CBX2 promotes cisplatin resistance in ovarian cancer via SIAH2-mediated β-catenin stabilization and ATG9B-dependent autophagy activation

Abstract

Background

Platinum-based chemotherapy is the first-line therapeutic strategy for ovarian cancer (OC), and resistance to it adversely affects most OC patients. CBX2 plays an essential role in cancer progression and is widely thought to be involved in cisplatin (DDP) resistance. This study aimed to investigate the molecular mechanism of CBX2 in DDP resistance in OC.

Materials and methods

The human normal ovarian epithelial cell line (IOSE80) and OC cell lines (SK-OV-3 and OVcar3) were used. The functions of CBX2 and ATG9B were explored by transfecting overexpression and silencing plasmids into OC cells. Drug-resistant DDP OC cell lines were established. Real-time quantitative PCR (qPCR), Western blotting, and immunofluorescence (IF) analysis were conducted to evaluate the molecular expression at the mRNA and protein levels. The Cell Counting Kit-8 (CCK-8) assay measured the IC₅₀ value of cells under DDP treatment. Flow cytometry was used to determine cell apoptosis, and a colony formation assay was used to measure cell proliferation. A co-immunoprecipitation (Co-IP) assay evaluated the binding relationship between SIAH2 and β-catenin.

Results

CBX2 was highly expressed in OC and was associated with DDP resistance. Besides, CBX2 overexpression enhanced DDP resistance and autophagy in OC cells. As expected, CBX2 silencing suppressed autophagy and DDP resistance in OC cells. Mechanistically, CBX2 stabilized β-catenin by inhibiting ubiquitin-mediated degradation. Inhibiting the Wnt/β-catenin pathway suppressed CBX2-induced autophagy and decreased DDP resistance in OC cells. Activating the Wnt/β-catenin pathway promoted CBX2-induced autophagy and increased DDP resistance in OC cells. ATG9B mediated CBX2-induced autophagy and DDP resistance. Finally, ATG9B inhibition rescued the effects of CBX2-mediated autophagy and DDP resistance.

Conclusion

CBX2 promotes cisplatin resistance in OC via SIAH2-mediated β-catenin stabilization and ATG9B-dependent autophagy activation. Overall, our study reveals a hitherto undocumented role of CBX2 in mediating DDP resistance, providing insights for potential therapeutic biomarkers to overcome DDP resistance in OC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13048-025-01944-4.

Introduction

Ovarian cancer (OC) represents a major gynecologic malignancy originating from the epithelial cells of the ovaries or fallopian tubes, germ cells, or stromal cells [1]. According to statistics from the Surveillance, Epidemiology, and End Results (SEER) database, the 5-year relative survival rate of ovarian cancer patients is less than 55% [2]. Unfortunately, OC patients often encounter drug resistance, with platinum drug resistance representing a significant challenge in the treatment of advanced OC [3]. Chemotherapy resistance and dissemination of OC cells are related to alterations in apoptosis, epithelial-mesenchymal transition (EMT), and autophagy [4]. Therefore, exploring the mechanisms underlying OC chemotherapy resistance is crucial for improving patient treatment.

Chromobox 2 (CBX2) is a member of the Polycomb group (PcG) family, which regulates gene expression through epigenetic mechanisms [5]. Previous reports have characterized CBX2 as an epigenetically modified transcriptional repressor, serving as a transcriptional activator in regulating the downstream genes [6, 7]. CBX2 is upregulated in OC and correlated with a poor prognosis [8]. Besides, deubiquitinating and stabilizing CBX2 can promote OC progression [9]. High CBX2 expression has been associated with platinum resistance in high-grade serous ovarian cancer [10], consistent with its established role as a key protein associated with tumor development and drug resistance [11–15]. However, its expression and functional role in affecting cisplatin (DDP) resistance of OC are not yet fully understood.

Previous research has suggested a relationship between CBX2 and SIAH2. As a ubiquitination regulatory gene of β-catenin protein, SIAH inhibits the Wnt/β-catenin pathway by degrading β-catenin [16]. Xu et al. [17]. indicated that CBX2-driven inhibition of SIAH2 could lead to the accumulation of WNK1, thereby enhancing glycolysis in hepatocellular carcinoma. Previous studies have elucidated the regulation between the Wnt/β-catenin pathway and CBX2 in cancer progression [11, 18, 19]. However, whether CBX2 plays an essential role in DDP resistance through the Wnt/β-catenin pathway warrants further investigation. Given the established crosstalk between Wnt/β-catenin signaling and autophagy [20], we hypothesized that CBX2 confers DDP resistance in OC through Wnt/β-catenin-mediated autophagy activation. However, the role of SIAH2, a key ubiquitin ligase for β-catenin, in DDP resistance remains unclear.

Autophagy-related protein 9B (ATG9B), a multi-spanning transmembrane protein, plays a fundamental role in mammalian autophagy by regulating autophagosome initiation and formation [21]. ATG9B has been associated with various diseases, including hypertension, primary and early menopause. The ATG9B gene plays a key role in regulating autophagy (a lysosomal degradation pathway), and it is involved in regulating the formation and fusion of autophagic vesicles [22]. ATG9B exhibits distinct tissue-specific expression, showing high abundance in placental and ovarian tissues while remaining minimally expressed in testicular, hepatic, pulmonary, muscular, pancreatic, and neural tissues [23]. As reported by Zhong et al. [24]. ATG9B plays a critical role in colorectal cancer metastasis. Considering the tissue-specific feature of ATG9B, we hypothesized that ATG9B could regulate OC.

