TRIM45 Suppresses Breast Cancer Progression by Regulating Nrf2/Keap1 Pathway through Degradation of p62

TRIM45 Suppresses Breast Cancer Progression by Regulating Nrf2/Keap1 Pathway through Degradation of p62 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article TRIM45 Suppresses Breast Cancer Progression by Regulating Nrf2/Keap1 Pathway through Degradation of p62 Mohan Su, can Cao, fulin sun, Liangqian jiang, Fanghao Yang, Huhu Zhang, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9167161/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Overcoming oxidative stress is a critical mechanism by which breast cancer cells sustain malignant progression and therapeutic resistance. However, the specific regulatory networks governing this adaptive response remain incompletely understood. Here, we demonstrate that TRIM45, a tumor-suppressive E3 ubiquitin ligase, acts as a key negative regulator of the antioxidant defense system in breast cancer. Clinically, TRIM45 is significantly downregulated in breast cancer tissues, and its low expression strongly correlates with poor patient prognosis. Functionally, ectopic expression of TRIM45 suppresses proliferation, migration, and epithelial-mesenchymal transition (EMT) in MCF-7 and BT549 cells. Mechanistically, we demonstrate that TRIM45 promotes p62 ubiquitination and degradation in breast cancer cells. By promoting the proteasomal degradation of p62, TRIM45 liberates Keap1. This is accompanied by enhanced Nrf2 ubiquitination and restricted nuclear translocation, suggesting a mechanism whereby TRIM45-mediated p62 degradation contributes to Nrf2 pathway suppression.This dismantling of the Keap1/Nrf2 signaling pathway not only suppresses downstream antioxidant targets (HO-1, GPX4) to induce lethal oxidative stress, characterized by elevated ROS and MDA levels alongside reduced GSH and GSH-Px activity, but also attenuates the IGF-1/IGF-1R/ERK signaling pathway. Collectively, our study uncovers a previously unrecognized TRIM45/p62/Keap1/Nrf2 regulatory signaling pathway, establishing TRIM45 as a promising therapeutic target that exploits oxidative vulnerability in breast cancer. Overcoming oxidative stress is a critical adaptive mechanism for breast cancer cells to sustain malignant progression and therapeutic resistance. This study identifies TRIM45, a tumor-suppressive E3 ubiquitin ligase, as a key negative regulator of the antioxidant defense system in breast cancer. Clinically, TRIM45 is significantly downregulated in breast cancer tissues, and its low expression strongly correlates with poor patient prognosis. Functionally, ectopic expression of TRIM45 suppresses proliferation, migration, and epithelial-mesenchymal transition in MCF-7 and BT549 cells. Mechanistically, we demonstrate that TRIM45 directly interacts with p62 and promotes its ubiquitination-dependent proteasomal degradation. Concomitant with p62 reduction, we observed the restoration of Keap1 protein levels, coupled with decreased protein abundance and impaired nuclear translocation of the transcription factor Nrf2. This led to the downregulation of downstream antioxidant genes (HO-1, GPX4) and attenuation of the IGF-1/IGF-1R/ERK signaling pathway. Collectively, these alterations induced lethal oxidative stress, characterized by ROS accumulation and depletion of the glutathione system. Our findings reveal that TRIM45 exerts a tumor-suppressive role in breast cancer by targeting p62 for degradation and thereby disrupting the Keap1/Nrf2 antioxidant axis, nominating TRIM45 as a novel candidate for therapeutic strategies aimed at exploiting oxidative vulnerability. Breast cancer TRIM45 p62 Ubiquitination Keap1/Nrf2 pathway oxidative stress IGF-1R Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction In 2022, approximately 2.3 million new cases were reported, accounting for 11.7% of all cancer diagnoses,and for the first time, its incidence surpassed that of lung cancer ( 1 ). Despite advances in adjuvant therapies, tumor recurrence and metastasis remain persistent clinical obstacles, underscoring the urgent need to identify novel therapeutic targets. The Tripartite Motif (TRIM) protein family has emerged as a promising candidate owing to its versatile biological roles. Many TRIM proteins function as E3 ubiquitin ligases that regulate cell cycle progression, signaling, and DNA repair ( 2 – 6 ). TRIM45 acts as a tumor suppressor in multiple malignancies, including glioblastoma and hepatocellular carcinoma, by inhibiting MAPK and NF-κB pathways, stabilizing p53, and modulating epithelial-mesenchymal transition (EMT)( 7 – 11 ).However, the specific role of TRIM45 in breast cancer, particularly in the context of oxidative stress, remains largely unexplored. Dysregulation of oxidative stress responses is a hallmark of cancer. The transcription factor Nrf2 serves as the master regulator of antioxidant defense ( 12 ). Under basal conditions, Nrf2 is constitutively ubiquitinated and degraded by its negative regulator Keap1, maintaining redox homeostasis ( 13 – 15 ). Upon oxidative stress, Nrf2 stabilizes and translocates to the nucleus, driving expression of cytoprotective genes including HO-1 and GPX4 ( 16 – 18 ). Notably, Nrf2 also upregulates Insulin-like Growth Factor 1 Receptor (IGF-1R) expression, thereby promoting tumor cell proliferation ( 19 , 20 ). p62 (SQSTM1) is a multifunctional scaffold protein that plays pivotal roles in autophagy and oxidative stress response. Mechanistically, p62 competitively binds to Keap1, disrupting Keap1-Nrf2 interaction and leading to non-canonical Nrf2 activation ( 21 ). Aberrant activation of this pathway is prevalent in various malignancies and is associated with tumor progression and therapeutic resistance. The stability and function of p62 are tightly regulated by multiple E3 ubiquitin ligases. For instance, TRIM21 ubiquitinates p62 to inhibit the p62-Keap1-Nrf2 pathway, while TRAF6 promotes p62 ubiquitination to enhance its inflammatory function ( 20 , 21 ). Emerging evidence suggests that multiple TRIM family members orchestrate the Keap1-Nrf2 pathway through diverse mechanisms, thereby modulating oxidative stress and tumorigenesis ( 22 ). However, whether TRIM45 regulates p62 and the Keap1/Nrf2 pathway in breast cancer remains unknown. Given the pivotal role of p62 in linking ubiquitination to the Keap1/Nrf2 oxidative stress pathway ( 23 ), we hypothesized that p62 might be a novel functional substrate of TRIM45 in breast cancer. In this study, we demonstrate that TRIM45 is significantly downregulated in breast cancer tissues and suppresses malignant phenotypes in breast cancer cells. Mechanistically, we identify p62 as a key ubiquitination substrate of TRIM45 ( 24 ). TRIM45-mediated p62 degradation restores Keap1-dependent suppression of Nrf2, thereby inducing lethal oxidative stress and concurrently attenuating IGF-1R/ERK signaling. These findings establish the TRIM45/p62/Keap1/Nrf2 signaling pathway as a critical regulator of oxidative stress defense in breast cancer. Breast cancer remains the most commonly diagnosed malignancy in women, with tumor recurrence, metastasis, and resistance to standard therapies posing persistent clinical challenges. A key mechanism enabling this therapeutic resistance is the ability of cancer cells to adapt by activating endogenous antioxidant defense systems, such as the Keap1/Nrf2 pathway, to overcome treatment-induced oxidative stress ( 25 – 28 ). Therefore, deciphering the precise upstream regulatory network of this pathway is crucial for developing novel strategies to overcome drug resistance. The Tripartite motif (TRIM) protein family, particularly members functioning as E3 ubiquitin ligases, has emerged as a promising group of candidates due to their versatile roles in regulating cell cycle progression, signaling, and DNA repair ( 29 ). TRIM45, one such member, has been established as a tumor suppressor in multiple malignancies, including glioblastoma and hepatocellular carcinoma, by inhibiting MAPK/NF-κB pathways, stabilizing p53, and modulating epithelial-mesenchymal transition (EMT) ( 7 – 10 ). However, it remains puzzling that the specific role and mechanism of TRIM45 in breast cancer, particularly within the context of oxidative stress, are largely unexplored. Dysregulation of oxidative stress response is a hallmark of cancer. The transcription factor Nrf2 serves as the master regulator of antioxidant defense ( 27 , 30 ), whose activity is negatively controlled by the adaptor protein Keap1 ( 31 ). p62 (SQSTM1) is a multifunctional scaffold protein that competitively binds to Keap1, disrupting the Keap1-Nrf2 interaction and leading to non-canonical activation of Nrf2 signaling ( 32 ). Aberrant activation of this pathway is prevalent in various cancers and is associated with tumor progression and therapeutic resistance ( 33 ). The stability and function of p62 are regulated by multiple E3 ubiquitin ligases (e.g., TRIM21, TRAF6) ( 34 ), and emerging evidence suggests that several TRIM family members orchestrate the Keap1-Nrf2 axis through diverse mechanisms ( 35 ). Nevertheless, a critical unanswered question persists: In breast cancer, does a key tumor-suppressive E3 ubiquitin ligase exist that negatively regulates the Keap1/Nrf2 pathway by targeting p62 for degradation, thereby inhibiting tumor progression? Given the pivotal role of p62 in linking the ubiquitination machinery to the Keap1/Nrf2 oxidative stress pathway, coupled with the established tumor-suppressive and E3 ligase functions of TRIM45, we hypothesized that TRIM45 acts as such a key regulator. We propose that TRIM45 functions as an upstream E3 ubiquitin ligase for p62, promoting its ubiquitination and degradation. This, in turn, restores Keap1-mediated suppression of Nrf2, ultimately dismantling the oxidative stress defense and associated pro-survival signaling in breast cancer cells. This study aims to systematically test this hypothesis through clinical data analysis, cellular functional assays, and molecular mechanism exploration, seeking to uncover a novel regulatory axis in breast cancer oxidative stress adaptation. 