation were used to determine the localization of circESRP1. CCK-8, EdU incorporation, colony formation, Transwell, and wound healing assays were applied to assess the effects of circESRP1 on cell proliferation, migration, and invasion. The mutual regulatory mechanism of ceRNAs was investigated using dual-luciferase reporter, RNA pulldown, RNA immunoprecipitation (RIP), and Western blot assays. The biological effects were further validated in vivo in nude mouse xenograft models.ResultscircESRP1 was highly expressed in EC tissues and cells and was mainly localized in the cytoplasm. Silencing circESRP1 inhibited the proliferation, migration, and invasion of EC cells in vitro and in vivo; however, overexpression of circESRP1 had the opposite effects. Mechanistically, circESRP1 sponged miR-874-3p to upregulate CPEB4 expression and ultimately contribute to EC cell proliferation and metastasis. Furthermore, circESRP1 regulated tumour growth in xenograft models.ConclusionsCircESRP1 can interact with miR-874-3p to regulate EMT in endometrial cancer via the miR-874-3p/CPEB4 axis. CircESRP1 may serve as a promising therapeutic target for endometrial cancer.