Endometrial Cancer Samples Research Articles

TWIST1 expression and clinical significance in type I endometrial cancer and premalignant lesions: A retrospective clinical study.

The aim of this study was to assess the correlation of TWIST1 expression with clinical parameters and the prognosis of type I endometrial cancer (EC).This retrospective study enrolled 345 patients. Immunohistochemical staining was performed on 55 normal endometrium (NE) samples, 27 atypical hyperplasia (AH) samples, and 263 type I EC samples. The association between TWIST1 staining and clinical characteristics and survival was evaluated by univariate and multivariate analyses.We found significantly higher TWIST1 expression in patients with AHs and type I ECs than NEs, but there was no significant difference between TWIST1 expression in AHs and type I ECs. Aberrant TWIST1 expression was significantly associated with clinical parameters, indicating poor prognosis and shorter patient survival. Pearsons Chi-Squared test showed that high TWIST1 expression was significantly associated with a shorter disease-free survival and overall survival. More importantly, multivariate analysis showed that high TWIST1 expression, in addition to myometrial invasion, lymph vascular space invasion, and lymph node metastasis, was an independent predictor of worse DFS in patients with type I ECs.Our findings suggest that TWIST1 might be useful in diagnosing ECs and predicting prognosis in patients with AHs and type I ECs.

Human Endometrial Carcinogenesis Is Associated with Significant Reduction in Long Non-Coding RNA, TERRA.

Telomeres are transcribed as long non-coding RNAs called TERRAs (Telomeric repeat containing RNA) that participate in a variety of cellular regulatory functions. High telomerase activity (TA) is associated with endometrial cancer (EC). This study aimed to examine the levels of three TERRAs, transcribed at chromosomes 1q-2q-4q-10q-13q-22q, 16p and 20q in healthy (n = 23) and pathological (n = 24) human endometrium and to examine their association with cellular proliferation, TA and telomere lengths. EC samples demonstrated significantly reduced levels of TERRAs for Chromosome 16p (Ch-16p) (p < 0.002) and Chromosome 20q (Ch-20q) (p = 0.0006), when compared with the postmenopausal samples. No significant correlation was found between TERRA levels and TA but both Ch-16p and Ch-20q TERRA levels negatively correlated with the proliferative marker Ki67 (r = −0.35, p = 0.03 and r = −0.42, p = 0.01 respectively). Evaluation of single telomere length analysis (STELA) at XpYp telomeres demonstrated a significant shortening in EC samples when compared with healthy tissues (p = 0.002). We detected TERRAs in healthy human endometrium and observed altered individual TERRA-specific levels in malignant endometrium. The negative correlation of TERRAs with cellular proliferation along with their significant reduction in EC may suggest a role for TERRAs in carcinogenesis and thus future research should explore TERRAs as potential therapeutic targets in EC.

Biological Function of PD-L2 and Correlation With Overall Survival in Type II Endometrial Cancer.

In cancer, upregulation of coinhibitory B7 ligands has been associated with immune evasion. So far, anti-programmed death-1 (PD-1) and anti-PD-ligand 1 (PD-L1) antibodies have been used in immuno-oncology, with promising outcomes; however, it is still needed to identify other markers, especially for endometrial cancer (EC). EC is a gynecological malignancy historically classified into two types: type I, with mostly estrogen-dependent endometrioid diseases, and the most aggressive type II, including mainly estrogen-independent and non-endometrioid tumors. PD ligand-2 (PD-L2) is known as the second ligand of the PD-1 receptor and, upon its binding, contributes to T-cell exhaustion. Up to now, very few information are available about PD-L2 in cancers, and no data have been reported for EC. The aim of this work was to characterize the PD-L1 and PD-L2 ligand expression profile in EC cell lines, focusing the attention on the biological role of PD-L2 and its prognostic impact in human type II EC biopsies. Using in silico analysis of TCGA data, we performed a molecular profiling in a cohort of 506 patients, both types I and II, and PD-1 ligands expression was also analyzed in different primary human EC cell lines. Moreover, PD-L2 staining was evaluated in a cohort of human type II EC samples and correlated with the overall survival (OS), progression-free survival (PFS), and additional clinicopathological data. From the in silico analysis, PD-L2 was more expressed than PD-L1 in EC cell lines. PD-L2 was found highly expressed in 64.44% of tumor specimens, predominantly in the serous subtype, in both stromal and epithelial components, while in peritumoral and normal tissues it was predominantly moderate or low. In vitro, we investigated the cell autonomous role of PD-L2 in controlling cell survival, migration, and chemoresistance.

