Abstract The incidence of breast cancer continues to increase annually, posing a significant challenge for countries worldwide in terms of its prevention and treatment. Therefore, identifying novel therapeutic targets for breast cancer is urgently needed. The peroxiredoxin (PRDX) family is regarded as a good diagnostic marker for various tumors. However, the expression and prognostic significance of PRDX family members in breast cancer remain unclear and require systematic investigation. By using bioinformatic tools such as UALCAN, TIMER2.0, Human Protein Atlas Project (HPA), Gene Set Cancer Analysis (GSCA), and the cBioportal database, we systematically analyzed the expression pattern, prognostic value, methylation status and immune infiltrating association of PRDX gene family members in breast cancer. Through comprehensive analysis, we found that PRDX4 has good prognostic value and is closely related to immune infiltration, and further exploration of its oncogenic function in breast cancer is warranted. Subsequently, we performed a series of cellular assays to explore the potential role of PRDX4 in the progression of breast cancer. We demonstrated that PRDX4 promoted the proliferation, invasion, metastasis, and inhibited the apoptosis of breast cancer cells. In addition, PRDX4 expression was associated with the half maximal inhibitory concentration (IC[50]) of neratinib which primarily targets human epidermal growth factor receptor 2 (HER2) and showed good binding in molecular docking. Our subsequent experiments showed that the PRDX4-HER2 axis may serve as a potential combined target for neratinib therapy. Our findings suggest that PRDX4 may be a potential diagnostic and prognostic marker for breast cancer, and targeting PRDX4 could represent a novel strategy to improve the efficacy of targeted therapy for patients with HER2-positive breast cancer. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-025-13361-0. Keywords: PRDX4, Breast cancer, Prognosis, Immune infiltration, Drug resistance, Therapeutic target Subject terms: Cancer, Biomarkers Introduction Breast cancer is a malignant tumor that occurs in the epithelium of the breast and develops when the growth of breast cells is uncontrolled due to genetic alterations or hormonal disorders^[38]1,[39]2. According to the degree of infiltration and metastasis, breast cancer can be categorised into non-invasive breast cancer, which includes carcinoma in situ, and invasive breast cancer, which has the potential to invade and metastasise^[40]3,[41]4. Currently, the incidence of breast cancer is rising at an alarming rate, surpassing the incidence of lung cancer worldwide^[42]5. Furthermore, it is predominantly diagnosed in females, with only a small proportion of cases occurring in males (approximately 1%). Breast cancer has become the malignant tumor with the highest morbidity and mortality rates among females worldwide^[43]6. Its increasing incidence and disease burden have made its prevention and treatment challenging in various countries^[44]7. Therefore, identifying additional drug targets and biomarkers for the diagnosis, prevention, and treatment of breast cancer is urgently needed. The peroxiredoxin (PRDX) family, which contains six members: PRDX1, PRDX2, PRDX3, PRDX4, PRDX5, and PRDX6, can utilise thioredoxin as an intermediate electron donor to reduce reactive oxygen species (ROS) accumulation, thus exerting antioxidant protective functions against oxidative stress. It is also involved in various oxidative signalling^[45]8 and exhibits multiple functions in regulating cell proliferation, differentiation, apoptosis, senescence, cancer, and other diseases^[46]9. Several recent studies have shown that PRDX family members are highly expressed in various tumors and function as oncogenes. For example, a study on gastric cancer (GC) found that inhibition of PRDX2 increased cellular ROS levels and led to ROS-dependent endoplasmic reticulum stress, mitochondrial dysfunction, and apoptosis in GC cells, highlighting the role of PRDX2 as a potential target for GC therapies^[47]10. A study on colorectal cancer (CRC) demonstrated that