Suppression of MCM2 reduces cancer stem cells and epithelial-mesenchymal transition-related phenotypes in breast cancer cells

Abstract

Purpose

Minichromosome maintenance protein 2 (MCM2) is a key regulator of DNA replication and has been implicated in tumor progression. While previous studies have demonstrated its role in lung cancer stem cells (CSCs), its role in breast cancer remains unclear. This study aimed to investigate whether MCM2 regulates CSCs and epithelial-mesenchymal transition (EMT) in breast cancer.

Methods

MCM2 expression and its prognostic significance were analyzed using public breast cancer datasets. MDA-MB-231 breast cancer cells were transfected with MCM2-specific small interfering RNA. CSC-associated markers and EMT-related proteins were evaluated by Western blotting. Sphere formation, migration, and invasion assays were performed to assess CSC properties and metastatic potential.

Results

MCM2 expression was significantly elevated in breast cancer tissues and correlated with poor prognosis. Knockdown of MCM2 reduced the expression of CSC-associated markers, including ALDH1A1, Sox2, Oct4, and CD44. Functional assays showed that MCM2 suppression significantly decreased sphere-forming capacity, indicating impaired CSC properties. In addition, MCM2 knockdown increased E-cadherin expression while decreasing N-cadherin, Slug, Twist, and ZEB1 expression, suggesting inhibition of EMT. Migration and invasion abilities were also markedly reduced.

Conclusion

Suppression of MCM2 was associated with reduced CSC and EMT-related phenotypes in MDA-MB-231 breast cancer cells.

INTRODUCTION

Breast cancer is the most diagnosed cancer among women worldwide and remains a leading cause of cancer-related mortality [1]. Among its subtypes, triple-negative breast cancer is particularly aggressive and is associated with high rates of metastasis and recurrence [2]. Cancer stem cells (CSCs) represent a subpopulation of tumor cells with the ability to self-renew and differentiate into heterogeneous cancer cell populations [3,4]. CSCs play critical roles in tumor initiation, metastasis, and therapeutic resistance [5,6]. The maintenance of CSC properties is closely associated with epithelial-mesenchymal transition (EMT), a process that enhances cellular motility and invasiveness [7,8]. Minichromosome maintenance protein 2 (MCM2) is a key component of the MCM2-7 helicase complex, which is essential for DNA replication licensing [9,10]. MCM2 is frequently overexpressed in various cancers and has been associated with poor prognosis [11]. Previous studies demonstrated that MCM2 contributes to CSC maintenance and EMT-associated phenotypes in lung cancer and glioma models [12]. However, its role in breast cancer, particularly in regulating CSCs, remains poorly understood. In this study, we investigated whether MCM2 regulates CSCs and EMT in breast cancer cells and evaluated its potential role in tumor progression.

METHODS

Study design

This study was designed to investigate the role of MCM2 in regulating CSCs and EMT in breast cancer. Bioinformatics analyses were first conducted to evaluate MCM2 expression and its prognostic significance. Subsequently, in vitro experiments were performed using MDA-MB-231 breast cancer cells to examine the effects of MCM2 knockdown on CSC characteristics, EMT-related protein expression, and metastatic potential.

Bioinformatics analysis

MCM2 expression levels in breast cancer tissues and normal tissues were analyzed using the GEPIA3 database (https://gepia3.bioinfoliu.com/genecard/#MCM2), which integrates RNA sequencing data from The Cancer Genome Atlas and Genotype-Tissue Expression projects. Differential expression analysis was performed using the default GEPIA3 settings. The prognostic significance of MCM2 was evaluated using the Kaplan-Meier Plotter database (https://kmplot.com/analysis/). Survival analysis was performed using the Affymetrix probe set 202107_s_at corresponding to MCM2. Breast cancer patients were divided into high- and low-expression groups using the median MCM2 expression value as the cutoff (cutoff value=935). The analysis included a total of 4,929 breast cancer patients, consisting of 2,465 patients in the high-expression group and 2,464 patients in the low-expression group. Relapse-free survival (RFS) was analyzed using Kaplan-Meier survival curves. Hazard ratios (HRs) and 95% confidence intervals (CIs) were automatically calculated by the database, and statistical significance was determined using the log-rank test.

