MAD2L1 overexpression promotes breast cancer progression and confers resistance to paclitaxel via proteasomal stabilization

Introduction

Breast cancer is the most prevalent malignancy among women in Taiwan, accounting for a notable proportion of cancer-related morbidity and mortality. According to data from the Taiwan Cancer Registry (2000–2021), the incidence of breast cancer has steadily increased over the past decades (1). Epidemiological studies have indicated that the majority of patients with breast cancer in Taiwan have luminal subtypes (estrogen receptor-positive/HER2-negative), followed by HER2-positive and triple-negative subtypes, with the latter being associated with more aggressive clinical behavior and poorer prognosis (2,3). Despite advances in early detection and surgical management, metastatic and recurrent disease remain major clinical challenges. Paclitaxel, a widely used chemotherapeutic agent, primarily exerts its antitumor effects by targeting microtubules (4). By stabilizing microtubules and preventing their depolymerization, paclitaxel disrupts normal mitotic spindle formation, leading to G2/M phase cell cycle arrest and activation of the mitotic checkpoint (5,6). This mitotic disruption subsequently induces apoptosis in rapidly proliferating cancer cells (7,8). In breast cancer, paclitaxel has been shown to inhibit cell proliferation, reduce the clonogenic potential and modulate the expression of key cell cycle regulators (9,10), including cyclin A and cyclin-dependent kinase 1 (CDK1) (11,12), which are essential for G2/M transition. However, the development of chemoresistance (13), through mechanisms such as alterations in spindle assembly checkpoint proteins (14) or dysregulation of mitotic regulators (15), remains a major limitation to its long-term efficacy of paclitaxel.

Among the proteins regulating specific mitotic stages of the spindle assembly checkpoint, mitotic arrest deficiency protein 2-like 1 (MAD2L1) serves a notable role in ensuring accurate chromosome segregation during mitosis (16,17). By recruiting to unattached kinetochores to monitor kinetochore-microtubule attachments, MAD2L1 prevents premature anaphase onset, thereby maintaining genomic stability. Dysregulation or overexpression of MAD2L1 has been associated with aberrant cell cycle progression (18), increased proliferation (19) and enhanced tumorigenic potential in breast cancer (20,21). Experimental evidence has indicated that elevated MAD2L1 levels can promote cell proliferation, enhance the clonogenic capacity and stabilize key cell cycle regulators such as cyclin A and CDK1 (22). Furthermore, MAD2L1 overexpression may attenuate the cytotoxic effects of chemotherapeutic agents such as paclitaxel, suggesting its pivotal role in modulating both tumor growth and drug responses (23). Given its central function in mitotic regulation and potential involvement in chemoresistance, it is essential to elucidate the role of MAD2L1 in breast cancer progression and its interaction with paclitaxel. To this end, the specific aims of the present study were to investigate the functional effects of MAD2L1 on tumor cell proliferation, unveil its post-translational regulation of key cell cycle targets and characterize its contribution to paclitaxel resistance.

By identifying a previously unrecognized post-translational mechanism wherein MAD2L1 uncouples cyclin A and CDK1 protein stability from their mRNA transcription via ubiquitin inhibition, the present study demonstrates how MAD2L1 drives breast cancer progression and paclitaxel resistance, underscoring its potential as both a prognostic biomarker and a therapeutic target.

Materials and methods

Analysis of data from The University of Alabama at Birmingham Cancer data analysis portal (UALCAN) and the Human Protein Atlas (HPA)

MAD2L1 expression in breast cancer was analyzed using the UALCAN web portal (http://ualcan.path.uab.edu), which provides access to publicly available, de-identified RNA-sequencing data from The Cancer Genome Atlas (TCGA). MAD2L1 expression levels were compared between breast cancer tissues (n=1,097) and normal breast tissues (n=114). Subgroup analyses were conducted according to pathological stage, lymph node metastasis status and molecular subtype, including luminal, HER2-positive and triple-negative breast cancer. The statistical significance of differential expression was calculated by the UALCAN platform using a two-tailed, paired Student's t-test, and P<0.05 was considered to indicate a statistically significant difference. The expression profile of MAD2L1 across breast cancer cell lines was further evaluated using the HPA database (https://www.proteinatlas.org). Expression data from 62 breast cancer cell lines were analyzed to assess relative MAD2L1 expression levels. In addition, Gene Effect scores from CRISPR knockout screens (Broad's Achilles and Sanger's SCORE projects) were analyzed to assess genetic dependencies (http://ualcan.path.uab.edu). Chronos-computed scores were used, where a median score of 0 represents non-essential genes and −1 represents common essential genes; lower negative scores indicate stronger growth inhibition or cell death upon knockout (24,25). All analyses were performed using datasets generated by the respective platforms, and no additional normalization or transformation was applied beyond the default processing pipelines provided by UALCAN and HPA.