knockdown of PRDX1 and PRDX2 increased ROS production in CRC cells, leading to mitochondrial dysfunction and decreased ATP production, which significantly inhibited the proliferation of CRC cells^[48]11. In addition, PRDX family members are highly expressed in hepatocellular carcinoma, prostate adenocarcinoma, glioma, and lung cancer. They are regarded as good prognostic indicators and potential therapeutic targets in these cancers^[49]12–[50]15. However, studies on the prognostic and therapeutic value of this gene family in invasive breast cancer have not yet been conducted. Therefore, in this study, we analyzed the role of PRDX family members as oncogenes in breast cancer from multiple aspects, including expression profiles, clinical significance, prognostic value, co-expression gene pathway enrichment, and immune cell infiltration. Consequently, we identified PRDX4 as a potential prognostic marker. In multiple myeloma, PRDX4 maintains immunoglobulin production and sensitivity to proteasome inhibitors through an OTUD1-dependent mechanism^[51]16. In endometrial cancer, overexpression of PRDX4 is associated with cell proliferation, migration and its high expression is significantly correlated with poor prognosis^[52]17. The role of PRDX4 as an oncogene in breast cancer has not been yet reported. Therefore, through this study, we aimed to further explore the carcinogenic role of PRDX4 in breast cancer through bioinformatics analysis and cellular functional experiments. Materials and methods XianTao tool The XianTao platform ([53]https://www.xiantaozi.com/) is a useful web tool for bioinformatics analysis. We used it to visualize the expression of PRDX family members in pan-cancer from the The Cancer Genome Atlas (TCGA) as well as GTEx databases of PRDX family members in invasive breast cancer. Statistical analysis were performed using the Wilcoxon rank sum test, with significant results defined as p < 0.05. Additionally, by integrating TCGA datasets available on the platform, we evaluated the prognostic value of PRDX family members in invasive breast cancer through Cox regression analysis. Furthermore, we integrated TCGA datasets from the platform, performed proportional hazards assumption tests using the survival package, and conducted multivariate Cox regression analysis. GSCA analysis Gene Set Cancer Analysis (GSCA) ([54]http://bioinfo.life.hust.edu.cn/) is an open and easily accessible online platform^[55]18. We accessed this database to obtain the expression of PRDXs family members in breast cancer as well as to explore the association of PRDX4 expression levels with the half maximal inhibitory concentration (IC[50]) of CTRP drugs. HPA analysis The Human Protein Atlas Project (HPA) ([56]http://www.proteinatlas.org/) is an open protein database based on proteomics, transcriptomics, and systems biology data^[57]19. We used it to analyze the protein expression of PRDXs family members in breast cancer tissues versus adjacent normal tissues. UALCAN analysis UALCAN ([58]http://ualcan.path.uab.edu), based on the TCGA database, is a convenient and comprehensive multi-omics analysis website that can help users explore the expression differences of target genes in tumor and normal tissues. It also facilitates the analysis of gene expression and the significance of tumor clinicopathological features^[59]20. We used this database to investigate the association between PRDX family members and lymph node metastasis and distant metastasis. cBioportal analysis Covering data from twenty-eight thousand specimens, cBioportal ([60]http://www.cbioportal.org/) integrates data from 126 tumor genome studies, including somatic mutations, DNA copy number alterations (CNAs), mRNA and microRNA (miRNA) expression, and DNA methylation, and allows for a variety of analysis, but most notably a variety of analysis related to mutations and their visualization^[61]21. We used it to acquire the co-expressed genes of PRDX family members in breast cancer. STRING analysis STRING ([62]https://cn.string-db.org/) is a database that searches for interactions between proteins and can be used to explore both direct physical