Cell culture

Human breast cancer cell line MDA-MB-231 was obtained from the American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Cat. No. SH30243.01; HyClone) supplemented with 10% fetal bovine serum (FBS; Cat. No. SH30919.03; HyClone) and 1% penicillin-streptomycin solution (Cat. No. SV30010; HyClone). Cells were maintained in a humidified incubator at 37 °C with 5% CO₂.

siRNA-mediated knockdown of MCM2

To suppress MCM2 expression, MDA-MB-231 cells were transfected with a single small interfering RNA (siRNA) duplex targeting MCM2. The sense sequence was 5′-CACUCAGUACCUUGGAUCA-3′ and the antisense sequence was 5′-UGAUCCAAGGUACUGAGUG-3′ (Bioneer Corporation). A non-targeting control siRNA was used as a negative control. Transfection was performed using Lipofectamine RNAi MAX reagent (Cat. No. 13-778-150; Invitrogen) according to the manufacturer’s instructions. After transfection, cells were incubated for 48 hours at 37 °C in a humidified incubator with 5% CO₂ before subsequent experiments.

Western blot analysis

For protein analysis, cells were lysed using RIPA lysis buffer (Cat. No. 20-188; Millipore) supplemented with protease inhibitor cocktail tablets (Cat. No. 11836153001; Roche) and phosphatase inhibitor cocktail tablets (Cat. No. 04906837001; Roche). Protein concentrations were measured using the Protein Assay Dye Reagent Concentrate (Cat. No. 5000006; Bio-Rad Laboratories). Equal amounts of protein were separated by SDS-PAGE (8%–15%) and transferred onto nitrocellulose membranes (Amersham Protran 0.2 μm NC membrane; Cat. No. 10600001; Cytiva). Membranes were blocked with 5% non-fat milk in Tris buffered saline containing 0.1% Tween-20 and incubated with primary antibodies overnight at 4 °C. Primary antibodies used included ALDH1A1 (Cat. No. ab6192; Abcam), Sox2 (Cat. No. 3579; Cell Signaling Technology), Oct4 (Cat. No. 2750; Cell Signaling Technology), CD44 (Cat. No. 3570; Cell Signaling Technology), E-cadherin (Cat. No. ab15148; Abcam), N-cadherin (Cat. No. 610920; BD Biosciences), Slug (Cat. No. sc-166476; Santa Cruz Biotechnology), Twist (Cat. No. sc-15393; Santa Cruz Biotechnology), and ZEB1 (Cat. No. sc-25388; Santa Cruz Biotechnology). After incubation with HRP-conjugated secondary antibodies (anti-rabbit IgG; Cat. No. 7074S and anti-mouse IgG; Cat. No. 7076S; Cell Signaling Technology), protein bands were visualized using enhanced chemiluminescence reagents (Cat. No. sc-2048; Santa Cruz Biotechnology). Western blot bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Protein expression levels were normalized to β-actin and expressed relative to the control group, which was set to 1.0.

Sphere formation assay

To evaluate CSC properties, sphere formation assays were performed using ultra-low attachment plates (Cat. No. 3474; Corning). Cells were cultured in serum-free medium consisting of DMEM/F12 (Cat. No. 11320-033; Invitrogen) supplemented with B27 serum-free supplement (Cat. No. 17504-044; Invitrogen), epidermal growth factor (EGF; Cat. No. E9644; Sigma-Aldrich), and basic fibroblast growth factor (bFGF; Cat. No. 13256-029; Invitrogen). Cells were incubated for 10–14 days, and sphere formation was assessed by measuring sphere diameter and calculating sphere-forming efficiency.

Migration and invasion assays

Cell migration and invasion abilities were evaluated using Transwell chambers with an 8 μm pore size (Cat. No. 3422; Corning). For migration assays, uncoated chambers were used, whereas for invasion assays, chambers were pre-coated with Matrigel (BD Biosciences) according to the manufacturer’s protocol. Cells were seeded in serum-free medium (Opti-MEM; Cat. No. 31985-070; Invitrogen) in the upper chamber, while medium containing 10% FBS was added to the lower chamber. After incubation for 24 hours at 37 °C, migrated or invaded cells were fixed and stained with crystal violet (Cat. No. HT90132-1L; Sigma-Aldrich). Cells were counted under a light microscope.