2. Materials and methods 2.1 Cell culture and transfection Human breast cancer cell lines (MCF-7 and BT549) were donated by the Affiliated Hospital of Qingdao University and cultured in high-glucose DMEM (Procell, China) supplemented with 10% fetal bovine serum (BI, China), 1% penicillin-streptomycin solution (Solarbio, China) in a 5% CO 2 incubator at 37°C. When cells reached 70%-80% confluence in T25 flasks, they were trypsinized and reseeded into six-well plates. After 20–24 h, cells were transfected with TRIM45 overexpression plasmid using liposomal transfection reagent (Genechem, China). The medium was replaced with fresh complete medium 6 h post-transfection. 2.2 Antibodies The following primary antibodies were used for Western blotting: anti-MMP2 (Proteintech, China, Cat#10373-2-AP), anti-MMP9 (Abcam, UK, Cat#ab74277), anti-N-cadherin (Zenbio, China, Cat#AF4039), anti-E-cadherin (Zenbio, China, Cat#20874-1-AP), anti-Vimentin (Zenbio, China, Cat#R22775), anti-p62 (Proteintech, China, Cat#184201AP150UL), anti-GAPDH (Abcam, UK, Cat#ab8245), anti-Nrf2 (Abclonal, China, Cat#A0674), anti-Keap1 (Abcam, UK, Cat#ab139729), anti-IGF-1R (Santa Cruz, USA, Cat#sc-390130), and anti-VEGF (Santa Cruz, USA, Cat#sc-53463). HRP-conjugated secondary antibodies (Proteintech, China, Cat# SA00001-1 for rabbit IgG, and Cat# SA00001-2 for mouse IgG) were used at a 1:5000 dilution. 2.3 Cell Proliferation Assays 2.3.1 CCK-8 Assay 48 h after transfection, breast cancer cells were re-plated in 96-well plates according to 8000 cells per well. After 24 h, 10 µL of CCK-8 solution was added to 100 µL of medium per well. Absorbance values at 450 nm were measured after incubation at 37°C for 2 h. The absorbance value was positively correlated with cell proliferation ability. 2.3.2 4, 5-Ethynyl-2’- deoxyuridine (EdU) determination Glass coverslips were placed in 24-well plates, and breast cancer cells were seeded onto them prior to transfection. After 24 h, the cells were treated with 50 µmol EdU for 2 h, fixed with 4% methanol for 15 min, and then 100 µL of 1× Apollo staining reaction solution was added to each well. Subsequently, 0.5% Triton X-100 was added and the cells were washed 2–3 times. Finally, the cell slides were removed and mounted with DAPI. The number of positively stained cells was observed under a fluorescence microscope. 2.4 Cell Migration Assays 2.4.1 Wound Healing Assay The transfected breast cancer cells (4×10 5 cells per well) were spread into the six-well plate and cultured in an incubator of 5% CO 2 and 37°C for 48 h. When the cell fusion rate reached approximately 80–90%, the cells were starved for 12 h. Subsequently, they were scraped using a 200 µL pipette tip and washed three times with PBS. Moreover, the cells were observed and photographed by phase contrast microscope at 0 h, 24 h and 48 h, and the mobility was calculated. 2.4.2 Transwell migration Prior to the experiment, transfected cells were serum-starved for 12 h. Transwell chambers (8.0 µm pore size, Corning, USA) were placed in 24-well plates. A serum-free suspension of breast cancer cells (1×10^4 cells/200 µL) was added to the upper chamber, ensuring no bubbles were introduced. Meanwhile, 600 µL DMEM culture medium containing 20% FBS was added in the lower chamber. Matrigel was not used for coating in the experiment. After 48 h, the transwell chamber was removed then fixed with 100% methanol for 30 min, and stained with 0.1% crystal violet solution for 15 min. Fluorescence microscopy was used to observe. 2.5 Western Blots Total protein was extracted from breast cancer cells using RIPA buffer supplemented with 10% phosphatase inhibitor and 10% protease inhibitor. The concentration of the protein samples was detected with the BCA Protein Assay Kit ((Beyotime Biotechnology, China)). Protein samples were mixed with loading buffer (Yamei, China) at a 4:1 ratio and boiled at 100°C for 15 min in a metal bath (Mona, China). The protein samples were then separated on 10% SDS-PAGE gel, transferred to PVDF membranes (Millipore) and incubated in 5% bovine serum albumin for 2 h at room temperature, followed by incubation with specific primary antibodies overnight at 4°C. After washing three times with TBS-Tween 20 (0.1%, v/v), the membranes were incubated with the appropriate secondary antibodies for 1 h. After TBST washing, blots were examined using enhanced chemiluminescence (Millipore). 2.6 Immunoprecipitation (IP) After treatment, total protein was extracted from transfected cells, and an aliquot was used as input control. Protein lysates were incubated overnight at 4°C with 2 µg of anti-Flag antibody or an appropriate IP antibody. Then, 20 µL of Protein A/G agarose beads (Santa Cruz Biotechnology) were added and incubated for 2 h. After centrifugation at 800 rpm to remove the supernatant, 30 µL of fresh beads were added and incubated for another 4 h at 4°C to form the immune complex. After centrifugation at 800 rpm for 4 min at 4°C and four washes with washing buffer, the beads were resuspended in 30 µL of 1× loading buffer and boiled at 100°C for 10 min. 2.7 Ubiquitination detection For the ubiquitination assay, MCF-7 and BT549 cells were co-transfected with Flag-TRIM45 (0.5 µg), Myc-p62 (0.5 µg), and HA-Ub (1.0 µg) or corresponding empty vectors using liposome transfection reagent (Genechem, China) in six-well plates. At 48 h post-transfection, cells were treated with the proteasome inhibitor MG132 (MedChemExpress, USA) at a final concentration of 20 µM for 6 h before harvest. Total cell lysates were immunoprecipitated with anti-Myc antibody (Proteintech, China) and protein A/G agarose beads (Santa Cruz, USA), followed by immunoblotting with anti-HA antibody (Proteintech, China) to detect ubiquitinated p62. To assess protein stability, cells transfected with Flag-TRIM45 or empty vector in six-well plates were divided into four time-point groups (0, 2, 4, and 8 h). At 48 h post-transfection, cycloheximide (CHX, Sigma-Aldrich, USA) was added to the culture medium at a final concentration of 25 µg/mL to inhibit de novo protein synthesis. Cells were harvested at the indicated time points, and total proteins were extracted for Western blot analysis of p62 expression. Protein half-life was determined by densitometric analysis of p62 bands normalized to GAPDH. 2.8 Determination of malondialdehyde (MDA) content, glutathione (GSH) content and Glutathione peroxidase (GSH-Px) activity. At 72 h of transfection, cells were washed three times with PBS. Cells were lysed and the lysates were sonicated on ice for 3 min. The supernatant was collected by centrifugation at 10,000 g for 15 min at 4°C. The levels of MDA, GSH, and GSH-Px activity in the supernatant were measured using commercial biochemical kits (Cat# S0131M for MDA, Cat# S0052 for GSH, and Cat# S0056 for GSH-Px; Beyotime Biotechnology, Shanghai, China) according to the manufacturer‘s instructions. 2.9 Detection of ROS Intracellular superoxide anion levels were detected using a DHE-based ROS assay kit (Cat# S0064S; Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. Briefly, 72 h after transfection, cells cultured in 6-well plates were washed gently with PBS and incubated with 1 mL of DHE staining solution (diluted to a final concentration of 50 µM with DMEM) at 37°C for 50 min in a cell culture incubator protected from light. Subsequently, the red fluorescence intensity (excitation/emission = 535/610 nm) was observed and captured under a fluorescence microscope. The DHE stock solution was stored at -20°C protected from light and aliquoted to avoid repeated freeze-thaw cycles. 2.10 Statistical analysis All data are presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, USA). For comparisons between two groups, unpaired two-tailed Student‘s t-test was used. For comparisons involving multiple groups, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was applied. For experiments with a 2 × 2 factorial design (e.g., TRIM45 overexpression combined with TGF-β treatment), two-way ANOVA followed by Sidak's multiple comparisons test was used to assess the main effects and interaction between factors. Image processing and densitometric analysis were performed using ImageJ software (NIH, USA). A value of *p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001)). 3. Results 3.1 TRIM45 expression is downregulated in breast cancer patients, which indicates poor prognosis of breast cancer To evaluate the expression profile of TRIM45 in breast cancer, we analyzed transcriptomic data obtained from the Gene Expression Omnibus (GEO) database. Specifically, we retrieved datasets GSE5364 and GSE3744 to compare TRIM45 levels between breast cancer tissues and adjacent normal tissues. As illustrated ( Fig. 1 A ) , TRIM45 expression was significantly downregulated in tumor tissues compared to normal controls. To investigate the prognostic significance of TRIM45 in breast cancer, we retrieved clinical data from the TCGA database via the UCSC Xena browser. We performed Kaplan-Meier analysis for Overall Survival (OS), Progression-Free Survival (PFS), Disease-Specific Survival (DSS), and Disease-Free Survival (DFS). The results revealed statistically significant differences in OS, PFS, DSS and DFS. Specifically, high TRIM45 expression was associated with significantly better survival outcomes, whereas low expression was predictive of a poor prognosis in breast cancer patients (Figs. 1 B-D ). The difference was not statistically significant in the disease-free survival group ( Fig. 1 E ) . These findings indicate that TRIM45 is downregulated in breast cancer tissues, suggesting that loss of TRIM45 expression may contribute to breast cancer progression. 3.2 TRIM45 inhibited the proliferation of breast cancer cells To explore the effect of TRIM45 on the malignant development of breast cancer cells, we established two TRIM45 overexpressed cell lines, MCF-7 and BT549. CCK-8 experiment showed that the proliferation rate of TRIM45 overexpression cell lines was significantly reduced compared to the control cells ( Figs. 2 A and 2 B ) . Meanwhile, EdU incorporation assays revealed significantly reduced fluorescence intensity in TRIM45-overexpressing cells compared to controls. ( Figs. 2 C-F ) . These results indicate that TRIM45 overexpression inhibits the proliferation of breast cancer cells. 