interactions and indirect functional correlations between proteins. We used this tool to explore interactions between PRDX family co-expressed genes in breast cancer. Cytoscape analysis Cytoscape v3.9.1 ([63]https://cytoscape.org/) is an open-source software platform with powerful capabilities to analyze protein-protein interactions, protein-DNA and genetic interactions^[64]22. Thus, we utilized Cytoscape v3.9.1 to construct PPI network maps of co-expressed genes of the PRDX family. Metascape analysis Metascape ([65]https://metascape.org/) is a convenient gene enrichment analysis database that can be used to accomplish pathway enrichment and biological process annotation^[66]21. We used it for pathway enrichment analysis of the PRDX family. TIMER2.0 analysis TIMER2.0 ([67]http://timer.cistrome.org/) is an open query tool for immune infiltration^[68]23. We used TIMER 2.0 to explore the association between PRDX family members and immune cell infiltration in breast cancer. CancerSEA analysis CancerSEA ([69]http://biocc.hrbmu.edu.cn) is a multifunctional website designed to comprehensively explore the different functional states of cancer cells at the single-cell level^[70]24. We used it to explore the correlation between PRDX4 expression and different functional states of breast cancer. PubChem analysis PubChem ([71]https://pubchem.ncbi.nlm.nih.gov) is a useful website for searching biological activity data of organic small molecules^[72]25. We used it to obtain information on the substitution 3D structure of neratinib. RCSB PDB analysis The RCSB PDB ([73]https://www.rcsb.org/) is a member of the World Protein Data Bank, which stores three-dimensional structural information of biological macromolecules such as DNA, RNA, and proteins^[74]26. We used it to access and download the protein structure information of PRDX4. Autodock4 analysis Autodock4 is an open source molecule-ligand docking software. By processing molecule and ligand files and then performing calculations, the corresponding molecular docking files can be obtained, which can be further visualized and analyzed^[75]27. We utilized Autodock4 to perform the prediction of molecular docking of PRDX4 with neratinib. PyMol analysis PyMol ([76]http://www.pymol.org) is a Python-based molecular 3D structure display software, integrating mapping, animation and presentation, which can be used to create high-quality 3D structure images of small molecules or biomolecules (especially proteins). We used PyMol to visualize the docking files of PRDX4 and neratinib. Discovery studio analysis We used it to analyze the interaction force of PRDX4 with neratinib, as well as to access the docking activity pocket. PLP analysis We used it to obtain the bonding profile of the active site of PRDX4 interaction with neratinib ([77]https://plip-tool.biotec.tu-dresden.de)^[78]28. BEST analysis We utilized the BEST database ([79]https://rookieutopia.com) to explore the associations between PRDX4 and human epidermal growth factor receptor 2 (HER2) expression in breast cancer, as well as the correlation between PRDX4 and the sensitivity to neratinib. Openbabel analysis We use Openbabel analysis to perform file format conversions, the methods as the previously study^[80]29. Human tissue collection, RNA extraction, and detection From January 2020 to December 2023, we retrospectively collected the formalin-fixed paraffin-embedded (FFPE) specimens of 29 individuals with breast cancer and paired normal tissues from Xiangya Hospital of Central South University. The 29 pairs of breast cancer FFPE specimens including 12 pairs of HER2-positive as well as 17 pairs of other subtypes of breast cancer and paracarcinoma paraffin tissue specimens. None of the patients received any form of treatment, including radiotherapy, chemotherapy, or immunotherapy, prior to resection. All patients provided informed consent. This study was approved by the Ethics Committee of Xiangya Hospital (Approval No. 202401013). After the deparaffinization, the 29 pairs of breast cancer FFPE specimens with xylene, total RNA was extracted using total RNA AmoyDx^® FFPE RNA Extraction Kit (Cat. #8.02.0019). Meanwhile, RNA was extracted from breast cancer cell lines and normal mammary epithelial cells using the RNA simple Total RNA Kit (TIANGEN, China). cDNA was obtained using a reverse transcription system. