MAZ-Mediated ubiquitin-conjugating enzyme E2C upregulation promotes breast cancer progression via the MAPK signaling pathway.

Introduction
Breast cancer (BC), which originates from the uncontrolled proliferation of breast epithelial cells, is one of the most prevalent malignant tumors in women, with high lethality and an increasing incidence over the years [1]. Among patients with breast cancer, those with metastases have worse survival rates than those with primary tumors, with a 5-year survival rate of only 26 % for metastatic patients, compared to 90 % for patients with breast cancer [2]. Despite numerous available treatment options for breast cancer—including surgical treatment, drug chemotherapy, radiation therapy, targeted therapy, and endocrine therapy—it remains the leading cause of cancer-related deaths among women globally. The prognosis for patients with breast cancer remains poor, primarily because many are diagnosed at advanced stages, missing the optimal window for timely treatment [3,4]. Consequently, it is crucial to investigate specific and sensitive biomarkers for the early detection and treatment of patients with breast cancer to enhance their prognosis. Therefore, identifying the underlying molecular mechanisms of breast cancer metastasis and developing novel therapeutic targets are essential.
The proteasome is a large multicatalytic protease complex located in the cytoplasm and nucleus of eukaryotic cells. The ubiquitin-proteasome system is responsible for the degradation of the majority of intracellular proteins, thus playing an important role in regulating essential cellular processes, including the cell cycle, proliferation, differentiation, angiogenesis, and apoptosis [5]. Ubiquitination refers to the covalent attachment of ubiquitin to target proteins, catalyzed by a series of enzymes that include three key types: the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, and the E3 ubiquitin ligase [6]. Ubiquitin-conjugating enzyme E2C (UBE2C, also known as UBCH10 or dJ447F3.2), a key member of the ubiquitin-proteasome system, is crucial for the regulation of cell cycle progression [7]. UBE2C was initially identified by Townsley, who elucidated that the gene is located on the long arm of chromosome 20 (20q13.12) [8]. Several previous studies employing various in vitro cell culture models have identified UBE2C as an oncogenic protein associated with malignant transformation [9]. For instance, ectopic overexpression of UBE2C promoted cell proliferation in multiple cancer cell lines [10,11]. To date, high expression levels of UBE2C have been found to be prevalent in malignant tumors, including lung, breast, thyroid, uterine, and gastrointestinal tumors, and are indicative of unfavorable outcomes for patients [[12], [13], [14], [15], [16]]. Two clinical studies have also revealed that UBE2C is highly expressed in patients with breast cancer, particularly among with triple-negative breast cancer, and is associated with poor survival rates [17,18]. Despite there have been many studies on the regulatory relationship between UBE2C and breast cancer, the specific molecular mechanisms underlying this relationship have yet to be fully elucidated.
Transcription factors (TFs), also known as trans-acting factors, are DNA-binding proteins that specifically interact with cis-acting elements of eukaryotic genes, thereby activating or inhibiting gene transcription [19]. MYC-associated zinc finger protein (MAZ) is a well-characterized zinc-finger transcription factor composed of two structural domains: the N-terminal domain and the C-terminal domain. The N-terminal domain comprises a zinc finger, a zinc-induced α-helix, and a zinc-bound hydrophilic helix, while the C-terminal domain consists of an α-helix, a β-sheet, and a short amino acid sequence [20,21]. The zinc finger is the most critical domain of MAZ and is capable of binding zinc ions to regulate various biological processes. Zinc-induced α-helices and zinc-bound hydrophilic helices enhance the stability of MAZ and influence its function. MAZ is involved in numerous biological processes, including cell proliferation, differentiation, and organogenesis [22,23]. MAZ is frequently found to be highly expressed in various tumors, and its transcriptional activation of multiple downstream target genes further promotes cancer development, progression, and metastasis [[24], [25], [26], [27]].
The mitogen-activated protein kinase (MAPK) signaling pathway consists of three kinases: MAP kinase kinase kinase (MAP3K), MAP kinase kinase (MKK or MAP2K), and MAP kinase (MAPK). This pathway is a critical component of the signal transduction network in eukaryotic organisms, facilitating the transmission of extracellular signals. The pathway coordinates extracellular stimuli with intracellular responses and serves as a key signaling mechanism for cell proliferation, differentiation, apoptosis, and stress responses under both normal and pathological conditions [28,29]. The MAPK family can be classified into four classical pathways based on its four subunits: ERK, JNK, p38/MAPK, and ERK5 [30,31]. Currently, the ERK/MAPK pathway is the most extensively studied pathway within the MAPK family pathway, with numerous cancers associated with its dysfunction [32,33]. The ERK/MAPK signaling pathway is involved not only in the regulation of cell biological functions but also in tumorigenesis. Elevated ERK expression has been observed in a variety of human tumors, including ovarian [34], colon [35], hepatocellular [36] and breast cancers [37].
In the present study, we aimed to investigate the role of UBE2C in the progression of BC. Our results demonstrate that UBE2C is significantly upregulated in breast cancer and promotes its malignant progression. Additionally, we identified the positive transcription factor MAZ, which not only regulates UBE2C expression but also exhibits pro-oncogenic functions. By analyzing the mechanism of action of UBE2C, we revealed that its role in breast cancer is partially mediated by the ERK/MAPK signaling pathway. These findings provide valuable insights into the role of UBE2C in the development of breast cancer and may highlight potential intervention points for future targeted therapies.