Bioinformatic screening for candidate biomarkers and their prognostic values in endometrial cancer

BackgroundEndometrial cancer is a common gynecological cancer with annually increasing incidence worldwide. However, the biomarkers that provide prognosis and progression for this disease remain elusive.ResultsTwo eligible human endometrial cancer datasets (GSE17025 and GSE25405) were selected for the study. A total of 520 differentially expressed mRNAs and 30 differentially expressed miRNAs were identified. These mRNAs were mainly enriched in cell cycle, skeletal system development, vasculature development, oocyte maturation, and oocyte meiosis signalling pathways. A total of 160 pairs of differentially expressed miRNAs and mRNAs, including 22 differentially expressed miRNAs and 71 overlapping differentially expressed mRNAs, were validated in endometrial cancer samples using starBase v2.0 project. The prognosis analysis revealed that Cyclin E1 (CCNE1, one of the 82 hub genes, which correlated with hsa-miR-195 and hsa-miR-424) was significantly linked to a worse overall survival in endometrial cancer patients.ConclusionsThe hub genes and differentially expressed miRNAs identified in this study might be used as prognostic biomarkers for endometrial cancer and molecular targets for its treatment.

Abstract 4251: A performance characterization of microsatellite instability and mismatch repair testing methods in endometrial samples

Abstract Microsatellite instability (MSI), a functional measure of mismatch repair (MMR) deficiency, and immunohistochemistry testing for MMR proteins are well established technologies traditionally used for Lynch Syndrome screening in colorectal cancer. Recently testing for MMR deficiency, by MSI analysis or MMR protein expression, has expanded from hereditary cancer risk testing to a new role predicting immunotherapeutic response in all solid tumors. With this increase in utility and awareness, mismatch repair deficiency is now relevant in a variety of other cancer tissue types. However, little data is available for the performance of these testing techniques in non-colorectal cancer samples. In this study, we examine performance of methods for detecting MSI and MMR protein expression in endometrial cancer samples. 200 endometrial cancer samples were obtained for testing with MSI by PCR followed by capillary electrophoresis, and immunohistochemistry for MMR protein expression. Instability at 2/5 microsatellite loci was used as criteria for MSI-H designation and loss of immunostaining for one or more MMR protein was considered dMMR. Performance of these analysis methods for identification of MMR protein mutations was measured by comparing results to sequencing of MMR proteins using NGS. MSI by PCR followed by capillary electrophoresis and immunohistochemistry for all four major MMR proteins was successfully performed on all (200/200) samples. 140/200 samples had matched adjacent tissue for reference when performing MSI analysis. MSI and MMR did not exhibit 100% concordance, as expected due to their measurement of different biological effects associated with MMR deficiency. We also describe instances of microsatellite loci exhibiting more subtle shifts than those commonly observed in colorectal cancer, especially at shorter nucleotide repeats, an important consideration for future studies with pan-tumor MSI testing. With the increasing utility of identifying mismatch repair defects in hereditary and immuno-oncology for pan-tumor applications, choosing a testing approach that has the highest sensitivity is key. Our findings suggest that for maximal identification of mismatch repair deficiency, MMR by IHC and MSI by PCR are complementary technologies and should not be considered interchangeable. Consistent with these findings, a number of guideline committees globally have begun recommending co-testing for MMR and MSI. These two cost effective tools can be used together for both a functional and informative approach prior to more costly downstream testing. Citation Format: Samantha R. Lewis, Kathryn Oostdik, Shaun Peterson, Heather Tomlinson. A performance characterization of microsatellite instability and mismatch repair testing methods in endometrial samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4251.

Identification of biomarkers correlated with diagnosis and prognosis of endometrial cancer using bioinformatics analysis.