3.3 TRIM45 inhibits breast cancer cell migration by suppressing EMT and metastasis-related protein expression To further investigate the effect of TRIM45 on breast cancer cell migration, we performed transwell assays using TRIM45-overexpressing MCF-7 and BT549 cells. Transwell assays showed that the number of migratory cells was significantly reduced in TRIM45-overexpressing MCF-7 and BT549 cells compared to controls ( Figs. 3 A-D ). Consistently, wound healing assays revealed that the migration rate was markedly decreased in TRIM45-overexpressing cells at 24 h and 48 h post-scratching ( Figs. 3 E-H ) . These results demonstrate that TRIM45 overexpression significantly impairs the migratory capacity of breast cancer cells. To elucidate the molecular basis underlying this phenotypic suppression, we examined the expression of epithelial-mesenchymal transition (EMT) markers and metastasis-related proteins. Western blot analysis showed that TRIM45 overexpression led to a significant increase in the epithelial marker E-cadherin, accompanied by decreased expression of mesenchymal markers ( Figs. 3 I and 3 J ) . Furthermore, the expression of matrix metalloproteinases (MMP2 and MMP9) and the pro-angiogenic factor VEGF were markedly downregulated upon TRIM45 overexpression ( Figs. 3 K and 3 L ) . Collectively, these findings indicate that TRIM45 suppresses breast cancer cell migration by inhibiting EMT progression and downregulating key metastasis-related proteases. 3.4 TRIM45 binds to p62 and promotes its ubiquitination-dependent degradation Previous studies have demonstrated that TRIM45 plays a significant role in the biological processes of breast cancer cells; however, the specific molecular mechanisms underlying its function as an E3 ubiquitin ligase remain incompletely understood. To further investigate TRIM45 function, we used MCF-7 and BT549 cells stably overexpressing TRIM45 or empty vector as controls. Co-immunoprecipitation (Co-IP) assays using FLAG antibody-conjugated agarose beads were performed to identify potential interacting proteins of TRIM45. The results revealed that p62 specifically interacts with TRIM45 in breast cancer cells ( Figs. 4 A and 4 B ) , suggesting a direct protein-protein interaction between the two. To assess the impact of TRIM45 on p62 protein stability, we performed cycloheximide (CHX) chase assays. The results demonstrated that the half-life of p62 was significantly shortened in TRIM45-overexpressing MCF-7 and BT549 cells ( Figs. 4 C and 4 D ) ,indicating that TRIM45 promotes p62 degradation. Consistently, TRIM45 overexpression markedly reduced the protein level of p62, and this effect was reversed by treatment with the proteasome inhibitor MG132 ( Fig. 4 E ) , suggesting that TRIM45 regulates p62 degradation via the ubiquitin-proteasome pathway. To further validate whether p62 is a direct ubiquitination substrate of TRIM45, we performed ubiquitination assays by immunoprecipitating Flag-tagged TRIM45 and then immunoblotting for ubiquitin. This approach allows us to specifically assess the ubiquitination status of p62 that is in complex with TRIM45. The results showed that the ubiquitination level of p62 co-immunoprecipitated with TRIM45 was significantly enhanced in cells expressing exogenous TRIM45 ( Figs. 4 F and 4 G ) , supporting the notion that p62 is a direct target of TRIM45-mediated ubiquitination. In summary, these findings suggest that TRIM45 promotes the degradation of p62 through interaction and ubiquitination, thereby regulating its stability via the ubiquitin-proteasome pathway. 3.5 TRIM45-mediated p62 degradation activates Keap1 and suppresses Nrf2 signaling Previous studies have established that the KIR domain of p62 binds to the Keap1-DC domain, the same region responsible for Nrf2 interaction, thereby competitively disrupting the Keap1–Nrf2 complex. As the Keap1/Nrf2 signaling pathway serves as a central signaling cascade governing cellular resistance to oxidative stress, we sought to determine whether TRIM45 influences this pathway. To this end, we evaluated the expression of Nrf2 and Keap1 following TRIM45 overexpression. Our results showed that TRIM45 upregulation led to decreased Nrf2 protein levels and a concomitant increase in Keap1 expression ( Fig. 5 A and 5 B ) . We hypothesize that this effect stems from TRIM45-mediated degradation of p62, which alleviates p62-dependent sequestration of Keap1 and thus enhances Nrf2 ubiquitination and degradation. Consistent with this mechanism, nuclear-cytoplasmic fractionation assays combined with Western Blot analysis confirmed a pronounced attenuation of Nrf2 nuclear translocation upon TRIM45 overexpression ( Figs. 5 C and 5 D ) . Collectively, these findings indicate that TRIM45 not only impairs cellular antioxidant defense but may also interfere with growth factor signaling pathways pivotal for cancer progression. 3.6 TRIM45 suppresses the p62/Keap1/Nrf2 pathway and downregulates IGF-1R expression through oxidative stress Given that TRIM45 suppresses breast cancer progression through p62 degradation, leading to Nrf2 destabilization and oxidative stress, we further examined its effect on the IGF system, a critical pathway for breast cancer proliferation and survival. Given the established crosstalk between oxidative stress and growth factor signaling, we asked whether TRIM45-driven oxidative stress influences IGF pathway activity. Indeed, TRIM45 overexpression markedly reduced both basal and TGF-β-induced expression of IGF-1 and its receptor IGF-1R ( Figs. 6 A and 6 B ) . Previous studies have demonstrated that Nrf2 can transcriptionally regulate growth factor receptors, including IGF-1R ( 36 , 37 ). In line with this, our results suggest that TRIM45-mediated Nrf2 degradation leads to decreased IGF-1R levels, likely through the attenuation of Nrf2-driven transcription. In parallel, TRIM45 downregulated phosphorylation of ERK (p-ERK), a signaling hub associated with cell proliferation ( Figs. 6 C and 6 D ) . To further assess the redox state under TRIM45 overexpression, we measured key oxidative stress markers. Results showed a significant increase in malondialdehyde (MDA) content, accompanied by decreased glutathione (GSH) levels and reduced activity of glutathione peroxidase (GSH-Px) ( Figs. 6 E-G ) . These alterations collectively indicate a compromised cellular antioxidant system and enhanced oxidative stress. Consistent with this, reactive oxygen species (ROS) were significantly elevated in TRIM45-overexpressing cells ( Fig. 6 H ) . Together, these data demonstrate that TRIM45 disrupts redox homeostasis and concomitantly attenuates key proliferative and survival signaling pathways in breast cancer cells. 4. Discussion The metabolic adaptation of cancer cells to survive under persistent oxidative stress represents a fundamental mechanism of tumor progression and therapeutic resistance. In this study, we identified a previously unrecognized regulatory cascade involving the TRIM45, p62, Keap1, Nrf2, and IGF-1R signaling axis, which governs the progression of breast cancer (38). Our findings demonstrate that TRIM45 functions as a tumor-suppressive E3 ubiquitin ligase that specifically targets p62 for proteasomal degradation. This event subsequently reinstates the Keap1-mediated suppression of Nrf2, thereby disrupting the antioxidant defense system and growth factor signaling pathways simultaneously (33). The master regulator of cellular redox homeostasis, Nrf2, is frequently hijacked by malignant cells to facilitate survival in hostile microenvironments. Under normal physiological conditions, Keap1 acts as a substrate adaptor for the Cul3-based E3 ligase complex to promote the constitutive degradation of Nrf2 (39, 40). However, the accumulation of p62 can competitively bind to the Kelch domain of Keap1 through its KIR motif, which leads to the non-canonical activation of Nrf2 (41, 42). Our data provide compelling evidence that the loss of TRIM45 in breast cancer tissues facilitates the accumulation of p62 (43), thereby sequestering Keap1 and allowing Nrf2 to translocate into the nucleus. This transcriptional reprogramming upregulates downstream antioxidant enzymes such as HO-1 and GPX4, which effectively neutralize reactive oxygen species and prevent oxidative damage (44). An intriguing finding of this study is the crosstalk between Nrf2-mediated antioxidant responses and the IGF-1R/ERK survival pathway. We observed that the stabilization of Nrf2 not only enhances redox capacity but also sustains the expression of IGF-1R (45). Although the precise transcriptional mechanism remains to be fully elucidated, the concomitant reduction of IGF-1R and phosphorylated ERK upon TRIM45 overexpression suggests a synergistic suppressive effect. The downregulation of the IGF-1R/ERK axis significantly impairs the epithelial-mesenchymal transition process (46, 47), as evidenced by the restoration of E-cadherin and the loss of mesenchymal markers. This logical progression from biochemical modification to phenotypic alteration provides a mechanistic explanation for the impaired migratory and invasive capabilities of TRIM45-overexpressing cells. The induction of lethal oxidative stress through the targeted degradation of p62 represents a promising form of synthetic lethality in breast cancer therapy. Our clinical analysis using TCGA and GEO datasets (Figures 7A and 7B) reinforces the relevance of this pathway, as patients with low TRIM45 expression and high p62/Nrf2 activity exhibit significantly poorer prognosis (48). These results suggest that the TRIM45 status could serve as a predictive biomarker for sensitivity to pro-oxidant therapies. Patients lacking this endogenous tumor suppressor may be particularly vulnerable to pharmacological agents that further challenge their compromised antioxidant defenses or inhibit the Nrf2-driven growth signaling. In conclusion, our study delineates a novel tumor-suppressive role for TRIM45 in breast cancer through the modulation of the p62/Keap1/Nrf2 axis. By promoting the ubiquitination of p62, TRIM45 effectively dismantles the cytoprotective shield provided by Nrf2 and attenuates oncogenic IGF-1R signaling. This dual mechanism not only inhibits primary tumor cell proliferation but also curtails metastasis by reversing the EMT program. Future investigations into the upstream signals that trigger TRIM45 downregulation will further enhance