In the present study, we proposed that the upregulation of CBX2 in OC mediates the transcription level of seven in absentia homolog 2 (SIAH2). The inhibition of SIAH2 maintains the activation of the Wnt/β-catenin pathway and promotes ATG9B-mediated autophagy, thereby promoting DDP resistance in OC.

Materials and methods

Cell culture and transfection

The human normal ovarian epithelial cell line (IOSE80) and OC cell lines (SK-OV-3 and OVcar3) were obtained from the American Type Culture Collection (Manassas, VA) and cultured under RPMI-1640 or high glucose Dulbecco’s MEM supplemented with 10% fetal bovine serum (Biological Industries, USA) in a 5% CO₂ incubator at 37℃.

Vector construction and transfection

Using standard molecular cloning techniques, we inserted PCR-amplified leptin promoter fragments (both truncated and inhibitory variants) into the pGL3-Basic reporter vector (Promega) via T4 DNA ligase-mediated (TaKaRa) ligation. The following transfection groups were constructed using the Lipofectamine 2000 kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 80%–90% confluency: vector (Vector group), overexpressed CBX2 plasmid (CBX2 group), overexpressed ATG9B (ATG9B group), silencing (si) negative control (NC, si-NC group), si-CBX2 plasmid (siCBX2 group), and si-ATG9B plasmid (siATG9B group). The transfections were treated with the Wnt/β-catenin pathway inhibitor (LGK974, 20nM) and activator (LiCl, 20mM).

After transfection of SK-OV-3 and OVcar3 cells with either the control vector or CBX2 for 0, 6, 12, 18, and 24 h, the expression levels of GAPDH were assessed by Western blotting.

Drug-resistant DDP OC cell culture

The SK-OV-3 and OVCAR3 cells in the logarithmic phase were treated with increasing concentrations of DDP (0, 10, 20, 30, 40, 50, 60 µg/ml) under standard culture conditions. After reaching the high concentration, continue culturing for 4 to 8 weeks to screen for stable drug-resistant cell lines.

Real-time quantitative PCR (qPCR)

The total RNA in cells was extracted using TRIzol reagent (Invitrogen, USA). Sangon (Shanghai, China) designed and synthesized the primers for CBX2, ATG9B, SIAH2, and GAPDH. Gene expression analysis was conducted by qPCR using the HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme) on an ABI 7900HT system (Applied Biosystems). Relative expression levels of target genes were normalized to GAPDH and calculated using the 2^−ΔΔCt method.

Western blotting

The cells were lysed in RIPA (Beyotime, Shanghai, China) and quantified by a BCA kit (Thermo, MA, USA). The proteins were fractionated on standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane at 4 ℃. The PVDF membranes were blocked with 5% skimmed milk powder at 4 ℃ overnight and cultured with primary antibodies against CBX2 (ab214512, 1:1000), SIAH2 (ab137073, 1:1000), β-catenin (ATG5, ab32572, 1:5000), ATG9B (ab108338, 1:1000), autophagy-related protein 5 (ab108327, 1:1000), microtubule-associated protein 1 A/1B-light chain 3B isoforms I and II (LC3B I/II, ab192890, 1:1000), G1/S-specific cyclin-D1 (cyclinD1, ab134175, 1:1000), cellular myelocytomatosis oncogene (C-myc, ab32072, 1:1000), axis inhibition protein 2 (Axin2, ab32197, 1:1000), Histone H3 (ab1791, 1:5000), and GAPDH (ab8245, 1:1000). The samples were then incubated for 1 h at room temperature with a horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody. For protein detection, the immunoblotted PVDF membrane was incubated with ECL substrate (Biomiga) for 1 min, followed by X-ray film exposure. Quantitative analysis was performed using Image-Pro Plus 6.0 software, with all band intensities normalized to GAPDH loading controls.

Immunofluorescence (IF) analysis

IOSE80, SK-OV-3, and OVcar3 cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Then, they were fixed with primary anti-CBX2, -LC3 overnight and secondary anti-FITC antibody (ab6785) for one hour. The cells were fixed again in paraformaldehyde and stained with 4’, 6-diamidino-2-phenylindole (DAPI). Finally, the fluorescence intensity was observed under a confocal laser microscope (Olympus Optical, Tokyo, Japan) and then quantified as Integrated Density/Area by ImageJ software.

Cell counting kit-8 (CCK-8) assay

The cells were plated into 24-well plates and treated with graded DDP at 0, 10, 20, 30, 40, 50, and 60μg/mL for 48h. Then, the cells were treated with CCK-8 reagents and incubated in a 5% CO2 incubator at 37 ℃ for 4 h. The absorbance was measured at 450 nm under a microplate reader. The DDP dose-response curves were plotted, and IC₅₀ values were calculated.