Endometrial cancer (EC) is one of the most common malignancies in the female genital system, characterized by high mortality and recurrence rates. This study attempted to screen key genes and potential prognostic biomarkers for EC using bioinformatics analysis. Twenty-seven normal endometrial tissues and 135 EC samples were collected from four Gene Expression Omnibus (GEO) databases, then we identified the differentially expressed genes (DEGs) and conducted downstream analyses. Moreover, we screened hub genes by constructing a protein-protein interaction (PPI) network. Finally, we assessed the prognostic values and molecular mechanism of the potential prognostic genes using the Kaplan-Meier curve and Gene Set Enrichment Analysis (GSEA). As a result, 28 upregulated and 94 downregulated genes were determined after gene integration of these four GEO data sets. Gene Ontology analysis indicated that DEGs were mainly involved in transcriptional regulation and cell proliferation. The Kyoto Encyclopedia of Gene and Genome pathway analysis primarily related to transcriptional misregulation and apoptosis. Moreover, the PPI analysis revealed 10 hub genes (JUN, UBE2I, GATA2, WT1, PIAS1, FOXL2, RUNXI, EZR, TCF4, and NR2F2) with a high degree of connectivity, among them, the expression tendency of nine genes except UBE2I were consistent with messenger RNA level from The Cancer Genome Atlas data. Furthermore, only FOXL2, TCF4, and NR2F2 were significantly correlated with prognosis of EC patients, and their low expression associated biological pathways were enriched in the cell cycle and fatty acid metabolism. In conclusion, this study identified three key genes as biomarkers and potential therapeutic targets of EC on the basis of integrated bioinformatics analysis. The findings will improve our comprehension of the molecular mechanisms underlying the pathogenesis and prognosis of EC.

The NF-κB signalling pathway regulates GLUT6 expression in endometrial cancer

BackgroundGene and protein expression of the glucose transporter GLUT6 are elevated in multiple cancers, including endometrial cancer. However, the extrinsic and intrinsic mechanisms that regulate GLUT6 expression in this malignancy are unknown. Herein we investigate the potential mechanisms regulating GLUT6 expression in endometrial cancer. MethodsData mining of the GLUT6 gene (SLC2A6) in The Cancer Genome Atlas (TCGA) PanCan datasets was performed in cBioPortal. A transcriptome PCR array was used to identify regulators of GLUT6 expression. The role of RELA in regulating GLUT6 gene and protein expression was investigated by overexpressing constitutively active and dominant-negative RELA in endometrial cells. Endometrial cells were treated with the pro-inflammatory cytokine TNFα and the expression of RELA, IκBα, TNFα, and GLUT6 were examined by Western blotting and RT-qPCR. ResultsGLUT6 is altered in 1% of all cancer samples (157 of 10, 967 samples) within TCGA datasets including 4.7% of uterine (endometrial) cancers. GLUT6 expression was positively co-expressed with multiple members of the NF-κB signalling pathway including NFKB2, RELB, NFKBIE, and TNF in endometrial cancer samples. A transcriptome PCR array identified RELA as the top potential transcriptional regulator of GLUT6 expression. Overexpression of constitutively active RELA increased GLUT6 gene expression in normal endometrial epithelial cells (hUE-Ts), while overexpression of dominant-negative RELA decreased GLUT6 expression in cancerous RL95–2 endometrial cells. TNFα treatment activated canonical NF-κB signalling and increased the expression of GLUT6, but not that of other glucose transporters (GLUTs 1, 3, 4, 8, 10, or 12) in endometrial cells. ConclusionsTNFα is a cytokine that is commonly increased in obesity-related endometrial cancer and the findings herein support a potential mechanism whereby TNFα may contribute to endometrial cancer initiation or progression by increasing GLUT6 expression. Furthermore, we identified RELA, an important downstream mediator of the TNFα signalling cascade, as a regulator of GLUT6 expression in endometrial cells. Future studies are warranted to determine how GLUT6 expression affects endometrial tumourigenesis or cancer progression.

ASH2L is involved in promotion of endometrial cancer progression via upregulation of PAX2 transcription.

