Estrogen receptor 1 regulates ovarian cancer cell stemness and ferroptosis through modulation of the canonical RhoA/ROCK signaling pathway

Abstract

BACKGROUND

This study investigated the role of the estrogen receptor 1 (ESR1) in ovarian cancer and its underlying mechanisms. Target genes associated with ovarian cancer, cell stemness, and ferroptosis were identified using DisGeNET and GeneCards databases.

AIM

To explore the effect of ESR1 on ovarian cancer and its potential molecular mechanism.

METHODS

ESR1 expression levels in HOSE, SKOV3, A2780, and OVCAR3 cells were assessed using real-time polymerase chain reaction and western blotting. ESR1 knockdown was performed in A2780 cells to evaluate its effects on cell viability, proliferation, apoptosis, aldehyde dehydrogenase activity, cell stemness, ferroptosis, and the Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase (ROCK) signaling pathway with or without treatment with CN03 (a Rho activator).

RESULTS

ESR1, a gene associated with ovarian cancer, cell stemness, and ferroptosis, was significantly upregulated in ovarian cancer cells and interacted with the RhoA/ROCK signaling pathway. ESR1 knockdown resulted in decreased levels of RhoA, ROCK1, and ROCK2, reduced cell viability and proliferation, and increased apoptosis, all of which were reversed by CN03 treatment. Additionally, si-ESR1 led to decreased aldehyde dehydrogenase activity and cell stemness, reduced expression of octamer-binding transcription factor 4 and Nanog, increased Fe²⁺ levels, and elevated expression of ferritin heavy chain, ferritin light chain, and transferrin receptor 1. These changes were reversed by CN03 treatment.

CONCLUSION

ESR1 inhibition regulates cell stemness and ferroptosis in ovarian cancer by modulating the RhoA/ROCK signaling pathway.

Key Words: Estrogen receptor 1; Ovarian cancer; Cell stemness; Ferroptosis; RhoA/ROCK

Core Tip: This study investigated the effects of estrogen receptor 1 (ESR1) on ovarian cancer. Our findings revealed that ESR1, an overexpressed gene associated with cell stemness and ferroptosis in ovarian cancer, interacts with the Ras homolog (Rho) gene family member A/Rho-associated coiled-coil containing protein kinase signaling pathway. ESR1 knockdown in A2780 cells significantly decreased cell viability and proliferation, while increasing apoptosis. ESR1 silencing also reduced cell stemness (lowered aldehyde dehydrogenase, octamer-binding transcription factor 4, and Nanog) and promoted ferroptosis (increased Fe²⁺, ferritin heavy chain, and ferritin light chain, and decreased transferrin receptor 1). These inhibitory effects were reversed by treatment with CN03 (a Rho activator), demonstrating that ESR1 regulates ovarian cancer stemness and ferroptosis via the Ras homolog gene family member A/Rho-associated coiled-coil containing protein kinase signaling pathway.

INTRODUCTION

Ovarian cancer is the fifth leading cause of cancer-related deaths among women aged 40 years and older[1]. By 2022, an estimated 324398 new cases of ovarian cancer were diagnosed globally, resulting in approximately 206839 deaths. China has a heavy disease burden, accounting for 18.82% of all new ovarian cancer cases and 15.8% of ovarian cancer-related deaths worldwide[2]. Epithelial ovarian cancer accounts for over 95% of all malignant ovarian tumors, with high-grade serous ovarian cancer (HGSOC) being the predominant histological subtype responsible for approximately 80% of all ovarian cancer deaths[3]. Over 70% of HGSOC cases are diagnosed at advanced stages primarily because of the nonspecific early symptoms of the disease[4]. The conventional therapies for ovarian cancer involve cytoreductive surgery combined with platinum-based chemotherapy[5]. Nearly 70% of patients with late-stage ovarian cancer experience recurrence and develop treatment resistance, with fewer than half surviving beyond five years post-diagnosis[6]. Despite the effectiveness of poly ADP-ribose polymerase inhibitors, effective therapeutic options for ovarian cancer remain limited. Therefore, there is an urgent need to uncover the molecular mechanisms that drive ovarian cancer progression and identify novel therapeutic targets.