Methods

Immunohistochemistry (IHC)
This study enrolled a total of 40 pairs of patients with previously diagnosed benign or malignant breast tumors who underwent surgical treatment at the Affiliated Hospital of Guilin Medical University to assess the expression of UBE2C in both benign and malignant tissues. Following dewaxing and rehydration, slides were treated to quench endogenous peroxidase activity and subjected to antigen retrieval. The sections were then blocked with 10 % bovine serum for 1 h. The slides were then incubated with the primary antibody at 4 °C overnight, followed by incubation with a secondary antibody at 37 °C for 1 h. Immunostaining was performed using the DAB chromogenic kit (ZSGB-BIO, Beijing, China, Catalog No. ZLI-9018) and counterstained with hematoxylin.
The protein expression level of UBE2C was assessed using the IHC score, which was calculated by multiplying the proportion score by the intensity score. The scores were categorized as level 1 (IHC score 0–3), level 2 (IHC score 4–6), level 3 (IHC score 7–9), or level 4 (IHC score greater than 9). The proportion score represented the fraction of positively stained cells (≤25 %: 1; 26–50 %: 2; 51–75 %: 3; ≥75 %: 4), while the intensity score indicated the staining intensity (0 = no staining; 1 = weak; 2 = intermediate; 3 = strong) [38]. The median score value was used to establish the cut-off. Cancers with scores exceeding the cut-off value were deemed to exhibit high expression of the indicated molecule, while those with scores below the cut-off were considered to exhibit low expression.

Cell culture and cell transfection
The cell lines utilized in this study included several breast cancer cell lines (including MDA-MB-231, MDA-MB-468, MCF-7, BT474, SK-BR-3 cell lines), a mammary epithelial MCF-10A cell line, and a 293T cell line, obtained from the Cell Bank/Stem Cell Bank of the Chinese Academy of Sciences. Both the breast cancer cell lines and 293T cell line were cultured in DMEM medium (Pricella, Wuhan, China, Catalog No. PM150210) supplemented with 10 % fetal bovine serum (Pricella, Catalog No. 164,210–50) and 1 % penicillin-streptomycin (Pricella, Catalog No. PB180120), while the MCF-10A cell line was cultured in MCF-10A-specific medium (Pricella, Catalog No. CM-0525). All cell lines were cultured at 37 °C in a 5 % CO2 incubator, according to ATCC guidelines.
All plasmids, small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs) were obtained from Gene Pharma (Suzhou, China), with sequences listed in Supplementary Table 1. For transient transfection of siRNAs, Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA, Catalog No. L3000015) was used according to the manufacturer’s instructions, and functional experiments were performed 48 h post-transfection. To generate stable cell lines, lentiviral particles containing shUBE2C or UBE2C-overexpressing constructs were used to infect cells. The resulting stable MDA-MB-231 (shUBE2C) and MCF-7 (UBE2C-overexpressing) cell lines were then selected using medium containing 2 μg/ml and 0.5 μg/ml puromycin, respectively.