Flow cytometry

Cell apoptosis was measured after the DDP treatment for 48 h at its IC₂₅ concentration (5 µg/mL) using the Annexin V FITC/PI staining Apoptosis Detection kit (Sigma-Aldrich, Germany) for the apoptosis analysis. About 3 × 10⁴ cells were suspended and incubated with annexin V-FITC and PI stains in the dark at room temperature. A binding buffer was added to the cells, which were then analyzed using a flow cytometer (Agilent, USA). The data were then analyzed using WinMDI 2.9.

Colony formation

Cell colony formation was also performed to evaluate cell proliferation after the DDP treatment at the IC₂₅ concentration. The cells were trypsinized, seeded into 96-well plates, and cultured in RPMI-1640 supplemented with 10% FBS. Following fixation in 4% paraformaldehyde (PFA) for 24 h at 4 °C, samples were stained with 0.1% crystal violet solution for 30 min and imaged under a light microscope (Nikon Eclipse Ti, 40× objective).

Co-Immunoprecipitation (Co-IP) assay

One day after transfection with siCBX2 and NC, the cells were collected to extract proteins, and the SIAH2/β-catenin protein binding ability was detected by western blotting after co-immunoprecipitation. Briefly, cells were lysed, and the lysates were incubated with a cross-linked resin, and 10 µg of anti-β-catenin and anti-SIAH2 overnight at 4 ℃. An elution buffer was used the following day to release the protein complexes bound to the antibodies. Western blotting assays were conducted to analyze the eluted complexes with an acetyl-lysine antibody (ab190479, Abcam). As for glycosylation, the proteins were stained with 0.1% Vicia Villosa Lectin (VVA) for 1 h, then washed with PBST three times.

Statistical analysis

Data were expressed as mean ± standard deviation (SD). Statistical analyses, including the unpaired Student’s t-test and one-way ANOVA analyses, were performed using GraphPad Prism 10 (San Diego, CA, USA). A p-value less than 0.05 was considered statistically significant.

Results

CBX2 is upregulated in OC and associated with DDP resistance

The expression level of CBX2 in the Human normal ovarian epithelial cell line (IOSE80) and OC cell lines (SK-OV-3 and OVcar3) was detected by qPCR, western blotting, and IF analyses. CBX2 expression was significantly upregulated at both the mRNA and protein levels in SK-OV-3 and OVCAR3 cells compared to the normal IOSE80 cell line (P < 0.01, P < 0.001; Figs. 1A, B). Consistent with this, the fluorescence intensity of CBX2 was also significantly higher in the OC cell lines (Fig. 1C-D), further confirming its elevated expression in ovarian cancer.

Fig. 1.

CBX2 was highly expressed in OC and was associated with DDP resistance. A qPCR assessment of the mRNA expression level of CBX2 in the Human normal ovarian epithelial cell line and OC cell lines. B Western blotting analysis assessment of the protein expression level of CBX2 in IOSE80, SK-OV-3, and OVcar3 cells. C, D IF assessment of the fluorescence intensity of CBX2 in IOSE80, SK-OV-3, and OVcar3 cells. E CCK-8 assessment of the IC50 value in wild-type and DDP-resistant OC cell lines. F qPCR assessment of the mRNA expression level of CBX2 in wild-type and DDP-resistant OC cell lines. G Western blotting assessment of the protein expression level of CBX2 in wild-type and DDP-resistant OC cell lines. H, I IF assessment of the fluorescence intensity of CBX2 in wild-type and DDP-resistant OC cell lines. **P < 0.01, ***P < 0.001

Subsequently, we measured the expression level of CBX2 in wild-type and DDP-resistant OC cell lines. The IC50 value was significantly increased in SK-OV-3/DDP and OVcar3/DDP cells compared with the SK-OV-3/W and OVcar3/W cells (P < 0.001, Fig. 1E), showing higher DDP resistance in OC DDP cells than in wild-type cells. The expression level of CBX2 was also quantified in DDP and wild-type cells by qPCR, western blotting, and IF methods. As expected, CBX2 was significantly highly expressed in SK-OV-3/DDP and OVcar3/DDP cells than in SK-OV-3/W and OVcar3/W cells at both mRNA and protein levels (P < 0.001, Figs. 1F-I). Taken together, our findings suggest that CBX2 is highly expressed in OC and is associated with DDP resistance.

CBX2 overexpression enhances DDP resistance and autophagy in OC cells

To further confirm the function of CBX2 on DDP resistance and autophagy, we constructed CBX2-overexpressing and silenced plasmids and transfected them into OC cells. First, CBX2 expression in overexpressed transfections was determined at both mRNA and protein levels. Higher expression was observed in SK-OV-3/W-CBX2 and OVcar3/W-CBX2 groups than in SK-OV-3/W-Vector and OVcar3/W-Vector groups (P < 0.001, Fig. 2A). At the protein level, CBX2 exhibited upregulation in overexpressed transfections compared to vector transfections (Fig. 2B). Compared with the vector groups, the cells transfected with overexpressed CBX2 showed a higher survival rate and IC₅₀ value (P < 0.001, Fig. 2C). Flow cytometry was conducted to evaluate the cell apoptosis ratio under DDP treatment at the IC₂₅ concentration (5 µg/mL). We found that the cell apoptosis rate was significantly reduced in CBX2 overexpression groups compared to the vector groups (P < 0.001, Fig. 2D). Colony formation analysis showed that colony formation was significantly increased in SK-OV-3/W-CBX2 and OVcar3/W-CBX2 groups compared with the SK-OV-3/W-Vector and OVcar3/W-Vector groups (P < 0.001, Fig. 2E). These results collectively indicate that CBX2 promoted cell proliferation and suppressed apoptosis in OC cells. To validate our hypothesis that SIAH2 regulates β-catenin stability via ubiquitination, thereby influencing ATG9B-mediated autophagy and DDP resistance, we examined the expression of key pathway components. SIAH2 expression was measured in transfections and exhibited a lower expression in SK-OV-3/W-CBX2 and OVcar3/W-CBX2 groups than that in SK-OV-3/W-Vector and OVcar3/W-Vector groups (P < 0.001, Figs. 2F and G).