our understanding of breast cancer pathogenesis and may lead to the development of innovative therapeutic strategies that exploit redox vulnerabilities (Figure 7C). Abbreviations TRIM45, Tripartite motif containing 45; Nrf2, Nuclear factor erythroid 2-related factor 2; Keap1, Kelch-like ECH-associated protein 1; HO-1, Heme oxygenase-1; GPX4, Glutathione peroxidase 4; EMT, Epithelial-mesenchymal transition; MMP, Matrix metalloproteinase; VEGF, Vascular endothelial growth factor; IGF-1, Insulin-like growth factor 1; IGF-1R, Insulin-like growth factor 1 receptor; ROS, Reactive oxygen species; GEO, Gene Expression Omnibus; TCGA, The Cancer Genome Atlas; UCSC Xena, University of California, Santa Cruz Xena; OS, Overall survival; PFS, Progression-free survival; DSS, Disease-specific survival; DFS, Disease-free survival; CCK-8, Cell counting kit-8; EdU, 4,5-Ethynyl-2'-deoxyuridine; DAPI, 4',6-Diamidino-2-phenylindole; IP, Immunoprecipitation; CHX, Cycloheximide; MDA, Malondialdehyde; GSH, Glutathione; GSH-Px, Glutathione peroxidase; DHE, Dihydroethidium; SD, Standard deviation; NSCLC, Non-small cell lung cancer; MAPK, Mitogen-activated protein kinase; NF-κB, Nuclear factor kappa-B; RING, Really interesting new gene; RBCC, RING finger, B-box, coiled-coil; ElK-1, Ets-like protein 1; AP-1, Activator protein 1; PKC, Protein kinase C; RACK1, Receptor for activated C kinase 1; GSEA, Gene Set Enrichment Analysis; DMEM, Dulbecco's Modified Eagle Medium; FBS, Fetal bovine serum; RIPA, Radioimmunoprecipitation assay; BCA, Bicinchoninic acid; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, Polyvinylidene fluoride; TBST, Tris-buffered saline with Tween 20; HRP, Horseradish peroxidase Declarations Author contributions M.S and C.C: writing original draft, data curation, resources; F.S: language correction, chart correction; F.Y, H.Z, and X.Z: formal analysis, data curation; Z.W and C.Y: formal analysis, resources; Y.W and R.W: resources; L.Y: validation, formal analysis; Z.Z and B.L: supervision, conceptualization, writing review and editing. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported by Grants from the National Natural Science Foundation of China (No. 81871231 and 31401068). Youth Innovation and Science and Technology Plan of Colleges and Universities in Shandong Province (No. 2019KJK016), Shandong Taishan Scholars Young Experts Program (No. tsqn202103056), The Natural Science Foundation of Shandong Province (No. ZR2023MH082). Qingdao Natural Science Foundation Key Project (24-8-4-zrjj-8-jch). Acknowledgments Thanks to the TCGA databases for open access to the data. Institutional Review Board Statement: No application. Data Availability Statement: The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request. 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IGF-1R promotes the expression of cyclin D1 protein and accelerates the G1/S transition by activating Ras/Raf/MEK/ERK signaling pathway. Int J Clin Exp Pathol. 2017;10(12):11652-8. Hayes JD, Dayalan Naidu S, Dinkova-Kostova AT. Regulating Nrf2 activity: ubiquitin ligases and signaling molecules in redox homeostasis. Trends Biochem Sci. 2025;50(3):179-205. Dahl-Wilkie H, Gomez J, Kelley A, Manjit K, Mansoor B, Kanumuri P, et al. Chronic IL-1-Exposed LNCaP Cells Evolve High Basal p62-KEAP1 Complex Accumulation and NRF2/KEAP1-Dependent and -Independent Hypersensitive Nutrient Deprivation Response. Cells. 2025;14(3). Jiang XY, Guo QQ, Wang SS, Guo R, Zou Y, Liu JW, et al. DNA damage response pathway regulates Nrf2 in response to oxidative stress. Sci Adv. 2025;11(30):eadu9555. Liu S, Pi J, Zhang Q. Signal amplification in the KEAP1-NRF2-ARE antioxidant response pathway. Redox Biol. 2022;54:102389. Yang T, Chen Z, Yi F, Yang Y, Chu Z, Yang C, et al. TRIM39 aggravates hepatocellular carcinoma growth through targeting the p62-KEAP1-NRF2 axis. Sci Rep. 2025;16(1):3160. Luo J, Zhi Q, Li D, Xu Y, Zhu H, Zhao L, et al. Low dose radiotherapy combined with immune checkpoint inhibitors induces ferroptosis in lung cancer via the Nrf2/HO-1/GPX4 axis. Front Immunol. 2025;16:1558814. Lyons A, Coleman M, Riis S, Favre C, O'Flanagan CH, Zhdanov AV, et al. Insulin-like growth factor 1 signaling is essential for mitochondrial biogenesis and mitophagy in cancer cells. J Biol Chem. 2017;292(41):16983-98. Cevenini A, Orrù S, Mancini A, Alfieri A, Buono P, Imperlini E. Molecular Signatures of the Insulin-like Growth Factor 1-mediated Epithelial-Mesenchymal Transition in Breast, Lung and Gastric Cancers. Int J Mol Sci. 2018;19(8). Wang C, Su K, Zhang Y, Zhang W, Zhao Q, Chu D, et al. IR-A/IGF-1R-mediated signals promote epithelial-mesenchymal transition of endometrial carcinoma cells by activating PI3K/AKT and ERK pathways. Cancer Biol Ther. 2019;20(3):295-306. Ning L, Huo Q, Xie N. Comprehensive Analysis of the Expression and Prognosis for Tripartite Motif-Containing Genes in Breast Cancer. Front Genet. 2022;13:876325. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 12 Apr, 2026 Editor assigned by journal 20 Mar, 2026 Submission checks completed at journal 20 Mar, 2026 First submitted to journal 19 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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16:55:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":189541,"visible":true,"origin":"","legend":"\u003cp\u003eTRIM45 expression was down-regulated in breast cancer tissues by database analysis. (A) The expression of TRIM45 in normal tissues and breast cancer tissues was obtained from GSE5365 and GSE3744 datasets of GEO database. (B) Survival curves for overall survival. (C) Survival curves for progression-free survival. (D) Survival curves for disease-specific survival. (E) Survival curves for disease-free survival. Statistical analyses were performed by two-tailed unpaired Student's t-test. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9167161/v1/716da6ab5c6a2e872516af26.png"},{"id":107355966,"identity":"b353c720-374b-4237-b53e-51800bca9c5f","added_by":"auto","created_at":"2026-04-20 16:55:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":377065,"visible":true,"origin":"","legend":"\u003cp\u003eTRIM45 inhibited the proliferation of breast cancer. (A, B) Cell proliferation was detected by CCK-8 assay in MCF-7 and BT549 cells after overexpression of TRIM45. (C, D) After transfection of MCF-7 and BT549 cells with TRIM45 and control plasmids for 48 h, the cells were treated with EdU staining solution, and the number of proliferating cells was visualized by fluorescence. Scale bar = 150 μm. (E, F) Quantitative analysis of the number of positive cells.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9167161/v1/ef0f169e947e9951dd919bb1.png"},{"id":107355965,"identity":"f1367c92-4fec-4bb3-9c30-934e005fd188","added_by":"auto","created_at":"2026-04-20 16:55:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":788361,"visible":true,"origin":"","legend":"\u003cp\u003eTRIM45 inhibits breast cancer cell migration by suppressing EMT and metastasis-related protein expression. (A, B) MCF-7 and BT549 cells were transfected with TRIM45 overexpression plasmid or empty vector for 48 h. Transwell assays were performed after 12 h starvation, and migrated cells were stained with crystal violet. Representative images are shown. Scale bar = 50 μm. (C, D) Quantification of migrated cells per field from three independent experiments. (E, F) Wound healing assays were performed on transfected MCF-7 and BT549 cells, and images were captured at 0, 24, and 48 h post-scratching. (G, H) Quantification of migration rate relative to 0 h. (I) Western blot analysis of TRIM45 expression in MCF-7 and BT549 cells after transfection, and EMT markers including E-cadherin, N-cadherin, and vimentin. (J) Quantification of protein expression levels from (I). (K) Western blot analysis of MMP2, MMP9, and VEGF expression after TRIM45 overexpression. (L) Quantification of protein expression levels from (K). Statistical analyses were performed by two-tailed unpaired Student's t-test. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001. All data are presented as mean ± SD from three independent experiments.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9167161/v1/27fb996237be70071ad7d4b6.png"},{"id":107355969,"identity":"7030fc63-2ea4-4417-b890-27700ae2bc61","added_by":"auto","created_at":"2026-04-20 16:55:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":334518,"visible":true,"origin":"","legend":"\u003cp\u003eTRIM45 binds to p62 and promotes its ubiquitination-dependent degradation. (A) After overexpression of TRIM45 and empty plasmid in MCF-7, the proteins were incubated with TRIM45 antibody and p62 antibody binding protein with agarose beads, respectively. The extracted proteins were verified by Western Blot with FLAG antibody and p62 antibody. (B) We did the same IP experimental treatment in BT549 cells as in MCF-7 cells. (C, D) Western Blot analysis of the level of p62 after 0 h, 2 h, 4 h and 8 h CHX treatment in MCF-7 and BT549 cells after overexpression of TRIM45. Quantification plot of changes in p62 protein expression levels at different times. (E) Western Blot analysis of the level of p62 after 6h MG132 treatment after overexpression of TRIM45. (F) After transfection of Flag-TRIM45, Myc-p62, HA-Ub and their corresponding empty plasmids into cells, agarose beads were used for IP experiments, and the results of Western blot were presented. Statistical analyses were performed by two-tailed unpaired Student's t-test. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9167161/v1/bf4c4ae5d606dd011ca1014c.png"},{"id":107355964,"identity":"7c79d784-1abe-430f-b4f6-afdb9e375847","added_by":"auto","created_at":"2026-04-20 16:55:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":201475,"visible":true,"origin":"","legend":"\u003cp\u003eTRIM45-mediated p62 degradation activates Keap1 and suppresses Nrf2 signaling (A) Western Blot analysis of the expression of p62, Nrf2, Keap1 after overexpression of TRIM45. (B) Quantification plot of p62, Nrf2, Keap1expression levels. (C) Western Blot analysis of Nrf2 protein expression in nuclear and cytoplasmic fractions after TRIM45 overexpression.