Cell lines and transfection

Human MDA-MB-231 and MCF-7 breast cancer cell lines were obtained from American Type Culture Collection. Cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO2. Cell lines were authenticated by short tandem repeat profiling and routinely tested to confirm the absence of mycoplasma contamination. For transient overexpression, cells were transfected with 1, 2 or 4 µg of MAD2L1 expression plasmid or an empty vector control using TurboFect Transfection Reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 48 h transfection at 37°C, cells were harvested and subjected to subsequent cell proliferation and colony formation assays.

BrdU cell proliferation assay

Cell proliferation was evaluated using a BrdU Cell Proliferation ELISA Kit (cat. no. ab126556; Abcam) according to the manufacturer's instructions. Briefly, cells were seeded into 96-well plates and cultured for 24 h at 37°C. Following the indicated treatments, BrdU reagent was added to each well at a final concentration of 10 µM and incubated with the cells for 24 h at 37°C. Absorbance was measured at 450 nm using an INNO microplate reader (LTEK Co., Ltd.). All experiments were performed in triplicate and data are presented as the mean ± SD.

Colony formation assay

For colony formation analysis, 500 transfected cells were seeded into 10-cm culture dishes and cultured under standard conditions for 10–14 days to allow colony development. Colonies were fixed with 4% paraformaldehyde for 1 h at 37°C and stained with 0.1% crystal violet for 30 min at 37°C. Colonies were counted manually, and those containing >50 cells were defined as viable colonies. Colonies were visualized and imaged using a CKX53 microscope (Olympus Corporation). Three independent biological replicates were performed for each experiment.

Cell cycle analysis

Cell cycle distribution was analyzed using flow cytometry. After transfection, cells were harvested, washed with PBS and fixed in 70% ethanol at 4°C overnight. Fixed cells were washed with PBS and stained with PI (MilliporeSigma) solution containing RNase A (Thermo Fisher Scientific, Inc.) for 30 min at room temperature in the dark. DNA content was measured using a Guava flow cytometer (Luminex Corporation), and the data were analyzed using GuavaSoft software (version 3.3; Luminex Corporation). Cell cycle distribution, including G0/G1, S and G2/M phases, was calculated based on PI fluorescence intensity. Doublets were excluded using appropriate gating strategies. Three independent biological replicates were performed for all experiments.

Immunoprecipitation and ubiquitination assays

To evaluate the ubiquitination levels of cyclin A and CDK1, cells were treated with 10 µM of the proteasome inhibitor MG132 (Sigma-Aldrich; Merck KGaA) at 37°C for 4 h prior to cell harvesting to inhibit proteasomal degradation and allow the accumulation of ubiquitinated proteins. Equal amounts of protein lysates (input) were incubated with specific primary antibodies against cyclin A or CDK1 overnight at 4°C with gentle rotation. Normal IgG was used as a negative control. Immune complexes were then captured by incubation with Protein G magnetic beads for 2 h at 4°C. The beads were washed thoroughly with lysis buffer to remove non-specifically bound proteins, and the isolated proteins were subjected to subsequent western blot analysis.

