Abstract
Background
Ovarian cancer (OC) is one of the most lethal gynecologic malignancies, largely due to late diagnosis, high metastatic potential, and chemoresistance. Cystatin S (CST4) has been implicated in the tumor progression of several cancers, but its role in OC remains unclear. This study aims to investigate the expression, clinical significance, and functional role of CST4 in OC, particularly its involvement in epithelial-mesenchymal transition (EMT) via the Wnt/β-catenin signaling pathway.
Methods
Immunohistochemistry (IHC) was performed on 102 °C tissues and 20 normal ovarian tissues to assess CST4 expression. Through Kaplan-Meier analysis and Cox regression analysis, the association between CST4 expression and clinical pathological features, prognosis and survival outcomes was investigated. To investigate the functional role of CST4, small interfering RNA (siRNA) was used to silence CST4 expression in HEY and HO-8910 cell lines. Following CST4 knockdown, cell proliferation was assessed using CCK-8 assays and colony formation assays, while migratory and invasive capacities were evaluated through wound healing and transwell invasion assays, respectively. In addition, western blotting was employed to examine the expression of epithelial-mesenchymal transition (EMT)-related markers and key proteins involved in the Wnt/β-catenin signaling pathway.
Results
Compared with normal ovarian tissue, the expression of CST4 in OC tissues was significantly increased, and it was associated with more advanced FIGO stages, elevated CA125 levels, platinum resistance, and poor prognosis. High CST4 expression was identified as an independent prognostic factor for reducing overall and progression-free survival. Functional assays showed that CST4 knockdown suppressed OC cell proliferation, migration, and invasion. Mechanistically, CST4 silencing inhibited the Wnt/β-catenin signaling pathway and reversed the expression of EMT markers.
Conclusions
CST4 is a novel oncogenic regulator in OC that promotes EMT and tumor aggressiveness via the Wnt/β-catenin signaling pathway. It may serve as an independent prognostic biomarker and potential therapeutic target for OC.
Supplementary Information
The online version contains supplementary material available at 10.1007/s12672-026-04704-y.
Keywords: Ovarian cancer, CST4, Wnt, EMT, Prognosis
Introduction
Ovarian cancer (OC) is one of the most common and lethal gynecologic malignancies worldwide, ranking eighth in the incidence among female cancers [1]. According to the most recent global cancer statistics, an estimated 324,398 new cases of OC and 206,839 related deaths occurred in 2022, corresponding to approximately 2.13% of total cancer mortality [2]. Due to the anatomical location of the ovaries deep within the pelvis, early-stage OC is typically asymptomatic or presents with nonspecific symptoms. Combined with the absence of effective early screening strategies, over 70% of OC patients are diagnosed at advanced stages [3]. OC is often referred to as a “silent killer” because of its aggressive nature, including widespread pelvic and abdominal metastases, high recurrence rate, and frequent resistance to chemotherapy [4, 5]. Notably, epithelial OC accounts for over 90% of all OC cases, and the main treatment approach is cytoreductive surgery followed by platinum-based chemotherapy and poly (ADP-ribose) polymerase (PARP) inhibitors as maintenance therapy [6]. Despite the progress in treatment methods, the overall 5-year survival rate for OC remains only 59.6% [7], highlighting the urgent need to elucidate the mechanisms underlying tumor progression, metastasis, and chemoresistance in order to develop novel therapeutic strategies.
A growing body of research has identified that epithelial-mesenchymal transition (EMT) is a key process in tumor progression, metastasis, and treatment resistance. EMT is a reversible transdifferentiation program that causes epithelial cells to lose their intercellular adhesion properties and acquire mesenchymal characteristics, thereby enhancing migration and invasion abilities [8]. EMT is recognized as a central driving factor for metastasis and adverse clinical outcomes in various malignancies. In breast cancer, EMT is essential for early spread and metastatic colonization [9]; in pancreatic ductal adenocarcinoma, EMT-like changes confer both invasiveness and stem-like properties [10]; and in non-small cell lung cancer, EMT is associated with resistance to EGFR inhibitors [11]. In the context of OC, EMT is recognized as a key driver of tumor progression and metastasis [12, 13]. Moreover, it is also closely associated with chemoresistance, especially resistance to the two cornerstone drugs for OC treatment, platinum and paclitaxel [14, 15]. Given its dual role in metastasis and drug resistance, EMT has become an extremely attractive therapeutic target in the treatment of OC. Identifying the upstream regulators of EMT may provide new insights for clinical intervention.
Cystatin S (CST4) is a member of the cystatin superfamily that functions as a secreted cysteine protease inhibitor. It regulates extracellular matrix degradation and modulates cancer cell invasion and metastasis by inhibiting proteases such as serine proteases [16]. CST4 has been previously reported to be associated with immune-mediated diseases such as Sjögren’s syndrome [17], dental caries [18], and asthma [19], and has recently been linked to the metastasis of various cancers, including gastric cancer [20] and colorectal cancer [21]. Similarly, CST4 has been shown to promote the invasion and EMT of gastric cancer by activating the Wnt signaling pathway [22]. Its expression has also been associated with tumor prognosis and the modulation of the tumor microenvironment. It is notable that in OC, the expression of CST4 in tumor tissues is observed to be elevated and is associated with the shaping of the tumor microenvironment [23]. Although the specific function and mechanism of action of CST4 have not been clarified in this study. Given the emerging role of CST4 in tumor progression and its capacity to induce EMT in other cancers, it can be hypothesized that CST4 may also regulate EMT and metastasis in OC via the Wnt/β-catenin pathway. However, the functional significance of CST4 in OC remains largely unexplored.
