Overexpression of FOS enhances the malignant potential of eutopic endometrial stromal cells in patients with endometriosis‑associated ovarian cancer
- Authors:
- Published online on: February 17, 2025 https://doi.org/10.3892/or.2025.8878
- Article Number: 45
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Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Endometriosis (EM) is defined as the presence of endometrium-like tissue outside the endometrium and myometrium and is often detected in ovaries as an endometrial cyst of the ovary (EMC) (1). Long-term development of EMC may lead not only to chronic pelvic pain and infertility but also to malignant transformation, which is considered EM-associated ovarian cancer (EAOC) (2). The incidence of malignant transformation has been reported to be 0.7–2.5% (3). The main pathological types of EAOC are clear cell carcinoma and endometrioid carcinoma, which are characterized by co-occurrence within the same lesion of EM and cancerous tissue (4). However, the pathogenesis of EAOC remains unclear.
For several decades, a wide range of mechanisms, including oxidative stress, inflammation and estrogen stimulation, involved in the malignant transformation of ovarian EM to EAOC, have been studied (5–7). However, most of these studies focused only on the effects of the external microenvironment on the malignant transformation of ovarian lesions and ignored the changes that had already occurred while the lesion was in the eutopic endometrium, which might be the origin of EAOC. Previous studies have indicated that EAOC can be caused by implantation of eutopic endometrial epithelium and mesenchymal cells containing oncogenic changes in ovaries via retrograde menstruation (8). These changes in the eutopic endometrium include DNA methylation aberrance, oncogene activation and antioncogene inactivation (7,8). Interestingly, several researchers have indicated that compared with eutopic endometrium in normal patients, abnormal eutopic endometrium in EM patients is characterized by gene mutations and increased adhesion, metastasis and angiopoiesis (8,9).
In this regard, it was hypothesized that the eutopic endometrium of EAOC patients may exhibit more profound and diverse oncogenic mutations than those of EM patients. These mutations could encompass a range of genetic and epigenetic alterations, including but not limited to changes in cytoskeletal and chromatin-remodeling proteins, activation of oncogenes such as KRAS, and inactivation of tumor suppressor genes. Additionally, the eutopic endometrium in EAOC might be characterized by increased expression of genes such as RRM2, which are implicated in abnormal cell proliferation and potential malignant transformation. These complex genetic and molecular changes suggest a multifactorial pathogenesis of EAOC, diverging significantly from the pathogenesis observed in EM (10–13), which provided new insight into the detection of EAOC. Based on the differences in mRNA or protein expression in the eutopic endometrium between EAOC patients and EMC patients, endometrial biopsy followed by specific biomarker detection might be employed for distinguishing EAOC from EM.
The aim of the present study was to elucidate the potential eutopic endometrium-related pathogenesis of EAOC and to identify a feasible biomarker of EAOC in the eutopic endometrium via RNA sequencing. Subsequently, the protein expression of the biomarker was tested by immunohistochemistry (IHC) in eutopic endometrial tissue. Finally, the molecular function of the biomarker was explored in eutopic endometrial cells from EAOC and EM patients.
Materials and methods
Specimens
The present study was approved (approval no. 2021202) by the Jilin University Second Hospital's Ethics Committee (Changchun, China). Informed consent was provided by all patients for participation in the study.
Between January 2022 and September 2022, five eutopic endometrial samples and four eutopic endometrial samples were taken from EAOC and EMC patients at the Second Hospital of Jilin University. Within 30 min of excision, the samples were collected in liquid nitrogen and utilized for RNA sequencing.
At the Second Hospital of Jilin University, paraffin-embedded eutopic endometrial samples were collected from 63 patients with EAOC, 95 patients with EMC, and 16 healthy controls who were diagnosed between January 2012 and September 2022. The baseline clinical characteristics of the EAOC and EMC patients are displayed in Table I. The detailed clinical information of the normal patients included in the present study is presented in Table SI.
The inclusion criteria for EAOC patients were as follows: In accordance with Sampson's diagnostic criteria (SAMPSON 1925) (14), the lesions were histologically identified as EAOC, and eutopic endometrium specimens were obtained.
The following criteria were applied to the included EMC patients: i) Eutopic endometrial specimens could be obtained; and ii) the lesions were histologically identified as EMC.
The following criteria were applied to the included normal controls: i) An EM diagnosis was ruled out through laparoscopic hysterectomy or hysteromyomectomy; ii) Specimens of the eutopic endometrium were obtained.