Western blotting

Total cellular proteins were extracted using RIPA lysis buffer supplemented with a protease inhibitor cocktail (Roche Diagnostics). Protein concentrations were determined using a BCA protein assay (Thermo Fisher Scientific, Inc.). Equal amounts of protein (30 µg per well) were separated by 10% SDS-PAGE and transferred onto PVDF membranes (MilliporeSigma). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 and incubated overnight at 4°C with the following primary antibodies: Anti-MAD2L1 (1:1,000; cat. no. 4636; Cell Signaling Technology, Inc.), anti-cyclin A (1:200; cat. no. ab185619; Abcam), anti-CDK1 (1:200; cat. no. ab131450; Abcam), anti-ubiquitin (1:1,000; cat. no. 10201-2-AP; Proteintech Group, Inc.) and anti-β-actin (1:1,000; cat. no. A5441; Sigma-Aldrich; Merck KGaA). After washing, membranes were incubated with appropriate HRP-conjugated secondary antibodies (1;1,000; cat. no. #58802 and cat. no. #93702; Cell Signaling Technology, Inc.) for 1 h at room temperature. Protein signals were detected using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Inc.) and visualized using a chemiluminescence imaging system. Three independent biological replicates were performed for all experiments.

Reverse transcription-quantitative PCR (RT-qPCR) analysis

Total RNA was extracted from cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Complementary DNA was synthesized using the PrimeScript RT Reagent Kit (Takara Bio, Inc.) at 37°C for 15 min, followed by a reverse transcriptase inactivation step at 85°C for 5 sec. RT-qPCR was performed using SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following thermal protocol: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min, and a final melting curve stage and a real-time PCR detection system.

The mRNA expression levels of cyclin A (forward, 5′-CTCTACACAGTCACGGGACAAAG-3′; reverse, 5′-CTGTGGTGCTTTGAGGTAGGTC-3′) and CDK1 (forward, 5′-AAATGTGTGTAGGTCTCAC-3′; reverse, 5′-ATGATTTAAGCCAACTCAAA-3′) were normalized to those of GAPDH (forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′; reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′) as an internal control. Relative gene expression was calculated using the 2−ΔΔCq method (26). Three independent biological replicates were performed for all experiments, with each reaction run in technical triplicates.

Apoptosis assay

Apoptosis was assessed using an annexin V-FITC/PI apoptosis detection kit (BD Biosciences). Following treatment with paclitaxel for 48 h, cells were harvested, washed with cold PBS and stained with annexin V-FITC and PI according to the manufacturer's instructions. Apoptotic cells were analyzed using an INNO microplate reader (LTEK, Co., Ltd.). Three independent biological replicates were performed for all experiments.

Statistical analysis

Statistical analyses and graphing were performed using GraphPad Prism 8.0 (Dotmatics). Data are presented as mean ± SD from three independent experiments (n=3). Differences between two independent groups were analyzed using the two-tailed unpaired Student's t-test. For comparisons among multiple groups, one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was performed. P<0.05 was considered to indicate a statistically significant difference. ImageJ software (National Institutes of Health) was used for the quantification of western blot proteins from three independent experiments (n=3).

Results

Elevated MAD2L1 expression is associated with poor prognosis and aggressive breast cancer phenotypes

Analysis of TCGA breast cancer data using the UALCAN database revealed that MAD2L1 expression was significantly upregulated in breast cancer tissues (n=1,097) compared with adjacent normal tissues (n=114) (Fig. 1A). Higher MAD2L1 levels were observed in advanced-stage tumors (Fig. 1B) and tumors with a high nodal metastasis stage (Fig. 1C), indicating its association with disease progression. Subtype analysis further demonstrated elevated MAD2L1 expression across luminal, HER2-positive and triple-negative breast cancer (Fig. 1D). Kaplan-Meier survival analysis demonstrated that patients with high MAD2L1 expression had a significantly worse overall survival compared with patients with low expression (P=0.015; Fig. 1E). Functional CRISPR knockout experiments (24,25) confirmed the oncogenic role of MAD2L1, as its knockout markedly reduced breast cancer cell proliferation, with the most pronounced effect observed in MDA-MB-231 triple-negative breast cancer cells and the mildest effect observed in MCF-7 cells (Fig. 1F). Consequently, these two cell lines were selected for further investigation. MAD2L1 overexpression was closely associated with aggressive tumor behavior and poor clinical outcomes in breast cancer, suggesting it may serve as a potential biomarker and therapeutic target.