In this study, we investigated the expression pattern, clinical significance, and functional role of CST4 in OC. We demonstrated that CST4 was significantly overexpressed in OC tissues and was associated with poor prognosis. Mechanistically, CST4 promoted the EMT of OC cells via activation of the Wnt/β-catenin signaling pathway. Our findings suggest that CST4 acts as a key driver of OC progression and may serve as a promising therapeutic target to suppress EMT and metastasis in OC.
Methods
Tissue samples
A total of 102 °C tissue specimens and 20 normal ovarian tissue samples were collected from patients who underwent surgical resection at the First Affiliated Hospital of Bengbu Medical University between January 2021 and December 2023. All OC samples were confirmed as epithelial OC through histopathological examination, according to the classification standards of the World Health Organization (WHO). Normal ovarian tissues were obtained from patients who underwent oophorectomy due to benign gynecological diseases and were confirmed to have no malignant lesions through pathological examination. Clinical and pathological data, including age, tumor stage, histological subtype, tumor grade, and residual tumor status after surgery, were retrieved from the medical records. Tumor stage was assessed according to the 2014 standards of the International Federation of Gynecology and Obstetrics (FIGO) [24]. Patients who received preoperative chemotherapy, radiotherapy, or had concurrent malignancies were excluded from the study to avoid the influence of confounding factors.
The tissue specimens were immediately fixed with 10% neutral buffered formalin solution, and then embedded in paraffin for histological analysis, or snap-frozen in liquid nitrogen and stored at −80 °C for molecular assays. This research protocol was approved by the Institutional Ethics Committee of the First Affiliated Hospital of Bengbu Medical University (approval number: 2023-088-NG), and informed consent was obtained from all patients or their legal guardians prior to tissue collection. All procedures adhered to the ethical principles outlined in the Declaration of Helsinki.
Immunohistochemistry (IHC)
The paraffin-embedded ovarian tissue section (4 μm thick) were deparaffinized in xylene and rehydrated through a series of gradually diluted ethanol solutions (100%, 95%, 85%, and 70%). Antigen retrieval was performed by boiling the sections in citrate buffer (pH 6.0) for 15 min, followed by natural cooling to room temperature. Endogenous peroxidase activity was blocked by incubating the sections with 3% hydrogen peroxide (H₂O₂) for 10 min at room temperature. After rinsing with PBS, sections were incubated with a primary antibody against CST4 (1/1000, ab151771; Abcam, UK) for 1 h at room temperature. Following the primary antibody incubation, the slides were treated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min. The sections were stained with 3,3’-diaminobenzidine (DAB) substrate and then counterstained with hematoxylin. The sections were then dehydrated through graded alcohols, cleared in xylene, and mounted using neutral resin. Microscopic visualization and image capture were performed using a Leica DM3000 light microscope (Leica Microsystems, Wetzlar, Germany).
IHC staining results were assessed independently by two experienced pathologists who were blinded to the clinical information. Semi-quantitative evaluation was based on both staining intensity and the percentage of positively stained tumor cells as described previously [25]. Staining intensity was scored as follows: 0 (no staining), 1 (light yellow), 2 (brown-yellow), and 3 (dark brown). The percentage of positive cells was scored as: 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). The final immunoreactivity score (IRS) was calculated by multiplying the intensity and percentage scores, with total scores ranging from 0 to 12. Based on the IRS, CST4 expression was classified as follows: 0, negative; 1–4, weakly positive; and ≥ 5, strongly positive. Samples with an IRS ≥ 5 were considered CST4 high-expression (CST4⁺), while those with an IRS < 5 were considered CST4 low-expression (CST4⁻).
Cell culture and transfection
Five human ovarian cancer (OC) cell lines, HO-8910, HEY, OVCAR3, SKOV3, and IOSE-80 (immortalized normal ovarian epithelial cells), were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were authenticated within the last three years by short tandem repeat (STR) profiling and tested negative for mycoplasma contamination. Cells were cultured in RPMI-1640 medium (Wuhan Punosai Life Sciences and Technology Co., Ltd.) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (100 U/mL penicillin and 100 µg/mL streptomycin). Cells were maintained at 37 °C in a humidified incubator containing 5% CO₂. For all experiments, cells within passage numbers 5 to 20 were used to ensure consistency and stability of phenotype.
