OTUD4 deubiquitination stabilizes EGFR and activates the PI3K/AKT pathway to promote the invasiveness of triple-negative breast cancer.

Introduction
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC), accounting for approximately 25% of cases, and is associated with the poorest prognosis. Treatment options remain limited, relying primarily on conventional chemotherapy and surgery [1–3]. Notably, TNBC frequently exhibits overexpression of epidermal growth factor receptor (EGFR), which is linked to adverse clinical outcomes [4–7]. EGFR promotes tumor progression by regulating cell proliferation, metastasis, apoptosis inhibition, and drug resistance through activation of downstream signaling pathways, particularly PI3K/AKT/mTOR [8–10].
Deubiquitinases (DUBs), a family of approximately 100 enzymes [11], counteract ubiquitin ligases (UBs) by removing ubiquitin chains from target proteins [12], thereby preventing proteasomal degradation and maintaining protein functions [13]. Under normal physiological conditions, cellular homeostasis is maintained through a dynamic balance between ubiquitination and deubiquitination. However, dysregulation of certain DUBs contributes to tumorigenesis [14].
OTU domain-containing 4 (OTUD4), a deubiquitinating enzyme of the ovarian tumor protease domain (OTUD) family [15], has been implicated in cancer progression. OTUD4 enhances tumor metastasis by collaborating with IRTKS to promote SETDB1-mediated H3K9 trimethylation, leading to E-cadherin suppression [16]. Additionally, OTUD4 stabilizes CDK1 by removing K11-, K29-, and K33-linked multiubiquitination and reduces K6- and K27-linked ubiquitination of FGFR1, thereby activating the MAPK pathway and promoting glioblastoma progression [17]. By stabilizing Snail1, OTUD4 regulates melanoma metastasis and chemoresistance [18]. Furthermore, in TNBC, OTUD4 stabilizes CD73 by counteracting TRIM21-mediated ubiquitination, contributing to immune evasion [19].
In this study, we demonstrate that OTUD4 is highly expressed in TNBC and correlates with poor prognosis. Functional analyses reveal that OTUD4 enhances TNBC aggressiveness both in vitro and in vivo through the EGFR-mediated PI3K/AKT pathway. Mechanistically, OTUD4 (568–1114aa) interacts with EGFR (1–600aa) and stabilizes EGFR independently and via NRP1 recruitment for deubiquitination. These findings provide new insights into TNBC pathogenesis and potential therapeutic targets.

Materials and Methods

Cell culture
The normal breast and BC cell lines used in this experiment were acquired from Procell Life Science and Technology (Wuhan, China). The breast cancer cells were cultured in a DMEM medium (Gibco, New York, USA). MCF10A cells were cultured in a specific mammary epithelial cell medium (CM0525, Procell Life Science and Technology). All media were supplemented with 10% fetal bovine serum (Zeta Life, USA) and incubated at 37 °C in a humidified atmosphere with 5% CO2.

Clinical samples
A total of 20 pairs of TNBC tissues and their corresponding adjacent non-cancerous tissues were collected. All participating patients were informed, and their written informed consent was obtained. The collected specimens were divided into two parts, with one part rapidly placed in liquid nitrogen and the other fixed in formalin. This study was approved by the Ethics Committee of the Affiliated Hospital of Guizhou Medical University.

Bioinformatics analysis
The analysis involved profiling the OTUD4 mRNA expression across different types of BC within the TCGA-BRCA phenotype data. The OTUD4 expression in TCGA normal samples served as a reference. The UALCAN online tool was utilized to analyze the protein expression of different types of BC within the CPTAC database, and the results were compared against the normal sample protein expression. The survival plots for OTUD4 were generated based on TCGA-BRCA data, along with linear correlation plots of the OTUD4 expression in relation to the EGFR and NRP1 expressions. Differential gene expression between high and low OTUD4 expressions was analyzed, and differential pathway enrichment analysis charts were constructed.

Antibodies
The antibodies used in the study are described in Supplementary Table 1.