The estrogen receptor 1 (ESR1) gene, located in the cell nucleus, forms homodimers or heterodimers with ESR2 to regulate various physiological processes[7]. Numerous studies have demonstrated an association of ESR1 with ovarian cancer, breast cancer, endometrial cancer, and other malignancies[8]. ESR1 is expressed in up to 60% of ovarian epithelial tumors, with higher expression levels observed than in normal ovaries[9]. Recent investigations have suggested that significant upregulation of ESR1 in ovarian tissue promotes ovarian cancer progression[10]. Elevated ESR expression facilitates lymphatic or vascular spread in HGSOC and correlates with poor clinical outcomes[11]. Thus, ESR1 holds promise as a potential biomarker for risk stratification and regional metastasis assessment in patients with ovarian cancer.

Mounting evidence suggests that cancer stem cells (CSCs) contribute to chemotherapy resistance and tumor recurrence[12]. As a rare subset of malignant cells, CSCs exhibit unique characteristics, such as self-renewal, drug resistance, and an epithelial-mesenchymal transition phenotype[13]. Ovarian CSCs can be identified by specific surface markers, including CD133, CD117, and CD44, as well as elevated aldehyde dehydrogenase (ALDH) activity[14]. Moreover, ovarian CSCs show increased levels of stem cell-associated factors such as Nanog homeobox (NANOG), SRY-box transcription factor 2, and Krüppel-like factor 4, which have been linked to tumor initiation, immune evasion, metastasis, and chemotherapy resistance[15]. Understanding the mechanisms underlying CSC properties may provide new strategies for improving the prognosis and treatment of ovarian cancer.

The Ras homolog (Rho) serves as a molecular switch cycling between the guanosine diphosphate- and guanosine triphosphate-bound forms[16]. In its guanosine triphosphate-bound state, Rho sequentially activates downstream kinases such as Rho-associated coiled-coil containing protein kinase (ROCK) and LIM domain kinase[17]. Activated LIM domain kinase phosphorylates cofilin, inhibiting actin severing, whereas non-phosphorylated cofilin promotes actin depolymerization[18]. Studies have suggested that adrenomedullin derived from ovarian cancer cells polarizes tumor-associated macrophages, thereby promoting ovarian cancer cell migration by activating the RhoA signaling pathway[19]. RhoA silencing significantly inhibits the growth, adhesion, migration, and invasion of ovarian cancer cells[20]. SMAD-specific E3 ubiquitin-protein ligase 1 enhances ovarian cancer cell migration and invasion by activating the RhoA/ROCK signaling pathway[21].

Therefore, we hypothesized that ESR1 promotes ovarian cancer cell proliferation, migration, invasion, and stemness while suppressing ferroptosis and apoptosis by enhancing the RhoA/ROCK signaling pathway. Using an in vitro ovarian cancer model, this study aimed to elucidate the role of ESR1 in ovarian cancer progression and to uncover the underlying mechanisms involved in this pathway.

MATERIALS AND METHODS

Reagents

Antibodies specifically targeting ESR1 (ab32063), B-cell lymphoma-2 (Bcl-2, ab182858), Bcl-2 associated X (Bax, ab32503), RhoA (ab187027), ROCK1 (ab134181), ROCK2 (ab125025), octamer-binding transcription factor 4, (OCT4, ab200834), Nanog (ab109250), ferritin heavy chain (FTH, ab75973), ferritin light chain (FTL, ab313563), transferrin receptor 1 (TFR1, ab109259), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ab181602) were procured from Abcam (Cambridge, MA, United States). The Cell Counting Kit-8 (CCK-8 kit, C0037), 5-ethynyl-2’-deoxyuridine (EdU) cell proliferation kit (C0071S), and Annexin V-FITC Apoptosis Detection Kit (C1062 L) were purchased from Beyotime Biotechnology (Shanghai, China). The ALDEFLUOR™ Kit (Catalog #01700) was obtained from STEMCELL Technologies (Vancouver, Canada). FerroOrange (product code: F374) was procured from Dojindo Molecular Technologies (Shanghai, China). CN03 (Rho Activator II, Catalog #CN03) was purchased from Cytoskeleton, Inc. (Denver, CO, United States).