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the RNA expression level of PRDX4 in 29 breast cancer patients, the expression of PRDX4 in breast cancer cell lines relative to normal epithelial tissues, and the expression level of HER2 in SKBR3 cells under different treatments. The primer sequences are shown in Table [81]1. Table 1. Primer sequence for qRT-PCR. Gene Primer (forward) Primer (reverse) PRDX4 AGAGGAGTGCCACTTCTACG GGAAATCTTCGCTTTGCTTAGGT U6 CTCGCTTCGGCAGCACAA AACGCTTCACGAATTTGCGT [82]Open in a new tab Cell culture The human normal mammary epithelial cells (mcf-10a) and breast cancer cell lines (SK-BR-3, MCF-7, ZR-75-1, and MDA-MB-231) used in the experiments were obtained from the Chinese Academy of Sciences (CAS) Type Culture Cell Bank. MDA-MB-231 cells were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin in a 5% CO[2] humidified incubator at 37 degrees Celsius. All cells were free from mycoplasma, bacterial, and fungal contamination. Small interference and cell transfection The small interference RNA (siRNA) was obtained commercially from Ribobio (China), the transfection reagent was lip3000, and the interfering sequence was listed in Table [83]2 below. The specific steps of cell transfection were referred to by Feng et al.^[84]30. Table 2. Sequence of PRDX4 SiRNA used in the study. siRNA name Sequence PRDX4siRNA-1 5′-GCAAAGCGAAGATTTCCAA-3′ PRDX4siRNA-2 5′-GCGACAGACTTGAAGAATT-3′ [85]Open in a new tab Drug sensitivity assay Cells were seeded into 6-well plates. After transfection with siPRDX4 and control siRNA according to the experimental design, the cells were incubated for 24 h. Subsequently, an appropriate concentration of neratinib (100 nM) was added, and after another 24-hour incubation, the cells were re-seeded into 96-well plates at a density of 10,000 cells per well. Following a 24-hour incubation period, cell viability was measured according to the standard experimental procedure of the Cell Counting Kit-8 (CCK-8). 5-Ethynyl-2′-deoxyuridine (EdU) assay RiboBio’s EdU kit (C10310-3) was used for the EdU assay. Experimental procedures were performed according to the kit instructions. Colony formation assay Each group was taken 1000 cells planted in six-well plates for clone formation assay, incubated at 37℃ for 10 days. After PBS washing, methanol was fixed for 15 min. Then, 1% crystal violet staining was performed for 30 min at room temperature. Apoptosis test The apoptosis kit used was AP101-100-kit (MULTI SCIENCE, China), and apoptotic cells were detected by flow cytometry by configuring each sample according to the steps in the kit instructions. Migration and invasion assay For cell migration ability assay, MDA-MB-231 cells transfected with siPRDX4 and NC were planted into the upper chamber in serum-free medium at a density of 2*10^4 cells/well, and the lower chamber was incubated with 600 µL of 20% FBS medium for 30 h at 37℃. For cell invasiveness assay, 10 µL of matrix gel (Corning) (serum-free medium ratio: matrix gel = 10:1) was added to the upper chamber to solidify, and then the same density of cells was planted in the upper chamber and the cells were incubated at 37 °C for 40 h. Remove the small chambers. Fix the transferred cells with 4% paraformaldehyde for 30 min. Then, dye the lower surface of the membrane with 1% crystal solution for 30 min. At last, count the cells on the lower side of the membrane. Statistical analysis Data were analyzed using GraphPad Prism 7.0 software. Survival analysis was conducted via the log-rank test, while Spearman’s correlation was employed to evaluate the connection between the PRDXs family and indicators of immune cell type and immunological infiltration. The two independent samples were contrasted utilizing Student’s t-test. p < 0.05 was set as the significance criterion. Results Abnormal expression of the PRDXs family in pan-cancer We used the Xiantao platform tool to explore TCGA data and