Quantitative real-time PCR (RT-qPCR)
Total RNA was extracted using TRIzol Reagent (Invitrogen, Catalog No. 15596018CN) and subsequently reverse transcribed with the PrimeScript RT kit (TOLOBIO, Shanghai, China, Catalog No. 22107). Quantitative real-time reverse transcription PCR (RT-qPCR) was conducted using SYBR Premix Ex Taq (Yugong Biotech, Jiangsu, China, Catalog No. EG20117M) on a LightCycler@ 96 Real-Time PCR System. RT-qPCR analysis was performed to quantify relative mRNA expression using GAPDH as an internal reference. The primer sequences used in this article were listed in Supplementary Table 2.

Western blot analysis
Western blot analysis was performed using standard laboratory protocols [39]. Briefly, cells were lysed in lysis buffer (Solarbio, Beijing, China, Catalog No. R0010). Protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA, Catalog No. ISEQ00010). Membranes were blocked with 5 % skim milk before being incubated with primary antibodies (4 °C, overnight) and subsequently with HRP-conjugated secondary antibodies. All antibodies used in this study are listed in Supplementary Table 3.

Cell proliferation assay
For the CCK-8 assay (MCE, New Jersey, USA, Catalog No. HY-K0301), cells were seeded in 96-well plates at a density of 2 × 10^3 cells per well. At designated time points, viability was measured at 450 nm according to the manufacturer’s protocol.
For the colony formation assay, cells were seeded in 6-well plates (2 × 10^3 cells/plate) and cultured for 2 weeks. The resulting colonies were fixed, stained with 0.1 % crystal violet, photographed, and quantified.

Wound healing assay
Cells were grown to 80–100 % confluency in 6-well plates. A sterile 200 µL pipette tip was used to create a “wound” scratch in the monolayer. After washing with PBS, cells were cultured in serum-free medium for 24 h. Images of the same marked wound areas were captured at 0 h and 24 h to assess cell migration.

Cell migration and invasion assays
Cell migration and invasion were assessed using Transwell chambers (Corning, New York, USA, Catalog No. 3422). For invasion assays, chambers were pre-coated with Matrigel (Corning, Catalog No. 356234); migration assays used uncoated chambers. Briefly, 5 × 10^4 cells in serum-free medium were seeded into the upper chamber, with medium containing 10 % serum in the lower chamber as a chemoattractant. After 24 h, cells that had migrated or invaded to the lower surface were fixed, stained, and quantified by microscopy.

Cycle assay
At 48 h post-transfection, cells were harvested and fixed in 70 % ethanol at 4 °C overnight. Cells were then stained with propidium iodide (PI) (Beyotime, Shanghai, China, Catalog No. C1052) according to the manufacturer’s instructions. The cell cycle distribution was subsequently analyzed by flow cytometry.

Apoptosis assay
Apoptosis was assessed using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Catalog No. C1062M) according to the manufacturer’s protocol. Briefly, 5 × 10^4 cells per sample were stained with Annexin V-FITC and propidium iodide (PI) and immediately analyzed by flow cytometry.

Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed on 293T cells 48 h after transfection with a MAZ plasmid or empty vector. The assay was conducted using the Pierce™ Magnetic ChIP Kit (Thermo Fisher Scientific, Massachusetts, USA, Catalog No. 26157) according to the manufacturer’s protocol. Briefly, chromatin was immunoprecipitated using 5 μg of anti-MAZ antibody (Proteintech, Wuhan, China, Catalog No. 39935) or 1 μg of normal IgG (Cell Signaling Technology, Boston, USA, Catalog No. 2729) as a control. The co-precipitated DNA was then analyzed by RT-qPCR to determine the enrichment of the UBE2C promoter.