Control of experimental bias

To minimize subjectivity and ensure reproducibility, all experiments were independently repeated at least three times. Quantitative analyses were performed using consistent threshold settings. Cell counting for migration and invasion assays was conducted in multiple randomly selected microscopic fields. Whenever possible, data analysis was performed in a blinded manner.

Statistical analysis

All experiments were performed at least three times independently. Quantitative data are presented as the mean±standard deviation (SD). Densitometric analyses of Western blot bands were performed using ImageJ software (National Institutes of Health), and the expression levels of target proteins were normalized to β-actin. The relative protein expression levels were calculated by setting the control group to 1.0. For sphere formation, migration, and invasion assays, quantitative analyses were performed using data obtained from three independent experiments. Cell counts and sphere measurements were analyzed from multiple randomly selected microscopic fields. Statistical analyses were performed using Microsoft 365 (Microsoft Corp.). Comparisons between two groups were conducted using a two-tailed Student t-test. A P-value of less than 0.05 was considered statistically significant.

Ethical considerations

This study did not involve human participants or animal experiments; therefore, institutional review board approval and informed consent were not required.

RESULTS

MCM2 expression is elevated in breast cancer and correlated with poor prognosis

To investigate the clinical relevance of MCM2 in breast cancer, we first analyzed its expression using public datasets. MCM2 expression was significantly higher in breast cancer tissues compared with normal breast tissues (Fig. 1A). Furthermore, Kaplan-Meier survival analysis demonstrated that patients with high MCM2 expression exhibited significantly poorer RFS than those with low MCM2 expression (Fig. 1B). The analysis included 4,929 breast cancer patients divided into high-expression (n=2,465) and low-expression (n=2,464) groups using the median expression value as the cutoff. The HR was 1.48 (95% CI, 1.34–1.64), with a log-rank P-value of 2.8×10⁻¹⁴, indicating a strong association between MCM2 overexpression and poor prognosis. These findings demonstrate that MCM2 is overexpressed in breast cancer tissues and that high MCM2 expression is associated with poor RFS.

Fig. 1.

MCM2 expression and prognostic significance in breast cancer. (A) Differential expression analysis of MCM2 between normal breast tissues and breast cancer tissues using the GEPIA3 database. (B) Kaplan-Meier RFS analysis using the Kaplan-Meier Plotter database. Patients were divided into high- and low-expression groups according to the median MCM2 expression value (probe set: 202107_s_at; cutoff value=935). A total of 4,929 breast cancer patients were included in the analysis. High MCM2 expression was significantly associated with poorer RFS. MCM2, minichromosome maintenance protein 2; RFS, relapse-free survival; HR, hazard ratio; CI, confidence interval.

Suppression of MCM2 reduces CSC-associated marker expression

To determine whether MCM2 is involved in the regulation of CSCs, MCM2 expression was suppressed using siRNA in MDA-MB-231 cells. Western blot analysis revealed that MCM2 knockdown significantly decreased the expression levels of CSC-associated markers, including ALDH1A1 and CD44, compared with the control group (Fig. 2A and B). Quantitative densitometric analysis further confirmed significant reductions in the protein levels of ALDH1A1 and CD44 following MCM2 suppression. Similarly, the expression of stemness-related transcription factors Sox2 and Oct4 was markedly reduced in MCM2-silenced cells (Fig. 2C and D). Densitometric quantification demonstrated that the decreases in Sox2 and Oct4 expression were statistically significant. These findings suggest that MCM2 contributes to the maintenance of CSC populations in breast cancer cells.

Fig. 2.

Suppression of MCM2 reduces cancer stem cell-associated marker expression in breast cancer cells. (A) Western blot analysis of MCM2, ALDH1A1, and CD44 expression following MCM2 knockdown. (B) Densitometric quantification of MCM2, ALDH1A1, and CD44 expression shown in panel A. (C) Western blot analysis of Sox2 and Oct4 expression following MCM2 knockdown. (D) Densitometric quantification of protein expression shown in panel C. Protein expression levels were normalized to β-actin. Data are presented as mean±SD from three independent experiments. MCM2, minichromosome maintenance protein 2; SD, standard deviation. *P<0.05, **P<0.01 vs. control.