investigate the expression patterns of the PRDX family members in various cancers. As shown in our results, the transcript expression levels of PRDX1, PRDX2, PRDX3, PRDX4, and PRDX5 were significantly upregulated in breast cancer, whereas that of PRDX6 was decreased (Fig. [86]1A). Based on normalised and batch-corrected RSEM mRNA expression, GSCA, which has over 10 paired tumor and normal samples, provides differential expression analysis for 14 TCGA cancer types. Thus, we explored the expression levels of PRDX family members in breast cancer using GSCA to make a complement. We found that the expression of PRDX1, PRDX2, and PRDX4 was significantly enhanced in breast cancer tissues compared with that in normal tissues. However, PRDX6 expression in breast cancer was considerably lower than that in normal samples (Fig. [87]1B). Fig. 1. [88]Fig. 1 [89]Open in a new tab Expression of PRDX family members in breast cancer. (A) Expression of each PRDX family gene in pan-cancer and adjacent normal tissues performed by Wilcoxon rank sum test in XianTao tool (n = 11,123). (B) Expression of the PRDXs family in 1,104 breast cancer and 114 normal samples was estimated using a t-test and further adjusted by FDR. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colorectal adenocarcinoma; DLBC, diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma. Immunohistochemical images were analyzed using the HPA database to verify these results. As shown in Fig. [90]2 shows that the protein levels of all PRDX family members in breast cancer were significantly elevated compared with those in normal samples. Fig. 2. [91]Fig. 2 [92]Open in a new tab Expression levels of the PRDX genes in breast cancer at protein levels. (A–F) the immunohistochemical images displaying the protein levels of PRDX1, PRDX2, PRDX3, PRDX4, PRDX5 and PRDX6 in breast cancer and normal samples. Association between the expression of PRDX family members and clinicopathological features in patients with breast cancer The UALCAN database was used to investigate the link between PRDX gene expression and breast cancer clinicopathological features, including pathological stage and nodal metastasis status. Our results illustrated that PRDX1, PRDX2, PRDX4, and PRDX5 had higher expression levels at all stages than normal tissues. However, no differences in PRDX3 expression were observed at the transcript level between breast cancer stages 1–4 and normal samples. In addition, the mRNA expression of PRDX6 at all breast cancer stages was downregulated compared with that in normal tissues (Fig. [93]3A). Moreover, we found that the mRNA levels of members of the PRDX family in breast cancer based on lymph nodule metastasis were consistent with the expression patterns based on breast cancer stages. As shown in Fig. [94]3B, the expression patterns of PRDX1, PRDX2, PRDX4, and PRDX5 were enhanced in all breast cancer nodal metastasis statuses, whereas no statistical differences in PRDX3 expression were observed between breast cancer with N0–4 status and normal breast tissues. Among the PRDX family members, PRDX1, PRDX2, and PRDX4 displayed the lowest expression in N4, which was highly correlated with the prognosis of patients with breast cancer. In addition, PRDX6 mRNA expression in all breast cancer nodal metastasis was lower than that in normal samples. These findings demonstrate that PRDX family members play important roles in breast cancer progression. Fig. 3. [95]Fig. 3 [96]Open in a new tab Relationship of PRDX gene family expression with clinicopathological features in patients with breast cancer. (A) Expression levels of PRDX family members in different breast cancer stages and normal samples. (B) PRDX gene expression levels in normal samples and breast cancer with different lymph nodal metastasis status. *p < 0.05, **p < 0.01, ***p < 0.001. BRCA, breast cancer. Prognostic value of PRDX family members in patients with breast cancer We used the Xiantao platform to investigate the association of PRDX genes with prognostic indicators of overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI). As shown in Fig. [97]4A higher PRDX1, PRDX3, and PRDX4 