Absent, small or homeotic 2‐like protein (ASH2L) is a core component of a multimeric histone methyltransferase complex that is involved in the maintenance of active transcription, participating in several cancers, however the biological function and molecular mechanism of ASH2L in endometrial cancer (ECa) are largely unknown. Endometrial cancer is a common malignant tumor in women and the incidence of this cancer is on the rise. Estrogen‐ERα signaling, as an oncogenic pathway, plays a crucial role in endometrial carcinogenesis. Therefore, further exploration of the molecular mechanisms around ERα‐mediated gene transcription in ECa would be helpful to the understanding of tumor development and to finding a new therapeutic target for ECa. Here, our study demonstrated that ASH2L was highly expressed in ECa samples, and higher expression of ASH2L was positively correlated with a poor prognosis. Moreover, we identified that ASH2L associated with ERα and that knockdown of ASH2L resulted in decreased expression of a subset of the estrogen‐induced target genes, including paired box 2 (PAX2), an oncogenic gene in ECa. ASH2L was recruited to cis‐regulatory elements in PAX2, thereby altering histone H3K4me3 and H3K27me3 levels, to enhance ERα‐mediated transactivation. Finally, depletion of ASH2L suppressed endometrial cancer cell proliferation and migration. Our findings suggest that ASH2L participates in the promotion of ECa progression, if not totally at least partially, via upregulation of PAX2 transcription.

The landscape of POLE variants in colorectal and endometrial tumors: Correlation with microsatellite instability (MSI) and tumor mutation burden (TMB).

e13538 Background: Polymerase epsilon (POLE) is a major replicative DNA polymerase. Somatic POLE pathogenic variants (PV) are prevalent in endometrial cancer (EC) and in germline predispose to colorectal cancer (CRC), EC, and possibly other cancers (CA). PVs in the exonuclease domain (ExoD) [amino acid (AA) 268-471] lead to CAs with exceptionally high TMB. PV and uncertain variants (VUS) outside ExoD are sometimes concurrent with an ExoD PV and/or MSI. We hypothesized that the presence of non-ExoD variants may further increase POLE-associated mutation rate and tumor mutational burden. Methods: We retrospectively examined 1870 CRC and 4481 EC genomic profiles conducted by Caris Life Sciences (6/2016-6/2019). All patients had a 592-targeted gene somatic panel. Profiles with a POLE variant (PV or VUS) were analyzed. Median TMB (TMB, in mutations/megabase) was dichotomized to low/intermed ( &lt; 17) vs high (&gt;17). Tumors were grouped by: single ExoD PV, ExoD PV plus another variant (PV or VUS), or no ExoD PV. Known CRC/EC ExoD PV drivers were identified (Campbell et al, Cell 2017): D275G, P286R, S297F/Y, F367C/L/V, V411L, L424F, P436R/S/Y, M444K/L, A456P, S459F/Y, S461L/P, A465V. Kruskal-Wallis and chi-square tests were used. Results: Overall 4.5% CRC (80/1870) and 6.5% EC (303/4481) samples had POLE variants (Table). High TMB was seen in 56.3% CRC and 53.3% EC. In both CRC/ECs, TMB was higher in tumors with an ExoD PV and a 2nd variant compared to those with a solitary ExoD PV or no ExoD PV (both p &lt; 0.001). MSI was more prevalent in CRC and EC with high TMB but no ExoD PV vs those with either high TMB and an ExoD PV, or low/intermed TMB and no ExoD PV (both p &lt; 0.001). In both CRC/ECs, several ExoD PV associated with very high TMB when non-ExoD regions of POLE contained recurrent variant clusters: AA 1906 (TMB 225); AA 1826-7 (TMB 243); AA 1380-2 (TMB 229). Conclusions: In CRC/ECs, POLE ExoD PV and MSI appear to drive TMB in distinct and largely non-overlapping ways. Non-ExoD POLE variants may synergize with ExoD PVs to further increase mutation rates. [Table: see text]

Prognostic significance of immune landscape in tumour microenvironment of endometrial cancer.

