Based on criteria such as sample size, data integrity, and unified platforms, we searched the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo) and selected GSE17025 and GSE39099 for bioinformatics analysis. Differentially expressed genes (DEGs) between EC and normal endometrial tissues were screened using the GEO2R online program, with screening criteria at $|$log₂fold change (FC)$|$ $>$3 and p 0.01. The intersecting DEGs from the two datasets were uploaded to the STRING database (https://string-db.org) to construct a protein-protein interaction (PPI) network. The top 30 key genes were identified using the 12 algorithms of the CytoHubba plug-in for Cytoscape software (Version 3.7.2, Cytoscape Consortium, San Diego, CA, USA), with genes screened in at least 7 out of 12 algorithms considered as key genes. The molecular complex module with the highest score was obtained using molecular complex detection (MCODE) plug-in of Cytoscape software, employing the following parameters: degree cutoff = 2, node score cutoff = 0.2, k-core = 10, and maximum depth = 100. The key genes identified by CytoHubba and the module of MCODE were intersected to obtain the training set genes, which were further validated using UALCAN database (http://ualcan.path.uab.edu/) based on the Cancer Genome Atlas (TCGA) data. The verified DEGs were then considered hub genes.

Human EC cell lines, AN3CA (catalog number: GDC0127) and KLE (catalog number: GDC0115), were purchased from the China Center for Type Culture Collection (Wuhan, Hubei, China). The cell lines used were tested for mycoplasma contamination and the results are negative (Supplementary Fig. 1) and verified by short tandem repeat (STR) verification. Cells were cultured in Minimum Essential Medium (MEM) or Dulbecco’s Modified Eagle Medium (DMEM)/F12 medium containing 10% (v/v) fetal bovine serum (all from Life Technologies, Grand Island, NY, USA) at 37 °C with 5% CO₂. Pro-TAME was purchased from Boston Biochem, Inc. (Cambridge, MA, USA). Apcin was purchased from Sigma–Aldrich Co. LLC. (St. Louis, MD, USA).

EC and normal endometrium tissues were fixed in formaldehyde and dehydrated in alcohol before being embedded in paraffin. Sections (5 µm thickness) were stained with HE and subsequently observed under a light microscope (Olympus IX71, Olympus Corp., Tokyo, Japan).

After deparaffinization and hydration through an ethanol gradient, the sections were boiled in EDTA buffer (Sigma–Aldrich Co. LLC., St. Louis, MO, USA). Next, the sections were treated with 3% hydrogen peroxide (H₂O₂, catalog number: 73113762, Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) to eliminate endogenous peroxidase activity. After blocking with 5% goat serum (catalog number: G1208, Servicebio, Wuhan, Hubei, China), the sections were incubated overnight at 4 °C with rabbit polyclonal antibody specific to CDC20 (1:200, catalog number: 10252-1-AP, Proteintech, Wuhan, Hubei, China). The sections were washed with phosphate-buffered saline (PBS) and subsequently incubated with a biotinylated secondary antibody. The immunohistochemical staining was visualized using a 3,3^′-Diaminobenzidine (DAB) detection kit (DAKO, Glostrup, Denmark), and the sections were counterstained with hematoxylin. The sections were observed under a light microscope (Olympus IX71, Olympus Corp., Tokyo, Japan). The optical density values were measured using Image J software (Version1.51j8, National Institutes of Health, Bethesda, MD, USA) for quantitative analysis.

Cells were lysed on ice using radio-immunoprecipitation assay (RIPA) buffer (catalog Number: G2002, Servicebio, Wuhan, Hubei, China) supplemented with a protease inhibitor. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (catalog number: G2043, Servicebio, Wuhan, Hubei, China) and transferred electrophoretically to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk (catalog number: GC310001, Servicebio, Wuhan, Hubei, China), the membranes were subsequently incubated overnight at 4 °C with primary antibodies specific to CDC20 (1:2000, catalog number: 10252-1-AP, Proteintech, Wuhan, Hubei, China) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2500, catalog number: ab9485, Abcam, Cambridge, MA, USA). The membranes were then washed with tris buffered saline tween-20 (TBST, catalog number: G2150, Servicebio, Wuhan, Hubei, China) and incubated with the secondary antibody (1:10000, catalog number: SA00001-2, Proteintech, Wuhan, Hubei, China) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection reagent (Sigma–Aldrich Co. LLC., St. Louis, MO, USA).

Cells were seeded into 96-well culture plates at a density of 2 $\times$ 10³ cells per well and cultured in complete media overnight. The following day, cells were treated with varying concentrations of pro-TAME (0, 5, 10, and 15 µM) or Apcin (100 µM) [18, 19, 21] and cultured for 24, 48 and 72 h. In addition, cells were also treated with both pro-TAME (5 and 10 µM) and Apcin (25 and 50 µM) [18, 19, 21]. Subsequently, Cell Counting Kit-8 (CCK-8) reagent (Abcam, Cambridge, MA, USA) was added to each well at a dilution of 1:10 and incubated for 2–4 h at 37 °C in the dark. The optical density values were measured using a microplate reader (Biotek, Winooski, VT, USA) at 450 nm wavelength.