Dual-luciferase reporter assay
To measure UBE2C promoter activity, cells were co-transfected with a MAZ expression plasmid and a UBE2C promoter-luciferase reporter construct, alongside a Renilla control plasmid. After 48 h, luciferase activity was measured using the Dual Luciferase Reporter Assay Kit (Vazyme, Jiangsu, China, Catalog No. DL101–01) according to the manufacturer’s instructions. Firefly luciferase activity was normalized to Renilla luciferase activity.

Xenograft model in nude mice
The female nude mice (5–6 weeks old) used in this study were obtained from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Guilin Medical University. Briefly, 5 × 10^6 MDA-MB-231 cells (stably expressing shCtrl or shUBE2C) and MCF-7 cells (stably expressing Vector or pcDNA3.1-UBE2C plasmid) were injected subcutaneously into the right subaxillary region of the mice. Tumor size was measured every 3 days over a period of 6 weeks. At the end of the sixth week, tumors were harvested and weighed. Tumor volumes were calculated using the formula: volume = length × width^2 × 0.5.

Statistical analysis
The experimental results of this study were obtained from at least three biologically independent samples. All data are expressed as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 9.0.2 software. Statistical analyses were performed by unpaired two-tailed Student's t-test or one-way analysis of variance (ANOVA). Pearson’s chi-square test was employed in SPSS 20.0 for correlation analysis. A P-value of < 0.05 was considered statistically significant.

Results

UBE2C is highly expressed in breast cancer and predicts poor prognosis
The expression of UBE2C in breast cancer was initially analyzed using the TCGA database, which revealed that UBE2C was highly expressed in various malignant tumors, including breast, lung, and hepatocellular carcinomas (Figs. 1A-C). Breast cancer patients with elevated UBE2C expression exhibit higher pathologic stages (Fig. 1D) and more aggressive molecular subtypes (Fig. 1E). Additionally, elevated UBE2C expression is associated with poorer overall survival, disease-specific survival, and progression-free interval in patients with breast cancer (Figs. 1F-H). Subsequently, a comparison of breast cancer cells available in our laboratory with human normal mammary epithelium cell showed that UBE2C expression was higher in breast cancer cell lines than that in normal mammary epithelial cell at both the mRNA and protein levels. UBE2C was upregulated in MDA-MB-231 and MDA-MB-468 cell lines, while it was expressed at relatively low levels in the MCF-7 and SK-BR-3 cell lines (Figs. 1I, J). The relationship between UBE2C expression and clinicopathological characteristics was further investigated through immunohistochemical staining of 40 pairs of clinical specimens obtained from normal and malignant breast cancer tissues. The results indicated that UBE2C expression was significantly higher in malignant tissues compared to normal tissues (Fig. 1K). The clinical and pathological characteristics of the 40 pairs of cases, along with the expression levels of UBE2C, are presented in Table 1. Positive correlations were found between UBE2C expression and T stage (P = 0.049), N stage (P = 0.001), pathologic stage (P = 0.008), histologic grade (P = 0.001), estrogen receptor (P = 0.012), progesterone receptor (P = 0.028), human epidermal growth factor receptor 2 (P = 0.004), and Ki-67 (P < 0.001). Therefore, UBE2C is highly expressed in breast cancer patients and cell lines, and its overexpression is regarded as a biomarker of poor survival in patients with breast cancer.

UBE2C promotes malignant progression of breast cancer cells
To investigate the role of UBE2C in breast cancer, we selected MDA-MB-231 and MDA-MB-468 cells, which exhibit high levels of UBE2C expression, for knockdown experiments followed by functional assays. The effectiveness of UBE2C knockdown was confirmed at both the mRNA and protein levels (Figs. 2A, B). The results showed that UBE2C knockdown resulted in a significant and time-dependent suppression of cell proliferation (Fig. 2C), a finding further confirmed by the colony formation assay (Fig. 2D), and inhibited the migration and invasive abilities of MDA-MB-231 and MDA-MB-468 cells, as demonstrated by wound healing and Transwell assays (Figs. 2E, F). Additionally, UBE2C knockdown inhibited epithelial-mesenchymal transition (EMT) (Fig. 2G). To explore the significance of UBE2C upregulation, we stably transfected MCF-7 and SK-BR-3 cells with the pcDNA3.1-UBE2C plasmid to induce UBE2C overexpression (Figs. 3A, B). The results demonstrated that UBE2C overexpression enhanced the growth (Figs. 3C, D), migration, and invasion capacities of these breast cancer cells (Figs. 3E-G), further confirming the oncogenic effects of UBE2C in breast cancer. Collectively, these results indicate that UBE2C promotes the proliferation, migration, and invasion of breast cancer cells, and its expression correlates with these malignant behaviors.