Fig. 2.

CBX2 overexpression enhances DDP resistance and autophagy in OC cells. A qPCR analysis assessment of the mRNA expression level of CBX2 in transfections. B Western blotting analysis assessment of the protein expression level of CBX2 in transfections. C CCK-8 determined the IC50 value in transfections. D Flow cytometry assessment of cell apoptosis in transfections. E Colony formation assessment of the cell proliferation in transfection. F qPCR analysis assessment of the mRNA expression level of SIAH2 in transfections. G, I Western blotting analysis assessment of the protein expression level of SIAH2, β-catenin in transfections. J qPCR analysis assessment of the mRNA expression level of ATG9B in transfections. K Western blotting analysis assessment of the protein expression level of ATG9B, ATG5, and LC3B I/II in transfections. L, M IF assessment of the fluorescence intensity of LC3 in transfections. *** P < 0.001

The protein expression of β-catenin in the total cell lysate and nuclear fraction was measured by western blotting. β-catenin expression was significantly increased by CBX2 overexpression in both cellular cytoplasmic and nuclear compartments (P < 0.001, Figs. 2H and I). These results revealed that CBX2 could inhibit SIAH2 expression, activating the Wnt/β-catenin pathway. The qPCR analysis revealed that CBX2 overexpression significantly increased ATG9B expression compared with the vector (P < 0.001, Figs. 2J and K). Consistent with our hypothesis that autophagy interacts with DDP resistance in OC, we evaluated autophagic activity in transfections by analyzing the expression levels of autophagy biomarkers (ATG5 and LC3B I/II) using western blotting. As expected, the protein levels of ATG5 and LC3B II were notably increased, and LC3B I was markedly decreased in SK-OV-3/W-CBX2 and OVcar3/W-CBX2 groups than in SK-OV-3/W-Vector and OVcar3/W-Vector groups (P < 0.001, Fig. 2K), showing increased autophagic flux. In the SK-OV-3/W-Vector and OVcar3/W-Vector groups, LC3 exhibited diffuse low-intensity signals. In contrast, in the SK-OV-3/W-CBX2 and OVcar3/W-CBX2 groups, many bright, cytoplasmic puncta (autophagosome accumulation) were observed in the cytoplasm (Fig. 2L-M), indicating an increase in the level of autophagy. These findings demonstrated that CBX2 overexpression enhanced DDP resistance by promoting proliferation, inhibiting apoptosis, and activating autophagy via the SIAH2/β-catenin/ATG9B axis.

CBX2 Silencing suppresses autophagy and DDP resistance in OC cells

To further assess the role of CBX2 in autophagy and DDP resistance, we silenced CBX2 in DDP-resistant OC cells. We confirmed CBX2 expression in transfections and found that it was significantly reduced in DDP-siCBX2 and DDP-siCBX2 groups compared with the DDP-NC group in both SK-OV-3 and OVcar3 cells (P < 0.001, Figs. 3A and B). Compared with the vector groups, the cells transfected with silenced CBX2 showed decreased cell activity and a lowered IC₅₀ value of DDP (P < 0.001, Fig. 3C). Besides, flow cytometry showed a significant increase in apoptosis, while colony formation assays demonstrated reduced proliferative capacity in CBX2-silenced cells (P < 0.001, Figs. 3D-E). These findings indicate that CBX2 depletion suppresses the malignant phenotype in DDP-resistant OC cells.

Fig. 3.

CBX2 silencing suppresses autophagy and DDP resistance in OC cells. A qPCR analysis assessment of the mRNA expression level of CBX2 in transfections. B Western blotting analysis assessment of the protein expression level of CBX2 in transfections. C CCK-8 assessment of the IC₅₀ value in transfections. D Flow cytometry assessment of cell apoptosis in transfections. E Colony formation assessment of the cell proliferation in transfection. F qPCR analysis assessment of the mRNA expression level of SIAH2 in transfections. G, I Western blotting assessment of the protein expression level of SIAH2, β-catenin in transfections. J qPCR analysis assessment of the mRNA expression level of ATG9B in transfections. K Western blotting assessment of the protein expression level of ATG9B, ATG5, LC3B I/II in transfections. L, M IF assessment of the fluorescence intensity of LC3 in transfections. *** P < 0.001