CCK-8 and EdU assay
To assess the impact of OTUD4 expression on the viability of triple-negative breast cancer (TNBC) cells, cell viability was measured at 450 nm using a CCK-8 assay kit (C0038; Beyotime Biotechnology, Shanghai, China) every 24 hours for a total of 4 days.
For proliferation analysis, TNBC cells were seeded in 96-well plates and examined using the Cell-Light EdU Apollo 488 or 567 in vitro imaging kit (RiboBio, Guangzhou, China). According to the manufacturer’s instructions, 50 µM EdU was added, the medium was discarded, and cells were incubated for 2 hours in a humidified incubator, followed by PBS washing. Cells were then fixed with 4% paraformaldehyde at room temperature and permeabilized with 0.5% Triton X-100. Nuclei were counterstained with 1× Hoechst 33342 working solution prepared by diluting the provided F solution with deionized water, and the 1× ApolloR staining solution was applied for 30 minutes. EdU-positive cells were counted in five randomly selected microscopic fields per well using a fluorescence microscope. All experiments were performed in triplicate. The EdU assay was conducted following the same procedures as previously described.

Proteomics
Three independent samples each of MDA-MB-231 NC and MDA-MB-231 OTUD4-SH1 were submitted to BGI (Shenzhen) for quantitative proteomic analysis. Each sample contained no fewer than 1 × 10^7 cells. Results were compared between MDA-MB-231 OTUD4-SH1 and MDA-MB-231 NC, with upregulation defined as FC > 2 and downregulation as FC < 1/2.

Plate colony formation assay
The experimental cells (200/well) were cultured in a 6-well plate for 14 days, after which the cells were fixed with paraformaldehyde, stained with crystal violet, and photographed.

Wound-healing assay
The cells were inoculated into a 6-well plate until they reached over 90% confluence. A scratch assay was then performed, followed by microscopic observation and photography. Images were taken again 24 hours later. The cell migration pattern was compared before and after the 24-h period.

Transwell assay
Serum-free cell suspension was inoculated in the upper chamber (CLS3422, Corning, Cambridge, USA) and serum-containing culture medium was added to the lower chamber. After 16 h, the cells were fixed with paraformaldehyde, stained with Giemsa, washed, air-dried, and photographed.

RT-qPCR
RNA from the experimental cells and tissues was extracted using a reagent kit (RE-03011, Foregene, Chengdu, China). Reverse transcription was performed into cDNA using a reverse transcription kit (Accurate Biology, Hunan, China). Quantitative PCR was conducted by using the CFX96 real-time system (Bio-Rad), and the gene expression was calculated by using the 2−ΔΔCT method. The primer sequences used in the study are shown in STable2.

Western blotting and immunoprecipitation
Western blotting was performed as per the standard techniques, which involved the steps of electrophoresis, transfer onto a membrane, blocking with milk at room temperature, incubation with primary and secondary antibodies, membrane washing, and visualization of the developed bands by using a chemiluminescence imaging system (Minichemi, Beijing, China).
In the co-immunoprecipitation (co-IP) experiments, COIP lysis buffer (PR20036; Sanying, Wuhan, China) was mixed with the indicated antibodies or normal IgG and incubated overnight at 4 °C. The mixtures were then incubated with magnetic beads (B23201; Sairuike Co., Shanghai, China) at room temperature for 30 min. After elution, samples were subjected to Western blotting. Silver staining was performed using a Fast Silver Stain Kit (P0017S; Beyotime Biotechnology) according to the manufacturer’s instructions; gels were fixed and imaged after staining. Gel bands were excised and submitted to BGI for mass spectrometric analysis.

Immunofluorescence (IF) assay
A total of 100,000 cells were uniformly seeded onto a confocal culture dish, fixed with paraformaldehyde, permeabilized with Triton X-100, blocked with goat serum, and incubated with antibodies overnight at 4 °C. The following day, fluorescent secondary antibodies were applied to the cells and incubated in the dark. Cell nuclei were stained with DAPI (blue), and observation was performed under a fluorescence microscope (ZEISS, Germany) under the same exposure conditions.

Half-Life assessment and ubiquitination
The experimental cells were incubated with cycloheximide (CHX) (30 μg/mL) (Selleck) and collected at appropriate time points for Western blotting. The protein density values were analyzed at different time points using ImageJ software and plotted as line graphs. For ubiquitination analysis, the cells were transfected with tagged plasmids, including HA-UB. Prior to the cell collection, the cells were treated with a proteasome inhibitor, MG132 (10 μM) (Selleck), for 4–6 h. Subsequently, Western blotting was performed to assess the levels of ubiquitination.

Transfection assay
The cell density employed for transient transfection was 60–70%, and the cells were transfected using 10 μL/well of Lipofectamine 3000 (Invitrogen Biotechnology, USA). Transfection with the designated siRNA or plasmids was performed for 48–72 h, depending on the experimental requirements. Subsequently, the cells were collected for further analysis.
The plasmid sequence for stable transfection was designed and synthesized by OBiO Technology. Slow viral infection was conducted as per the transfection instructions, and efficiency validation was performed at the protein and mRNA levels. The shRNA and siRNA sequences are shown in STable3.