Small interfering RNA (siRNA) targeting CST4 (si-CST4) and negative control siRNA (si-NC) were synthesized by Geno Meditech (Shanghai, China). Transfections were performed in the HEY and HO-8910 cell lines, which exhibited relatively high CST4 expression. Cells were seeded into 6-well plates at a density of 2 × 10⁵ cells/well and cultured until they reached approximately 70–80% confluence. Transfection was carried out using Lipofectamine™ 2000 reagent (Invitrogen, USA) according to the manufacturer’s protocol. Briefly, siRNA and Lipofectamine 2000 were diluted in a serum-free Opti-MEM medium, incubated for 5 min at room temperature, and then mixed and incubated for another 20 min to form complexes. The complexes were added to the cells and incubated for 6 h, after which the medium was replaced with fresh complete medium. Transfection efficiency was assessed after 48 h using qRT-PCR to confirm CST4 knockdown. After 48 h post-transfection, cells were harvested for subsequent assays.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cultured cells using TRIzol™ Reagent (Invitrogen, USA) according to the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). Complementary DNA (cDNA) was synthesized using a reverse transcription kit (Nearshore Proteins, Shanghai, China) following the manufacturer’s protocol. Quantitative real-time PCR was performed using the NovoStart® SYBR qPCR SuperMix Kit (2×, Novoprotein, China) on a real-time fluorescence PCR system (BD Biosciences, USA). The thermal cycling conditions were as follows: initial denaturation at 95 °C for 1 minute, followed by 40 cycles of denaturation at 95 °C for 20 seconds and annealing/extension at 60 °C for 60 seconds. The primer sequences used were as follows: CST4 forward: 5‘-CCCTCTGTGTACCCTGCTACTC-3’; CST4 reverse: 5‘-CTTCGGTGGGCCTTGTTGTACT-3’; GAPDH forward: 5’- GGAGCGAGATCCCTCCAAAAT-3‘, and GAPDH reverse: 5’-GGCTGTTGTCATACTTCTCATGG-3’. Primer amplification efficiencies were validated by standard curve analysis and were between 95% and 105% for all primer sets. Each reaction was run in triplicate. Relative gene expression levels were calculated using the 2−ΔΔCt method, with GAPDH used as the endogenous control.
Western blot analysis
Cells were lysed using RIPA lysis buffer (Beyotime, China) supplemented with 1% phenylmethylsulfonyl fluoride (PMSF) and incubated on ice for 30 min. Lysates were centrifuged at 12,000 × g for 15 min at 4 °C to remove cellular debris, and the supernatant was collected. Total protein concentration was determined using a BCA Protein Assay Kit (Beyotime, China) according to the manufacturer’s instructions. Protein samples were adjusted to equal concentrations and mixed with 5× SDS loading buffer at a ratio of 4:1. The samples were denatured by boiling at 100 °C for 5 min. Equal amounts of protein (30 µg per lane) were separated by SDS-PAGE on 10% polyacrylamide gels and subsequently transferred onto PVDF membranes (Millipore, USA) using a wet transfer method. Membranes were blocked with 5% non-fat milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies diluted in TBST. The primary antibodies used included: CST4 (ab151771; Abcam, UK), E-cadherin (ab314063; Abcam, Cambridge, UK), N-cadherin (ab76011; Abcam, Cambridge, UK), Vimentin (ab20346; Abcam, Cambridge, UK), β-catenin (ab6302; Abcam, UK), Cyclin D1 (ab226977; Abcam, UK), histone H1 (H1; ab71594; Abcam, UK) and GAPDH (ab32539; Abcam, Cambridge, UK). After washing with TBST, membranes were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody for 1 h at room temperature. Finally, protein bands were visualized using an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific, USA) and imaged using a chemiluminescence imaging system (Bio-Rad ChemiDoc MP). Band intensities were quantified using ImageJ software, and relative protein expression was normalized to GAPDH as a loading control.
Cell viability assay
Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan), following the manufacturer’s protocol. Briefly, the transfected HEY and HO-8910 ovarian cancer cells were seeded into 96-well plates at a density of 2 × 10³ cells per well in 100 µL of complete medium, with three replicate wells per condition. At designated time points (0-, 24-, 48-, and 72-hours post-transfection), 10 µL of CCK-8 solution was added to each well and incubated for 2 h at 37 °C. Following incubation, the absorbance (OD value) at 450 nm was measured using a microplate reader (BioTek Instruments, USA) to evaluate cell viability.
Colony formation assay
Colony formation ability was assessed to evaluate the long-term proliferative capacity of OC cells following CST4 knockdown. HEY and HO-8910 cells were seeded into 6-well plates at a density of 2 × 10³ cells per well and then maintained at 37 °C in a humidified atmosphere with 5% CO₂. The culture medium was replaced every 3 days. After 14 days, when visible colonies (≥ 50 cells) had formed, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30–60 min at room temperature. Following fixation, cells were rinsed again with PBS and stained with 0.1% crystal violet solution for 30 min. Excess stain was removed by washing with distilled water. Colonies were imaged and counted using ImageJ software.
Wound healing assay
A wound-healing assay was performed to assess the migratory capacity of OC cells following CST4 knockdown. Briefly, HEY and HO-8910 cells were seeded at a density of 5 × 10⁵ cells per well and incubated overnight at 37 °C in a 5% CO₂ incubator to allow for full confluency. After 24 h, a sterile 200 µL pipette tip was used to create a straight scratch in each well. Following scratching, the wells were gently washed 2- times with PBS to remove cell debris. The culture medium was then replaced with a serum-free medium to minimize cell proliferation and focus on migration. Afterwards, cells were incubated at 37 °C with 5% CO₂, and images of the wound area were captured at 0-, 24-, 48-, and 72-hours post-scratch using an inverted microscope (Leica DMi1, Wetzlar, Germany). The wound closure was quantified by measuring the remaining wound area at each time point using ImageJ software.