Cell culture and transfection

The human ovarian cancer cell lines SKOV3 (ATCC #HTB-77), A2780 (Sigma #93112519), and OVCAR3 (ATCC #HTB-161), and the normal ovarian epithelial cell line HOSEpiC (ScienCell #7310) were obtained from the indicated repositories. Cells were cultured in complete Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (final concentration 100 U/L) in a conventional incubator at 37 °C with 5% CO₂. Cells in the logarithmic growth phase with trypan blue exclusion rates exceeding 95% were selected for subsequent experiments. A2780 cells were transfected with si-ESR1, with or without CN03 (Rho activator) at a concentration of 0.1 M/L. siRNA transfection was performed using 20 μmol/L siRNA and Lipofectamine^® 2000 (Catalog #11668019, Thermo Fisher Scientific, Inc., MA, United States) following the manufacturer’s guidelines. The sequences used were as follows: SiRNA-ESR1, 5’-GGAUGAAGCUGAAGUAGAUTT-3’; siRNA-negative control (siRNA-NC), 5’-UUCUCCGAACGUGUCACGUTT-3’ (GenePharma, Shanghai, China). After 24 hours of treatment, the cells were harvested for functional assays, and RNA and protein were extracted for real-time polymerase chain reaction (PCR) and western blot analyses.

Disease gene targets

Targets associated with ovarian cancer, cell stemness, and ferroptosis were retrieved from DisGeNET (https://www.disgenet.org/home/) and GeneCards (https://www.genecards.org/) databases. The search query “ovarian cancer” was used, with an exclusive focus on humans.

Gene expression analysis

The Gene Expression Profiling Interactive Analysis (GEPIA) database contains RNA sequencing expression data from 8587 normal and 9736 tumor samples derived from the Genotype-Tissue Expression dataset and The Cancer Genome Atlas. The GEPIA database was used to determine ESR1 expression levels in 426 patients with ovarian cancer and 88 healthy controls. The GEPIA database is available at http://gepia.cancer-pku.cn/detail.php. A significance threshold of P < 0.05 was used for statistical validation.

Cell viability

Cell viability was assessed using the CCK-8 kit according to the manufacturer’s instructions. Cells subjected to different treatments were seeded at a density of 5 × 10³ cells/well in 96-well plates containing 100 μL of complete Dulbecco’s modified Eagle medium. After cell attachment, 10 μL of CCK-8 reagent was added to each well. The optical density was measured at 450 nm after 24 hours, 48 hours, and 72 hours following a 2-hour incubation with the reagent.

Colony formation assay

For the colony formation assay, cells were seeded in 6-well plates and cultured overnight. After various treatments, the culture medium was replaced and maintained with dimethyl sulfoxide three times per week until colonies became visible. The cells were then fixed with 4% paraformaldehyde and stained with crystal violet solution for approximately 10-15 days.

EdU assay

The EdU assay kit (C0071S; Beyotime, Beijing, China) was used to assess cell proliferation. The cells were seeded, treated, and exposed to EdU buffer. After fixation and permeabilization, the cells were stained with DAPI. Fluorescence images were captured and analyzed using ImageJ software (V1.8.0, NIH, Bethesda, MD, United States) to determine the proliferation rate.

Cell apoptosis analysis using flow cytometry

Cell apoptosis was evaluated using an Annexin V-FITC Apoptosis Detection Kit (C1062 L; Beyotime, Beijing, China) according to the manufacturer’s protocol. Following trypsinization, the cells were washed twice with cold phosphate-buffered saline and once with medium. The pellet was resuspended in 400 μL of 1 × binding buffer and transferred into 1.5-mL tubes. Subsequently, 3 μL of Annexin V-FITC and 5 μL of propidium iodide were added, and the cells were incubated for 15 minutes at room temperature in the dark. Analyses were performed using the Guava easyCyte system (United States) and FlowJo 7.6 software.