The exclusion criteria for patients were as follows: The use of hormone medications prior to surgery, diabetes or other endocrine illnesses, abnormal uterine hemorrhage, or major systemic diseases.
All the samples gathered, including those preserved in liquid nitrogen and those embedded in paraffin, fulfilled the aforementioned criteria.
RNA extraction and sequencing
Suzhou PANOMIX Biomedical Tech Co., Ltd., handled the RNA extraction and sequencing. Briefly, the whole RNA was extracted from the aforementioned samples. After that, oligo (DT) magnetic beads were used to select for mRNAs with polyA structures in total RNA. The enriched mRNA was then cut into pieces of ~300 bp in length by ion interruption, which were subsequently used as the template for reverse transcription into cDNA and PCR amplification. The cDNA library was subsequently sequenced using Illumina HiSeq and paired-end sequencing via next-generation sequencing (NGS).
RNA-seq data analysis
FPKM was used to normalize the raw counts. In Bayesian analysis, the R package ‘limma’ was utilized to identify the differentially expressed genes (DEGs) across eutopic endometrial samples from EAOC and EMC patients. The threshold for DEGs was defined as a |log2 Fold Change (logFC)|>1 and an adjusted P-value of 0.05. The STRING database (https://string-db.org/) was used to visualize the DEGs (15), and protein-protein interaction (PPI) analysis of the DEGs was performed using Cytoscape software (https://cytoscape.org/) network analysis. Gene functional enrichment analysis was based on Gene Ontology (GO) and gene set enrichment analysis (GSEA) using the R packages ‘clusterProfiler’ and ‘org.Hs.eg.db’, respectively (16). An adjusted P-value <0.05 or a false discovery rate (FDR) <0.25 were considered to indicate significant enrichment. c2.cp.v7.2. symbols. gmt was used as the reference gene set for GSEA. This gene set is part of the Molecular Signatures Database [MSigDB(https://www.gsea-msigdb.org/gsea/msigdb)] and is extensively utilized in GSEA for identifying biologically meaningful patterns within gene expression data. The choice of this particular gene set was influenced by its extensive coverage of known biological pathways and processes. This comprehensive scope is invaluable for accurately interpreting our RNA-seq data, especially in the context of eutopic endometrium-related pathogenesis in EAOC.
IHC
IHC staining was carried out as previously described (17). The paraffin-embedded materials were divided into 3-µm thick sections. The sections were heated in a microwave after being deparaffinized with xylol and then rehydrated with a succession of decreasing alcohol concentrations, starting with 100% ethanol, followed by 95, 85, 70%, and finally 50% ethanol for gradual rehydration. After incubation at room temperature for 20 min, non-specific binding was inhibited with 5% bovine serum albumin (cat. no. AR1006; Boster Biological Technology). The histological sections were then incubated at 4°C overnight with rabbit anti-FOS antibody (Wuhan Servicebio Technology Co., Ltd.; cat. no. GB12069; 1:400). The secondary antibody used was goat anti-rabbit IgG coupled with horseradish peroxidase (Proteintech Group, Inc.; cat. no. SA00001-2; 1:200), and the staining process was performed at 37°C for 30 min. Sections were counterstained with 0.1% hematoxylin (Boster Biological Technology) at room temperature for 2 min to detect reactive reactions using the chromogen 3,3′-diaminobenzidine (Boster). With an objective magnification of ×200 or ×400, histological images were recorded using a light microscope (Motic Incorporation, Ltd.; cat. no. AE2000). The program Image-Pro Plus 6.0 (Media Cybernetics, Inc.) was used to determine the positive cell density, and the results are shown as the mean optical density (MOD) values (17).
Isolation, identification and culture conditions for human endometrial stromal cells (hEnSCs)
As previously mentioned, the hEnSCs were separated from 3 EAOC patients and 3 EMC patients. Briefly, eutopic endometrial samples were received and dissected from the scalpels. Collagenase IV (MilliporeSigma) was then used to lyse the tissues for 1 h at 37°C. Dulbecco's modified Eagle's medium (DMEM)/nutrient combination F12 medium (DMEM/F12; Gibco; Thermo Fisher Scientific, Inc.) was used to cultivate the hEnSCs after they had been filtered via a 40-µm filter. A total of 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (cat. no. P1400; Beijing Solarbio Science & Technology Co., Ltd.) were added as supplements. Using flow cytometry (BD Biosciences), the homogeneity of the cultures was assessed based on morphological traits and confirmed by surface-positive (CD73, CD90 and CD105) and negative (HLA-II) markers.