Tumour microenvironment (TME) is crucial to tumorigenesis. This study aimed to uncover the differences in immune phenotypes of TME in endometrial cancer (EC) using Uterine Corpus Endometrial Carcinoma (UCEC) cohort and explore the prognostic significance. We employed GVSA enrichment analysis to cluster The Cancer Genome Atlas (TCGA) EC samples into immune signature cluster modelling, evaluated immune cell profiling in UCEC cohort (n = 538) and defined four immune subtypes of EC. Next, we analysed the correlation between immune subtypes and clinical data including patient prognosis. Furthermore, we analysed the expression of immunomodulators and DNA methylation modification. The profiles of immune infiltration in TCGA UCEC cohort showed significant difference among four immune subtypes of EC. Among each immune subtype, natural killer T cells (NKT), dendritic cells (DCs) and CD8+T cells were significantly associated with EC patients survival. Each immune subtype exhibited specific molecular classification, immune cell characterization and immunomodulators expression. Moreover, the expression immunomodulators were significantly related to DNA methylation level. In conclusion, the identification of immune subtypes in EC tissues could reveal unique immune microenvironments in EC and predict the prognosis of EC patients.

Computational analysis for identification of the extracellular matrix molecules involved in endometrial cancer progression.

Recurrence and poorly differentiated (grade 3 and above) and atypical cell type endometrial cancer (EC) have poor prognosis outcome. The mechanisms and characteristics of recurrence and distal metastasis of EC remain unclear. The extracellular matrix (ECM) of the reproductive tract in women undergoes extensive structural remodelling changes every month. Altered ECMs surrounding cells were believed to play crucial roles in a cancer progression. To decipher the associations between ECM and EC development, we generated a PAN-ECM Data list of 1516 genes including ECM molecules (ECMs), synthetic and degradation enzymes for ECMs, ECM receptors, and soluble molecules that regulate ECM and used RNA-Seq data from The Cancer Genome Atlas (TCGA) for the studies. The alterations of PAN-ECM genes by comparing the RNA-Seq expressions profiles of EC samples which have been grouped as tumorigenesis and metastasis group based on their pathological grading were identified. Differential analyses including functional enrichment, co-expression network, and molecular network analysis were carried out to identify the specific PAN-ECM genes that may involve in the progression of EC. Eight hundred and thirty-one and 241 PAN-ECM genes were significantly involved in tumorigenesis (p-value <1.571e-15) and metastasis (p-value <2.2e-16), respectively, whereas 140 genes were in the intersection of tumorigenesis and metastasis. Interestingly, 92 of the 140 intersecting PAN-ECM genes showed contrasting fold changes between the tumorigenesis and metastasis datasets. Enrichment analysis for the contrast PAN-ECM genes indicated pathways such as GP6 signaling, ILK signaling, and interleukin (IL)-8 signaling pathways were activated in metastasis but inhibited in tumorigenesis. The significantly activated ECM and ECM associated genes in GP6 signaling, ILK signaling, and interleukin (IL)-8 signaling pathways may play crucial roles in metastasis of EC. Our study provides a better understanding of the etiology and the progression of EC.

Frequent promoter methylation of HOXD10 in endometrial carcinoma and its pathological significance.

Homeobox D 10 (HOXD10) is important in cell differentiation and morphogenesis and serves as a tumor suppressor gene (TSG) in a number of malignancies. The present study investigated its promoter methylation status and association with the clinicopathological features of endometrial cancer (EC), and measured HOXD10 protein expression levels. EC samples (n=62), including 50 endometroid adenocarcinoma (EA) and 12 mucinous endometrial carcinoma samples (EC) and 70 non-cancerous samples were collected. All samples were evaluated for the methylation status of several TSGs, including HOXD10, using methylation-specific PCR. HOXD10 expression level was evaluated using immunohistochemistry. 5-Aza-2-deoxycytidine treatment was performed in the EC cell line Ishikawa to observe the change in HOXD10 expression levels. HOXD10 promoter methylation was more frequent in cancer samples (P<0.001). Downregulation of HOXD10 in EC samples was confirmed at the protein level using immunohistochemistry (P<0.001) and immunohistochemical staining was negatively associated with methylation status (P<0.05). Less HOXD10 protein was expressed in MEC compared with EA samples (P<0.001). The HOXD10 promoter was hypermethylated in both EA and MEC, causing decreased HOXD10 protein expression levels in EC cells. HOXD10 expression levels were partially reversed by 5-Aza-2-deoxycytidine treatment. The results of the present study demonstrated that epigenetic silencing of HOXD10 putatively contributed to the tumorigenesis of EA. Although there was no significant difference in HOXD10 methylation between EA and MEC, HOXD10 protein expression levels differed between these two diseases, indicating that it may be a useful protein biomarker for distinguishing between these two lesions.