Cells were seeded in 6-well culture plates and cultured in complete media overnight. The following day, 15 µM pro-TAME alone or a combination of 10 µM pro-TAME with 25 µM Apcin was added to the culture media. After being treated with pro-TAME and Apcin for 72 h, cells were stained with Annexin V-Fluorescein Isothiocyanate/Propidium Iodide (FITC/PI) (BD Biosciences, Bedford, MA, USA) and analyzed using a flow cytometer (Beckman, DxFLEX, Brea, CA, USA) to evaluate the cell apoptosis.

Cells were seeded in 6-well culture plates and cultured until they reached approximately 90% confluence. After a 24 h incubation in serum-free basal media for starvation, the cell monolayers were scratched with a 200 µL pipette tip to create linear wounds, and the media were replaced to fresh serum-free basal media, with or without pro-TAME (15 µM). Images of the wounds were captured under a light microscope (Olympus IX71, Olympus Corp., Tokyo, Japan) at 0 and 20 h after pro-TAME treatment. The migration rates of the cells were determined as follows: (width of wound at 0 h – width of wound at 20 h)/width of wound at 0 h $\times$ 100%.

Cells (1 $\times$ 10⁵ per well) were seeded into the upper compartments of Transwell chambers (8-mm pores, Corning, NY, USA) pre-coated with Matrigel (BD Biosciences, MA, USA) in a 24-well culture plate. Serum-free media with or without pro-TAME (15 µM) were added to the upper chambers, while complete media served as chemoattractant in the lower chambers. After incubation at 37 °C for 24 h, the residual cells in the upper chambers were removed with cotton swabs, and the invaded cells were fixed in formaldehyde for 30 min. Next, the fixed cells were stained with 0.1% crystal violet and counted under a light microscope (Olympus IX71, Olympus Corp., Tokyo, Japan) in 10 random fields of view per well.

The results are presented as the mean $\pm$ standard deviation (SD) from three independent experiments. Statistical analyses were conducted using GraphPad Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA). One-way Analysis of Variance (ANOVA) or unpaired t-test was applied to determine the statistical significance. The p-value 0.05 was considered significant.

GSE17025 contained 92 samples of EC and 12 samples of nonmalignant endometrium, while GSE39099 included data from 169 patients with EC and 20 patients with nonmalignant endometrium. A total of 4442 and 2447 DEGs were identified from GSE17025 and GSE39099, respectively. Among these, 832 genes were common to both datasets, including 305 upregulated and 527 downregulated DEGs (Fig. 1A,B). Upon analyzing the PPI network of the 832 genes, we identified 10 key genes that appeared in at least 7 out of the 12 algorithms within the CytoHubba plug-in, including KIF11, EGFR, MKI67, CDC20, FOXM1, RHOA, CHEK1, BIRC5, KIF23, and ZWINT (Fig. 1C). Additionally, the molecular complex module with the highest score of 48.423 was derived from MCODE plug-in (Fig. 1D), consisting of 53 genes. 8 of the 10 key genes, excluding EGFR and RHOA, were intersected with the module genes (Fig. 1C). As a hub gene, CDC20 has been reported to have oncogenic functions and was chosen for further investigation.

Fig. 1.

Identification of CDC20 as a novel target for EC treatment. (A) A total of 832 genes were identified through the intersection of GSE17025 and GSE39099 using the GEO2R online program, applying the screening criteria of $|$log₂FC$|$ $>$3 and p 0.01. (B) Volcano plot of the 832 intersected genes between GSE17025 and GSE39099. The red dots indicate upregulated genes and the blue dots indicate downregulated genes. (C) 10 key genes were identified in at least 7 out of the 12 algorithms of CytoHubba plug-in of Cytoscape software. The molecular complex module with the highest score was obtained using MCODE plug-in, which included 53 genes. Among these, the 10 genes screened by CytoHubba shared 8 common genes with the module. (D) The molecular complex module with the highest score is presented. The black box indicates CDC20 is a hub gene of the molecular complex module. CDC20, cell division cycle protein 20; EC, endometrial carcinoma; MCODE, molecular complex detection; FC, fold change.

To investigate the potential roles of CDC20 in EC, we analyzed its expression levels using data from TCGA. The mRNA level of CDC20 was higher in the EC tissues compared to nonmalignant tissues (Fig. 2A). The histological morphology of nonmalignant and EC tissues was observed by HE staining. Compared with nonmalignant tissues, the crowded and densely-packed glands vary in size and are arranged without polarity in EC tissues (Fig. 2B). The immunohistochemical analysis indicated increased expression of CDC20 at protein level in EC tissues compared to nonmalignant tissues (Fig. 2B,C). These results suggest that CDC20 may play a significant role in EC.

Fig. 2.