Furthermore, as previously described, immunofluorescence staining was performed to detect the stromal and epithelial markers Vimentin and Src (18). The main antibodies utilized were rabbit anti-Src (Wuhan Servicebio Technology Co., Ltd.; cat. no. GB111035; 1:500) and mouse anti-vimentin (Wuhan Servicebio Technology Co., Ltd.; cat. no. GB12192; 1:500). The secondary antibodies used were goat anti-rabbit IgG (H+L) conjugated with Cy3 (Wuhan Servicebio Technology Co., Ltd.; cat. no. GB21303; 1:300) and goat anti-mouse IgG (H+L) conjugated with FITC (Wuhan Servicebio Technology Co., Ltd.; cat. no. GB22301; 1:100). Slides were examined under a fluorescence microscope (Olympus Corporation). In the resulting images, Src protein is indicated by red fluorescence, Vimentin protein by green, and cell nuclei were counterstained with DAPI, appearing blue.
Generation of stably transfected hEnSCs
LV-FOS and LV-NC were purchased from Hunan Fenghui Biological Co., Ltd. The lentivirus had titers of 1.8 and 1.5×108 TU/ml, respectively. To prevent frequent freezing and thawing, the samples were often kept in a low-temperature refrigerator at −80°C and put at 4°C before use. The suspended initial cells were injected into six-well plates (5×105 cells/well). After the cells had adhered to the wall for 12 h, MOI=200 cell culture media containing LV-FOS or LV-NC was added. Polybrene (5 g/ml) was added to the medium to increase the rate of infection. After 12 h of incubation at 37°C, 5% CO2, and saturated humidity, the medium was replaced. A total of 72 h after infection, the expression of FOS was assessed using an inverted fluorescence microscope. Green luminous cells with the FOS signature were positive cells. To calculate the transfection efficiency, the number of fluorescent positive cells was counted and this value was divided by the total number of cells present in the field of view. This ratio was then multiplied by 100 to express the efficiency as a percentage. Specifically, fields were randomly selected, and both fluorescent (indicating successful transfection) and non-fluorescent cells were counted using a fluorescence microscope. Then, cells with the virus that encodes the gene for puromycin resistance were chosen from 1.5 g/ml puromycin. Prior to cell collection and analysis, puromycin (0.5 g/ml) screening was carried out for an additional week.
Cell Counting Kit-8 (CCK-8) assay
hEnSCs were seeded onto 96-well plates at a density of 3,000 cells/well along with FOS inhibitor (T-5224; cat. no. HY-12270; MedChemExpress) at various concentrations (0, 5, 10 and 20 M). CCK-8 reagent (10 µl/well; cat. no. K009; Zeta Life Inc.) was then added to each well 48 and 72 h later. Using a microplate reader (cat. no. E0226; Nanjing DeTie Laboratory Equipment Co., Ltd.), the absorbance of each well was assessed at 450 nm after 1.5 h of culture at 37°C.
Colony formation assay
A total of 100 cells per well of 6-well plates were plated with the appropriate cells. A total of 10 days later, the development of a typical cell clone was observed. The cells were stained with 10% Giemsa (Biotopped; http://www.bjbiotopped.com/) at room temperature for 10 min after being fixed with methanol at room temperature for 30 min. To assess the colony production capacity of the cells, visible colonies were counted. Traditionally, a dense conglomerate of cells is regarded as a colony when the number of cells exceeds 50. The software ImageJ [Fiji (https://imagej.net/software/fiji/downloads) (National Institutes of Health)] was used to count the number of colonies.
Cell scratch assay
The specified cells were seeded onto 6-well plates (6×105 cells/well) for the cell scratch test. T-5224 was introduced to hEnSCs at concentrations of 0, 5, 10 and 20 M after the cells had been cultured at 37°C for 24 h. A plastic pipette tip was used to scrape the cell layer. After 48 h, the rate of cell migration near the scratch edge was examined. The cells were serum-starved during the assay.