UBE2C promotes cell cycle progression and inhibits apoptosis in breast cancer cells
Cell cycle distribution analysis indicated that the proportion of cells in the G1 phase significantly increased following UBE2C knockdown in both MDA-MB-231 and MDA-MB-468 cells, while the proportion of cells in the S phase markedly decreased (Fig. 4A). Additionally, the proportion of apoptotic cells increased in these cells (Fig. 4B). These findings were further corroborated by increased cleavage of caspase-3, elevated levels of Bik, Bad, Bid, and Puma, and decreased levels of Bcl-2 in MDA-MB-231 and MDA-MB-468 cells, as detected by western blot analysis (Fig. 4C). Conversely, a significant reduction in the proportion of cells in the G1 phase and a notable accumulation of cells in the S phase were observed in MCF-7 and SK-BR-3 cells with UBE2C overexpression (Fig. 4D). In these cells, the proportion of apoptotic cells was inhibited (Fig. 4E), as indicated by the opposing western blot results, which showed decreased cleavage of caspase-3 compared to those in MDA-MB-231 and MDA-MB-468 cells (Fig. 4F). Collectively, these results suggest that UBE2C can enhances cell cycle progression through the S phase and inhibits apoptosis.

MAZ enhances the transcriptional expression of UBE2C
To elucidate the mechanism by which UBE2C influences breast cancer cell function, we first investigated the transcription factors that regulate UBE2C expression. Using the GTRD (https://gtrd20–06.biouml.org/) and PROMO (https://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) databases, we predicted potential transcription factors, including E2F8, YY1, VDR, SRF, ATF3, MAZ, AR, STAT5A, USF1 (Fig. 5A). An experimental screening via RT-qPCR and western blot identified MAZ as the primary regulator (Figures S1, S2).
We subsequently confirmed that MAZ expression positively correlates with UBE2C (Fig. 5B) and is highly expressed in triple-negative breast cancer (TNBC) cell lines, consistent with UBE2C’s expression pattern (Fig. 5C). RT-qPCR and western blot analyses revealed that MAZ expression was significantly downregulated in TNBC cells following knockdown, and UBE2C expression was also significantly suppressed (Figs. 5D-F). To further verify whether MAZ plays a regulatory role by binding to the UBE2C promoter, we conducted a dual luciferase assay and found that MAZ significantly enhanced luciferase activity (Fig. 5G). Additionally, ChIP-qPCR analysis confirmed that MAZ binds to the UBE2C promoter, thereby enhancing its transcriptional activity (Fig. 5H). Through JASPAR (https://jaspar2022.genereg.net/) database, we identified potential MAZ binding sites within the UBE2C promoter and selected three with high prediction scores for further study. We then constructed luciferase reporter gene vectors containing the full-length wild-type (WT) and three mutants (Mut1–3) of the UBE2C promoter, which demonstrated that MAZ enhances luciferase activity in both the wild-type and mutant-1 UBE2C promoters (Figs. 5I, J). In summary, our data suggests that MAZ binds to the UBE2C promoter and positively regulates its expression at the transcriptional level. We confirmed the clinical relevance of this axis using public databases; MAZ mRNA levels were significantly elevated in TCGA primary tumors compared to normal tissue and remained high across all cancer stages (Figure S3A). Furthermore, Kaplan-Meier analysis confirmed that the MAZ/UBE2C axis is associated with a significantly poorer prognosis, showing that the “High MAZ/High UBE2C” group had significantly worse OS (P = 0.00029), relapse-free survival (RFS) (P < 0.0001), and distant metastasis-free survival (DMFS) (P = 0.0017) than the “Low MAZ/Low UBE2C” group (Figures S3B-D).