Transwell invasion assay
Transwell assay was performed to evaluate the invasive capacity of OC cells. Transwell chambers with 8 μm pore-size polycarbonate membranes (Corning, USA) were used. Prior to seeding, the upper chamber was coated with 50 µL of Matrigel (Corning, USA) diluted in serum-free medium to a final concentration of 250 µg/mL (1:4 dilution of the stock) and incubated at 37 °C for 2 h to allow gel solidification. After that, 500 µL of complete medium containing 10% FBS was added to the lower chamber as a chemoattractant. A total of 1 × 10⁴ HEY or HO-8910 cells, previously transfected with either si-CST4 or si-NC, were resuspended in 200 µL of serum-free medium and seeded into the upper chamber. Cells were incubated at 37 °C with 5% CO₂ for 24 h. After incubation, the non-invading cells on the upper surface of the membrane were gently removed using a cotton swab moistened with PBS. The invaded cells on the lower surface were fixed with 4% paraformaldehyde for 20 min at room temperature, washed with PBS, and stained with 0.1% crystal violet for 30 min. Excess stain was removed by washing with PBS, and membranes were air-dried. Stained cells were visualized and photographed under an inverted light microscope (Olympus, Japan). For quantification, five randomly selected fields per membrane were imaged at 200× magnification, and the number of invaded cells was counted using ImageJ software.
Statistical analysis
All statistical analyses were performed using SPSS software version 25.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 8.0 (GraphPad Software, USA). Quantitative data are presented as mean ± standard deviation (SD). Differences between the two groups were analyzed using the Student’s t-test, while comparisons among multiple groups were performed using one-way ANOVA followed by Tukey’s post hoc test. The association between CST4 protein expression and clinicopathological characteristics was evaluated using the Chi-square test (χ² test) or Fisher’s exact test, as appropriate. Spearman correlation analysis was used to assess correlations between CST4 expression and continuous clinical variables. Kaplan–Meier survival curves were generated to evaluate overall survival (OS) and progression-free survival (PFS), and differences between groups were compared using the log-rank test. Univariate and multivariate survival analyses were performed using the Cox proportional hazards regression model. The proportional hazards assumption was verified by inspection of log-minus-log survival plots and by testing Schoenfeld residuals; no significant violations of the assumption were detected for variables included in the final models (all p > 0.05). Hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) were calculated. A two-sided p value < 0.05 was considered statistically significant.
Results
CST4 is highly expressed in OC tissues
IHC analysis was performed on paraffin-embedded tissue sections to assess the expression of CST4 protein in OC and normal ovarian tissues. CST4 expression, characterized by staining intensity and the percentage of positive cells, was significantly higher in OC tissues compared to normal controls (Fig. 1). Among 102 °C samples, 6 (5.9%) were negative for CST4, 54 (52.9%) showed low expression, and 42 (41.2%) exhibited high expression. In contrast, among the 20 normal ovarian tissues, 16 (80%) were negative, and only 4 (20%) showed low expression, with no cases showing high CST4 expression. Statistical analysis revealed a significant difference in CST4 expression between tumor and normal tissues (χ² = 12.599, p < 0.001; Table 1). These findings indicate that CST4 is markedly overexpressed in OC tissues compared to normal ovarian tissues.
Fig. 1.
CST4 is highly expressed in OC tissues. Schematic diagram of IHC staining of OC paraffin-embedded tissue and normal tissues. Bar scale = 5 μm, bar scale = 10 μm
Table 1.
IHC quantification of CST4 expression in 102 °C paraffin-embedded tissues and 20 normal tissues
| Types | Total | Negative expression | Positive expression | ||
|---|---|---|---|---|---|
| Low | High | ||||
| Tumor | 102 | 6 | 54 | 42 | |
| Normal | 20 | 16 | 4 | 0 | |
| Types | Total | CST4 | X2 | P | |
|---|---|---|---|---|---|
| Low expression (%) | High expression (%) | ||||
| Tumor | 102 | 60 (58.82%) | 42 (41.18%) | 12.559 | < 0.001 |
| Normal | 20 | 20 (100%) | 0 (0.00%) | ||
CST4 expression is associated with poor clinicopathological features and prognosis in OC
To explore the clinical relevance of CST4, we analyzed its association with clinicopathological features in 102 °C patients. One-way Chi-square analysis showed that high CST4 expression was significantly associated with advanced FIGO stage (p < 0.001), histological type (p = 0.048), elevated CA125 levels (p = 0.020), platinum resistance (p = 0.004), and mortality (p < 0.001), but not with patient age, tumor grade, ascites, lymph node status, tumor size, CA153, or CA199 levels (p > 0.05; Table 2).
Table 2.