mRNA expression levels were closely linked to poorer OS. As shown in Fig. [98]4B, enhanced expression of PRDX4 was associated with poor DSS and PFI (Fig. [99]4C). Among all the PRDX family genes, PRDX4, whose high expression is statistically correlated with poor OS, DSS, and PFI, was closely associated with the prognosis of breast cancer. Furthermore, multivariate Cox regression analysis also showed that PRDX4 could serve as an independent prognostic marker for breast cancer (Fig. [100]S2). Thus, we speculate that PRDX4 can serve as a biomarker for breast cancer prognostic detection. Fig. 4. [101]Fig. 4 [102]Open in a new tab Prognostic analysis of the PRDX family members in patients with breast cancer. (A–C) Statistical association of the PRDX genes mRNA expression levels with OS (n = 1086), DSS (n = 1066), and PFI (n = 1086) in breast cancer determined by using the Wilcoxon rank sum test. TCGA samples were split into high- and low-expression groups based on the mRNA expression value of the mean cutoff. Prognostic analysis was assessed by log-rank tests, and p < 0.05 meant statistical significance. OS, overall survival; DSS, disease-specific survival; PFI, progression-free interval. Enrichment analysis of PRDX family genes The cBioportal website tool was also used to identify the co-expressed genes of the PRDX family with threshold values of |log[2] fold-change[FC]| ≥ 1 and p < 0.05 (Supplementary Table 1). To better exhibit the correspondence between these genes, we used Cytoscape to visualize them in the form of a protein-protein interaction network (PPI) (Fig. [103]S1A). Metascape was used to perform a range of pathway enrichment analyses for PRDX co-expressed genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the co-expressed genes were mainly related to protein digestion and absorption, neuroactive ligand-receptor interaction, and ABC transporters (Fig. [104]S1B). Gene Ontology (GO) analysis revealed that the co-expressed genes were mainly associated with the extracellular matrix, calcium ion binding, and circulatory system processes (Fig. [105]S1C). Molecular function (MF) analysis demonstrated that these genes were widely involved in extracellular matrix structural constituents, calcium ion binding, and monoatomic ion-gated channel activity (Fig. [106]S1D). Biological process (BP) analysis suggested that the corresponding genes were mainly linked to circulatory system processes, kidney development, and cell–cell adhesion (Fig. [107]S1E). Cellular component (CC) analysis showed that co-expressed genes frequently participated in the extracellular matrix, dendritic tree, and postsynaptic specialisation membrane (Fig. [108]S1F). These findings demonstrate that members of the PRDX family influence the progression and drug resistance of patients with breast cancer and may serve as potential targets for breast cancer treatment. Relationship between immune cells infiltration and mRNA levels of PRDX family members in breast cancer TIMER2.0 was applied to explore the correspondence between immune cell infiltration and the expression of PRDX family members. The immune cell infiltration landscape is shown in Fig. [109]5, expression of PRDX1 was significantly associated with infiltration of B cells (correlation coefficient [cor] = − 0.173, p < 0.05), CD4 + Th2 cells (cor = 0.407, p < 0.05), macrophage (cor = 0.247, p < 0.05), and dendritic cells (DCs) (cor = 0.162, p < 0.05). PRDX2 mRNA was majorly associated with the infiltration of CD8 + cells (cor = − 0.113, p < 0.05), CD4 + Th2 cells (cor = 0.138, p < 0.05), and DCs (cor = 0.142, p < 0.05). PRDX3 mRNA expression was mainly correlated with DC infiltration (cor = 0.128, p < 0.05). PRDX4 mRNA levels significantly correlated with B cell infiltration (cor = 0.22, p < 0.05), CD8 + cells (cor = 0.129, p < 0.05), CD4 + Th2 cells (cor = 0.519, p < 0.05), macrophages (cor = 0.268, p < 0.05), and DCs (cor = 0.137, p < 0.05). PRDX6 mRNA expression was significantly associated with CD4 + Th2 cell infiltration (cor = 0.32, p < 0.05). However, the expression of PRDX5 was not closely linked to immune cell infiltration. These findings indicate