MAZ promotes breast cancer cells proliferation and invasion and rescues UBE2C-induced breast cancer progression
Knockdown of MAZ caused a significant and time-dependent inhibition of cell proliferation (Fig. 6A),which was further corroborated by the colony formation assay (Fig. 6B), and decreased migration and invasion capacities in MDA-MB-231 and MDA-MB-468 cells (Figs. 6C, D). We co-transfected plasmids for MAZ knockdown and UBE2C overexpression. The results indicated that UBE2C level could be partially down-regulated after MAZ knockdown (Fig. 6E), and that MAZ knockdown significantly rescued breast cancer cells proliferation induced by UBE2C overexpression (Figs. 6F, G). Similarly, the migration and invasion capacities of breast cancer cells driven by UBE2C overexpression were partially but significantly rescued upon knockdown of MAZ (Figs. 6H, I). Overall, these results demonstrate that MAZ not only promotes breast cancer cells proliferation, migration, and invasion, but also partially rescues the phenomenon of breast cancer cells progression promoted by UBE2C overexpression. In subsequent experiments, following co-transfection of plasmids for MAZ knockdown and UBE2C overexpression, we found that MAZ level was partially upregulated after UBE2C overexpression (Figure S4A). And UBE2C overexpression partially rescued the inhibition of breast cancer cells proliferation, migration, and invasion caused by MAZ knockout (Figures S4B-E). Taken together, these results show that MAZ may partially promotes breast cancer cells progression by upregulating UBE2C expression.

UBE2C promotes breast cancer cells progression via activating the MAPK pathway
To investigate the signaling pathways potentially involved in UBE2C-induced breast cancer progression, we conducted RNA sequencing. KEGG pathway analysis identified that the MAPK signaling pathway as a key downstream target of UBE2C (Fig. 7A). Given its critical role in regulating various cellular activities, including proliferation, differentiation, survival, and death, it was prioritized for further investigation. As illustrated in Fig. 7B, knockdown of UBE2C inhibited MAPK signaling activity in both MDA-MB-231 and MDA-MB-468 cells. Similarly, an increase in MAPK signaling activity was observed in MCF-7 and SK-BR-3 cells following UBE2C overexpression (Fig. 7C). To further elucidate the significance of the MAPK pathway, UBE2C knockdown cells were treated with the MAPK activator Ro67–7476, which restored MAPK phosphorylation level (Fig. 7D). Additionally, proliferation rates of UBE2C knockdown cells were partially restored after treatment with Ro67–7476, as assessed based on the CCK-8 and the colony formation assays (Figs. 7E, F). Consistently, a partial enhancement of cell migration and invasion capacities was also observed in UBE2C knockdown cells treated with Ro67–7476 (Figs. 7G, H). These results suggest that UBE2C may mediate the proliferation, migration and invasion capacities of breast cancer cells through activation of the MAPK signaling pathway.

UBE2C enhances tumorigenicity in breast cancer cell xenografts
Significantly lower tumor volume and weight were observed in the UBE2C knockdown group compared to the control group (Figs. 8A-C). UBE2C mRNA and protein levels were significantly reduced in tumor tissues of UBE2C knockdown group compared to the control group (Figs. 8D, E). Ki67 expression was consistently suppressed in transplanted tumors due to UBE2C knockdown (Fig. 8F). The effect of UBE2C overexpression on breast cancer growth was also investigated. Consistent with in vitro results, UBE2C overexpression significantly promoted breast cancer growth, with both the tumor volume and weight in the UBE2C overexpression group being significantly higher than those in the control group (Figs. 8G-I). Furthermore, the expression levels of UBE2C and Ki67 were significantly elevated in the tumor tissues of the UBE2C overexpression group (Figs. 8J-L). We also observed a strong positive association between UBE2C and MAZ expression in vivo, where UBE2C knockdown significantly reduced MAZ at both the mRNA and protein levels, and its overexpression conversely increased them (Figures S5A, B). Moreover, UBE2C promoted EMT in these tumors, as its knockdown inhibited this process while its overexpression induced it (Figure S5C).