ALDEFLUOR assay

The ALDH assay was performed using an ALDEFLUOR Kit (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s instructions. Briefly, 2.5 × 10⁵ cells were suspended in 500 μL of ALDEFLUOR assay buffer containing the ALDH substrate and incubated for 45 minutes at 37 °C. N,N-diethylaminobenzaldehyde, a specific ALDH inhibitor, was used as a control. The cells were then analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, United States), and the data were analyzed using FlowJo software (BD Biosciences, San Jose, CA, United States).

Cell spheroidization assay

Cells in the logarithmic growth phase were cultured in a serum-free medium. The cells were then harvested and seeded at 1000 cells/well in low-attachment 6-well plates. Following static incubation for 7-10 days, neurospheres containing more than 50 cells were examined under a microscope, and their sizes were assessed.

Measurement of Fe²⁺ content

Cells were seeded at a concentration of 1 × 10⁵ cells/mL in 12-well culture plates. Intracellular Fe²⁺ levels were measured using the FerroOrange kit (Dojingo, Molecular Technologies Inc., Shanghai, China) according to the manufacturer’s instructions[22]. After various treatments, cells were incubated in serum-free medium for 4 hours and then stained with 1 μmol/L FerroOrange in HBSS for 30 minutes at 37 °C. The fluorescence intensity was immediately measured using an automated microplate spectrophotometer (Synergy H1, BioTek Instruments, VT, United States) at an excitation wavelength of 543 nm and an emission wavelength of 580 nm.

Quantitative reverse transcription PCR

Total RNA was extracted from the cells using TRIzol reagent (Beyotime, Beijing, China) according to the manufacturer’s protocol. The RNA was used for cDNA synthesis using reagents from Beyotime Biotechnology. PCR was performed using an ABI 7900 fluorescence quantitative PCR instrument (ABI, United States). The primer sequences were as follows: ESR1 (human) forward 5’-CATGATGAATCTGCAGGGAG-3’ and reverse 5’-GACAGAAATGTGTACACTCCAG-3’; GAPDH (human) forward 5’-TCAAGATCATCAGCAATGCC-3’ and reverse 5’-CGATACCAAAGTTGTCATGGA-3’. GAPDH expression was used to normalize mRNA expression levels, and relative fold changes were calculated using the 2^-ΔΔCt method[23].

Western blotting

Proteins extracted from cells were transferred onto polyvinylidene fluoride membranes and blocked with 5% skim milk. The membranes were then incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used at a 1:2000 dilution: ESR1 (ab32063), Bax (ab32503), Bcl-2 (ab182858), RhoA (ab187027), ROCK1 (ab134181), ROCK2 (ab125025), OCT4 (ab200834), Nanog (ab109250), FTH (ab75973), FTL (ab313563), TFR1 (ab109259), and GAPDH (ab181602). All the primary antibodies were purchased from Abcam (Cambridge, MA, United States). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-human secondary antibody (A0201, 1:2000; Beyotime, Beijing, China). Protein bands were visualized using enhanced chemiluminescence, with GAPDH as the internal control. Protein levels were quantified using ImageJ software (V1.8.0, National Institutes of Health, Bethesda, MD, United States).

Statistical analysis

Data were analyzed using SPSS Statistics (Version 20, Chicago, IL, United States)[24]. Continuous data are presented as mean ± SD. Differences between the two groups were examined using t-tests. For comparisons involving three or more groups, a one-way analysis of variance followed by the LSD test was used. Statistical significance was set at P < 0.05.