Next, SIAH2, ATG9B, and β-catenin expression levels were determined in DDP-siCBX2 and DDP-NC groups through qPCR and western blotting analyses. Consistent with our hypothesis, CBX2 silencing upregulated SIAH2 expression while downregulating β-catenin (both cytoplasmic and nuclear fractions) and ATG9B (P < 0.001, Figs. 3H-K), suggesting inhibition of the Wnt/β-catenin pathway. We next examined autophagy markers (ATG5 and LC3B-I/II) to assess autophagic activity. Western blotting revealed decreased ATG5 and LC3B-II levels alongside increased LC3B-I in siCBX2 cells (P < 0.001, Fig. 3K). IF analysis further confirmed attenuated autophagy, with siCBX2-treated cells exhibiting diminished LC3 puncta formation compared to controls (Fig. 3L-M). Furthermore, another knockdown experiment targeting CBX2 with siRNA-2 yielded consistent results, demonstrating that si-CBX2-2 suppressed both DDP resistance and autophagy. (Figure S1). These results collectively demonstrate that CBX2 silencing suppresses DDP resistance by inhibiting proliferation, promoting apoptosis, and attenuating autophagy via the SIAH2/β-catenin/ATG9B axis.

CBX2 stabilized β-catenin by inhibiting ubiquitin-mediated degradation

In the above experiments, we established that CBX2 influences the Wnt/β-catenin pathway. However, whether it plays an essential role in the ubiquitination and degradation of β-catenin protein remains unclear. Next, we explored the effects of CBX2 on ubiquitination and degradation of β-catenin protein. Following transfection to overexpress β-catenin, the protein was subsequently degraded in a time-dependent manner. However, the degradation rate was significantly reduced in SK-OV-3/W-CBX2 and OVcar3/W-CBX2 groups than in SK-OV-3/W-Vector and OVcar3/W-Vector groups at 12, 18, and 24 h after transfections (Fig. 4A), suggesting CBX2-mediated stabilization of β-catenin.

Fig. 4.

CBX2 stabilized β-catenin by inhibiting ubiquitin-mediated degradation. A Western blotting analysis determined the expression level of β-catenin after CBX2 transfection at 0, 6, 12, 18, and 24 h. B Co-IP detected the binding relationship between SIAH2 and β-catenin. ***P < 0.001

Given that SIAH2 is a ubiquitin ligase for β-catenin, we examined its interaction through Co-IP. The expression level of SIAH2 was remarkably increased, and β-catenin was decreased in SK-OV-3/DDP-siCBX2 and OVcar3/DDP-siCBX2 groups compared with the SK-OV-3/DDP-NC and OVcar3/DDP-NC groups (P < 0.001). Consistent with our hypothesis, CBX2 knockdown significantly increased SIAH2 expression while decreasing β-catenin levels (P < 0.001). The Co-IP assay revealed strong SIAH2 enrichment by β-catenin immunoprecipitation in the siCBX2 group compared to NC controls (Fig. 4B), confirming their physical interaction was enhanced by CBX2 knockdown. These results demonstrate that CBX2 protects β-catenin from SIAH2-mediated ubiquitination and subsequent degradation, providing a mechanistic basis for CBX2’s regulation of Wnt/β-catenin signaling.

Inhibiting the Wnt/β-catenin pathway suppressed CBX2-induced autophagy and decreased DDP resistance in OC cells

To functionally validate the regulation of CBX2 on the Wnt/β-catenin pathway in autophagy and DDP resistance, we employed LGK974, a specific Wnt/β-catenin pathway inhibitor. qPCR and western blotting were used to analyze the expression levels of CBX2 in the vector and in cells overexpressing CBX2 following treatment with LGK974. These experiments confirmed successful CBX2 overexpression in both SK-OV-3 and OVcar3 cells, which was not significantly altered by LGK974 treatment (P < 0.001, Figs. 5A-B). Next, the cells were treated with DDP at its IC₂₅ concentration (5 µg/mL), and the cell apoptosis and proliferation were evaluated. Compared with the Vector group, the cell apoptosis rate was significantly increased in the Vector + LGK974 group (P < 0.001, Fig. 5C). Compared with the CBX2 group, the cell apoptosis rate was significantly increased in the CBX2 + LGK974 group (P < 0.001, Fig. 5C). Conversely, LGK974 remarkably reduced the cell colony abundance in Vector and CBX2 transfections (P < 0.001, Fig. 5D). Western blotting measured the protein expression of β-catenin in the cell and nucleus after the LGK974 treatment. LGK974 significantly reduced the expression level of β-catenin in the nucleus of both cells, but did not affect the total level (P < 0.001, Fig. 5E). Similarly, LGK974 significantly decreased the ATG9B expression in Vector and CBX2 transfections (P < 0.001, Figs. 5F and G). Cyclin D1, c-Myc, and axin2 have been established as target genes of the Wnt/β-catenin pathway, and their expression levels were assessed following LGK974 treatment. Compared with the expression levels of cyclin D1, c-Myc, and axin2 in Vector and CBX2 groups, they were significantly decreased in Vector + LGK974 and CBX2 + LGK974 groups (P < 0.001, Fig. 5G). As for ATG5 and LC3B II autophagy biomarkers, their expression levels were also significantly inhibited by LGK974 supplementation in CBX2 transfections (P < 0.001, Fig. 5G). IF analysis of LC3 showed that the fluorescence intensity in Vector + LGK974 and CBX2 + LGK974 groups was remarkably decreased compared to the Vector and CBX2 groups (Fig. 5H). Inhibiting the Wnt/β-catenin pathway influenced the expression levels of SIAH2, ATG9B, autophagy, and DDP resistance. The above results demonstrated that the inhibition of the Wnt/β-catenin pathway reversed CBX2-mediated autophagy and DDP resistance in OC cells.