Figure 1. MAD2L1 expression is upregulated in breast cancer and associated with poor prognosis. (A) Expression levels of MAD2L1 in breast cancer tissues (n=1,097) and normal breast tissues (n=114) based on The Cancer Genome Atlas data analyzed using The University of Alabama at Birmingham Cancer Data Analysis Portal database. (B) MAD2L1 expression according to tumor stage. (C) MAD2L1 expression according to nodal metastasis status. (D) Subtype-specific expression of MAD2L1 in luminal, HER2-positive and triple-negative breast cancer. (E) Kaplan-Meier survival analysis showing that patients with high MAD2L1 expression exhibited significantly worse overall survival (P=0.015). (F) CRISPR-mediated knockout of MAD2L1 reduced proliferation in breast cancer cell lines. ****P<0.0001. MAD2L1, mitotic arrest deficiency protein 2-like 1.

MAD2L1 overexpression promotes proliferation and colony formation in breast cancer cells

To further investigate the functional impact of MAD2L1 in breast cancer, 62 breast cancer cell lines (Fig. 2A), including MCF-7 and MDA-MB-231 cells (Fig. 2B), were analyzed using the HPA database. This analysis aimed to characterize the endogenous expression landscape of MAD2L1 across diverse breast cancer cell lines by quantifying baseline mRNA levels using normalized transcripts per million (nTPM) values. The biological effects of MAD2L1 overexpression were subsequently assessed by transfecting 1, 2 and 4 µg of MAD2L1 plasmid into these cells, which led to a dose-dependent increase in MAD2L1 expression levels (Figs. 2C and S1A and B). The results further revealed that cell proliferation increased proportionally with the amount of MAD2L1 plasmid transfected, indicating a dose-dependent proliferative effect (Fig. 2D and E). In addition, colony formation assays demonstrated that transfection of 2 µg of MAD2L1 plasmid significantly enhanced colony formation in both MCF-7 (Fig. 2F and G) and MDA-MB-231 (Fig. 2H and I) cells. Overexpression of MAD2L1 enhanced breast cancer cell proliferation and the clonogenic potential, supporting its role as a promoter of tumor cell proliferation.

Figure 2. MAD2L1 overexpression promotes breast cancer cell proliferation and colony formation. (A) Expression levels of MAD2L1 across 62 breast cancer cell lines based on data obtained from the Human Protein Atlas database. (B) Representative expression profiles of MAD2L1 in MCF-7 and MDA-MB-231 cells. (C) Western blotting and (D and E) BrdU assays showing that cell proliferation was increased in a dose-dependent manner following transfection with 1, 2 or 4 µg of MAD2L1 plasmid in MCF-7 and MDA-MB-231 cells. Colony formation assay images and quantification of (F and G) MCF-7 and (H and I) MDA-MB-231 cells treated with 2 µg of MAD2L1. Data are presented as the mean ± SD of three independent experiments. **P<0.01; ****P<0.0001. MAD2L1, mitotic arrest deficiency protein 2-like 1; nTPM, number of transcripts per million.

MAD2L1 overexpression decreases G2/M phase accumulation by stabilizing cyclin A and CDK1 through inhibition of ubiquitination

To determine the impact of MAD2L1 on the cell cycle, 2 µg of MAD2L1 plasmid was transfected into MCF-7 and MDA-MB-231 cells, followed by cell cycle analysis using flow cytometry. The results demonstrated that MAD2L1 overexpression led to a marked decrease in the G2/M phase population in both cell lines (Fig. 3A and B). Western blot analysis further revealed that MAD2L1 overexpression enhanced the protein levels of cyclin A and CDK1 (Fig. 3C), whereas their mRNA levels remained unchanged, as determined by RT-qPCR (Fig. 3D). These findings suggested that MAD2L1 may regulate cyclin A and CDK1 at the post-translational level. Consistently, treatment with the proteasome inhibitor MG132 elevated cyclin A and CDK1 expression, and co-treatment with MG132 and MAD2L1 overexpression resulted in the highest protein levels (Fig. 3E and F). To further investigate whether MAD2L1 regulated cyclin A and CDK1 through the ubiquitin-proteasome pathway, in vitro ubiquitination assays were performed. As shown in Fig. 3G and H, immunoprecipitation of cyclin A and CDK1 followed by western blotting for ubiquitin revealed that MAD2L1 overexpression markedly reduced the ubiquitination levels of both proteins. These findings demonstrated that MAD2L1 stabilizes cyclin A and CDK1 protein levels by inhibiting their ubiquitination rather than directly altering proteasomal activity.