RESULTS

Expression of ESR1 in human ovarian cancer and cell lines

Target genes associated with ovarian cancer, cell stemness, and ferroptosis were screened using DisGeNET and GeneCards databases. A total of 21 genes were identified using these platforms (Figure 1A). GEPIA confirmed the significant overexpression of ESR1 in ovarian cancer patients (P < 0.05, Figure 1B). ESR1 expression levels in HOSE, SKOV3, A2780, and OVCAR3 cells were evaluated using real-time PCR and western blotting. Comparative analysis revealed significantly higher ESR1 levels in SKOV3, A2780, and OVCAR3 cells than in HOSE cells (P < 0.001), with A2780 cells exhibiting the highest ESR1 expression (Figure 1C and D). Therefore, A2780 cells were selected for further investigation.

Figure 1 Expression of estrogen receptor 1 in ovarian cancer patients and cell lines. A: Screening of targeted genes associated with ovarian cancer, cell stemness, and ferroptosis using DisGeNET and GeneCards databases; B: GEPIA analysis of estrogen receptor 1 (ESR1) in ovarian cancer patients and health controls; C: Real-time polymerase chain reaction analysis revealed increased ESR1 levels in ovarian cancer cell lines; D: Western blot analysis revealed increased ESR1 levels. Data was expressed as mean ± SD (n = 3). ^aP < 0.01; ^bP < 0.01 vs HOSE group. ESR1: Estrogen receptor 1.

Effect of ESR1 downregulation on cell function in A2780 cells

The transfection efficiency of si-ESR1 in A2780 cells was evaluated by examining ESR1 expression levels using real-time PCR. A decrease in ESR1 mRNA levels was observed in A2780 cells (P < 0.001; Figure 2A), indicating successful transfection. After transfection with si-ESR1, cell viability significantly decreased in A2780 cells at 72 hours (P < 0.001; Figure 2B). Colony formation and EdU assays revealed reduced cell proliferation (P < 0.001, Figure 2C; P < 0.01, Figure 2D). Additionally, flow cytometric analysis revealed an increase in apoptosis (Figure 2E). In the si-ESR1 group, there was a marked increase in Bax expression (P < 0.001) and a reduction in Bcl-2 expression (P < 0.001, Figure 2F).

Figure 2 Effect of estrogen receptor 1 downregulation on cell function in A2780 cells. A: Real-time polymerase chain reaction analysis revealed a significant decrease in estrogen receptor 1 (ESR1) levels in A2780 cells following transfection with si-ESR1. Then, A2780 cells were transfected with si-NC and si-ESR1; B: Cell viability was notably reduced by Cell Counting Kit-8 assay; C: Cell proliferation was notably reduced by colony formation assay; D: Cell proliferation was notably reduced by 5-ethynyl-2’-deoxyuridine assay; E: Cell apoptosis was increased by flow cytometry; F: Expression levels of B-cell lymphoma-2 associated X were significantly elevated, while B-cell lymphoma-2 expression was decreased. Data was expressed as mean ± SD (n = 3). ^bP < 0.01 vs si-NC group. ESR1: Estrogen receptor 1; EDU: 5-ethynyl-2’-deoxyuridine assay; Bcl-2: B-cell lymphoma-2; Bax: B-cell lymphoma-2 associated X.

Effect of ESR1 downregulation on cell stemness and ferroptosis in A2780 cells

After transfection with si-ESR1, ALDH activity was significantly reduced in A2780 cells (P < 0.001; Figure 3A). Cell stemness, as indicated by the spheroidization assay, decreased in the si-ESR1 group (P < 0.001; Figure 3B). Similarly, the protein levels of OCT4 and Nanog were significantly decreased in the si-ESR1 group (P < 0.001; Figure 3C). Fe²⁺ content increased in A2780 cells after si-ESR1 transfection (P < 0.001, Figure 3D). Furthermore, the expression of FTH and FTL was significantly increased (P < 0.01), whereas that of TFR1 was decreased in the si-ESR1 group (P < 0.001, Figure 3E).