In vivo experiments
The animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Guizhou Medical University. Four-week-old female nude mice were obtained from the Guangdong Medical Laboratory Animal Center. The mice were housed under SPF conditions and acclimatized to the facility a week prior to administering the injections. The mice received subcutaneous injections into the left axillary region with 3 × 106 cells of either OTUD4-knockdown lentivirus or control vector. The tumor volume and mouse body weight were measured weekly. The mice were euthanized via carbon dioxide inhalation 30 days after injection. The tumors were excised and weighed. The following formula was used to calculate the volume: (length × width^2) / 2.

Immunohistochemistry
The clinical specimens and animal experimental samples were embedded in paraffin, sliced into sections, deparaffinized, rehydrated in different gradients of alcohol, subjected to antigen retrieval, incubated with H2O2 to eliminate endogenous peroxidase activity, blocked with goat serum, and finally incubated with primary antibodies overnight at 4 °C. The next day, the sections were incubated with secondary antibodies, developed with DAB, counterstained, dehydrated, and mounted. Once dried, the slides were observed and documented by using a microscope.

Statistical analysis
All statistical analyses in this study were performed using GraphPad Prism software (version 10.0). The results are presented as the mean ± standard deviation of three independent experiments. The differences between the two groups were evaluated using two-tailed unpaired Student’s t-tests, while differences between multiple groups were assessed by one-way analysis of variance (ANOVA). The significance level was set as follows: p < 0.05 was considered to indicate statistical significance, denoted as *p < 0.05, **p < 0.01, and ***p < 0.001 for the respective levels of significance.

Results

High Expression of OTUD4 in TNBC is Associated with Poor Prognosis
To evaluate OTUD4 expression in breast cancer, we analyzed data from the TCGA-BRCA and UALCAN databases. OTUD4 mRNA and protein levels were elevated in triple-negative breast cancer, HER2-positive breast cancer, luminal A, and luminal B subtypes compared with normal breast tissue (Fig. 1A, B). Consistently, OTUD4 mRNA and protein expression in breast cancer specimens were significantly higher than in normal tissues (Fig. S1A, B). Additionally, OTUD4 expression was significantly higher in BC samples than in normal tissues at both the mRNA and protein levels (Figs. S1A, B). Comparative analysis of TNBC tumor tissues and adjacent non-tumor tissues revealed significantly increased OTUD4 expression in TNBC (Fig. 1C). Similarly, TNBC cell lines (MDA-MB-231, BT-549, HCC1937, and HS578T) exhibited higher OTUD4 protein levels compared to the normal mammary epithelial cell line MCF10A and non-TNBC BC cell lines (MCF7, T47D, SKBR3, BT474) (Fig. 1D). TCGA database analysis further indicated that elevated OTUD4 expression correlates with poor prognosis in TNBC (Fig. 1E). Immunohistochemical analysis of 10 paired clinical samples confirmed higher OTUD4 expression in TNBC compared to adjacent tissues (Fig. 1F), consistent with findings from the Human Protein Atlas (Fig. S1C). Additionally, public datasets showed that OTUD4 mRNA levels were higher in TNBC cell lines than in other BC subtypes (Fig. S1D). Based on the above results, OTUD4 may be associated with prognosis in TNBC.

OTUD4 promotes the invasiveness of TNBC cells in vitro
To investigate the role of OTUD4 in TNBC, we generated stable OTUD4-knockdown cell lines in two TNBC models (Fig. 2A, S2A). EDU, CCK-8, and colony formation assays demonstrated that OTUD4 knockdown significantly reduced TNBC cell proliferation (Fig. 2B–D). Consistent with previous reports indicating that OTUD4 silencing suppresses TNBC metastasis [20], our findings showed that OTUD4 knockdown impaired wound closure in wound healing assays and reduced TNBC cell migration in Transwell assays (Fig. 2E, F). To further validate its oncogenic function, we established OTUD4-overexpressing cell lines in the same TNBC models (Fig. 2G, S2B). OTUD4 overexpression enhanced TNBC cell proliferation, colony formation (Fig. 2H–J), and migration (Figs. S2C, D). These results demonstrate that OTUD4 enhances the invasiveness of TNBC.