Figure 3. MAD2L1 overexpression reduces G2/M phase accumulation and stabilizes cyclin A and CDK1 proteins. Flow cytometry analysis showing that MAD2L1 overexpression decreased the G2/M phase population in (A) MCF-7 and (B) MDA-MB-231 cells. (C) Western blot analysis revealing elevated cyclin A and CDK1 protein expression following MAD2L1 overexpression. (D) Reverse transcription-quantitative PCR analysis demonstrating the mRNA levels of cyclin A and CDK1 in MCF-7 and MDA-MB-231 cells. (E and F) Western blotting results revealing the protein levels of cyclin A and CDK1 following co-treatment with MG132 and MAD2L1 overexpression. (G and H) Following immunoprecipitation, western blotting demonstrated that ubiquitination of cyclin A and CDK1 was decreased following MAD2L1 overexpression. Data are presented as the mean ± SD of three independent experiments. *P<0.05; **P<0.01; ****P<0.0001. MAD2L1, mitotic arrest deficiency protein 2-like 1; CDK1, Cyclin-dependent kinase 1.

MAD2L1 attenuates the antitumor effects of paclitaxel by modulating the cell cycle and apoptosis

To explore the interaction between MAD2L1 and paclitaxel, a chemotherapeutic drug known to affect cell cycle progression, MCF-7 and MDA-MB-231 cells were treated with increasing concentrations of paclitaxel (1, 2 and 4 µM). BrdU assays demonstrated dose-dependent inhibition of proliferation by paclitaxel in both cell lines (Fig. 4A and B). Western blot analysis revealed that paclitaxel suppressed the protein expression levels of MAD2L1, cyclin A and CDK1, consistent with its role in inducing G2/M arrest. However, overexpression of MAD2L1 mitigated the inhibitory effects of paclitaxel, maintaining higher levels of cyclin A and CDK1 proteins (Figs. 4C and D and S1C and D). Functionally, MAD2L1 overexpression reduced paclitaxel-induced growth inhibition (Fig. 4E and F) and attenuated G2/M phase arrest in both cell lines (Fig. 4G and H). Furthermore, annexin V staining indicated that MAD2L1 overexpression decreased paclitaxel-induced apoptosis (Fig. 4I), suggesting a cytoprotective effect against paclitaxel-mediated cytotoxicity. Taken together, these results indicated that MAD2L1 overexpression diminished the anticancer efficacy of paclitaxel by counteracting its effects on cell cycle arrest and apoptosis.

Figure 4. MAD2L1 attenuates the inhibitory effects of paclitaxel on breast cancer cells. BrdU assays showing the dose-dependent inhibitory effect of paclitaxel (1, 2 and 4 µM) on the proliferation of (A) MCF-7 and (B) MDA-MB-231 cells. (C and D) Western blot analysis of MAD2L1, cyclin A and CDK1 expression following paclitaxel treatment, with or without MAD2L1 overexpression. (E and F) BrdU analysis showing that MAD2L1 overexpression mitigated paclitaxel-induced inhibition of cell proliferation. (G and H) Flow cytometry analysis of the cell cycle distribution demonstrating that MAD2L1 overexpression reduced paclitaxel-induced G2/M phase arrest in MCF-7 and MDA-MB-231 cells. (I) Annexin V-FITC/PI apoptosis assay showing that MAD2L1 decreased paclitaxel-induced apoptosis. Data are presented as the mean ± SD of three independent experiments. *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001. BrdU, bromodeoxyuridine; MAD2L1, mitotic arrest deficiency protein 2-like 1; CDK1, Cyclin-dependent kinase 1.

Discussion

The present findings unveil a novel post-translational mechanism wherein MAD2L1 dictates breast cancer progression by stabilizing the cell cycle machinery. Notably, MAD2L1 uncouples cyclin A and CDK1 protein abundance from their mRNA transcription. By suppressing ubiquitin-mediated proteasomal degradation, MAD2L1 extends the half-life of these core G2/M regulators, shifting the conventional paradigm from transcriptional wiring to post-translational stabilization in mitotic control. Clinically, this biochemical shield directly translates into paclitaxel resistance. While paclitaxel normally triggers apoptosis by downregulating MAD2L1, cyclin A and CDK1, forced MAD2L1 expression overrides this suppression. By maintaining these cell cycle engines under drug pressure, MAD2L1-overexpressing cells successfully evade G2/M arrest and apoptotic cascades. Collectively, these insights establish MAD2L1 as an active driver of chemoresistance and a promising therapeutic target to sensitize refractory breast cancers.