Western blot analysis
RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) was used to separate the soluble proteins from the different concentrations (0, 5, 10 and 20 M) of T-5224-treated or stably transfected hEnSCs. A Detergent Compatible Bradford Protein Assay Kit (cat. no. P0006C; Beyotime Institute of biotechnology) was used to quantify the protein concentrations. Then, 15 µg of protein was run through each lane of a 4–20% Precast Bis-Tris Gel (Absin; cat. no. abs9384;). Preserved standards, which are molecular weight markers, (Beijing Transgen Biotech Co., Ltd.; cat. no. DM141-01) were employed. The separated proteins were electrophoretically transferred onto polyvinylidene difluoride membranes (iBlot system; Invitrogen; Thermo Fisher Scientific, Inc.). The membranes were then blocked with 5% skim milk for 1 h at room temperature. Subsequently, membranes were incubated with primary antibodies, including anti-FOS (Wuhan Servicebio Technology Co., Ltd.; cat. no. GB12069; 1:1,000), anti-P21 (Proteintech Group, Inc.; cat. no. 10355-1-AP; 1:2,000), anti-CDK4 (Proteintech Group, Inc.; cat. no. 11026-1-AP; 1:2,000), anti-CyclinD1 (Proteintech Group, Inc.; cat. no. 26939-1-AP; 1:5,000), anti-phosphorylated (p-)Stat3 (BIOSS; cat. no. bs-1658R; 1:1,000), anti-Stat3 (BIOSS; cat. no. bsm-52235R; 1:750), anti-MMP2 (BIOSS; cat. no. bs-0412R; 1:500), anti-MMP9 (BIOSS; cat. no. bs-4593R; 1:500) and anti-GAPDH (Bioworld Technology, Inc.; cat. no. BS65656; 1:20,000), at room temperature for 1.5 h. After 1 h of room temperature incubation with secondary-HRP antibodies (Bioworld Technology, Inc.; cat. nos. BS13278 and BS12478; 1:20,000), the protein levels were assessed using an image densitometer (Clinx Science Instruments Co., Ltd.) and an ECL kit (cat. no. KF8005; Affinity Biosciences).
Statistical analysis
The statistical analysis utilized the mean of three independent tests together with the standard deviation (SD). When performing the statistical analyses with SPSS 23.0 IBM Corp._ or R version 3.6.3 (https://www.r-project.org/), one-way analysis of variance (ANOVA) was used to examine group differences, followed by Dunnett's post hoc test, the Kruskal-Wallis test, or unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
Results
Identification of DEGs via RNA sequencing
The RNA-seq data were used to analyze the significant (|logFC|>1, adjusted P-value <0.05) differences between the eutopic endometrium of EAOC patients and those of EMC patients, and 249 DEGs were identified, including 202 significantly upregulated and 47 downregulated genes (Fig. 1A). The data of eutopic endometrium samples from EMC patients were considered controls. FOS mRNAs with significant differences are indicated in the volcano plot. The interactions among the DEGs are demonstrated in the PPI network (Fig. 1B). PPI network analysis revealed that genes such as JUN, MKI67, BUB1B, TOP2A, EXO1, UBE2C, KIF4A and CENPF occupied central positions in the network, indicating a greater functional correlation with the FOS gene. Investigating the roles of these genes could be instrumental in elucidating the potential pathways and functional mechanisms through which the FOS gene may contribute to the progression from EMC to EAOC.
Pathway enrichment of DEGs
A total of 15 signaling pathways related to the 249 DEGs were enriched according to GO analysis. Notably, it was found that the enrichment pathways were associated mainly with mitotic nuclear division, vasculogenesis, the ERK1 and ERK2 cascades, cell-cell adhesion and epithelial cell proliferation (Fig. 1C). The interactions among these pathways are revealed in Fig. 1D.
Moreover, to explore the relevant pathways more comprehensively, the data of all genes were further subjected to GSEA. The GSEA results indicated that DNA methylation, HDACS deacetylase histones, meiotic recombination, cell cycle checkpoints and the AP1 pathway were enhanced in the samples from EAOC patients (Fig. 1E), whereas some pathways were inhibited, including oxidative phosphorylation (OXPHOS), complement activation, the No2-IL12 pathway, and the electron transport chain OXPHOS system (Fig. 1F).
IHC verification of FOS expression in endometrial clinical samples
The endometrium exhibits notable differences between the proliferative and secretory phases. Specifically, the proliferative phase typically spans from the 5th to the 14th day of the menstrual cycle and is predominantly regulated by estrogen. The pathological characteristics of this phase include: i) Progressive thickening of the endometrium; ii) glandular structures appearing as straight, elongated tubes, uniformly distributed; iii) glandular cells exhibiting a columnar shape with evident signs of mitosis; iv) proliferation of stromal cells, presenting a loose arrangement; and v) vascular proliferation without notable coiling.