OTUD4 promotes the progression of TNBC in vitro via the EGFR/PI3K/AKT pathway
To elucidate the molecular mechanisms underlying OTUD4-mediated TNBC invasiveness, we performed enrichment analysis, which identified a strong association between OTUD4 upregulation and the EGFR and PI3K/AKT pathways in TNBC (Fig. 3A). Correlation analysis further confirmed a significant positive correlation between OTUD4 and EGFR expression (Fig. 3B). Quantitative proteomics analysis revealed that OTUD4 knockdown in MDA-MB-231 cells reduced both OTUD4 and EGFR protein levels compared to controls (Fig. 3D). KEGG enrichment analysis indicated downregulation of the PI3K/AKT pathway following OTUD4 silencing (Fig. 3C). The activation of the EGFR signaling pathway primarily depends on EGFR levels and its phosphorylation status (Fig. S2E). Additionally, western blots demonstrated decreased total and phosphorylated levels of key EGFR/PI3K/AKT pathway components upon OTUD4 knockdown (Fig. 3E), while OTUD4 overexpression activated this pathway (Fig. 3F). To confirm that OTUD4 exerts its oncogenic effects via EGFR, we performed rescue experiments. Knockdown of EGFR in OTUD4-overexpressing cells abolished the tumor-promoting effects of OTUD4 overexpression (Fig. 3G–I). At the protein level, EGFR knockdown counteracted OTUD4-induced EGFR stabilization, while OTUD4 stability remained unaffected by EGFR knockdown (Fig. 3J) (Fig. S3A). These results demonstrate that OTUD4 promotes TNBC progression by activating the PI3K/AKT pathway through EGFR.

The interaction between OTUD4 and EGFR is a driving factor for the progression of TNBC
To identify target genes affected by OTUD4 in the EGFR/PI3K/AKT pathway, we performed immunoprecipitation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify OTUD4-interacting proteins (Fig. 4A). The analysis revealed EGFR as a potential binding partner of OTUD4 (Fig. 4B). Given this interaction, we hypothesized that OTUD4 modulates the PI3K/AKT pathway through EGFR. Co-immunoprecipitation experiments confirmed endogenous OTUD4-EGFR interaction in TNBC cells (Fig. 4C, D), while IF analysis showed co-localization of OTUD4 and EGFR (Fig. 4E). To further delineate their specific binding regions, we constructed plasmids with truncated and deletion variants labeled with Flag for OTUD4: full-length 1-1114aa, 1-449aa, 450-1114aa, Δ1-155aa, Δ568-1114aa; and MYC-tagged truncations and deletions for EGFR: 1-1210aa, Δ621-1210aa、958-1210aa、645-957aa. Following transfection in HEK-293T cells and subsequent CO-IP analysis confirmed the interaction between OTUD4 (568-1114aa) and EGFR (958-1210aa) (Fig. 4F, G).

OTUD4 stabilizes EGFR expression through deubiquitination
To investigate the mechanism by which OTUD4 regulates EGFR expression, we considered its role as a deubiquitinating enzyme and its potential effect on EGFR protein stability. Given the reported half-life of EGFR [21, 22], we performed CHX chase assays to assess EGFR degradation over time. The results showed that OTUD4 knockdown significantly reduced the half-life of EGFR (Fig. 5A, B, S3B, C). However, RT-qPCR analysis confirmed that OTUD4 knockdown did not alter EGFR mRNA levels (Fig. 5C). To further explore this, we treated cells with MG132, a proteasome inhibitor, and observed that MG132 restored EGFR protein levels in OTUD4-knockdown cells (Fig.5D). The catalytically inactive C45S andΔOTU-OTUD4 plasmids both bind EGFR (Fig. S4D) but have lost deubiquitination activity (Fig. S4E). Moreover, ubiquitination assays demonstrated that MG32-stabilized EGFP polyubiquitination (Fig. 5E). To confirm the deubiquitinating activity of OTUD4, we co-transfected EGFR with either wild-type OTUD4 or the catalytically inactive OTUD4 C686S mutant. Only wild-type OTUD4 reduced EGFR ubiquitination, while the mutant form had no effect (Fig. S3E), indicating that OTUD4 regulates EGFR stability via deubiquitination. Among the seven lysine residues in ubiquitin (K6, K11, K27, K29, K33, K48, and K63), K48-linked ubiquitination typically mediates proteasomal degradation, whereas K63-linked ubiquitination is associated with cellular signaling [23]. Our results demonstrated that OTUD4 overexpression specifically reduced K48-linked polyubiquitination of EGFR, without affecting K63-linked ubiquitination (Fig. 5F, G). To identify the functional domain responsible for EGFR deubiquitination, we performed ubiquitination assays using OTUD4 truncation constructs. The results indicated that full-length OTUD4 and its 549-1114aa region effectively reduced EGFR ubiquitination. We speculate that the 568-1114aa region is critical for OTUD4-mediated EGFR deubiquitination (Fig. 5H).