The present study demonstrated that MAD2L1 expression was upregulated in breast cancer and associated with aggressive tumor features, including higher stage, nodal metastasis and poor overall survival. Functionally, MAD2L1 overexpression promoted breast cancer cell proliferation, enhanced the clonogenic capacity and modulated G2/M phase progression by stabilizing key cell cycle regulators such as cyclin A and CDK1. Notably, MAD2L1 attenuated the cytotoxic effects of paclitaxel, reducing drug-induced growth inhibition, G2/M arrest and apoptosis in both MCF-7 and MDA-MB-231 cells. These findings collectively suggested that MAD2L1 serves a dual role in driving tumor progression and mediating chemoresistance, highlighting its potential as both a prognostic biomarker and a therapeutic target in breast cancer. Previous studies have demonstrated that MAD2L1 functions as a pro-tumorigenic regulator, and its overexpression suppressed G2/M phase accumulation, whereas MAD2L1 inhibition induced G2/M arrest, which was accompanied by decreased expression levels of cyclin A and cyclin E1 (22,27). These reports are consistent with the present observations.

The present results indicated that MAD2L1 overexpression maintained high levels of cyclin A and CDK1 proteins through post-translational regulation involving proteasomal degradation pathways. Cyclin A and CDK1 are tightly regulated during the cell cycle, and their degradation is primarily mediated by the ubiquitin-proteasome system, in which E3 ubiquitin ligases tag proteins for proteasomal degradation during specific cell cycle phases (28,29). In the present study, MAD2L1 overexpression did not alter cyclin A or CDK1 mRNA levels, indicating that the increased protein abundance resulted from inhibition of proteasome-mediated degradation rather than transcriptional upregulation. Treatment with the proteasome inhibitor MG132 further increased cyclin A and CDK1 levels (30), and the combination of MAD2L1 overexpression with MG132 treatment resulted in the highest protein accumulation, suggesting that MAD2L1 may interfere with ubiquitination or recognition by the proteasome. Mechanistically, MAD2L1, as a core spindle assembly checkpoint (SAC) protein, could influence the activity of E3 ubiquitin ligases (31), which are responsible for targeting cyclin A and CDK1 for degradation at the metaphase-anaphase transition (32). Previous studies have also revealed that MAD2L1 possesses the capability to modulate oligoubiquitination or short-chain polyubiquitination. The research demonstrated that APC/C-mediated ubiquitination operates via a hierarchical two-step process: Initial chain nucleation or priming driven by Ube2C (or Ube2D) to conjugate the first few ubiquitin molecules onto the substrate, followed by processive chain elongation mediated by Ube2S, which specifically extends K11-linked polyubiquitin long chains to assemble high-molecular-weight complexes (33). Crucially, when MAD2L1 is activated as a core SAC component, it suppresses the E3 ligase complex by selectively blocking the recruitment and elongation capacity of Ube2S, while leaving the upstream, Ube2C-driven priming nucleation partially permissible (34). Consequently, the catalytic activity of the APC/C complex becomes restricted exclusively to the synthesis of short-chain ubiquitin conjugates, strategically placing a molecular cap on polyubiquitin chain topography and leading to the characteristic accumulation of these short conjugates. The present experimental findings align with this enzymatic paradigm. Notably, because MAD2L1 acts at the furthest upstream level to physically constrain chain extension, this characteristic low-molecular-weight topology remains consistent even when downstream proteasomal clearance is thoroughly blocked by MG132. Therefore, rather than reflecting a technical artifact, this uniform band distribution provides direct evidence that MAD2L1 structurally caps polyubiquitin chain topology, capturing these core cell cycle components as functional, short-chain intermediates to delay their degradation. By delaying E3 ubiquitin ligase activation, MAD2L1 may extend the half-life of cyclin A and CDK1, thereby sustaining their protein levels during G2/M progression. This stabilization ensures continuous CDK1/cyclin A kinase activity, promoting mitotic entry and progression even under conditions of stress or chemotherapeutic challenge, such as paclitaxel treatment.