(D) Statistical analyses were performed by two-tailed unpaired Student's t-test. *P \u0026lt; 0.05, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9167161/v1/b4e84fd3fb9a7e5b564830e5.png"},{"id":107356014,"identity":"270bf15f-cc88-4d14-9f2f-7efb5fa2b4e2","added_by":"auto","created_at":"2026-04-20 16:55:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":260846,"visible":true,"origin":"","legend":"\u003cp\u003eTRIM45 suppresses the p62/Keap1/Nrf2 pathway and downregulates IGF-1R expression through oxidative stress. (A) Western blot analysis of IGF-1 and IGF-1R expression after TRIM45 overexpression with or without TGF-β treatment. (B) Quantification of IGF-1 and IGF-1R protein levels from (A). Data were analyzed by two-way ANOVA followed by Šídák's multiple comparisons test. (C) Western blot analysis of p-ERK and total ERK expression. (D) Quantification of p-ERK/ERK ratio. Unpaired two-tailed Student's t-test was used for (D). (E) Quantification of GPX4 and HO-1 protein expression levels. (F) Representative western blots of HO-1 and GPX4. (G) Measurement of GSH-Px activity, GSH and MDA content. (H) Representative fluorescence images of ROS detection (scale bar = 50 μm) and quantification of relative ROS fluorescence intensity. Data in (E, G, H) were analyzed by unpaired two-tailed Student's t-test. All data are presented as mean ± SD from three independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9167161/v1/5dcb2bf363dc7c48e80c03fe.png"},{"id":107487471,"identity":"bc7d3f33-6149-48d7-9fe1-3768972ceef4","added_by":"auto","created_at":"2026-04-22 02:41:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":323913,"visible":true,"origin":"","legend":"\u003cp\u003eClinical correlation and proposed mechanistic model of the TRIM45-p62-Keap1-Nrf2 signaling pathway in breast cancer. (A) GSEA method was used to identify the gene distribution of TRIM45 and oxidative stress related genes, Nrf2 signaling related genes in breast cancer cells.(B) The expression differences of p62, Keap1 and Nrf2 were classified according to the expression level of TRIM45 in the TCGA database. (C) Diagram of the mechanism of TRIM45 on p62/Keap1/Nrf2 pathway in breast cancer cells.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9167161/v1/e96d69be41a8aeaf61315c0e.png"},{"id":107489019,"identity":"9f4dcf32-41d5-4755-83c0-9fd107e717ad","added_by":"auto","created_at":"2026-04-22 02:46:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2666324,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9167161/v1/e16f1af8-d709-414c-a5f5-4c997cec8e9e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"TRIM45 Suppresses Breast Cancer Progression by Regulating Nrf2/Keap1 Pathway through Degradation of p62","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn 2022, approximately 2.3\u0026nbsp;million new cases were reported, accounting for 11.7% of all cancer diagnoses,and for the first time, its incidence surpassed that of lung cancer (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Despite advances in adjuvant therapies, tumor recurrence and metastasis remain persistent clinical obstacles, underscoring the urgent need to identify novel therapeutic targets.\u003c/p\u003e \u003cp\u003eThe Tripartite Motif (TRIM) protein family has emerged as a promising candidate owing to its versatile biological roles. Many TRIM proteins function as E3 ubiquitin ligases that regulate cell cycle progression, signaling, and DNA repair (\u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). TRIM45 acts as a tumor suppressor in multiple malignancies, including glioblastoma and hepatocellular carcinoma, by inhibiting MAPK and NF-κB pathways, stabilizing p53, and modulating epithelial-mesenchymal transition (EMT)(\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).However, the specific role of TRIM45 in breast cancer, particularly in the context of oxidative stress, remains largely unexplored.\u003c/p\u003e \u003cp\u003eDysregulation of oxidative stress responses is a hallmark of cancer. The transcription factor Nrf2 serves as the master regulator of antioxidant defense (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Under basal conditions, Nrf2 is constitutively ubiquitinated and degraded by its negative regulator Keap1, maintaining redox homeostasis (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Upon oxidative stress, Nrf2 stabilizes and translocates to the nucleus, driving expression of cytoprotective genes including HO-1 and GPX4 (\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Notably, Nrf2 also upregulates Insulin-like Growth Factor 1 Receptor (IGF-1R) expression, thereby promoting tumor cell proliferation (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ep62 (SQSTM1) is a multifunctional scaffold protein that plays pivotal roles in autophagy and oxidative stress response. Mechanistically, p62 competitively binds to Keap1, disrupting Keap1-Nrf2 interaction and leading to non-canonical Nrf2 activation (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Aberrant activation of this pathway is prevalent in various malignancies and is associated with tumor progression and therapeutic resistance.\u003c/p\u003e \u003cp\u003eThe stability and function of p62 are tightly regulated by multiple E3 ubiquitin ligases. For instance, TRIM21 ubiquitinates p62 to inhibit the p62-Keap1-Nrf2 pathway, while TRAF6 promotes p62 ubiquitination to enhance its inflammatory function (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Emerging evidence suggests that multiple TRIM family members orchestrate the Keap1-Nrf2 pathway through diverse mechanisms, thereby modulating oxidative stress and tumorigenesis (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). However, whether TRIM45 regulates p62 and the Keap1/Nrf2 pathway in breast cancer remains unknown.\u003c/p\u003e \u003cp\u003eGiven the pivotal role of p62 in linking ubiquitination to the Keap1/Nrf2 oxidative stress pathway (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), we hypothesized that p62 might be a novel functional substrate of TRIM45 in breast cancer. In this study, we demonstrate that TRIM45 is significantly downregulated in breast cancer tissues and suppresses malignant phenotypes in breast cancer cells. Mechanistically, we identify p62 as a key ubiquitination substrate of TRIM45 (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). TRIM45-mediated p62 degradation restores Keap1-dependent suppression of Nrf2, thereby inducing lethal oxidative stress and concurrently attenuating IGF-1R/ERK signaling. These findings establish the TRIM45/p62/Keap1/Nrf2 signaling pathway as a critical regulator of oxidative stress defense in breast cancer.\u003c/p\u003e \u003cp\u003eBreast cancer remains the most commonly diagnosed malignancy in women, with tumor recurrence, metastasis, and resistance to standard therapies posing persistent clinical challenges. A key mechanism enabling this therapeutic resistance is the ability of cancer cells to adapt by activating endogenous antioxidant defense systems, such as the Keap1/Nrf2 pathway, to overcome treatment-induced oxidative stress (\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Therefore, deciphering the precise upstream regulatory network of this pathway is crucial for developing novel strategies to overcome drug resistance.\u003c/p\u003e \u003cp\u003eThe Tripartite motif (TRIM) protein family, particularly members functioning as E3 ubiquitin ligases, has emerged as a promising group of candidates due to their versatile roles in regulating cell cycle progression, signaling, and DNA repair (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). TRIM45, one such member, has been established as a tumor suppressor in multiple malignancies, including glioblastoma and hepatocellular carcinoma, by inhibiting MAPK/NF-κB pathways, stabilizing p53, and modulating epithelial-mesenchymal transition (EMT) (\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). However, it remains puzzling that the specific role and mechanism of TRIM45 in breast cancer, particularly within the context of oxidative stress, are largely unexplored.\u003c/p\u003e \u003cp\u003eDysregulation of oxidative stress response is a hallmark of cancer. The transcription factor Nrf2 serves as the master regulator of antioxidant defense (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), whose activity is negatively controlled by the adaptor protein Keap1 (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). p62 (SQSTM1) is a multifunctional scaffold protein that competitively binds to Keap1, disrupting the Keap1-Nrf2 interaction and leading to non-canonical activation of Nrf2 signaling (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Aberrant activation of this pathway is prevalent in various cancers and is associated with tumor progression and therapeutic resistance (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The stability and function of p62 are regulated by multiple E3 ubiquitin ligases (e.g., TRIM21, TRAF6) (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), and emerging evidence suggests that several TRIM family members orchestrate the Keap1-Nrf2 axis through diverse mechanisms (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Nevertheless, a critical unanswered question persists: In breast cancer, does a key tumor-suppressive E3 ubiquitin ligase exist that negatively regulates the Keap1/Nrf2 pathway by targeting p62 for degradation, thereby inhibiting tumor progression?\u003c/p\u003e \u003cp\u003eGiven the pivotal role of p62 in linking the ubiquitination machinery to the Keap1/Nrf2 oxidative stress pathway, coupled with the established tumor-suppressive and E3 ligase functions of TRIM45, we hypothesized that TRIM45 acts as such a key regulator. We propose that TRIM45 functions as an upstream E3 ubiquitin ligase for p62, promoting its ubiquitination and degradation. This, in turn, restores Keap1-mediated suppression of Nrf2, ultimately dismantling the oxidative stress defense and associated pro-survival signaling in breast cancer cells. This study aims to systematically test this hypothesis through clinical data analysis, cellular functional assays, and molecular mechanism exploration, seeking to uncover a novel regulatory axis in breast cancer oxidative stress adaptation.