Fig. 5.

Inhibiting the Wnt/β-catenin pathway mediates CBX2-induced autophagy and DDP resistance in OC cells. A qPCR assessment of the mRNA expression level of CBX2 in transfections under the LGK974 treatment. B Western blotting assessment of the protein expression level of CBX2 in transfections under the LGK974 treatment. C Flow cytometry assessment of cell apoptosis in transfections under LGK974 treatment. D Colony formation assay assessing the cell proliferation in transfection under the LGK974 treatment. E Western blotting assessment of the protein expression level of β-catenin in cells and the nucleus in transfection under the LGK974 treatment. F qPCR assessment of the mRNA expression level of ATG9B in transfections under the LGK974 treatment. G Western blotting assessment of the protein expression level of cyclin D1, c-Myc, axin2, ATG9B, ATG5, and LC3B I/II in transfections under the LGK974 treatment. H IF assessment of the fluorescence intensity of LC3 in transfections under the LGK974 treatment. *** P < 0.001. ns, no significant difference

Activating the Wnt/β-catenin pathway promoted CBX2-induced autophagy and increased DDP resistance in OC cells

LiCl is an activator of the Wnt/β-catenin pathway. In this study, we assessed the role of the Wnt/β-catenin pathway on autophagy and DDP resistance by supplementing LiCl in DDP-NC and DDP-siCBX2 transfections. CBX2 expression level was not influenced by LiCl treatment in DDP-NC and DDP-siCBX2 transfections of SK-OV-3 and OVcar3 cells (P > 0.05, Figs. 6A and B). In addition, CBX2 exhibited lower expression in DDP-siCBX2 groups than in the DDP-NC group, demonstrating the success of transfections (P < 0.001, Figs. 6A and B). Next, the cells were treated with DDP at its IC₂₅ concentration (5 µg/mL), and the cell apoptosis and proliferation rates were evaluated. Compared with the NC group, the cell apoptosis rate was significantly decreased in the NC + LiCl group (P < 0.001, Fig. 6C). Compared with the siCBX2 group, the cell apoptosis rate was significantly decreased in the siCBX2 + LiCl group (P < 0.001, Fig. 6C). LiCl remarkably increased the cell colony abundance in siCBX2 transfections (P < 0.001, Fig. 6D). Taken together, these findings suggest that LiCl could inhibit cell apoptosis and promote cell proliferation. The protein expression of β-catenin in the cell and nucleus under the LiCl treatment was subsequently quantified. LiCl treatment induced the nuclear translocation of β-catenin without altering its total cellular levels (P < 0.001, Fig. 6E). Besides, LiCl significantly increased the ATG9B expression in NC and siCBX2 transfections (P < 0.001, Figs. 6F and G). Cyclin D1, c-Myc, and axin2 expression levels were evaluated under LiCl treatment. The expression levels of cyclin D1, c-Myc, and axin2 were significantly increased in NC + LiCl and siCBX2 + LiCl groups compared with the NC and siCBX2 groups (P < 0.001, Fig. 6G). ATG9B, ATG5 and LC3B II expression levels were significantly upregulated by LiCl (P < 0.001, Fig. 6G). IF analysis revealed that the fluorescence intensity of LC3 in siCBX2 + LiCl groups was significantly increased compared with the siCBX2 groups (Fig. 6H). Finally, activating the Wnt/β-catenin pathway promoted CBX2-induced autophagy and increased DDP resistance in OC cells.

Fig. 6.

Activating the Wnt/β-catenin pathway promoted CBX2-induced autophagy and increased DDP resistance in OC cells. A qPCR assessment of the mRNA expression level of CBX2 in transfections under the LiCl treatment. B Western blotting assessment of the protein expression level of CBX2 in transfections under the LiCl treatment. C Flow cytometry assessment of cell apoptosis in transfections under the LiCl treatment. D Colony formation assessment of cell proliferation in transfection under the LiCl treatment. E Western blotting assessment of the protein expression level of β-catenin in cells and the nucleus in transfection under the LiCl treatment. F qPCR analysis assessment of the mRNA expression level of ATG9B in transfections under the LiCl treatment. G Western blotting analysis assessment of the protein expression level of cyclin D1, c-Myc, axin2, ATG9B, ATG5, and LC3B I/II in transfections under the LiCl treatment. H IF assessment of the fluorescence intensity of LC3 in transfections under the LiCl treatment. *P < 0.05. ** P < 0.01. *** P < 0.001. ns, no significant difference

ATG9B mediated CBX2-induced autophagy and DDP resistance through the Wnt/β-catenin pathway