MiRNAs in endometrioid endometrial cancer metastatic loci derived from positive lymph nodes.

MicroRNAs (miRNAs) take part in tumorigenesis and show aberrant expression levels in cancerous tissues. We aimed to perform miRNA profiling of endometrioid endometrial cancer (EEC) metastatic loci derived from lymph nodes. Identification of aberrant miRNAs in positive lymph nodes could contribute to establishing new diagnostic markers and therapeutic targets. During the screening phase of the study, we performed profiling of 754 human miRNAs in endometrioid endometrial cancer tissues, microdissected metastatic loci from lymph nodes and healthy lymph nodes (Taqman Array). Selection of candidate miRNAs and subsequent validation using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 50 tissue samples were performed. After the screening phase of the study, five miRNAs were selected (hsa-miR-18b, hsa-miR-148a-5p, hsa-miR-204, hsa-miR-424, hsa-miR-129-1-3p). Validation revealed that miRNA-204 and miRNA-424 were highly downregulated in metastatic tissues compared with endometrial cancer samples (hsa-miR-204-P=.0008; hsa-miR-424-P=.0001). Receiver operating characteristic curves, which were constructed to compare endometrioid endometrial cancer and positive endometrioid endometrial cancer lymph nodes yielded the following area under the curves (AUCs): hsa-miR-204-.802 (96% confidence interval CI 0.676-0.927), hsa-miR-424-.84 (95% CI 0.711-0.969). Compared with primary endometrioid endometrial cancer tissue, metastatic loci derived from positive lymph nodes are characterized by profound downregulation of miRNA-204 and miRNA-424.

CDK4/RB/E2Fs axis as potential therapeutic target of endometrial cancer

The increasing incidence rate and decreasing patients’ five-year survival rate for endometrial cancer (EC) in recent decades highlight the necessity for further investigation of the molecular characteristics involved in cancer initiation and progression. In this study, we found that the pathways associated with mitotic cell cycle were enriched in primary EC samples versus normal endometrial samples through analyzing RNA-seq data of The Cancer Genome Atlas (TCGA). The messenger RNA (mRNA) expression of three activator E2Fs (E2F1, E2F2, and E2F3) and their target genes increased significantly in EC samples. Additionally, the high transcriptional activity of activator E2Fs was associated with poor survival, advanced clinical stage, high histologic grade, and aggressive histological type. We further demonstrated that E2Fs hyperactivation correlated with DNA hypomethylation and high cyclin-dependent kinase 4 (CDK4) expression. Moreover, abemaciclib, a selective CDK4 inhibitor, significantly inhibited the proliferation rates of human EC cell lines in vitro. And, abemaciclib also obviously inhibited EC cell growth in nude mice model. Collectively, our data suggest that the misregulation of CDK4/RB/E2Fs axis is associated with EC oncogenesis, and abemaciclib is a potential targeted drug for EC.

Changes in the expression of Notch and Wnt signalling molecules in human endometrial cancer.