One-way chi-square analysis of factors affecting CST4 expression using CST4 expression level as the end event, n (%)
| N | CST4 | X2 | P | ||
|---|---|---|---|---|---|
| Low expression | High expression | ||||
| Age | 1.213 | 0.271 | |||
| < 60 | 64 | 35 (54.69) | 29 (45.31) | ||
| ≥ 60 | 38 | 25 (65.79) | 13 (34.21) | ||
| FIGO staging | 13.069 | < 0.001* | |||
| Ⅰ+Ⅱ | 46 | 36 (78.26) | 10 (21.74) | ||
| Ⅲ+Ⅳ | 56 | 24 (42.86) | 32 (57.14) | ||
| Histological type | 3.893 | 0.048* | |||
| Non-plasma adenocarcinoma | 33 | 24 (72.73) | 9 (27.27) | ||
| Plasma adenocarcinoma | 69 | 36 (52.17) | 33 (47.83) | ||
| Histological grading | 1.086 | 0.297 | |||
| G1 + G2 | 62 | 39 (62.9) | 23 (37.1) | ||
| G3 | 40 | 21 (52.5) | 19 (47.5) | ||
| Preoperative ascites | 3.152 | 0.076 | |||
| NO | 52 | 35 (67.31) | 17 (32.69) | ||
| YES | 50 | 25 (50) | 25 (50) | ||
| Lymphatic node transfer | 0.843 | 0.359 | |||
| NO | 93 | 56 (60.22) | 37 (39.78) | ||
| YES | 9 | 4 (44.44) | 5 (55.56) | ||
| Tumor diameter | 0.068 | 0.794 | |||
| < 8 | 47 | 27 (57.45) | 20 (42.55) | ||
| ≥ 8 | 55 | 33 (60) | 22 (40) | ||
| CA125 | 5.399 | 0.020* | |||
| < 500 | 48 | 34 (70.83) | 14 (29.17) | ||
| ≥ 500 | 54 | 26 (48.15) | 28 (51.85) | ||
| CA153 | 2.153 | 0.142 | |||
| ≤ 28 | 69 | 44 (63.77) | 25 (36.23) | ||
| > 28 | 33 | 16 (48.48) | 17 (51.52) | ||
| CA199 | 0.045 | 0.831 | |||
| ≤ 37 | 89 | 52 (58.43) | 37 (41.57) | ||
| > 37 | 13 | 8 (61.54) | 5 (38.46) | ||
| Platinum-sensitive | 8.446 | 0.004* | |||
| NO | 22 | 7 (31.82) | 15 (68.18) | ||
| YES | 80 | 53 (66.25) | 27 (33.75) | ||
| Outcomes | 20.255 | < 0.001* | |||
| Survival | 49 | 40 (81.63) | 9 (18.37) | ||
| Death | 53 | 20 (37.74) | 33 (62.26) | ||
| Residual tumor size | 0.545 | 0.460 | |||
| R0/R1 | 65 | 40 (61.54) | 25 (38.46) | ||
| R2 | 37 | 20 (54.05) | 17 (45.95) | ||
*P < 0.05
To determine whether CST4 expression was prognostically significant, we conducted univariate Cox regression analysis using patient outcome as the endpoint. FIGO stage (p < 0.001), histological type (p = 0.029), CA125 (p = 0.018), and CST4 expression (p < 0.001) were found to be significant predictors of poor prognosis (Table 3).
Table 3.
Factors influencing OC patient outcomes by univariate Cox analysis with patient outcome as the endpoint event, n (%)
| N | Outcomes | X2 | P | ||
|---|---|---|---|---|---|
| Survival (n = 49) | Death (n = 53) | ||||
| Age | 0.090 | 0.760 | |||
| < 60 | 64 | 30 (46.9) | 34 (53.1) | ||
| ≥ 60 | 38 | 19 (50) | 19 (50) | ||
| FIGO staging | 12.571 | < 0.001* | |||
| Ⅰ+Ⅱ | 46 | 31 (67.4) | 15 (32.6) | ||
| Ⅲ+Ⅳ | 56 | 18 (32.1) | 38 (67.9) | ||
| Histological type | 4.754 | 0.029* | |||
| Non-plasma adenocarcinoma | 33 | 21 (63.6) | 12 (36.4) | ||
| Plasma adenocarcinoma | 69 | 28 (40.6) | 41 (59.4) | ||
| Histological grading | 0.809 | 0.368 | |||
| G1 + G2 | 62 | 32 (51.6) | 30 (48.4) | ||
| G3 | 40 | 17 (42.5) | 23 (57.5) | ||
| Lymphatic node transfer | 0.331 | 0.565 | |||
| NO | 93 | 46 (49.5) | 47 (50.5) | ||
| YES | 9 | 3 (33.3) | 6 (66.7) | ||
| Tumor diameter | 0.394 | 0.530 | |||
| < 8 | 47 | 21 (44.7) | 26 (55.3) | ||
| ≥ 8 | 55 | 28 (50.9) | 27 (49.1) | ||
| CA125 | 5.565 | 0.018* | |||
| < 500 | 48 | 29 (60.4) | 19 (39.6) | ||
| ≥ 500 | 54 | 20 (37) | 34 (63) | ||
| CA153 | 2.664 | 0.103 | |||
| ≤ 28 | 69 | 37 (53.6) | 32 (46.4) | ||
| > 28 | 33 | 12 (36.4) | 21 (63.6) | ||
| CA199 | 0.201 | 0.654 | |||
| ≤ 37 | 89 | 42 (47.2) | 47 (52.8) | ||
| > 37 | 13 | 7 (53.8) | 6 (46.2) | ||
| CST4 | 20.255 | < 0.001* | |||
| Low expression | 60 | 40 (66.7) | 20 (33.3) | ||
| High expression | 42 | 9 (21.4) | 33 (78.6) | ||
| Residual tumor size | 1.307 | 0.252 | |||
| R0/R1 | 65 | 34 (52.31) | 31 (47.69) | ||
| R2 | 37 | 15 (40.54) | 22 (59.46) | ||
*P < 0.05
Multivariate Cox regression analysis confirmed that high CST4 expression (HR = 2.351, 95% CI: 1.278–4.326, p = 0.006) and advanced FIGO stage (III/IV) (HR = 2.877, 95% CI: 1.460–5.671, p = 0.002) were independent prognostic factors for poor overall survival in OC (Table 4).
Table 4.