The expression of CDC20 in EC tissues. (A) Analysis of CDC20 mRNA levels in EC tissues and paired nonmalignant tissues from TCGA database. *p 0.05. (B) The HE staining and immunohistochemical analysis of CDC20 protein levels in EC tissues and nonmalignant tissues. Scale bar, 50 µm. (C) Quantitative results for CDC20 protein levels in the immunohistochemical analysis. *p 0.05. CDC20, cell division cycle protein 20; EC, endometrial carcinoma; TCGA, The Cancer Genome Atlas; HE, hematoxylin and eosin.

Western blot results revealed stable expression of CDC20 in EC cell lines (Fig. 3A). The AN3CA and KLE cells were treated with increasing concentrations of pro-TAME. The formula and structure of pro-TAME and Apcin are shown in Fig. 3B,C. As shown in Fig. 3D, the proliferation of EC cells was significantly suppressed by pro-TAME in a time- and dose-dependent pattern. Treatment with 5–10 µM pro-TAME resulted in slight growth inhibition in EC cells between 24 and 48 h. Low concentration (10 µM) of pro-TAME inhibited KLE cell proliferation significantly (cell survival rate 0.8) until 72 h, whereas high concentration (15 µM) of pro-TAME led to growth inhibition as early as 24 h, with the inhibitory effects becoming more pronounced over time.

Fig. 3.

The effect of pro-TAME treatment on the growth of human EC cells. (A) Validation of CDC20 protein levels by Western blot in AN3CA and KLE cell lines. (B,C) The formula and structure of pro-TAME and Apcin. (D) A CCK-8 assay was conducted to detect EC cell proliferation following treatment with increasing concentrations (0, 5, 10, and 15 µM) of pro-TAME at different time point (24, 48 and 72 h). #p 0.05, AN3CA, compared with 0 µM. *p 0.05, KLE, compared with 0 µM. pro-TAME, pro-Tosyl-L-Arginine Methyl Ester; CDC20, cell division cycle protein 20; EC, endometrial carcinoma; CCK-8, Cell Counting Kit-8.

Next, we conducted flow cytometry assay to detect cell apoptosis. Treatment with high concentrations of pro-TAME (15 µM) for 72 h resulted in significantly increased percentage of early apoptotic cells (lower right quadrant, LR) and dead cells (upper right quadrant, UR), compared with the control groups (p 0.05) (Fig. 4).

Fig. 4.

The effect of pro-TAME treatment on apoptosis of EC cells. (A) EC cells were treated with either 15 µM pro-TAME alone or 10 µM pro-TAME in combination with 25 µM Apcin for 72 h. Cell apoptosis was analyzed using flow cytometry. (B) Quantitative results for apoptosis rates. *p 0.05. pro-TAME, pro-Tosyl-L-Arginine Methyl Ester; EC, endometrial carcinoma; PE-A, phycoerythrin area; FITC-A; fluorescein isothiocyanate area; Q1-UL, upper left quadrant; Q1-UR, upper right quadrant; Q1-LL, lower left quadrant; Q1-LR, lower right quadrant; A, apcin; T, pro-TAME.

To explore the effects of pro-TAME treatment on metastasis ability of EC cells, we conducted wound healing assay and invasion assay. No significant differences were detected in the migration rates or the average numbers of migrated cells per high-power field between pro-TAME-treated EC cells and control cells (p $>$ 0.05, Fig. 5). Thus, pro-TAME treatment did not alter the migration or invasive ability of EC cells.

Fig. 5.

The effect of pro-TAME treatment on the migration and invasive ability of human EC cells. (A,B) The migration ability of pro-TAME-treated EC cells was assessed using a wound healing assay. (A) AN3CA cells. (B) KLE cells. The images were captured under a light microscope at 0 and 20 h. Scale bar, 200 µm. (C) The invasive ability of pro-TAME-treated EC cells was assessed using a transwell assay. The images were captured under a light microscope at 24 h. Scale bar, 50 µm. (D) Quantitative results for wound healing assay. (E) Quantitative results for transwell assay. n.s, not significant. pro-TAME, pro-Tosyl-L-Arginine Methyl Ester; EC, endometrial carcinoma.

Since pro-TAME and Apcin inhibit APC/C^CDC20 through different mechanisms, we next treated the EC cells with both inhibitors. Interestingly, the combination of pro-TAME and Apcin significantly enhanced the inhibition of EC cell growth. Furthermore, the administration of lower concentrations of pro-TAME (5–10 µM) and Apcin (25–50 µM) resulted in equal even stronger suppression of EC cells compared to the highest concentration of each single inhibitor (15 µM pro-TAME or 100 µM Apcin) (Fig. 6). Moreover, the administration of both pro-TAME (10 µM) and Apcin (25 µM) at lower concentrations contributed to higher percentage of apoptotic and dead cells overall (p 0.05) (Fig. 4).

Fig. 6.

The effect of pro-TAME combined with Apcin on the growth of human EC cells. A CCK-8 assay was conducted to assess EC cell proliferation following treatment with various combinations of pro-TAME and Apcin at different time point (24, 48, and 72 h). #p 0.05, AN3CA, compared with 0 µM. *p 0.05, KLE, compared with 0 µM. pro-TAME, pro-Tosyl-L-Arginine Methyl Ester; EC, endometrial carcinoma; CCK-8, Cell Counting Kit-8.