Conversely, the secretory phase generally occurs from the 15th to the 28th day of the menstrual cycle and is influenced primarily by progesterone. The pathological hallmarks of this phase include the following: i) continued thickening of the endometrium, reaching its peak; ii) glands acquiring a ‘serrated’ or spiral configuration, becoming more convoluted and dilated; iii) enlargement of glandular cells, with the presence of secretory granules in the cytoplasm and nuclei migrating basally (indicative of subnuclear vacuolation); iv) densification of stromal cells and the matrix, possibly involving pre-decidual cells; and v) increased coiling of blood vessels, forming a distinctive spiral pattern.
To validate the expression of FOS, samples from 57 patients with EMC and 55 patients with EAOC during the proliferative phase and 38 EMC and 8 EAOC patient samples during the secretory phase were utilized. The control group comprised 16 patients, including 3 in the secretory phase and 13 in the proliferative phase. FOS expression in proliferative endometrial tissues was assessed via IHC staining. The results indicated that FOS expression was upregulated in the endometrial tissues of patients with EAOC compared with that in patients with EMC (Fig. 2A and B). FOS expression levels in secretory endometrial tissues were measured by the same method. As demonstrated in Fig. 2C and D, FOS expression was upregulated in the endometrial tissues of patients in the EAOC group compared with that of patients in the EMC group.
The expression of FOS in all endometria is shown in Fig. 2E. These results were consistent with previous results. FOS expression was higher in EAOC than in EMC, suggesting that FOS might be closely related to the conversion from EMC to EAOC.
Identification of human endometrial stromal cells
hEnSCs were cultured from fresh endometrial tissues from EMC and EAOC patients, and flow cytometry and immunofluorescence staining were used to identify the endometrial cells cultured in vitro. The cell surface markers CD105, CD73, CD90 and HLA-II were detected via flow cytometry. The results showed that the expression of CD105, CD73 and CD90 on the surface of the endometrial membrane was positive in primary culture, while the expression of HLA-II was negative (Fig. 3A).
The immunofluorescence staining results are displayed in Fig. 3B. The endometrial cell markers Src and Vimentin could be detected in the cytoplasm. These results confirmed that the extracted cells were endometrial cells.
Detection of differences in FOS expression levels and proliferation and migration abilities of EMC- and EAOC-derived hEnSCs
Western blot assays were performed using hEnSCs from EMC and EAOC, respectively, to measure the expression levels of FOS protein in the cells, and it was found that the expression of FOS in EAOC was significantly higher than that in EMC (Fig. 3C and D). Subsequently, CCK-8 was performed to detect the cell viability and proliferation ability of hEnSCs from different tissues, and it was found that the proliferation ability of hEnSCs from EAOC was significantly stronger than that of hEnSCs from EMC (Fig. 3E). Finally, the migration ability of hEnSCs was examined using a scratch assay. The migration ability of hEnSCs from EAOC was still significantly higher than that of hEnSCs from EMC (Fig. 3F and G).
The effect of inhibiting FOS on the proliferation of hEnSCs
hEnSCs derived from EAOC were treated with different concentrations of the FOS inhibitor T-5224, and cell viability was determined via a CCK-8 assay. After treatment with T-5224 ≥10 µM, the viability of hEnSCs significantly decreased with increasing FOS inhibitor concentration for 48 or 72 h (P<0.01), and the effect was dose-dependent (Fig. 4A).
The effect of the FOS inhibitor T-5224 on the proliferation of hEnSCs derived from EAOC was further detected by a colony formation assay (Fig. 4B and C). The colony formation ability of hEnSCs was significantly reduced after treatment with 10 or 20 µM T-5224 (P<0.01).
The effect of inhibiting FOS on the migration of hEnSCs
A cell scratch assay was performed to determine the effect of the FOS inhibitor T-5224 on the migration of hEnSCs derived from EAOC. As demonstrated in Fig. 4D and E, 5, 10 and 20 µM T-5224 inhibited the migration of hEnSCs in a dose-dependent manner.