Multivariate Cox regression analysis of overall survival in OC patients
| B | SE | Wald | P | HR | 95% CI | ||
|---|---|---|---|---|---|---|---|
| UCL | LCL | ||||||
| FIGO staging | 1.507 | 0.342 | 9.556 | 0.002 | 2.877 | 1.460 | 5.671 |
| Histological type | 0.272 | 0.298 | 0.833 | 0.361 | 1.312 | 0.735 | 2.342 |
| CA125 | 0.432 | 0.298 | 2.102 | 0.149 | 1.541 | 0.856 | 2.775 |
| CST4 | 0.855 | 0.311 | 7.551 | 0.006 | 2.351 | 1.278 | 4.326 |
Kaplan–Meier survival analysis confirmed that patients with high CST4 expression had significantly shorter progression-free survival (PFS) and overall survival (OS) (Table 5; Fig. 2A-B, p < 0.05). Together, these results suggest that CST4 overexpression is associated with aggressive disease features and may serve as an independent predictor of poor prognosis in OC.
Table 5.
Number of patients at risk of OC
| Time (months) | 0 | 20 | 40 | 60 | 80 | 100 |
|---|---|---|---|---|---|---|
| Low-CSF4 | 60 | 58 | 52 | 45 | 38 | 35 |
| High-CSF4 | 42 | 38 | 32 | 25 | 18 | 15 |
Fig. 2.
Association of CST4 expression with clinicopathological features of OC and correlation with poor prognosis. A, B Kaplan-Meier survival analysis of PFS and OS in patients with high versus low CST4 expression
Silencing CST4 suppresses proliferation, migration, and invasion of OC cells
To investigate the functional significance of CST4 in OC, we first assessed its expression levels in a panel of OC cell lines (SKOV3, HEY, OVCAR3, and HO-8910) compared to a normal human ovarian epithelial cell line (IOSE-80). qRT-PCR analysis revealed that CST4 mRNA was significantly upregulated in all four OC cell lines relative to IOSE-80 (Fig. 3A). Among them, HEY and HO-8910 cells exhibited the highest CST4 expression levels and were thus selected for subsequent loss-of-function studies. To evaluate the effects of CST4 silencing, we designed three independent small interfering RNAs (siRNAs) targeting CST4. Transfection efficiency was confirmed by qRT-PCR, which showed that siCST4-1 achieved the most potent knockdown in both HEY and HO-8910 cells (Fig. 3B). Therefore, siCST4-1 was selected for use in all subsequent experiments. We next examined the impact of CST4 knockdown on cell proliferation. CCK-8 assays demonstrated that CST4 silencing significantly reduced cell viability in a time-dependent manner over 72 h (Fig. 3C, D), indicating impaired proliferative capacity. Consistently, colony formation assays revealed a marked decrease in both the number and size of colonies formed by CST4-silenced cells compared to control siRNA-transfected cells (Fig. 3E), further supporting a role for CST4 in promoting long-term cell growth. To assess the role of CST4 in cell migration, we performed wound-healing assays. CST4 knockdown significantly impaired the migratory ability of both HEY and HO-8910 cells, as evidenced by a reduced wound closure rate at 24- and 48-hours post-scratch (Fig. 3F-G). Additionally, Transwell invasion assays showed that silencing CST4 led to a significant decrease in the number of cells invading through Matrigel-coated membranes, indicating a diminished invasive potential (Fig. 3H-I). Collectively, these results suggest that CST4 plays a critical oncogenic role in ovarian cancer by promoting cell proliferation, migration, and invasion in vitro. Its silencing effectively suppresses these malignant phenotypes, supporting CST4 as a potential therapeutic target in OC.
Fig. 3.
Silencing of CST4 inhibits OC cell proliferation, migration, and invasion. A qRT-PCR analysis of CST4 mRNA expression in OC cell lines (SKOVR3, HEY, OVCR3, HO-8910) and a normal human ovarian epithelial cell line (IOSE-80). B qRT-PCR analysis was performed to assess the expression of CST4 in OC cells that had been transfected with si-CST4. C CCK-8 analyses of the effect of silencing CST4 on HEY and HO-8910 cell viability. D, E Colony formation assay was conducted to analyze the effect of silencing CST4 on HEY and HO-8910 cell proliferation. F, G Wound-healing assays were performed to analyze the effect of silencing CST4 on HEY and HO-8910 cell migration rates. H, I Transwell assays were conducted to analyze the effect of silencing CST4 on HEY and HO-8910 cell invasion capacities. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
CST4 promotes EMT through activation of the Wnt/β-catenin signaling pathway
Given previous reports linking CST4 to EMT via Wnt signaling in other tumor types [26], we next explored whether CST4 exerts similar regulatory effects on EMT in OC cells through the Wnt/β-catenin signaling pathway. To investigate this, we performed western blot analysis to examine the expression of key components of the Wnt/β-catenin pathway and EMT-associated markers in HEY and HO-8910 cells following CST4 knockdown. As shown in Fig. 4A, B, the expression of β-catenin and its downstream effector Cyclin D1 was significantly higher in HEY and HO-8910 cells, and silencing CST4 resulted in a marked decrease in their expression level. We next assessed the effect of CST4 knockdown on EMT marker expression. Western blot results revealed that silencing CST4 led to a significant reduction in the mesenchymal markers (N-cadherin and Vimentin) in both HEY and HO-8910 cells. Conversely, expression of the epithelial marker (E-cadherin) was notably upregulated upon CST4 depletion (Fig. 4A, B). To explore whether CST4 regulated β-catenin cellular localization, cytoplasmic and nuclear protein extracts were separately prepared. Western blotting demonstrated that transfection with the si-CST4 significantly increased the proportion of β-catenin in the cytoplasm and decreased the proportion of β-catenin in the nucleus, compared with the cells transfected with the negative control (Fig. 4C and D). Taken together, these findings suggest that CST4 facilitates EMT in OC cells at least in part by activating the Wnt/β-catenin signaling pathway. By promoting mesenchymal characteristics and repressing epithelial traits, CST4 may enhance the invasive and metastatic potential of OC cells.