Figure 3 Effect of estrogen receptor 1 downregulation on cell stemness and ferroptosis in A2780 cells. A2780 cells were transfected with si-NC and si-ESR1. A: Aldehyde dehydrogenas activity was reduced following transfection with si-ESR1; B: Cell spheroidization assay demonstrated a decrease in cell stemness; C: Protein levels of octamer-binding transcription factor 4 and Nanog were notably decreased; D: Fe²⁺ content increased; E: Expression levels of ferritin heavy chain and ferritin light chain were significantly elevated, while the expression of transferrin receptor 1 was decreased. Data was expressed as mean ± SD (n = 3). ^bP < 0.01 vs si-NC group. ALDH: Aldehyde dehydrogenas; ESR1: Estrogen receptor 1; OCT4: Octamer-binding transcription factor 4; FTH: Ferritin heavy chain; FTL: Ferritin light chain; TFR1: Transferrin receptor 1.

Effect of ESR1 downregulation on the RhoA/ROCK signaling pathway in A2780 cells

Protein-protein interaction network analysis revealed that ESR1 interacts with RhoA, ROCK1, and ROCK2 (Figure 4A). The expression levels of RhoA, ROCK1, and ROCK2 were assessed using real-time PCR and western blotting. In the si-ESR1 group, the mRNA levels of RhoA, ROCK1, and ROCK2 decreased (P < 0.05) but were partially restored by CN03 treatment (P < 0.05, Figure 4B). Similarly, the protein levels of RhoA, ROCK1, and ROCK2 were decreased in the si-ESR1 group (P < 0.05) and were partially rescued by CN03 (P < 0.05, Figure 4C).

Figure 4 Effect of estrogen receptor 1 downregulation on the Ras homolog gene family member A/Rho-associated coiled-coil containing protein kinase signaling pathway in A2780 cells. A: Protein-protein interaction network analysis. A2780 cells were transfected with si-NC and si-ESR1, and treated with CN03; B: Real-time polymerase chain reaction analyzed the mRNA levels of RhoA, ROCK1, and ROCK2 in A2780 cells transfected with si-ESR1 with or without CN03; C: Western blot analyzed the protein levels of RhoA, ROCK1, and ROCK2. Data was expressed as mean ± SD (n = 3). ^bP < 0.01 vs si-NC group; ^dP < 0.01 vs si-ESR1 group. ESR1: Estrogen receptor 1; RHOA: Ras homolog gene family member A; ROCK: Rho-associated coiled-coil containing protein kinase.

Effect of ESR1 downregulation on cell function in A2780 cells via inhibition of the RhoA/ROCK signaling pathway

The effects of ESR1 downregulation on ovarian cancer cells were examined with a focus on the RhoA/ROCK signaling pathway. Following transfection with si-ESR1, a significant decrease in cell viability was observed in A2780 cells (P < 0.001), which was counteracted by CN03 treatment (P < 0.01; Figure 5A). The colony formation assay showed reduced cell proliferation in the si-ESR1 group (P < 0.001), which was reversed by CN03 treatment (P < 0.001; Figure 5B). EdU assay revealed decreased cell proliferation in the si-ESR1 group (P < 0.01), and this suppression was alleviated by CN03 (P < 0.05, Figure 5C). Flow cytometry assessed cell apoptosis, revealing increased apoptosis after si-ESR1 transfection, which was inhibited in the si-ESR1 + CN03 group (Figure 5D). Similarly, Bax expression increased significantly (P < 0.01), Bcl-2 expression decreased in the si-ESR1 group (P < 0.001), and both changes were reversed by CN03 treatment (P < 0.05, Figure 5E).