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell culture and transfection\u003c/h2\u003e \u003cp\u003eHuman breast cancer cell lines (MCF-7 and BT549) were donated by the Affiliated Hospital of Qingdao University and cultured in high-glucose DMEM (Procell, China) supplemented with 10% fetal bovine serum (BI, China), 1% penicillin-streptomycin solution (Solarbio, China) in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C. When cells reached 70%-80% confluence in T25 flasks, they were trypsinized and reseeded into six-well plates. After 20\u0026ndash;24 h, cells were transfected with TRIM45 overexpression plasmid using liposomal transfection reagent (Genechem, China). The medium was replaced with fresh complete medium 6 h post-transfection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Antibodies\u003c/h2\u003e \u003cp\u003eThe following primary antibodies were used for Western blotting: anti-MMP2 (Proteintech, China, Cat#10373-2-AP), anti-MMP9 (Abcam, UK, Cat#ab74277), anti-N-cadherin (Zenbio, China, Cat#AF4039), anti-E-cadherin (Zenbio, China, Cat#20874-1-AP), anti-Vimentin (Zenbio, China, Cat#R22775), anti-p62 (Proteintech, China, Cat#184201AP150UL), anti-GAPDH (Abcam, UK, Cat#ab8245), anti-Nrf2 (Abclonal, China, Cat#A0674), anti-Keap1 (Abcam, UK, Cat#ab139729), anti-IGF-1R (Santa Cruz, USA, Cat#sc-390130), and anti-VEGF (Santa Cruz, USA, Cat#sc-53463). HRP-conjugated secondary antibodies (Proteintech, China, Cat# SA00001-1 for rabbit IgG, and Cat# SA00001-2 for mouse IgG) were used at a 1:5000 dilution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell Proliferation Assays\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 CCK-8 Assay\u003c/h2\u003e \u003cp\u003e48 h after transfection, breast cancer cells were re-plated in 96-well plates according to 8000 cells per well. After 24 h, 10 \u0026micro;L of CCK-8 solution was added to 100 \u0026micro;L of medium per well. Absorbance values at 450 nm were measured after incubation at 37\u0026deg;C for 2 h. The absorbance value was positively correlated with cell proliferation ability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 4, 5-Ethynyl-2\u0026rsquo;- deoxyuridine (EdU) determination\u003c/h2\u003e \u003cp\u003eGlass coverslips were placed in 24-well plates, and breast cancer cells were seeded onto them prior to transfection. After 24 h, the cells were treated with 50 \u0026micro;mol EdU for 2 h, fixed with 4% methanol for 15 min, and then 100 \u0026micro;L of 1\u0026times; Apollo staining reaction solution was added to each well. Subsequently, 0.5% Triton X-100 was added and the cells were washed 2\u0026ndash;3 times. Finally, the cell slides were removed and mounted with DAPI. The number of positively stained cells was observed under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell Migration Assays\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Wound Healing Assay\u003c/h2\u003e \u003cp\u003eThe transfected breast cancer cells (4\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well) were spread into the six-well plate and cultured in an incubator of 5% CO\u003csub\u003e2\u003c/sub\u003e and 37\u0026deg;C for 48 h. When the cell fusion rate reached approximately 80\u0026ndash;90%, the cells were starved for 12 h. Subsequently, they were scraped using a 200 \u0026micro;L pipette tip and washed three times with PBS. Moreover, the cells were observed and photographed by phase contrast microscope at 0 h, 24 h and 48 h, and the mobility was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Transwell migration\u003c/h2\u003e \u003cp\u003ePrior to the experiment, transfected cells were serum-starved for 12 h. Transwell chambers (8.0 \u0026micro;m pore size, Corning, USA) were placed in 24-well plates. A serum-free suspension of breast cancer cells (1\u0026times;10^4 cells/200 \u0026micro;L) was added to the upper chamber, ensuring no bubbles were introduced. Meanwhile, 600 \u0026micro;L DMEM culture medium containing 20% FBS was added in the lower chamber. Matrigel was not used for coating in the experiment. After 48 h, the transwell chamber was removed then fixed with 100% methanol for 30 min, and stained with 0.1% crystal violet solution for 15 min. Fluorescence microscopy was used to observe.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Western Blots\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from breast cancer cells using RIPA buffer supplemented with 10% phosphatase inhibitor and 10% protease inhibitor. The concentration of the protein samples was detected with the BCA Protein Assay Kit ((Beyotime Biotechnology, China)). Protein samples were mixed with loading buffer (Yamei, China) at a 4:1 ratio and boiled at 100\u0026deg;C for 15 min in a metal bath (Mona, China). The protein samples were then separated on 10% SDS-PAGE gel, transferred to PVDF membranes (Millipore) and incubated in 5% bovine serum albumin for 2 h at room temperature, followed by incubation with specific primary antibodies overnight at 4\u0026deg;C. After washing three times with TBS-Tween 20 (0.1%, v/v), the membranes were incubated with the appropriate secondary antibodies for 1 h. After TBST washing, blots were examined using enhanced chemiluminescence (Millipore).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunoprecipitation (IP)\u003c/h2\u003e \u003cp\u003eAfter treatment, total protein was extracted from transfected cells, and an aliquot was used as input control. Protein lysates were incubated overnight at 4\u0026deg;C with 2 \u0026micro;g of anti-Flag antibody or an appropriate IP antibody. Then, 20 \u0026micro;L of Protein A/G agarose beads (Santa Cruz Biotechnology) were added and incubated for 2 h. After centrifugation at 800 rpm to remove the supernatant, 30 \u0026micro;L of fresh beads were added and incubated for another 4 h at 4\u0026deg;C to form the immune complex. After centrifugation at 800 rpm for 4 min at 4\u0026deg;C and four washes with washing buffer, the beads were resuspended in 30 \u0026micro;L of 1\u0026times; loading buffer and boiled at 100\u0026deg;C for 10 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Ubiquitination detection\u003c/h2\u003e \u003cp\u003eFor the ubiquitination assay, MCF-7 and BT549 cells were co-transfected with Flag-TRIM45 (0.5 \u0026micro;g), Myc-p62 (0.5 \u0026micro;g), and HA-Ub (1.0 \u0026micro;g) or corresponding empty vectors using liposome transfection reagent (Genechem, China) in six-well plates. At 48 h post-transfection, cells were treated with the proteasome inhibitor MG132 (MedChemExpress, USA) at a final concentration of 20 \u0026micro;M for 6 h before harvest. Total cell lysates were immunoprecipitated with anti-Myc antibody (Proteintech, China) and protein A/G agarose beads (Santa Cruz, USA), followed by immunoblotting with anti-HA antibody (Proteintech, China) to detect ubiquitinated p62.\u003c/p\u003e \u003cp\u003eTo assess protein stability, cells transfected with Flag-TRIM45 or empty vector in six-well plates were divided into four time-point groups (0, 2, 4, and 8 h). At 48 h post-transfection, cycloheximide (CHX, Sigma-Aldrich, USA) was added to the culture medium at a final concentration of 25 \u0026micro;g/mL to inhibit de novo protein synthesis. Cells were harvested at the indicated time points, and total proteins were extracted for Western blot analysis of p62 expression. Protein half-life was determined by densitometric analysis of p62 bands normalized to GAPDH.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Determination of malondialdehyde (MDA) content, glutathione (GSH) content and Glutathione peroxidase (GSH-Px) activity.\u003c/h2\u003e \u003cp\u003eAt 72 h of transfection, cells were washed three times with PBS. Cells were lysed and the lysates were sonicated on ice for 3 min. The supernatant was collected by centrifugation at 10,000 g for 15 min at 4\u0026deg;C. The levels of MDA, GSH, and GSH-Px activity in the supernatant were measured using commercial biochemical kits (Cat# S0131M for MDA, Cat# S0052 for GSH, and Cat# S0056 for GSH-Px; Beyotime Biotechnology, Shanghai, China) according to the manufacturer\u0026lsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Detection of ROS\u003c/h2\u003e \u003cp\u003eIntracellular superoxide anion levels were detected using a DHE-based ROS assay kit (Cat# S0064S; Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. Briefly, 72 h after transfection, cells cultured in 6-well plates were washed gently with PBS and incubated with 1 mL of DHE staining solution (diluted to a final concentration of 50 \u0026micro;M with DMEM) at 37\u0026deg;C for 50 min in a cell culture incubator protected from light. Subsequently, the red fluorescence intensity (excitation/emission\u0026thinsp;=\u0026thinsp;535/610 nm) was observed and captured under a fluorescence microscope. The DHE stock solution was stored at -20\u0026deg;C protected from light and aliquoted to avoid repeated freeze-thaw cycles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, USA). For comparisons between two groups, unpaired two-tailed Student\u0026lsquo;s t-test was used. For comparisons involving multiple groups, one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post-hoc test was applied. For experiments with a 2 \u0026times; 2 factorial design (e.g., TRIM45 overexpression combined with TGF-β treatment), two-way ANOVA followed by Sidak's multiple comparisons test was used to assess the main effects and interaction between factors. Image processing and densitometric analysis were performed using ImageJ software (NIH, USA). A value of *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001)).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 TRIM45 expression is downregulated in breast cancer patients, which indicates poor prognosis of breast cancer\u003c/h2\u003e \u003cp\u003eTo evaluate the expression profile of TRIM45 in breast cancer, we analyzed transcriptomic data obtained from the Gene Expression Omnibus (GEO) database. Specifically, we retrieved datasets GSE5364 and GSE3744 to compare TRIM45 levels between breast cancer tissues and adjacent normal tissues. As illustrated \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, TRIM45 expression was significantly downregulated in tumor tissues compared to normal controls.