Building upon our findings that Wnt/β-catenin signaling regulates ATG9B expression, we next investigated whether ATG9B serves as the critical effector in CBX2-mediated autophagy and DDP resistance. Overexpressed and intervention plasmids of ATG9B were constructed and transfected into OC cells. First, the effects of ATG9B interference were investigated on CBX2 overexpression-induced autophagy and drug resistance in OC cells. CBX2 exhibited significant upregulation in CBX2 and CBX2 + siATG9B groups compared to the Vector and Vector + siATG9Please check if Supplementary Materials were captured correctly. Otherwise, kindly amend if necessary.Please check if Supplementary Materials were captured correctly. Otherwise, kindly amend if necessary.Please check if Supplementary Materials were captured correctly. Otherwise, kindly amend if necessary.Please check if Supplementary Materials were captured correctly. Otherwise, kindly amend if necessary.B groups (P < 0.001, Figs. 7A and B). SiATG9B exhibited limited ability to influence the expression level of CBX2. We found that siATG9B significantly enhanced cell apoptosis rate and slowed cell proliferation (P < 0.001, Figs. 7C and D). Western blotting was used to quantify the protein expression of β-catenin in the cell and nucleus following co-transfection with siATG9B. SiATG9B showed no significant effect on β-catenin expression in both compartments (P < 0.001, Fig. 7E). This result demonstrated that the Wnt/β-catenin pathway regulates ATG9B unidirectionally. In the co-transfections with siATG9B, ATG9B exhibited lower expression than without siATG9B transfections (P < 0.001, Figs. 7F and G), revealing the success of transfections. Western blotting analysis was conducted to assess the effect of siATG9B knockdown on the Wnt/β-catenin pathway and autophagy. We found that the expression levels of Cyclin D1, c-Myc, axin2, and ATG5 exhibited no significant differences upon siATG9B knockdown (Fig. 7G). The expression levels of LC3B I were significantly decreased by siATG9B, and that of LC3B II was significantly increased, suggesting ATG9B regulates autophagy independent of ATG5. Overall, this study provides mechanistic evidence that therapeutic targeting of ATG9B could overcome CBX2-mediated DDP resistance in OC.

Fig. 7.

ATG9B mediated CBX2-induced autophagy and DDP resistance through the Wnt/β-catenin pathway. A qPCR assessment of the mRNA expression level of CBX2 in co-transfections. B Western blotting assessment of the protein expression level of CBX2 in co-transfections. C Flow cytometry assessment of cell apoptosis in co-transfections. D Colony formation assay assessing the cell proliferation in co-transfections. E Western blotting assessment of the protein expression level of β-catenin in cells and the nucleus in co-transfections. F qPCR assessment of the mRNA expression level of ATG9B in co-transfections. G Western blotting assessment of the protein expression level of cyclin D1, c-Myc, axin2, ATG9B, ATG5, and LC3B I/II in co-transfections. *P < 0.05. ** P < 0.01. *** P < 0.001. ns, no significant difference

ATG9B overexpression rescued CBX2-mediated autophagy and DDP resistance

In overexpressed ATG9B co-transfections, CBX2 expression was quantified. Similar to that of siATG9B, overexpressed ATG9B exhibited no significant effect on regulating the expression of CBX2 (P > 0.05, Figs. 8A and B). Compared with the siCBX2 group, the cell apoptosis rate was significantly decreased, and the cell colony abundance was significantly increased in siCBX2 + ATG9B (P < 0.001, Figs. 8C and D). The cell apoptosis rate and cell colony formation were not influenced by overexpressed ATG9B without siCBX2 transfection. These results demonstrated that ATG9B can reverse the role of siCBX2 in promoting cell apoptosis and reducing cell proliferation. Overexpression of ATG9B did not significantly alter β-catenin levels in either the total cell lysate or the nuclear fraction (P > 0.05, Fig. 8E), confirming the unidirectional regulation of ATG9B by the Wnt/β-catenin pathway. The success of the transfection was validated, as ATG9B expression was significantly higher in the co-transfected groups compared to controls (P < 0.001, Figs. 8F and G). Western blotting analysis was next conducted to assess the role of ATG9B on the Wnt/β-catenin pathway and autophagy. We found that the expression levels of cyclin D1, c-Myc, axin2, and ATG5 exhibited no significant differences upon ATG9B overexpression (Fig. 8G). However, LC3B II were significantly promoted by ATG9B overexpression (P < 0.001, Fig. 8G). Overall, the above findings suggest that ATG9B overexpression enhances autophagy and targeting ATG9B may represent a viable strategy to overcome CBX2-mediated DDP resistance in OC.

Fig. 8.

ATG9B overexpression rescued CBX2-mediated autophagy and DDP resistance. A qPCR assessment of the mRNA expression level of CBX2 in co-transfections. B Western blotting assessment of the protein expression level of CBX2 in co-transfections. C Flow cytometry assessment of cell apoptosis in co-transfections. D Colony formation assays measuring the cell proliferation in co-transfections. E Western blotting assessment of the protein expression level of β-catenin in cells and the nucleus in co-transfections. F qPCR assessment of the mRNA expression level of ATG9B in co-transfections. G Western blotting assessment of the protein expression level of cyclin D1, c-Myc, axin2, ATG9B, ATG5, and LC3B I/II in co-transfections. *P < 0.05. ** P < 0.01. *** P < 0.001. ns, no significant difference

Discussion

Platinum-based chemotherapy, particularly DDP, remains the cornerstone of OC treatment, leading to treatment failure and recurrence [25, 26]. However, the development of chemoresistance significantly compromises clinical outcomes, underscoring the urgent need to elucidate underlying mechanisms and identify novel therapeutic targets [27, 28]. Our study revealed a hitherto unreported role of CBX2 in mediating DDP resistance through the Wnt/β-catenin/ATG9B-autophagy axis activation in OC cells.