Suppression of MCM2 impairs sphere-forming ability

To further evaluate the functional role of MCM2 in CSCs, sphere formation assays were performed. As shown in Fig. 3A, MCM2 knockdown significantly reduced sphere size, indicating impaired proliferative capacity of CSCs. In addition, quantitative analysis revealed a marked decrease in sphere-forming efficiency in the si-MCM2-treated group compared with the control group (Fig. 3B). Sphere-forming efficiency was significantly reduced following MCM2 suppression, suggesting impaired CSC-associated functional properties.

Fig. 3.

Effect of MCM2 knockdown on sphere-forming ability in breast cancer cells. (A) Representative images of tumor spheres and quantification of sphere diameter following MCM2 knockdown. (B) Single-cell assay showing sphere-forming efficiency of si-Control and si-MCM2-treated MDA-MB-231 cells. Data are presented as mean±SD from three independent experiments. MCM2, minichromosome maintenance protein 2; SD, standard deviation. *P<0.05 vs. control.

Suppression of MCM2 alters EMT-related marker expression

Given the close relationship between CSCs and EMT, we next investigated whether MCM2 regulates EMT-related processes. Western blot analysis showed that MCM2 knockdown was associated with increased E-cadherin expression and reduced N-cadherin expression. Densitometric quantification confirmed that these changes were statistically significant (Fig. 4A and B). Furthermore, the expression levels of EMT-associated transcription factors, including Slug, Twist, and ZEB1, were markedly decreased following MCM2 suppression (Fig. 4C and D). Quantitative analysis demonstrated significant reductions in all three EMT-related proteins compared with the control group. These findings suggest that MCM2 contributes to the maintenance of mesenchymal characteristics in breast cancer cells.

Fig. 4.

Suppression of MCM2 alters epithelial-mesenchymal transition-related marker expression in breast cancer cells. (A) Western blot analysis of E-cadherin and N-cadherin expression following MCM2 knockdown. (B) Densitometric quantification of protein expression shown in panel A. (C) Western blot analysis of Slug, Twist, and ZEB1 expression following MCM2 knockdown. (D) Densitometric quantification of Slug, Twist, ZEB1, and MCM2 expression shown in panel C. Protein expression levels were normalized to β-actin. Data are presented as mean±SD from three independent experiments. MCM2, minichromosome maintenance protein 2; SD, standard deviation. *P<0.05, **P<0.01 vs. control.

MCM2 enhances migration and invasion

To assess the functional consequences of EMT regulation, migration and invasion assays were performed. As shown in Fig. 5, MCM2 knockdown significantly reduced both migration and invasion abilities of MDA-MB-231 cells. Quantitative analysis demonstrated a substantial decrease in the number of migrated and invaded cells in the si-MCM2 group compared to the control group. These results suggest that MCM2 is associated with migratory and invasive phenotypes in breast cancer cells.

Fig. 5.

Suppression of MCM2 reduces migration and invasion in breast cancer cells. (A) Invasion and migration assay images comparing si-Control and si-MCM2 treated cells. (B) Quantification of migrated and invaded cells showing significant reduction in MCM2 knockdown cells. Data are presented as mean±SD from three independent experiments. MCM2, minichromosome maintenance protein 2; SD, standard deviation. *P<0.05, **P<0.01 vs. control.

DISCUSSION

In the present study, suppression of MCM2 was associated with reduced CSC- and EMT-related phenotypes in breast cancer cells. Bioinformatics analysis revealed that MCM2 is significantly overexpressed in breast cancer tissues and is strongly associated with poor patient survival. The HR of 1.48 indicates that elevated MCM2 expression is clinically meaningful and may serve as a prognostic biomarker. These findings are consistent with previous studies reporting that MCM2 is overexpressed in various cancers and is associated with tumor progression [11].