NRP1 recruits OTUD4 to deubiquitinate EGFR
Mass spectrometry analysis identified NRP1 as a potential OTUD4 interactor (Fig. 4A, B). Previous studies have reported that NRP1 knockdown reduces EGFR protein and phosphorylation levels in TNBC [24], though the underlying mechanisms remain unclear. We confirmed this finding, showing that NRP1 downregulation in TNBC cell lines led to decreased EGFR and p-EGFR protein levels (Fig. 6A). Correlation analysis further demonstrated a positive association between OTUD4 and NRP1 expression in TNBC (Fig. 6B). Co-immunoprecipitation (CO-IP) experiments confirmed the interaction between OTUD4 and NRP1 (Figs. 4C, D, 6C, D), and IF analysis showed their colocalization in the cytoplasm (Figs. 4E, 6E, S4A). These findings suggest that, in addition to its independent role in deubiquitinating EGFR, OTUD4 may be recruited by NRP1 to stabilize EGFR expression. To assess the impact of NRP1 on EGFR stability, we conducted ubiquitination assays. CHXchase experiments revealed a significantly shorter EGFR half-life in NRP1-knockdown TNBC cells compared to controls, without affecting OTUD4 expression (Fig. 6F–G). RT-qPCR analysis confirmed that NRP1 did not regulate EGFR at the mRNA level (Fig. 6H). Following siNRP1, EGFR co-precipitated in OTUD4 immunoprecipitates (IP-OTUD4) was significantly reduced (Fig. S4B). Moreover, overexpression of OTUD4 rescued the reduction in EGFR half-life caused by NRP1 knockdown (Figs. S4C, D), and MG132 treatment similarly restored EGFR stability (Fig. 6I). Immunoprecipitation and Western blot analyses further demonstrated that NRP1-dependent deubiquitination by OTUD4 regulates EGFR stability (Fig. 6J). Additionally, neither EGFR nor OTUD4 knockdown affected NRP1 expression (Figs. S4E, F). These results suggest that NRP1 recruits OTUD4 to mediate EGFR deubiquitination.

OTUD4 promotes TNBC proliferation in vivo via the EGFR/PI3K/AKT pathway
To evaluate the role of OTUD4 in TNBC proliferation in vivo, we subcutaneously injected stable SHNC, SHOTUD4#1, and SHOTUD4#2 MDA-MB-231 cells into the axilla of female nude mice. Tumor growth measurements showed that OTUD4 knockdown significantly inhibited tumor progression (Fig. 7A–C). Immunohistochemical analysis revealed reduced expression of OTUD4, EGFR, PI3K, AKT, mTOR, and Ki67 in OTUD4-knockdown tumors compared to controls, indicating that OTUD4 promotes TNBC tumor growth via the EGFR-mediated PI3K/AKT pathway (Fig. 7D).