Endometrial cancer is the sixth most frequent type of cancer among women worldwide. Type I adenocarcinomas account for 80-85% of endometrial cancer cases and sometimes require more aggressive treatment than theremaining part of this group. Therefore, molecular markers to stratify adenocarcinomas are needed. In this study, we analysed Notch and Wnt signalling in human endometrial cancer cases to evaluate these pathway elements as potential biomarkers for type I endometrial cancer. Endometrial samples were obtained from 47 women undergoing surgery for stageI-IV endometrial cancer in theNational Cancer Institute (Vilnius, Lithuania) in 2015-2016. Theexpression at themRNA level of signalling molecules genes (NOTCH1, NOTCH2, NOTCH3, NOTCH4, JAG1, JAG2, DLL1, HES1, AXIN2 and CTNNB1) was analysed by thequantitative real-time polymerase chain reaction. Relative expression of NOTCH1, NOTCH4, HES1 and β-catenin proteins in endometrioid adenocarcinoma was evaluated by theWestern blot method. The expression level of Notch receptors, ligands, and thetarget gene, as well as CTNNB1 and AXIN2, was reduced in stage I endometrioid adenocarcinoma if compared to theadjacent non-tumour tissue. Theexpression of all receptors, ligands, and target molecules was reduced in adenocarcinomas of later stages. Thestatistically significant correlations between transcript amounts of Notch receptors and ligands were found. There was astatistically significant difference in the gene expression of Notch signalling pathway components between different tumour differentiation grade samples. Apositive correlation between mRNA and protein theexpression level of NOTCH1, NOTCH4, HES1 was determined in stage I samples. Analysis of 47 human endometrial cancer samples revealedareduction in thetranscript levels of Notch and Wnt signalling molecule compared to theadjacent non-tumour tissue. These results suggest tumour suppressor function of Notch and Wnt signalling in human endometrial cancer. More detailed research on these signalling pathways should reveal their importance as potential biomarkers.

Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) Promotes Uterine Serous Cancer Cell Proliferation and Cell Cycle Progression.

Uterine serous carcinoma (USC) is the most aggressive form of endometrial cancer, with poor survival rates and high recurrence risk. Therefore, the purpose of this study was to identify therapeutic targets that could aid in the management of USC. By analyzing endometrial cancer samples from The Cancer Genome Atlas (TCGA), we found Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) to be highly expressed in USC and to correlate with poorer overall survival. UCHL1 silencing reduced cell proliferation in vitro and in vivo, cyclin B1 protein levels and cell cycle progression. Further studies showed that UCHL1 interacts with cyclin B1 and increases cyclin B1 protein stability by deubiquitination. Treatment of USC-bearing mice with the UCHL1-specific inhibitor reduced tumor growth and improved overall survival. Our findings suggest that cyclin B1 is a novel target of UCHL1 and targeting UCHL1 is a potential therapeutic strategy for USC.

KLHL14, an ovarian and endometrial-specific gene, is over-expressed in ovarian and endometrial cancer.

Ovarian cancer (OC) and endometrial cancer (EC) are two types of the most frequent gynecological malignancies worldwide. However, the prognosis of OC and EC patients remained gloomy. Therefore, there was still an urgent need to identify new biomarkers for early diagnosis and treatment of OC and EC. TCGA datasets were used to screen the KLHL14 expression levels in 18 different types of human cancers. TCGA datasets were also used to analyze the association between KLHL14 expression levels and OS/PFS in OC and EC. Human Protein Atlas was used to detected the KLHL14 protein levels in OC and EC. Kaplan-Meier plotter was used to evaluate the prognostic values of KLHL14 in Ovarian cancer. MAS 3.0 was used to perform GO and KEGG pathway analysis. STRING was used to perform PPI network. KLHL14 was specially expressed in OC and EC samples. Moreover, KLHL14 was found to be up-regulated in all stage of OC and EC samples. By analyzing Kaplan-Meier plotter and TCGA datasets, we found higher KLHL14 expression level was associated with shorter overall and progression-free survival in both OC and EC patients. Furthermore, GO and KEGG analysis of KLHL14 co-expressing genes indicated it played important roles in OC and EC progression. We for the first time reported KLHL14 was specially over-expressed in ovarian and endometrial cancer, up-regulation of KLHL14 was positively associated with worse outcome. Finally, we found knockdown of KLHL14 suppressed OC cell proliferation. KLHL14 could be a potential biomarker and therapy target for OC and EC.

Relationship between polycomb-group protein BMI-1 and phosphatases regulating AKT phosphorylation level in endometrial cancer.