CBX2 is reportedly involved in cancer progression, facilitating cancer cell proliferation, enhancing cancer stemness, and suppressing cell apoptosis in various cancers [29–32]. Although the function of CBX2 in regulating cancer progression has been widely studied, its role in facilitating DDP resistance and autophagy in OC has rarely been revealed. This study found that CBX2 was highly expressed in OC cells and was further upregulated in the DDP-resistant OC cells. Besides, overexpressing CBX2 enhances autophagy and chemoresistance in normal OC cells. Conversely, CBX2 inhibition suppressed autophagy and chemoresistance in DDP-resistant OC cells. These results identify CBX2 as a critical target for alleviating DDP resistance. Our finding that CBX2 contributes to chemoresistance is consistent with previous research, which demonstrated that CBX2 exhibits an antiapoptotic effect, inducing resistance to DDP and ionizing radiation in cervical cancer cells [32]. However, the role of CBX2 in autophagy was previously unestablished, a gap our study now addresses. Emerging evidence indicates that autophagic activation confers DDP resistance in malignant cells through multiple molecular mechanisms [33], primarily via cytoprotective autophagy that antagonizes apoptotic pathways. Therefore, the present study is the first to connect CBX1-mediated chemoresistance and autophagy and provides a mechanistic explanation for this function.

Wnt/β-catenin is a known pathway involved in autophagy and chemoresistance. Pérez-Plasencia et al. [34]. reported an interplay between the Wnt/β-catenin pathway and autophagy in drug repositioning, highlighting their therapeutic potential in cancer. Song et al. [35]. revealed that miR-219-5p/HMGA2 could reduce DDP resistance in OC by suppressing Wnt/β-catenin signaling and autophagy. This study found that the Wnt/β-catenin pathway can promote CBX2-induced autophagy and increase DDP resistance. Besides, the regulatory role of CBX2 on the Wnt/β-catenin pathway has been suggested in several studies. For example, the Wnt/β-catenin pathway, regulated by miR-342-5p, targets CBX2, thereby reducing the proliferation, invasion, migration, and viability of OC cell lines SKOV3 and OVCAR3 [11]. Similarly, in glioma pathogenesis, miR-149-3p demonstrates anti-tumorigenic properties by targeting CBX2 to inhibit Wnt/β-catenin pathway activation [19]. Therefore, we speculated Wnt/β-catenin pathway might mediate the regulation of CBX2 on autophagy and DDP-resistance in OC. Our results validated that CBX2 inhibits the key regulator of β-catenin, SIAH2 [16]. Accordingly, CBX2 enhanced the expression and nuclear localization of β-catenin. The transcriptional target of β-catenin, including CyclinD1, C-myc and Axin2, also demonstrated a similar trend to β-catenin, confirming the activation of the Wnt/β-catenin pathway by CBX2. More importantly, LGK974, a Wnt/β-catenin pathway inhibitor, reversed the effect of CBX2 overexpression on autophagy and DDP-resistance in OC. Conversely, LiCl, an activator of the Wnt/β-catenin pathway, restored the effect of CBX2 inhibition. These results highlighted the critical role of the Wnt/β-catenin pathway in the regulation of CBX2 on the DDP-resistance of OC.

This study further identified ATG9B as the downstream molecular of the CBX2-Wnt/β-catenin pathway mediating autophagy and DDP-resistance of OC cells. ATG9B is one of the key proteins in the autophagy process and belongs to the ATG9 family, which plays a vital role in the initiation and expansion of autophagic membranes [22]. He et al. [36]. reported that selamectin can reduce autophagy through inhibiting the expression of ATG9B in uveal melanoma. Although previous research has suggested the function of CBX2 and the Wnt/β-catenin pathway on autophagy, their regulation of ATG9B has not been reported. In our study, both the intervention of CBX2 and the Wnt/β-catenin pathway changed the expression of ATG9B and the autophagy level in OC. Furthermore, rescue experiments demonstrated that the regulation of CBX2 on autophagy and DDP-resistance of OC is dependent on ATG9B expression. Based on these results, we confirmed that the CBX2-Wnt/β-catenin axis modulates DDP-resistance of OC by mediating ATG9B-dependent autophagy process.

Conclusion

This study explored the molecular mechanism of CBX2 in autophagy and DDP resistance in OC. We found that CBX2 enhances ATG9B-mediated autophagy and DDP-resistance in OC by regulating the SIAH2/Wnt/β-catenin pathway. These findings identify a potential therapeutic target for overcoming DDP resistance.

Authors’ contributions

A: Conceptualization: Xinxin KouB: Funding acquisition, Project administration and Resources: Yuanjing HuC: Investigation and Methodology, Validation and Visualization: Xinxin Kou, Lijie Dong, Zheng Zhao, Xiaoxia YangD: Data curation and Formal analysis: Xinxin Kou, Lijie DongE: Visualization and Writing – original draft: Xinxin KouF: Writing –review & editing: Yuanjing Hu.

Funding

This project was supported by Tianjin Key Specialized Department Construction Project of Integrated Traditional Chinese and Western Medicine (Sino-Western Collaborative ‘Flagship’ Department) and Tianjin Key Medical Discipline (Specialty) Construction Project (No. TJYXZDXK-043 A).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.