Effect of FOS inhibition on the expression of cell cycle-related proteins in hEnSCs
Western blotting was used to detect the expression of cell cycle-related proteins in hEnSCs derived from EAOC after treatment with the FOS inhibitor T-5224. As the T-5224 concentration increased, the expression of P21 gradually increased, and the expression of CDK4 and Cyclin D1 gradually decreased (Fig. 4F and G).
Effect of FOS inhibition on the expression of proteins associated with endometrial cell invasion
Western blotting was used to detect the expression of invasion-related proteins in EAOC-derived hEnSCs treated with the FOS inhibitor T-5224. As the FOS inhibitor concentration increased, the expression of p-Stat3, MMP2 and MMP9 gradually decreased (Fig. 4H and I).
Effect of the upregulation of FOS on the proliferation of hEnSCs
The pLKO1-FOS plasmid was transfected into EMC hEnSCs using a lentiviral vector, after which the stably transfected cell lines were screened. As revealed in Fig. 5A, the transfection efficiency was ~80–90%. Further western blot detection of FOS expression in stably transfected cell lines identified that FOS expression was significantly upregulated in stably transfected cell lines compared with that in the parent strains (Fig. 5G and H).
CCK-8 and colony formation assays were subsequently used to evaluate the effect of upregulated FOS expression on cell proliferation. The results demonstrated that, compared with that of the control group, the viability of hEnSCs from EMC was significantly increased after the upregulation of FOS expression (Fig. 5B). The results of the colony formation assay also suggested that upregulated FOS expression could promote the proliferation of hEnSCs (Fig. 5C and D).
Effect of upregulated FOS on the migration of hEnSCs
Cell scratch assays were used to detect the effect of stable transfection of FOS on the migration of EMC-derived hEnSCs. As indicated in Fig. 5E and F, stable transfection of FOS promoted the migration of endometrial cells.
Effect of upregulated FOS on the expression of proliferation-related proteins in hEnSCs
Western blotting was used to detect the expression of cell cycle-related proteins in hEnSCs from EMC after stable transfection with FOS. It was identified that with the upregulation of FOS expression, P21 expression was downregulated, while CDK4 and Cyclin D1 expression was upregulated (Fig. 5G and H).
Effect of upregulated FOS on the expression of migration-related proteins in hEnSCs
The expression of invasion-related proteins in hEnSCs derived from EMC was detected by western blotting. The results demonstrated that p-Stat3, MMP2 and MMP9 expression was upregulated along with the upregulation of FOS expression (Fig. 5I and J). The mechanism through which the FOS protein regulates the proliferation and migration ability of hEnSCs is shown in Fig. 6.
Discussion
In the present study, RNA sequencing technology was utilized to identify 249 DEGs between the eutopic endometrium of patients with EMC and those with EAOC. Notably, a core group of genes, FOS, JUN, MKI67, TOP2A, BUB1B, EXO1, KIF4A, KIF20A, CENPF, NEK2 and KIF18B, was highlighted as central to the present PPI analysis, which indicated that the functions of these genes are strongly related to each other.
Further analysis revealed that these genes are notably involved in critical cellular processes. Specifically, FOS and JUN are key components of the MAPK signaling pathway and are known for their roles in cell proliferation, differentiation and apoptosis, and for their link to tumor development (19,20). MKI67 is implicated in cell proliferation and maintaining the organization of chromosomes during cell division (21). TOP2A plays a crucial role in cell division and growth, particularly in ovarian cancer (22). Along with CENPE and NEK2, BUB1B ensures accurate chromosome separation during cell division (23–26). In previous years, EXO1 has been shown to be overexpressed in various cancers, contributing to the survival of cancer cells through its DNA repair function (27–29). Additionally, the KIF family of proteins is known for its involvement in cell cycle regulation and cell division (30,31).
Notably, FOS was found to be overexpressed in the eutopic endometrium of EAOC patients compared with that in the eutopic endometrium of EMC patients. The overexpression of FOS suggested its potential role in modulating the behavior of human endometrial stromal cells (hEnSCs), specifically by influencing their proliferation and migration abilities.
These findings provide a detailed picture of the molecular interactions and processes involved in the transformation of EMC to EAOC. Understanding these dynamics provides valuable insights into the biological pathways involved in this transition, setting the stage for further discussion on the results of gene functional enrichment analysis, where the broader implications of these gene expression changes on cellular functions will be we explored.