Fig. 4.
CST4 promotes OC cell proliferation, migration, and epithelial mesenchymal transition (EMT) via regulating Wnt signalling pathway. A, B Western blot assay was performed to detect the effect of silencing CST4 on the expression of Wnt pathway-related proteins β-catenin and cyclin D1, and mesenchymal markers N-cadherin, Vimentin, and epithelial marker E-cadherin. C, D Western blot assay was performed to detect the effect of CST4 silencing on the expression of β-catenin protein in the cytoplasmic and nuclear fraction *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Discussion
OC is one of the most lethal malignancies of the female reproductive system. Despite the progress made in surgical and chemotherapy interventions, the overall prognosis remains poor. Therefore, identifying novel molecular markers and therapeutic targets is critical for improving early diagnosis, predicting patient outcomes, and developing effective treatment strategies. In this study, we demonstrate that CST4 is significantly overexpressed in OC tissues and cell lines, and that high CST4 expression is significantly associated with advanced clinical stage, platinum resistance, and poor prognosis. Functionally, CST4 promotes proliferation, migration, invasion, and EMT of OC cells through activation of the Wnt/β-catenin signaling pathway.
In recent years, the cystatin family of cysteine protease inhibitors has emerged as a group of molecules that play significant regulatory roles in the development and progression of cancer [27]. Among them, CST4, a type II cystatin, is associated with multiple malignancies [28, 29], but its role in OC remains unclear. In this study, we demonstrated for the first time that CST4 was significantly upregulated in OC tissues compared to that in normal ovarian tissues. These findings are consistent with the previous results of Wang et al., study based on bioinformatics [23], which pointed out that CST4 is one of the key genes associated with poor prognosis and immune infiltration in the tumor microenvironment of OC. Here, we provide the first comprehensive validation of CST4 overexpression in clinical OC samples, highlighting its potential as a diagnostic biomarker.
While the bioinformatics study by et al. [23] offers valuable prognostic insights, it lacks experimental validation and mechanistic exploration. Moreover, although CST4 has been reported to promote EMT and metastasis in esophageal cancer [26], its functional role and underlying molecular mechanisms in OC were previously unknown. The present study therefore represents a significant conceptual and experimental advance in the field. First, we move beyond correlative analyses by providing the first direct pathological and molecular evidence of CST4 overexpression in OC tissues and cell lines. Second, through gain- and loss-of-function experiments, we establish CST4 as a bona fide oncogenic driver that actively promotes malignant phenotypes, including proliferation, migration, invasion, and EMT, in OC cells. Most importantly, we uncovered a previously unrecognized mechanistic link between CST4 and the Wnt/β-catenin signaling pathway. We demonstrate that CST4 activates this pathway, while CST4 knockdown reverses key molecular events, including β-catenin accumulation, Cyclin D1 expression, and EMT marker dysregulation. Collectively, these findings transform CST4 from a prognostic signature into a validated functional effector with a defined signaling mechanism in OC progression.
Further, we found that CST4 expression was significantly associated with adverse clinicopathological parameters, including a higher FIGO stage and elevated CA125 levels, and was more prevalent in platinum-resistant tumors. These findings further reinforce the notion that CST4 is related to the invasiveness of the disease and treatment failure. Our survival analysis further supports the clinical significance of CST4. High CST4 expression was significantly associated with shortened PFS and OS, and multivariate Cox regression analysis identified CST4 as an independent prognostic factor. These results align with those of other cancer studies. For example, in colorectal cancer and gastric cancer, elevated CST4 expression predicts a lower survival rate of patients and is associated with invasiveness and metastatic diseases [21, 22].
Metastasis is a significant characteristic of malignant tumors and an important factor contributing to poor prognosis in OC. Migration and invasion of tumor cells are crucial steps in the metastasis process and are closely related to recurrence and chemotherapy resistance [30]. Previous studies have shown that CST4 promotes the proliferation, migration, and metastasis of gastric cancer and colorectal cancer, while CST4 silencing reduces the metastatic behavior [21, 22]. In line with these findings, we demonstrated that CST4 knockdown significantly reduced proliferation, colony-forming ability, migration, and invasion of OC cells in vitro. These results highlight the tumor-promoting function of CST4 in OC and its potential as a therapeutic target for inhibiting the progression of metastasis.