Paclitaxel is one of the most widely used chemotherapeutic agents for breast cancer treatment in both adjuvant and metastatic settings (35) due to its potent ability to disrupt mitotic spindle formation (36) and induce G2/M cell cycle arrest (37). Clinically, paclitaxel exhibits marked benefits, including improved overall response rates, enhanced progression-free survival and efficacy across multiple breast cancer subtypes compared to the control group, particularly in combination regimens with anthracyclines or targeted therapies (38,39). Its predictable mechanism of action and established dosing schedules have made it a cornerstone of breast cancer chemotherapy. However, the development of chemoresistance is a major clinical challenge, often leading to relapse or disease progression despite initial responsiveness. Resistance mechanisms include alterations in microtubule dynamics (40), overexpression of drug efflux pumps (41) and dysregulation of spindle assembly checkpoint proteins (14), which can attenuate paclitaxel-induced mitotic arrest and apoptosis. In addition, some breast cancer subtypes, including triple-negative tumors, may exhibit intrinsic resistance, limiting the therapeutic benefit of paclitaxel as a monotherapy (42,43). These clinical benefits and limitations underscore the need for an improved understanding of molecular determinants of the response to paclitaxel. Targeting regulators of the mitotic checkpoint or protein stability pathways, such as MAD2L1, could enhance paclitaxel efficacy, reduce chemoresistance and potentially expand its clinical utility.

The present findings revealed a functional interaction between MAD2L1 and paclitaxel in regulating G2/M phase dynamics. While paclitaxel is well known to induce G2/M arrest by stabilizing microtubules and preventing mitotic progression (44), overexpression of MAD2L1 had the opposite effect, reducing the proportion of cells in the G2/M population. This reduction suggested that excessive MAD2L1 could override the checkpoint-mediated arrest normally triggered by paclitaxel. Mechanistically, MAD2L1 overexpression stabilized cyclin A and CDK1, which are two essential regulators that drive G2/M transition, thereby promoting continued cell cycle progression despite paclitaxel treatment. As a result, cells were able to partially escape microtubule damage-induced cell cycle arrest, diminishing the cytostatic and cytotoxic impact of paclitaxel. This phenomenon offered a mechanistic explanation for why MAD2L1-overexpressing cells displayed attenuated G2/M arrest and reduced apoptosis following paclitaxel exposure. When cyclin A/CDK1 levels remain elevated, cells may bypass the canonical spindle-assembly checkpoint, enter mitosis prematurely or undergo aberrant mitotic exit (45), all of which lessen the efficacy of paclitaxel-induced cell cycle arrest. Consequently, MAD2L1 functions as a resistance factor that counteracts the mechanism of action of paclitaxel, enabling sustained proliferation even under chemotherapeutic pressure.

The expression level of MAD2L1 may serve as a key determinant of paclitaxel responsiveness. Understanding how MAD2L1 orchestrates the balance between mitotic arrest, proteasomal degradation and apoptotic signaling could provide valuable insights for overcoming paclitaxel resistance. Furthermore, targeting of MAD2L1-mediated regulatory pathways may represent a promising strategy to enhance the therapeutic efficacy of taxane-based chemotherapy in breast cancer. These findings suggested that MAD2L1 links mitotic checkpoint control to the proteasomal degradation machinery, enabling breast cancer cells to maintain the levels of G2/M regulatory proteins and enhancing the proliferative capacity. Targeting of the MAD2L1-proteasome axis may therefore represent a potential strategy to sensitize tumor cells to mitotic inhibitors or chemotherapy.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was funded by the Ministry of Science and Technology of Taiwan (grant nos. NSTC 112-2314-B-650-004-MY3 and 115-2314-B-650-003) and by E-Da Hospital/E-Da Cancer Hospital (grant nos. EDCHP115009, EDCHS115004 and EDCHJ114001).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

SHW, CHY and THH conceived and designed the experiments. CWW, MWL and CYH performed the experiments. EMT, CMH, MWL and THH analyzed the data. SHW and THH wrote the manuscript. CWW and THH confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References