Figure 5 Effect of estrogen receptor 1 downregulation on cell function in A2780 cells by inhibiting the Ras homolog gene family member A/Rho-associated coiled-coil containing protein kinase signaling pathway. A2780 cells were transfected with si-NC and si-ESR1, and treated with CN03. A: Cell viability significantly decreased in A2780 cells following transfection with si-ESR1, which was rescued by CN03 treatment; B: Colony formation assay showed reduced cell proliferation in the si-ESR1 group, with restoration upon CN03 introduction; C: 5-ethynyl-2’-deoxyuridine assay revealed decreased cell proliferation in the si-ESR1 group, which was alleviated by CN03 treatment; D: Flow cytometry analysis demonstrated increased apoptosis in A2780 cells post si-ESR1 transfection, with apoptosis inhibition observed in the si-ESR1+ CN03 group; E: Expression levels of B-cell lymphoma-2 associated X and B-cell lymphoma-2 were altered in the si-ESR1 group, with an increase in B-cell lymphoma-2 associated X expression and a decrease in B-cell lymphoma-2 expression, both of which were reversed by CN03 treatment. Data was expressed as mean ± SD (n = 3). ^bP < 0.01 vs si-NC group; ^cP < 0.05 vs si-ESR1 group; ^dP < 0.01 vs si-ESR1 group. ESR1: Estrogen receptor 1; EDU: 5-ethynyl-2’-deoxyuridine assay; Bcl-2: B-cell lymphoma-2; Bax: B-cell lymphoma-2 associated X.

Effect of ESR1 downregulation on cell stemness and ferroptosis in A2780 cells via inhibition of the RhoA/ROCK signaling pathway

After si-ESR1 transfection into A2780 cells, a significant decline in ALDH activity was observed (P < 0.001), which was mitigated by CN03 treatment (P < 0.01; Figure 6A). In the si-ESR1 group, cell stemness, as measured by the spheroidization assay, was decreased (P < 0.001); however, this reduction was reversed by CN03 (P < 0.001, Figure 6B). The protein levels of OCT4 and Nanog were significantly decreased in the si-ESR1 group (P < 0.001) and were restored by CN03 (P < 0.001, Figure 6C). Upon si-ESR1 transfection, Fe²⁺ content increased in A2780 cells (P < 0.001) but decreased with CN03 supplementation (P < 0.01, Figure 6D). Furthermore, FTH and FTL expression increased significantly, whereas TFR1 expression decreased in the si-ESR1 group (P < 0.001), and these changes were reversed by CN03 (P < 0.01, Figure 6E).

Figure 6 Effect of estrogen receptor 1 downregulation on cell stemness and ferroptosis in A2780 cells by inhibiting the Ras homolog gene family member A/Rho-associated coiled-coil containing protein kinase signaling pathway. A2780 cells were transfected with si-NC and si-ESR1, and treated with CN03. A: Aldehyde dehydrogenas activity significantly decreased in A2780 cells post si-ESR1 transfection, which was partially restored by CN03 treatment; B: Cell spheroidization assay demonstrated a decrease in cell stemness in the si-ESR1 group, with a reversal observed upon CN03 introduction; C: Protein levels of octamer-binding transcription factor 4 and Nanog decreased significantly in the si-ESR1 group, but were restored by CN03 treatment; D: Fe²⁺ content in A2780 cells increased following si-ESR1 transfection, and decreased with CN03 supplementation; E: Expression levels of ferritin heavy chain and ferritin light chain increased, while transferrin receptor 1 expression decreased in the si-ESR1 group, and these changes were reversed by CN03 treatment. Data was expressed as mean ± SD (n = 3). ^bP < 0.01 vs si-NC group; ^dP < 0.01 vs si-ESR1 group. ALDH: Aldehyde dehydrogenas; ESR1: Estrogen receptor 1; OCT4: Octamer-binding transcription factor 4; FTH: Ferritin heavy chain; FTL: Ferritin light chain; TFR1: Transferrin receptor 1.

DISCUSSION

Ovarian cancer remains a major health concern worldwide owing to its high mortality rate and limited treatment options, particularly in advanced stages[25]. ESR1 has been implicated in various cancers, including ovarian cancer, as it plays a crucial role in cell proliferation, survival, and differentiation[26]. In this study, we investigated ESR1 expression in ovarian cancer and its effects on cell function, stemness, and ferroptosis in A2780 cells. Additionally, we investigated the involvement of the RhoA/ROCK signaling pathway in mediating the effects of ESR1 downregulation.