Discussion
TNBC poses a significant treatment challenge due to the absence of effective therapeutic targets, limiting the use of endocrine and targeted therapies. Additionally, its pronounced tumor heterogeneity reduces the efficacy of conventional chemotherapy, leading to rapid tumor progression, early metastasis, and drug resistance. These challenges underscore the need for deeper investigation into TNBC pathogenesis.
This study integrates public database analysis with experimental validation to demonstrate that OTUD4 is highly expressed in TNBC, enhancing its invasiveness in vitro and highlighting its potential as a prognostic marker. Although public databases show considerable differences in OTUD4 mRNA expression between TNBC and normal tissues, the CPTAC database currently includes relatively few protein-level samples; therefore, the relationship between OTUD4 expression and TNBC prognosis requires further validation in larger cohorts. Under normal physiological conditions, ubiquitination and deubiquitination maintain cellular homeostasis. However, dysregulated deubiquitinating enzymes contribute to tumor progression [25]. For example, USP26 enhances colorectal cancer invasiveness by deubiquitinating PRKN, inhibiting PRKN-mediated mitophagy, and promoting tumorigenesis [26]. Similarly, aberrant upregulation of deubiquitinating enzymes in TNBC has been widely reported. STAMBP stabilizes RAI14 by inhibiting its K48-linked ubiquitination, promoting TNBC invasiveness, and correlating with poor prognosis [27]. OTUD2 is overexpressed in TNBC and linked to reduced patient survival by deubiquitinating and stabilizing CDK1, enhancing tumor proliferation and metastasis [28]. OTUD5 deubiquitinates K48-linked polyubiquitin chains, stabilizing SLC7A11 and inhibiting ferroptosis, contributing to TNBC progression [29]. Moreover, UCHL1 stabilizes KLF5 through deubiquitination, leading to increased EGFR expression and endocrine resistance via ESR1 downregulation [30]. These findings suggest that targeting deubiquitinating enzymes to regulate oncogene stability may offer a therapeutic approach for TNBC.
Our proteomic analysis identified EGFR as a potential interactor of OTUD4, with subsequent quantitative proteomics revealing that OTUD4 downregulation reduces EGFR expression and its associated signaling pathways. Enrichment analysis further indicated that OTUD4 upregulation correlates with EGFR-mediated activation of the PI3K/AKT pathway. Both in vitro and in vivo experiments confirmed that OTUD4 knockdown decreases the expression of EGFR and its downstream PI3K/AKT markers. Notably, EGFR protein overexpression and gene amplification occur in approximately 58% of TNBC cases [31], and EGFR overexpression is associated with poorer overall survival (OS) and disease-free survival (DFS) in patients with TNBC compared to those with normal EGFR expression [32]. EGFR is a critical receptor regulating cellular growth and survival, and its increased expression is often associated with enhanced tumor invasiveness and drug resistance. EGFR maintains cell proliferation through its downstream signaling pathways [33], and its overexpression activates downstream signaling pathways, leading to tumor proliferation and metastasis [34]. In particular, the PI3K/AKT/mTOR pathway, frequently dysregulated in TNBC, has emerged as a promising therapeutic target [35, 36].
Although the EGFR-mediated PI3K/AKT pathway has been extensively studied in preclinical models, clinical trials using single or multiple small-molecule inhibitors, as well as tyrosine kinase inhibitors (TKIs), have yielded unsatisfactory therapeutic outcomes [37, 38]. One possible reason is that TKIs are generally more effective in cancers harboring tyrosine kinase mutations, which are relatively rare in TNBC [39, 40]. Additionally, EGFR signaling is not confined to a single pathway but involves multiple interacting networks. For instance, EGFR knockdown can induce a negative feedback mechanism, increasing HER3 activation and promoting EGFR/HER3 dimerization [41]. Similarly, inhibition of downstream molecules such as mTOR can trigger compensatory activation of upstream components, ultimately sustaining TNBC progression [38]. A paradoxical role of EGFR in TNBC has been proposed: in primary tumors with EGFR amplification, EGF stimulation enhances proliferation, making these cells highly sensitive to EGFR inhibitors. However, upon epithelial-mesenchymal transition (EMT) and metastasis, particularly to the lungs, TNBC cells develop resistance to EGFR inhibitors, and EGF signaling shifts from promoting growth to exerting an inhibitory effect [42, 43]. These observations underscore the heterogeneity of TNBC and the complexity of targeting EGFR. In this study, we report two novel mechanisms by which OTUD4 regulates EGFR stability. First, OTUD4 directly deubiquitinates EGFR by removing K48-linked polyubiquitin chains, thereby preventing its degradation. Second, NRP1 recruits OTUD4 to further stabilize EGFR expression (Fig. 7E). This reveals a previously unrecognized regulatory mechanism in which EGFR activity is modulated not only through its tyrosine kinase function but also via ubiquitination dynamics. The recruitment of OTUD4 by NRP1 suggests that EGFR stability is controlled by complex protein interactions, highlighting multiple potential intervention points for TNBC therapy.
In conclusion, our study demonstrates that OTUD4 is overexpressed in TNBC and enhances its invasiveness by stabilizing EGFR, leading to activation of the PI3K/AKT signaling pathway. OTUD4 stabilizes EGFR through direct deubiquitination and via recruitment by NRP1, providing new insights into EGFR regulation in TNBC. These findings offer theoretical support for future therapeutic strategies targeting OTUD4. However, this study has certain limitations. It remains unclear whether targeting OTUD4 to inhibit EGFR could trigger compensatory mechanisms due to crosstalk among multiple downstream pathways. Additionally, we did not screen for OTUD4 inhibitors or explore combinatorial treatment approaches involving OTUD4 targeting. These aspects warrant further investigation in future studies.

Supplementary information