To further reveal the potential changes in signaling pathways caused by differences in gene expression between eutopic endometria from EMC patients and those from EAOC patients, differential GO analysis and GSEA was performed in the present study. Notably, GO analysis revealed that the following signaling pathways were significantly enriched: Angiogenesis, the ERK1 and ERK2 cascades, and cell-cell adhesion. Angiogenesis is an important factor that supports malignant tumor transformation, development and metastasis (32,33). ERKs ensure cell proliferation, differentiation, survival and metastasis by receiving a large number of growth factors (EGF, NGF and PDGF) and acting on nuclear transcription factors such as AP-1 and NF-κB (34,35). In addition, dysfunction of cell adhesion molecules also plays an important role in tumor metastasis (36). Interestingly, GSEA revealed that DNA methylation, HDACS-mediated deacetylation of histones, the cell cycle checkpoint, and the AP1 pathway were enhanced in the eutopic endometrium of EAOC patients, while complement activation was inhibited (37–39). AP-1, an intracellular transcriptional activator, is a dimer composed of c-FOS and c-JUN. Previous studies have shown that the AP-1 pathway mediates tumor cell proliferation and metastasis (40,41). These results showed that the proliferation and metastasis abilities of eutopic endometrium in patients with EAOC were enhanced, which was consistent with the PPI prediction, implying that the endometrium has more potential for malignant transformation in patients with EAOC than in patients with EMC. The malignant transformation caused by dysfunction of these pathways might increase the likelihood of developing EAOC in the eutopic endometrium. However, further experiments are needed to confirm these predictions.
Considering the important role of the AP1 pathway in cell proliferation and metastasis and the high expression of FOS mRNA in eutopic endometrium of EAOC patients, FOS protein expression in eutopic endometria of patients with EAOC and EMC was further analyzed using IHC staining. The current results revealed that the FOS protein was overexpressed in the eutopic endometrium of EAOC patients compared with that in the endometrium of EMC patients. Notably, previous studies have reported that FOS, a nuclear protein transcription factor, plays an important role in cervical cancer, prostate cancer and other tumors by regulating cell growth, proliferation and metastasis (42–45). Hence, it was suggested that the FOS protein might be involved in enhancing the malignant potential of the eutopic endometrium.
To test this hypothesis, primary hEnSCs were extracted from the eutopic endometrial tissues of EMC and EAOC patients to further verify the regulatory mechanism of FOS protein on the eutopic endometrium in EAOC. According to previous studies, primary hEnSCs cultured to the 3rd generation exhibit a fibroblast-like morphology, with positive expression of the cell surface markers CD105, CD73 and CD90 and negative expression of HLA-II (46,47). Moreover, Src and Vimentin proteins were positively expressed in hEnSCs (48,49). The present identification results for hEnSCs were consistent with previous studies, indicating that the extraction of primary hEnSCs was successful and could be used for subsequent experimental studies.
Subsequently, in the present study, using hEnSCs extracted from endometrial tissues of EMC and EAOC patients, western blot experiments revealed differential expression of FOS protein. The results of CCK-8 and scratch assays also suggested that the proliferation and migration ability of hEnSCs from endometrial tissues of EAOC patients were significantly stronger than that of hEnSCs from endometrial tissues of EMC patients. The potential mechanism of the proliferation and migration ability of hEnSCs needs to be further investigated.
In the present study, the authors delved deeper into the multifaceted role of the FOS protein in modulating the cellular dynamics of hEnSCs derived from EAOC patients. The inhibition of FOS protein activity by T-5224 revealed a dose-dependent decrease in the proliferation and migration of hEnSCs from EAOC patients, highlighting the central role of FOS in these processes. Moreover, the overexpression of FOS in hEnSCs from EMC patients significantly augmented their proliferative and migratory capabilities, corroborating the findings from the inhibition experiments and underscoring the dual regulatory nature of FOS.
The decision to use inhibitors instead of interfering plasmids to suppress FOS was informed by several considerations. Primarily, the focus on the short-term functions and mechanisms of FOS necessitated a methodology that provided swift action and easy control over effects, attributes aptly met by the inhibitors. Their rapid onset and the ability to fine-tune the dosage offered a precise way to study FOS's immediate impact. By contrast, for experiments involving FOS overexpression, interfering plasmids were used due to the lack of suitable FOS activators. Furthermore, the use of inhibitors aligns with previous research (50), where FOS inhibitors effectively blocked human chorionic gonadotropin-induced increases in PGE2 and the expression of prostaglandin synthases and transporters in human granulosa cells. This approach, supported by literature, allowed for a more straightforward and controllable operation, avoiding the variables potentially introduced by the varying transfection efficiencies of interfering plasmids. Thus, the authors' methodology harnessing the specificity and controllability of inhibitors significantly contributed to the understanding of FOS's role in the pathogenesis of EAOC.