\u003c/p\u003e \u003cp\u003eTo investigate the prognostic significance of TRIM45 in breast cancer, we retrieved clinical data from the TCGA database via the UCSC Xena browser. We performed Kaplan-Meier analysis for Overall Survival (OS), Progression-Free Survival (PFS), Disease-Specific Survival (DSS), and Disease-Free Survival (DFS). The results revealed statistically significant differences in OS, PFS, DSS and DFS. Specifically, high TRIM45 expression was associated with significantly better survival outcomes, whereas low expression was predictive of a poor prognosis in breast cancer patients (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D\u003cb\u003e).\u003c/b\u003e The difference was not statistically significant in the disease-free survival group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. These findings indicate that TRIM45 is downregulated in breast cancer tissues, suggesting that loss of TRIM45 expression may contribute to breast cancer progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 TRIM45 inhibited the proliferation of breast cancer cells\u003c/h2\u003e \u003cp\u003eTo explore the effect of TRIM45 on the malignant development of breast cancer cells, we established two TRIM45 overexpressed cell lines, MCF-7 and BT549. CCK-8 experiment showed that the proliferation rate of TRIM45 overexpression cell lines was significantly reduced compared to the control cells \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Meanwhile, EdU incorporation assays revealed significantly reduced fluorescence intensity in TRIM45-overexpressing cells compared to controls. \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-F\u003cb\u003e)\u003c/b\u003e. These results indicate that TRIM45 overexpression inhibits the proliferation of breast cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 TRIM45 inhibits breast cancer cell migration by suppressing EMT and metastasis-related protein expression\u003c/h2\u003e \u003cp\u003eTo further investigate the effect of TRIM45 on breast cancer cell migration, we performed transwell assays using TRIM45-overexpressing MCF-7 and BT549 cells. Transwell assays showed that the number of migratory cells was significantly reduced in TRIM45-overexpressing MCF-7 and BT549 cells compared to controls \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D\u003cb\u003e).\u003c/b\u003e Consistently, wound healing assays revealed that the migration rate was markedly decreased in TRIM45-overexpressing cells at 24 h and 48 h post-scratching \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-H\u003cb\u003e)\u003c/b\u003e. These results demonstrate that TRIM45 overexpression significantly impairs the migratory capacity of breast cancer cells.\u003c/p\u003e \u003cp\u003eTo elucidate the molecular basis underlying this phenotypic suppression, we examined the expression of epithelial-mesenchymal transition (EMT) markers and metastasis-related proteins. Western blot analysis showed that TRIM45 overexpression led to a significant increase in the epithelial marker E-cadherin, accompanied by decreased expression of mesenchymal markers \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e. Furthermore, the expression of matrix metalloproteinases (MMP2 and MMP9) and the pro-angiogenic factor VEGF were markedly downregulated upon TRIM45 overexpression \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL\u003cb\u003e)\u003c/b\u003e. Collectively, these findings indicate that TRIM45 suppresses breast cancer cell migration by inhibiting EMT progression and downregulating key metastasis-related proteases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 TRIM45 binds to p62 and promotes its ubiquitination-dependent degradation\u003c/h2\u003e \u003cp\u003ePrevious studies have demonstrated that TRIM45 plays a significant role in the biological processes of breast cancer cells; however, the specific molecular mechanisms underlying its function as an E3 ubiquitin ligase remain incompletely understood. To further investigate TRIM45 function, we used MCF-7 and BT549 cells stably overexpressing TRIM45 or empty vector as controls. Co-immunoprecipitation (Co-IP) assays using FLAG antibody-conjugated agarose beads were performed to identify potential interacting proteins of TRIM45. The results revealed that p62 specifically interacts with TRIM45 in breast cancer cells \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, suggesting a direct protein-protein interaction between the two.\u003c/p\u003e \u003cp\u003eTo assess the impact of TRIM45 on p62 protein stability, we performed cycloheximide (CHX) chase assays. The results demonstrated that the half-life of p62 was significantly shortened in TRIM45-overexpressing MCF-7 and BT549 cells \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e,indicating that TRIM45 promotes p62 degradation. Consistently, TRIM45 overexpression markedly reduced the protein level of p62, and this effect was reversed by treatment with the proteasome inhibitor MG132 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e, suggesting that TRIM45 regulates p62 degradation via the ubiquitin-proteasome pathway.\u003c/p\u003e \u003cp\u003eTo further validate whether p62 is a direct ubiquitination substrate of TRIM45, we performed ubiquitination assays by immunoprecipitating Flag-tagged TRIM45 and then immunoblotting for ubiquitin. This approach allows us to specifically assess the ubiquitination status of p62 that is in complex with TRIM45. The results showed that the ubiquitination level of p62 co-immunoprecipitated with TRIM45 was significantly enhanced in cells expressing exogenous TRIM45 \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e, supporting the notion that p62 is a direct target of TRIM45-mediated ubiquitination.\u003c/p\u003e \u003cp\u003eIn summary, these findings suggest that TRIM45 promotes the degradation of p62 through interaction and ubiquitination, thereby regulating its stability via the ubiquitin-proteasome pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 TRIM45-mediated p62 degradation activates Keap1 and suppresses Nrf2 signaling\u003c/h2\u003e \u003cp\u003ePrevious studies have established that the KIR domain of p62 binds to the Keap1-DC domain, the same region responsible for Nrf2 interaction, thereby competitively disrupting the Keap1\u0026ndash;Nrf2 complex. As the Keap1/Nrf2 signaling pathway serves as a central signaling cascade governing cellular resistance to oxidative stress, we sought to determine whether TRIM45 influences this pathway. To this end, we evaluated the expression of Nrf2 and Keap1 following TRIM45 overexpression. Our results showed that TRIM45 upregulation led to decreased Nrf2 protein levels and a concomitant increase in Keap1 expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. We hypothesize that this effect stems from TRIM45-mediated degradation of p62, which alleviates p62-dependent sequestration of Keap1 and thus enhances Nrf2 ubiquitination and degradation. Consistent with this mechanism, nuclear-cytoplasmic fractionation assays combined with Western Blot analysis confirmed a pronounced attenuation of Nrf2 nuclear translocation upon TRIM45 overexpression \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Collectively, these findings indicate that TRIM45 not only impairs cellular antioxidant defense but may also interfere with growth factor signaling pathways pivotal for cancer progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6 TRIM45 suppresses the p62/Keap1/Nrf2 pathway and downregulates IGF-1R expression through oxidative stress\u003c/h2\u003e \u003cp\u003eGiven that TRIM45 suppresses breast cancer progression through p62 degradation, leading to Nrf2 destabilization and oxidative stress, we further examined its effect on the IGF system, a critical pathway for breast cancer proliferation and survival. Given the established crosstalk between oxidative stress and growth factor signaling, we asked whether TRIM45-driven oxidative stress influences IGF pathway activity. Indeed, TRIM45 overexpression markedly reduced both basal and TGF-β-induced expression of IGF-1 and its receptor IGF-1R \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Previous studies have demonstrated that Nrf2 can transcriptionally regulate growth factor receptors, including IGF-1R (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In line with this, our results suggest that TRIM45-mediated Nrf2 degradation leads to decreased IGF-1R levels, likely through the attenuation of Nrf2-driven transcription. In parallel, TRIM45 downregulated phosphorylation of ERK (p-ERK), a signaling hub associated with cell proliferation \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo further assess the redox state under TRIM45 overexpression, we measured key oxidative stress markers. Results showed a significant increase in malondialdehyde (MDA) content, accompanied by decreased glutathione (GSH) levels and reduced activity of glutathione peroxidase (GSH-Px) \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-G\u003cb\u003e)\u003c/b\u003e. These alterations collectively indicate a compromised cellular antioxidant system and enhanced oxidative stress. Consistent with this, reactive oxygen species (ROS) were significantly elevated in TRIM45-overexpressing cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. Together, these data demonstrate that TRIM45 disrupts redox homeostasis and concomitantly attenuates key proliferative and survival signaling pathways in breast cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe metabolic adaptation of cancer cells to survive under persistent oxidative stress represents a fundamental mechanism of tumor progression and therapeutic resistance. In this study, we identified a previously unrecognized regulatory cascade involving the TRIM45, p62, Keap1, Nrf2, and IGF-1R signaling axis, which governs the progression of breast cancer (38). Our findings demonstrate that TRIM45 functions as a tumor-suppressive E3 ubiquitin ligase that specifically targets p62 for proteasomal degradation. This event subsequently reinstates the Keap1-mediated suppression of Nrf2, thereby disrupting the antioxidant defense system and growth factor signaling pathways simultaneously (33).