Discussion
Malignant proliferation and metastasis frequently lead to treatment failure and poor prognosis for patients with breast cancer. The findings of this study provide novel insights into the molecular mechanisms underlying the malignant progression of breast cancer, specifically emphasizing the significance of UBE2C. This research demonstrates that UBE2C significantly contributes to breast cancer pathogenesis, mediated by its regulation through the transcription factor MAZ and its involvement in the MAPK signaling pathway.
UBE2C expression has been extensively reported to be elevated in various malignant tumors, including head and neck squamous cell carcinoma [40], esophageal squamous cell carcinoma [41], gastric cancer [42], cervical cancer [43], pancreatic cancer [44], endometrial cancer [45], and lung cancer [46]. The upregulation of UBE2C in these tumors is associated with tumorigenesis and progression, including breast cancer [47]. This study corroborates previous findings that UBE2C is overexpressed in breast cancer tissues compared to normal breast tissues [48]. Additionally, we demonstrated that both the upregulation and downregulation of UBE2C expression in breast cancer cells promote and inhibit cell proliferation, migration, invasion, and EMT, respectively. Collectively, these findings suggest that UBE2C may function as a critical oncogene and could be a potential therapeutic target in breast cancer.
Additionally, our study found that elevated UBE2C expression was associated with several aggressive features in BC patients, including advanced TNM stage, high histological grade, negativity for hormone receptor (ER and PR), HER2 positivity, and high Ki67 level. Moreover, elevated level of UBE2C correlated with poorer survival outcomes in BC patients. Our findings are consistent with previous studies indicating that increased expression of UBE2C is linked to poor prognosis for patients with invasive breast cancer [1,2]. The prognostic significance of UBE2C has also been validated in node-positive breast cancer and high-risk early breast cancer cohorts [49,50]. Our study identifies UBE2C as a potential biomarker for poor prognosis in breast cancer. Concurrently, biophysical properties are emerging as critical indicators of cell invasiveness. For example, Abdolahad et al. utilized a carbon nanotube-based nanobioelectronic platform to measure shear force as a determinant of cancer cell invasiveness [51]. A future approach that integrates UBE2C expression analysis with this nanotube technology could offer a more comprehensive prediction of invasiveness by assessing cells from both molecular and biomechanical standpoints. Taken together, these results indicate that UBE2C may serve as a valuable prognostic biomarker for patients with breast cancer.
To elucidate the specific mechanism underlying the overexpression of UBE2C in breast cancer, we predicted its upstream transcription factor using bioinformatics analysis and employed dual-luciferase reporter and ChIP assays to confirm the binding interaction. Our study identified that MAZ binds to the UBE2C promoter, thereby enhancing its transcriptional expression. As a key transcription factor containing six C2H2 zinc finger domains, MAZ exerts dual regulatory effects on both the initiation and termination of gene transcription. For instance, MAZ can activate transcription by binding to the promoters of various genes, including NEIL3 [52], ERK1/2 [53], BCKDK [54], NDUFS3 [55], MAP2K2 [56], and members of the Ras gene family [25,57], and can exert a transcriptional inhibitory effect on genes such as ENOS [58], SP4 [59], and telomerase [60]. Furthermore, numerous studies have demonstrated that MAZ is highly expressed in various malignant tumors and plays a critical role in tumor occurrence and progression, including lung adenocarcinoma [52], thyroid cancer [53], melanoma [54], prostate cancer [25], and clear cell renal cell carcinoma [27]. In breast cancer, MAZ acts as a pivotal regulatory factor for the tumor-specific expression of PPARγ1 in breast cancer cells, and its overexpression may elevate PPARγ1 level by activating the tumor-specific promoter pA1, thereby promoting cell proliferation and inhibiting apoptosis [24]. Additionally, MAZ can transcriptionally activate VEGF, thereby directly driving angiogenesis in TNBC [61]. Consistently, our study found that MAZ was highly expressed in BC cell lines, particularly in TNBC cells. Furthermore, MAZ can modulate UBE2C transcription and promote BC cells proliferation, migration, and invasion, providing new insights into the role of MAZ in regulating breast cancer progression at the transcriptional level. We further investigated the clinical relevance of this newly identified axis. Our prognostic analysis highlights the MAZ/UBE2C axis as a potential novel biomarker for aggressive breast cancer, as co-expression was significantly associated with a poor patient prognosis. In addition to MAZ, UBE2C can also be transcriptionally regulated by the transcription factors FOXM1 [11,62] and MYBL2a [63].
The MAPK pathway, recognized as a key oncogenic signaling cascade, regulates various cellular activities, including proliferation, differentiation, apoptosis or survival, inflammation, and innate immunity, and plays a crucial role in the development of numerous cancers, including BC [64]. The Ras/Raf/MEK/ERK (MAPK) pathway, regarded as the most classical MAPK signaling pathway, is essential for tumor cell growth, survival and differentiation [65]. Although the ERK/MAPK pathway is involved in the development of breast cancer, the signaling cascade mediated by UBE2C remains to be fully elucidated. In this study, we found that UBE2C expression level is transcriptionally activated by MAZ, and that UBE2C contributes to the malignant progression of BC cells by positively regulating the ERK/MAPK pathway. Furthermore, concomitant treatment with the MAPK activator Ro67–7476 in UBE2C-knockdown BC cells partially reversed the inhibition of BC cells proliferation, migration, and invasion. Therefore, we hypothesize that the regulatory effect of UBE2C on the malignant progression of BC cells is mediated, at least in part, through the ERK/MAPK pathway. Consistent with our findings, two previous studies have demonstrated that overexpression of UBE2C can regulate tumor progression in non-small cell lung cancer and gastric cancer by activating the ERK pathway [66,67]. In clear cell renal cell carcinoma, MAZ binds to the MAP2K2 (MEK2) promoter and increases MAP2K2 transcription, thereby activating the ERK signaling pathway [68]. In thyroid cancer, MAZ regulates transcriptional reprogramming and ERK1/2 activation by activating the transcription of BRAF, KARS, and LOC547 [53]. A previous study demonstrated that the existence of a positive feedback regulatory loop between MAZ and Ras, wherein Ras activates MAZ through the ERK/MAPK signaling pathway, and activated MAZ further promotes the expression of Ras, thereby establishing a feedforward regulatory mechanism. This mechanism drives angiogenesis in breast cancer [69]. Consistent with previous reports, in this study, we demonstrated the relationship between MAZ and the ERK/MAPK signaling pathway, which MAZ activates the MAPK signaling pathway (Figure S6).