At the molecular level, FOS has been implicated in the transition from the G1 phase to the S phase of the cell cycle, a critical juncture for cell division and proliferation. This regulation is orchestrated through the suppression of P21, a cyclin-dependent kinase inhibitor, and the concurrent upregulation of CDK4 and CyclinD1, proteins that are pivotal in driving cell cycle progression (51–53). The modulation of these molecular players by FOS underscores its integral role in the proliferation of not only tumor cells but also hEnSCs, as evidenced by the present findings.
Furthermore, FOS has been demonstrated to bolster cell migration, an attribute pivotal for tissue remodeling and potentially metastasis. This migration facilitation is mediated through the upregulation of MMP2 and MMP9, which are matrix metalloproteinases that modulate the extracellular matrix and facilitate cellular movement (53). The present observations resonate with this mechanism, as the FOS protein in the current study was shown to regulate the Stat3/MMP2/MMP9 signaling pathway, aligning with its established role in promoting cell migration.
Notably, the interplay between P21, CDK4/CyclinD1 and FOS orchestrates a delicate balance between cell cycle arrest and progression. P21 acts as a brake, halting the cell cycle in response to various stimuli, including DNA damage. FOS, by inhibiting P21, effectively releases this brake, allowing CDK4/CyclinD1 to drive the cell cycle forward. This mechanism is central not only to normal cellular functions but also to the pathology of cancer, where dysregulated cell cycle progression is a hallmark.
In the context of cell migration, the Stat3/MMP2/MMP9 pathway regulated by FOS is indicative of its role in cellular architecture remodeling. In particular, MMP2 and MMP9 are crucial for degrading the extracellular matrix, a process essential for cell movement and invasion. This capability, while vital for normal processes such as wound healing, can be coopted by cancer cells to facilitate metastasis.
Thus, the present study elucidated the pivotal role of FOS in regulating the proliferation and migration of hEnSCs through the P21/CyclinD1/CDK4 and Stat3/MMP2/MMP9 signaling pathways. These findings not only shed light on the molecular underpinnings of FOS function but also highlight its potential as a therapeutic target in conditions characterized by aberrant cellular proliferation and migration.
In conclusion, the DEGs-enriched signal pathways regulating proliferation and migration were promoted in eutopic endometrium of EAOC patients compared with those in the eutopic endometrium of EMC patients, indicating that the malignant potential of the eutopic endometrium of EAOC patients was enhanced. FOS protein was overexpressed in the eutopic endometrium of EAOC patients and could enhance the malignant potential of hEnSCs by regulating the P21/CyclinD1/CDK4 and Stat3/MMP2/MMP9 signaling pathways.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
The present study was supported by the Department of Science and Technology of Jilin (grant no. 3D5214067433) and the project of Jilin Provincial Development and Reform Commission (grant no. 3J1196620433).
Availability of data and materials
The datasets generated and/or analyzed during the current study are available in the Gene Expression Omnibus Repository (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE226575).
Authors' contributions
MC and LZ conceived and designed the study. KH, JC and MW acquired the data and performed the statistical analysis. XL, KW, WJ, ZY and ZW performed the experiments and analyzed the data. KH and JC drafted the manuscript. JC, KH and ZY contributed to revising the manuscript for intellectual content and language editing. All authors read and approved the final version of the manuscript. MC and JC confirm the authenticity of all the raw data.
Ethics approval and consent to participate
The present study was approved by the Ethics Committee of Jilin University Second Hospital (approval no. 2021202; Changchun, China). Informed consents were obtained from patients for participation in the EAOC-relative study.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
|
EM |
endometriosis |
|
EMC |
endometrial cyst of ovary |
|
EAOC |
endometriosis-associated ovarian cancer |
|
DEGs |
differential expressed genes |
|
PPI |
protein-protein interaction |
|
GO |
Gene Ontology |
|
GSEA |
Gene set enrichment analysis |
|
IHC |
immunohistochemical |
|
CCK-8 |
Cell Counting Kit-8 |
|
hEnSCs |
human endometrial stromal cells |
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