\u003c/p\u003e\n\u003cp\u003eThe master regulator of cellular redox homeostasis, Nrf2, is frequently hijacked by malignant cells to facilitate survival in hostile microenvironments. Under normal physiological conditions, Keap1 acts as a substrate adaptor for the Cul3-based E3 ligase complex to promote the constitutive degradation of Nrf2 (39, 40). However, the accumulation of p62 can competitively bind to the Kelch domain of Keap1 through its KIR motif, which leads to the non-canonical activation of Nrf2 (41, 42). Our data provide compelling evidence that the loss of TRIM45 in breast cancer tissues facilitates the accumulation of p62 (43), thereby sequestering Keap1 and allowing Nrf2 to translocate into the nucleus. This transcriptional reprogramming upregulates downstream antioxidant enzymes such as HO-1 and GPX4, which effectively neutralize reactive oxygen species and prevent oxidative damage (44).\u003c/p\u003e\n\u003cp\u003eAn intriguing finding of this study is the crosstalk between Nrf2-mediated antioxidant responses and the IGF-1R/ERK survival pathway. We observed that the stabilization of Nrf2 not only enhances redox capacity but also sustains the expression of IGF-1R (45). Although the precise transcriptional mechanism remains to be fully elucidated, the concomitant reduction of IGF-1R and phosphorylated ERK upon TRIM45 overexpression suggests a synergistic suppressive effect. The downregulation of the IGF-1R/ERK axis significantly impairs the epithelial-mesenchymal transition process (46, 47), as evidenced by the restoration of E-cadherin and the loss of mesenchymal markers. This logical progression from biochemical modification to phenotypic alteration provides a mechanistic explanation for the impaired migratory and invasive capabilities of TRIM45-overexpressing cells.\u003c/p\u003e\n\u003cp\u003eThe induction of lethal oxidative stress through the targeted degradation of p62 represents a promising form of synthetic lethality in breast cancer therapy. Our clinical analysis using TCGA and GEO datasets\u0026nbsp;(Figures 7A and 7B) reinforces the relevance of this pathway, as patients with low TRIM45 expression and high p62/Nrf2 activity exhibit significantly poorer prognosis (48). These results suggest that the TRIM45 status could serve as a predictive biomarker for sensitivity to pro-oxidant therapies. Patients lacking this endogenous tumor suppressor may be particularly vulnerable to pharmacological agents that further challenge their compromised antioxidant defenses or inhibit the Nrf2-driven growth signaling.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study delineates a novel tumor-suppressive role for TRIM45 in breast cancer through the modulation of the p62/Keap1/Nrf2 axis. By promoting the ubiquitination of p62, TRIM45 effectively dismantles the cytoprotective shield provided by Nrf2 and attenuates oncogenic IGF-1R signaling. This dual mechanism not only inhibits primary tumor cell proliferation but also curtails metastasis by reversing the EMT program. Future investigations into the upstream signals that trigger TRIM45 downregulation will further enhance our understanding of breast cancer pathogenesis and may lead to the development of innovative therapeutic strategies that exploit redox vulnerabilities\u0026nbsp;(Figure 7C).\u003cbr clear=\"all\"\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTRIM45, Tripartite motif containing 45; Nrf2, Nuclear factor erythroid 2-related factor 2; Keap1, Kelch-like ECH-associated protein 1; HO-1, Heme oxygenase-1; GPX4, Glutathione peroxidase 4; EMT, Epithelial-mesenchymal transition; MMP, Matrix metalloproteinase; VEGF, Vascular endothelial growth factor; IGF-1, Insulin-like growth factor 1; IGF-1R, Insulin-like growth factor 1 receptor; ROS, Reactive oxygen species; GEO, Gene Expression Omnibus; TCGA, The Cancer Genome Atlas; UCSC Xena, University of California, Santa Cruz Xena; OS, Overall survival; PFS, Progression-free survival; DSS, Disease-specific survival; DFS, Disease-free survival; CCK-8, Cell counting kit-8; EdU, 4,5-Ethynyl-2'-deoxyuridine; DAPI, 4',6-Diamidino-2-phenylindole; IP, Immunoprecipitation; CHX, Cycloheximide; MDA, Malondialdehyde; GSH, Glutathione; GSH-Px, Glutathione peroxidase; DHE, \u0026nbsp;Dihydroethidium; SD, Standard deviation; NSCLC, Non-small cell lung cancer; MAPK, Mitogen-activated protein kinase; NF-κB, Nuclear factor kappa-B; RING, Really interesting new gene; RBCC, RING finger, B-box, coiled-coil; ElK-1, Ets-like protein 1; AP-1, Activator protein 1; PKC, Protein kinase C; RACK1, Receptor for activated C kinase 1; GSEA, Gene Set Enrichment Analysis; DMEM, Dulbecco's Modified Eagle Medium; FBS, Fetal bovine serum; RIPA, Radioimmunoprecipitation assay; BCA, Bicinchoninic acid; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, Polyvinylidene fluoride; TBST, Tris-buffered saline with Tween 20; HRP, Horseradish peroxidase\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.S and C.C: writing original draft, data curation, resources; F.S: language correction, chart correction; F.Y, H.Z, and X.Z: formal analysis, data curation; Z.W and C.Y: formal analysis, resources; Y.W and R.W: resources; L.Y: validation, formal analysis; Z.Z and B.L: supervision, conceptualization, writing review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Grants from the National Natural Science Foundation of China (No. 81871231 and 31401068). Youth Innovation and Science and Technology Plan of Colleges and Universities in Shandong Province (No. 2019KJK016), Shandong Taishan Scholars Young Experts Program (No. tsqn202103056), The Natural Science Foundation of Shandong Province (No. ZR2023MH082). Qingdao Natural Science Foundation Key Project (24-8-4-zrjj-8-jch).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to the TCGA databases for open access to the data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo application.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author upon reasonable request. 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Front Genet. 2022;13:876325.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, TRIM45, p62, Ubiquitination, Keap1/Nrf2 pathway, oxidative stress, IGF-1R ","lastPublishedDoi":"10.21203/rs.3.rs-9167161/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9167161/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOvercoming oxidative stress is a critical mechanism by which breast cancer cells sustain malignant progression and therapeutic resistance. However, the specific regulatory networks governing this adaptive response remain incompletely understood. Here, we demonstrate that TRIM45, a tumor-suppressive E3 ubiquitin ligase, acts as a key negative regulator of the antioxidant defense system in breast cancer. Clinically, TRIM45 is significantly downregulated in breast cancer tissues, and its low expression strongly correlates with poor patient prognosis. Functionally, ectopic expression of TRIM45 suppresses proliferation, migration, and epithelial-mesenchymal transition (EMT) in MCF-7 and BT549 cells. Mechanistically, we demonstrate that TRIM45 promotes p62 ubiquitination and degradation in breast cancer cells. By promoting the proteasomal degradation of p62, TRIM45 liberates Keap1. This is accompanied by enhanced Nrf2 ubiquitination and restricted nuclear translocation, suggesting a mechanism whereby TRIM45-mediated p62 degradation contributes to Nrf2 pathway suppression.This dismantling of the Keap1/Nrf2 signaling pathway not only suppresses downstream antioxidant targets (HO-1, GPX4) to induce lethal oxidative stress, characterized by elevated ROS and MDA levels alongside reduced GSH and GSH-Px activity, but also attenuates the IGF-1/IGF-1R/ERK signaling pathway. Collectively, our study uncovers a previously unrecognized TRIM45/p62/Keap1/Nrf2 regulatory signaling pathway, establishing TRIM45 as a promising therapeutic target that exploits oxidative vulnerability in breast cancer.\u003c/p\u003e \u003cp\u003eOvercoming oxidative stress is a critical adaptive mechanism for breast cancer cells to sustain malignant progression and therapeutic resistance. This study identifies TRIM45, a tumor-suppressive E3 ubiquitin ligase, as a key negative regulator of the antioxidant defense system in breast cancer. Clinically, TRIM45 is significantly downregulated in breast cancer tissues, and its low expression strongly correlates with poor patient prognosis. Functionally, ectopic expression of TRIM45 suppresses proliferation, migration, and epithelial-mesenchymal transition in MCF-7 and BT549 cells. Mechanistically, we demonstrate that TRIM45 directly interacts with p62 and promotes its ubiquitination-dependent proteasomal degradation. Concomitant with p62 reduction, we observed the restoration of Keap1 protein levels, coupled with decreased protein abundance and impaired nuclear translocation of the transcription factor Nrf2. This led to the downregulation of downstream antioxidant genes (HO-1, GPX4) and attenuation of the IGF-1/IGF-1R/ERK signaling pathway. Collectively, these alterations induced lethal oxidative stress, characterized by ROS accumulation and depletion of the glutathione system. Our findings reveal that TRIM45 exerts a tumor-suppressive role in breast cancer by targeting p62 for degradation and thereby disrupting the Keap1/Nrf2 antioxidant axis, nominating TRIM45 as a novel candidate for therapeutic strategies aimed at exploiting oxidative vulnerability.\u003c/p\u003e","manuscriptTitle":"TRIM45 Suppresses Breast Cancer Progression by Regulating Nrf2/Keap1 Pathway through Degradation of p62","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-20 16:54:20","doi":"10.21203/rs.3.rs-9167161/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-13T03:50:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-20T13:11:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-20T13:10:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Cell International","date":"2026-03-19T08:36:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"215f8bb0-1f0c-4372-9ce0-75c1edd801f9","owner":[],"postedDate":"April 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T16:54:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-20 16:54:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9167161","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9167161","identity":"rs-9167161","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}