At the cellular level, suppression of MCM2 was associated with reduced CSC-associated marker expression. Knockdown of MCM2 resulted in decreased expression of CSC markers, including ALDH1A1 and CD44, as well as key transcription factors such as Sox2 and Oct4. These factors are essential for maintaining stem cell identity and self-renewal capacity [3,5]. Therefore, the reduction of these markers suggests that MCM2 contributes to the maintenance of CSC-associated characteristics in breast cancer cells. Functionally, MCM2 knockdown significantly impaired sphere formation, a commonly used assay for CSC-associated functional properties. Both sphere size and sphere-forming efficiency were markedly reduced, suggesting that MCM2 contributes to CSC-associated functional properties. These findings suggest that MCM2 is associated with both the molecular and functional characteristics of CSCs.

In addition to its role in CSC regulation, MCM2 suppression was associated with alterations in EMT-related marker expression. Suppression of MCM2 was associated with reduced expression of mesenchymal markers and EMT-related transcription factors, suggesting a partial attenuation of the mesenchymal phenotype in MDA-MB-231 cells. Furthermore, EMT-related transcription factors such as Slug, Twist, and ZEB1 were significantly downregulated. These findings suggest a potential association between MCM2 expression and EMT-related phenotypes in breast cancer cells [7,8]. Importantly, EMT is closely associated with CSC properties and metastatic potential [13]. Therefore, the observed association of MCM2 suppression with both CSC-related and EMT-related phenotypes suggests a potential role for MCM2 in maintaining aggressive tumor characteristics. Consistent with this, functional assays demonstrated that MCM2 enhances migration and invasion. The suppression of MCM2 significantly reduced both migratory and invasive abilities of breast cancer cells, indicating that MCM2 contributes to metastasis. These results suggest that MCM2 may contribute to aggressive tumor phenotypes associated with migration and invasion.

Our interest in MCM2 was based not only on its established role in DNA replication but also on our recent findings in lung cancer [12]. In a previous study, we demonstrated that MCM2 contributes to the maintenance of CSC properties through the modulation of CXCL1 secretion in lung cancer cells [12]. Unlike other MCM family members, MCM2 had already shown a functional association with CSC-related phenotypes, making it a strong candidate for further investigation in breast cancer. These findings suggested that MCM2 may possess functions beyond replication licensing and may contribute to the maintenance of aggressive tumor phenotypes. Therefore, the present study was designed to determine whether the CSC-related function of MCM2 could be extended to breast cancer cells.

Several studies have highlighted the clinical significance of MCM2 in breast cancer. Abe et al. [14] reported that inhibition of MCM2 suppressed the growth of highly malignant breast cancer cells and suggested MCM2 as a potential therapeutic target in aggressive breast tumors. Samad et al. [15] further demonstrated that elevated MCM2 expression is significantly associated with poor prognosis in breast cancer patients through computational analyses. More recently, Unal et al. [16] reported that MCM2 may serve as a useful biomarker for identifying high-risk breast cancer patients according to TAILORx classification. Collectively, these studies established the prognostic and clinical relevance of MCM2 in breast cancer. However, despite accumulating evidence supporting MCM2 as a biomarker, its functional role in regulating CSC properties and EMT has remained largely unexplored. In the present study, suppression of MCM2 reduced CSC-associated markers, sphere-forming ability, EMT-related proteins, and invasive phenotypes in breast cancer cells. These findings suggest that the biological functions of MCM2 in maintaining aggressive tumor characteristics may be conserved across multiple cancer types [3,5,13]. Therefore, our results extend previous observations and provide evidence that MCM2 is associated with CSC- and EMT-related phenotypes in breast cancer cells [7,8,13]. Taken together, our findings support the possibility that MCM2 contributes to breast cancer progression through the regulation of stem cell-like and mesenchymal characteristics.

Despite these findings, this study has several limitations. First, only a single breast cancer cell line (MDA-MB-231) was used. Second, in vivo validation was not performed. Future studies should include additional breast cancer cell lines and animal models to confirm the role of MCM2 in tumor progression and metastasis. In conclusion, suppression of MCM2 reduced CSC-associated markers, sphere-forming ability, EMT-related phenotypes, and migratory/invasive capacities in MDA-MB-231 breast cancer cells. These findings suggest that MCM2 may contribute to the maintenance of aggressive breast cancer phenotypes.