Conclusion
In conclusion, our study underscores the important role of the MAZ/UBE2C/MAPK signaling axis in pathogenesis of breast cancer. We demonstrate that the overexpression of UBE2C, driven by the transcription factor MAZ, not only promotes the invasive phenotypes of breast cancer cells through the activation of the ERK/MAPK signaling pathway but also serves as a potential biomarker for this malignancy. These findings highlight the critical role of UBE2C in tumorigenesis and EMT, suggesting that this axis may represent a promising therapeutic target in breast cancer.

Funding
This work was supported by the National Natural Science Foundation of China (grant number 82060483), and Natural Science Foundation of Guangxi Province (grant number 2024GXNSFAA010396).

Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Institutional review board statement
The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Affiliated Hospital of Guilin Medical University (protocol number 2023YJSLL-119) on (Dec 22, 2023), and written informed consent was obtained from all patients involved in this research. The animal study protocol was approved by the Animal Care Committee of Guilin Medical University (protocol number 20241103) on (Dec 30, 2024).

CRediT authorship contribution statement
Jinhui Bai: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Conceptualization. Mei Deng: Writing – review & editing, Visualization, Supervision, Resources, Methodology, Funding acquisition, Conceptualization. Xueting Wu: Validation, Methodology, Investigation, Formal analysis. Chao Xiong: Validation, Methodology, Investigation, Formal analysis, Conceptualization. Huixian Wu: Visualization, Supervision, Project administration, Conceptualization. Li Wang: Visualization, Conceptualization. Jie Qin: Validation, Methodology, Formal analysis, Conceptualization.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.