One of the key mechanisms behind cancer metastasis is EMT, which enables epithelial tumor cells to acquire mesenchymal characteristics, thereby enhancing their mobility, invasiveness, and resistance to treatment [31]. EMT is characterized by the downregulation of epithelial markers (E-cadherin) and the upregulation of mesenchymal markers (N-cadherin, Vimentin), which enables tumor cells to detach from the primary lesion, acquire migratory properties, and disseminate to distant sites [32–34]. Among the signaling cascades implicated in EMT, the Wnt/β-catenin pathway is one of the most extensively studied. Activation of this pathway leads to stabilization and nuclear translocation of β-catenin, which in turn drives the transcription of downstream targets such as cyclin D1 and c-Myc, key regulators of cell proliferation, survival, and stemness [35]. Dysregulation of the Wnt signaling pathway has been reported in multiple cancers, including OC, where it not only facilitates EMT but also contributes to the development of chemoresistance [36–38]. Notably, approximately 51% of endometrioid OC cases have mutations in β-catenin or other components of the Wnt pathway, including APC [39]. Moreover, hyperactivation of Wnt/β-catenin signaling has been implicated in therapeutic resistance in high-grade serous ovarian cancer, and pharmacological interventions targeting this pathway have demonstrated potential in overcoming resistance [40]. In line with these reports, we found that silencing CST4 in OC cells downregulates the expression of β-catenin and its downstream effector Cyclin D1. Furthermore, CST4 knockdown suppressed the expression of mesenchymal markers (N-cadherin and Vimentin) and promoted the expression of epithelial markers (E-cadherin). These findings suggest that CST4 may enhance OC cell migration, invasion and EMT by activating the Wnt/β-catenin signaling pathway. Apoptosis, a naturally programmed cell death mechanism, eliminates abnormal cells produced during mitosis. Cancer may develop when this equilibrium is disrupted, either by excessive cell proliferation or reduced apoptosis [41]. However, the role of CST4 in regulating OC cell apoptosis remains unclear. In addition, mutations in the TP53 gene are the most common genetic alterations in human cancers, leading to the accumulation of mutant p53 protein [42]. The relationship between CST4 and mutant p53 in OC cells remains to be further explored. Moreover, multi-omics approaches offer significant potential in drug discovery, particularly in predicting drug sensitivity. With continuing technological advancements, the ability of multi-omics to transform disease treatment and improve patient survival continues to grow, making it a key focus in the ongoing fight against cancer [43, 44]. In the future, by integrating artificial intelligence and machine learning technologies, we plan to construct CST4-related multi-omics prediction models to enhance the accuracy of cancer diagnosis and prognosis assessment.
Limitations
Despite the strengths of this study, several limitations should be acknowledged. First, although our in vitro experiments provide compelling evidence for the oncogenic role of CST4 in OC, in vivo validation using orthotopic or metastatic OC models is required to confirm its effects on tumor growth and metastasis. Second, while we demonstrated that CST4 knockdown suppresses β-catenin and Cyclin D1 expression and alters β-catenin localization, the causal relationship between CST4 and Wnt/β-catenin pathway activation would be further strengthened by rescue experiments, such as ectopic β-catenin expression in CST4-silenced cells. Third, the precise molecular mechanism by which CST4 regulates Wnt signaling remains to be elucidated, including whether CST4 influences Wnt ligand production, receptor expression, or β-catenin stability and degradation. In this context, Wnt-specific reporter assays (e.g., TOP/FOP Flash) would provide direct evidence of pathway activation downstream of CST4. Finally, large-scale clinical validation is needed to confirm the predictive value of CST4 and to explore its feasibility as a therapeutic target, for example through antibody-based strategies or RNA interference approaches.
Future directions
Beyond the scope of the present study, further investigation into the tumor microenvironment is warranted. In particular, the relationship between CST4 expression and immune cell infiltration has not yet been explored. Advanced computational approaches, such as immune deconvolution algorithms, could be employed to characterize immune cell composition, functional states, and exhaustion profiles in relation to CST4 expression. Additionally, pathway-centric analyses may uncover CST4-associated signaling networks involved in drug resistance and immune evasion. Subgroup analyses based on OC histological subtypes and treatment response status, particularly platinum-resistant versus platinum-sensitive disease, will be essential to determine whether CST4 can serve as a stratified predictive biomarker. Such studies, ideally conducted in large and independent cohorts, will be critical for advancing the clinical translation of CST4-targeted strategies.
Conclusion
In summary, this study identifies that CST4 is a novel oncogenic driver in OC, which promotes EMT and metastasis by activating the Wnt/β-catenin pathway. High CST4 expression is associated with poor prognosis, advanced disease stage, and platinum resistance. These findings provide a strong theoretical basis for further development of CST4 as a prognostic biomarker and therapeutic target for OC.
Supplementary Information
Acknowledgements
Not applicable.
Author contributions
Conceptualization, Shanshan Ma; Data curation, Junjun Shao; Funding acquisition, Shanshan Ma; Investigation, Junjun Shao, and Wei Gao; Methodology, Junjun Shao, Wei Gao, and Yuzhi Li; Project administration, Bo Yang; Resources, Software, Suyang Guo, and Qingsong Zhang; Supervision, Shanshan Ma; Validation, Li Zhou; Writing – original draft, Junjun Shao; Writing – review & editing, Shanshan Ma. All authors have read the final manuscript and approved it.
Funding
This work was supported by the 2022 Bengbu Medical College Natural Science Key Project (No. 2022byzd038).
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Declarations
Ethics approval and consent to participate
The study involving human subjects complied with the Declaration of Helsinki and was approved by the ethical committee of the First Affiliated Hospital of Bengbu Medical University (approval number: 2023-088-NG). All participants provided written informed consent. There were no animal studies involved in this report.
Consent for publication
Not applicable, as no identifiable or sensitive patient data are included in this manuscript.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Supplementary Materials
Data Availability Statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.