The PI3K/AKT pathway is frequently activated in endometrial carcinoma. BMI‐1 (B‐lymphoma Mo‐MLV insertion region 1) protein affects expression of PTEN (phosphatase and tensin homolog) in some cancers, but its significance for endometrial tumorigenesis is not known. The objective of this study was to determine the relationship between BMI‐1 and expression of factors affecting AKT (protein kinase B) phosphorylation level in endometrial cancer. The expression of proteins and mRNAs was investigated in endometrial cancer specimens and samples of non‐neoplastic endometrial tissue by Western blot and RT‐PCR, respectively. The impact of BMI‐1 down‐regulation on AKT phosphorylation and expression of genes coding for several phosphatases were studied in HEC1A cells. The results showed that BMI‐1 depletion caused increase in PHLPP1 and PHLPP2 (PH domain and leucine‐rich repeat protein phosphatases 1/2) expression and decrease in phospho‐AKT (pAKT) level. In more advanced tumours with higher metastatic potential, the expression of BMI‐1 was lower compared to tumours less advanced and without lymph node metastasis. There were significant inverse correlations between BMI‐1 and PHLPPs, especially PHLPP1 in normal endometrial samples. The inverse correlation between BMI‐1 and PHLPP1/PHLPP2 expression was observed in PTEN positive but not PTEN negative cancers. Low PHLPP2 expression in tumours predicted poorer overall survival. BMI‐1 impacts on AKT phosphorylation level in endometrial cells by regulation of PHLPP expression.

ASH2L Acts As a Co-Activator of ERα to Participate in the Promotion of Endometrial Cancer Progression

Background: Estrogen-ERα signaling is an oncogenic pathway plays a crucial role in endometrial carcinogenesis. However, modulation of ERα action in Endometrial cancer (ECa) remains to be elusive. Absent, small or homeotic 2-like protein (ASH2L) is a core component of multimeric histone methyltransferase complex, which is involved in maintenance the active chromatin states. What is the function of ASH2L on modulation of E2-ERα induced transactivation in ECa needs to be further understood. Methods: Immunoprecipitation, Immunofluorescence assay, luciferase assay, quantitative RT-PCR, and western blot were performed to investigate how ASH2L regulates the transcriptional activity of ERα. ChIP assay was performed to determine the recruitment of ASH2L to cis-regulatory elements. Lentiviral infection, colony formation, transwell experiments, and mice xenograft experiments were performed to evaluate the function of ASH2L on cell growth and migration. Findings: Our study demonstrated that ASH2L was highly expressed in ECa samples, and the higher expression of ASH2L is positively correlated with the poor prognosis. ASH2L associates with ERα and upregulates ERα-induced transactivation in ECa cells. ASH2L is recruited to cis -regulatory elements at ERα regulated gene, thereby modulating histone H3K4me3 and H3K27me3 levels, to maintain the active transcription. ASH2L depletion suppresses cell growth and migration in ECa. Interpretation: Our findings suggest that ASH2L associates with ERα to be involved in regulation of ERα-induced transactivation. ASH2L participates in promotion of ECa progression. Funding Statement: This study was supported by National Natural Science Foundation of China [31871286 for Yue Zhao, 81872015 for Chunyu Wang, 31701102 for Shengli Wang, 81702800 for Renlong Zou, 81502438 for Tingting Zhou, 81902889 for Wensu Liu]. Declaration of Interests: The authors declare no conflict of interests. Ethics Approval Statement: All procedures of animal experiments have been carried out in compliance with ethical regulations approved by the Animal Ethics Committee of China Medical University. This study was approved by the Ethics Committee of the Medical Faculty of the China Medical University.

Loss of MDC1 in Endometrial Carcinoma Is Associated With Loss of MRN Complex and MMR Deficiency.

To evaluate the frequency of loss of mediator of DNA damage checkpoint protein 1 (MDC1) protein expression in endometrial cancer (EC) and to determine whether loss of MDC1 is associated with certain clinicopathological parameters. MDC1 expression was examined in 426 samples of EC. The nuclear immunoreactivity of the protein was defined as: negative, weak, moderate and strong. Loss of MDC1 expression (defined as negative nuclear staining) was found in 8.9% (38/426) of ECs and was significantly associated with the loss of MRE11 homolog, double-strand break repair nuclease, RAD50 double-strand break repair protein and nibrin complex components. In addition, loss of expression of MDC1 showed a significant correlation with any mismatch repair deficiency, with endometrioid histological subtype and low tumour grading. Based on these findings, we suggest that MDC1 loss frequently occurs in ECs with microsatellite instability. Due to deficient homologous recombination DNA repair, MDC1-negative ECs might show an increased sensitivity to poly(ADP-ribose) polymerase-inhibitory therapy.