Our findings revealed a significant overexpression of ESR1 in patients with ovarian cancer and elevated ESR1 levels in SKOV3, A2780, and OVCAR3 cells. A2780 cells exhibited the highest ESR1 levels among all the cell lines studied. These results align with previous studies highlighting increased ESR1 expression in ovarian cancer and its association with disease progression and poor prognosis[26,27].

Downregulation of ESR1 in A2780 cells resulted in reduced cell viability and proliferation and increased apoptosis. These observations indicate that ESR1 plays a pivotal role in promoting the survival and growth of ovarian cancer cells. Moreover, the increase in Bax and decrease in Bcl-2 further support the pro-apoptotic influence of ESR1, as changes in the Bax/Bcl-2 ratio are known to regulate apoptotic mechanisms[28].

Similarly, our study showed that ESR1 downregulation led to a decline in cell stemness, as evidenced by reduced ALDH activity and decreased levels of the stemness markers OCT4 and Nanog. These results suggest that ESR1 is involved in maintaining the stemness characteristics of ovarian cancer cells, which are vital for tumor initiation and progression[29,30].

Furthermore, ESR1 downregulation increased Fe²⁺ content and altered the expression of key ferroptosis-related proteins, including FTH, FTL, and TFR1. These findings indicate that ESR1 influences ferroptosis[31], a form of regulated cell death characterized by iron-dependent lipid peroxidation[32]. The dysregulation of ferroptosis has been implicated in cancer development and resistance to therapy, highlighting the potential significance of ESR1 in the modulation of this process in ovarian cancer.

Our study identified interactions between ESR1 and key proteins in the RhoA/ROCK signaling pathway, including RhoA, ROCK1, and ROCK2. Downregulation of ESR1 decreased the expression of RhoA, ROCK1, and ROCK2 at both mRNA and protein levels. These findings suggest that ESR1 regulates the RhoA/ROCK pathway in ovarian cancer cells, which is known to play a role in cell migration, invasion, and cytoskeletal dynamics[33].

Inhibition of the RhoA/ROCK signaling pathway using CN03 partially rescued the effects of ESR1 downregulation on cell function, stemness, and ferroptosis in A2780 cells. CN03 treatment reversed the decrease in cell viability, proliferation, and stemness observed after ESR1 downregulation. Additionally, CN03 supplementation mitigated the alterations in Fe²⁺ content and expression of ferroptosis-related proteins induced by ESR1 downregulation. These results suggested that the RhoA/ROCK pathway may mediate, at least in part, the effects of ESR1 on ovarian cancer cell behavior.

This study provides valuable insights into the role of ESR1 in ovarian cancer progression and suggests that targeting ESR1 or its downstream signaling pathways, such as the RhoA/ROCK pathway, could represent a promising therapeutic approach. Further preclinical and clinical investigations are warranted to confirm these findings and explore the potential of ESR1 as a therapeutic target for ovarian cancer management.

CONCLUSION

This study demonstrates that ESR1 is significantly overexpressed in ovarian cancer cells and plays a pivotal role in promoting cell viability, proliferation, and stemness, while inhibiting apoptosis and ferroptosis. Mechanistically, ESR1 appears to exert these effects, at least in part, by activating the RhoA/ROCK signaling pathway, as evidenced by the reversal of si-ESR1-induced phenotypes upon treatment with the Rho activator CN03. These findings suggest that targeting ESR1 or the downstream RhoA/ROCK axis is a potential therapeutic strategy for ovarian cancer. However, it must be acknowledged that this study was primarily based on in vitro experiments using a single cell line (A2780), and the conclusions are therefore limited in their direct applicability to in vivo physiological and clinical settings. Future studies employing animal models, patient-derived samples, and additional ovarian cancer cell lines are required to validate the translational relevance of these findings. Furthermore, the precise molecular mechanisms by which ESR1 regulates ferroptosis and stemness independent of the RhoA/ROCK pathway warrant further investigation.