the potential influence of PRDX family members on immune responses in the tumor microenvironment (TME) of patients with breast cancer. Fig. 5. [110]Fig. 5 [111]Open in a new tab Correlation between mRNA expression levels of the PRDX family members and immune cells infiltration. (A–F) The tumor immune environment landscape displayed the association between PRDX1-6 mRNA expression and various immune cell infiltration levels in patients with breast cancer (n = 1100). The partial Spearman’s correlation was used to perform association analysis. Analysis of potential biological functions of PRDX4 in breast cancer Based on a series of analyses, we identified PRDX4 as a potential prognostic and therapeutic target for patients with breast cancer. To assess the major role of PRDX4 in breast cancer, we used the CancerSEA database. The results demonstrated that PRDX4 mainly contributed to the processes of invasion, epithelial-mesenchymal transformation (EMT), and hypoxia in most tumors (Fig. [112]6A), including breast cancer (Fig. [113]6B). The t-distributed stochastic neighbor embedding (t-SNE) plot was used to display the expression distribution of PRDX4 in breast cancer at the single-cell level (Fig. [114]6C). Fig. 6. [115]Fig. 6 [116]Open in a new tab Single-cell analysis of PRDX4 in breast cancer. (A) A heatmap containing the correlation of PRDX4 with functional status in various malignancies. (B) The functional status is highly related to PRDX4 in breast cancer. (C) T-SNE plots displayed the single-cell distribution of PRDX4 expression. (D) PRDX4 expression level in different breast cancer cell lines. (E) The mRNA knockdown efficiency of PRDX4 in MDA-MB-231 cells. Each group was set up with three replicates. Student’s t-test was used for to compare groups, and the data were presented as the mean ± standard deviation (SD). **p < 0.01, ***p < 0.001, ***p < 0.0001. t-SNE, t-distributed stochastic neighbor embedding; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myeloid leukemia; CRC, colorectal cancer; BRCA, breast cancer; AST, astrocytoma; GBM, glioblastoma multiforme; HGG, high-grade glioma; ODG, oligodendroglioma; HNSCC, head and neck Squamous Cell Carcinoma; RCC, renal cell carcinoma; LUAD, lung adenocarcinoma; NSCLC, non-small cell lung cancer; OV, ovarian Cancer; PC, pancreatic cancer; MEL, melanoma; RB, retinoblastoma; UM, uveal melanoma. Oncogenic function determination of PRDX4 in breast cancer cells To further investigate the oncogenic function of PRDX4 in breast cancer, we performed qRT-PCR to determine the expression levels of PRDX4 in normal human mammary epithelial cells MCF-10a and breast cancer cell lines (HCC1806, MCF-7, SKBR3, ZR75-1, and MDA-MB-231). The results revealed that the mRNA levels of PRDX4 were significantly higher in various breast cancer cell lines (HCC1806, MCF-7, SKBR3, ZR-75-1, and MDA-MB-231) than in normal mammary epithelial cells. Notably, the mRNA level of PRDX4 in MDA-MB-231 cells was even higher than that in other breast cancer cell lines (Fig. [117]6D). Therefore, we characterized the oncogenic function of PRDX4 in these cell lines. qRT-PCR was used to characterize the efficiency of PRDX4 knockdown by each interfering fragment. The results showed that the knockdown efficiency of the PRDX4-2 interfering fragment was better and could be used for subsequent experiments (Fig. [118]6E). In the EdU assay, the proliferation rate of MDA-MB-231 cells decreased by 20% after PRDX4 knockdown (Fig. [119]7A, B). In the clone formation assay, the clone formation ability of MDA-MB-231 cells decreased by approximately 15% compared to that of the control group, indicating that the expression of PRDX4 regulates the proliferation of breast cancer cells (Fig. [120]7C, D). In the apoptosis assay, the apoptotic rate of MDA-MB-231 cells increased by only 1.5% after PRDX4 inhibition (Fig. [121]7H, I). Invasion and migration assays showed that the invasion rate of MDA-MB-231 cells decreased to 57% of that of the control group, and the migration rate decreased to 29.6% of that of the control group after PRDX4 silencing (Fig. [122]7E, G). These results demonstrate that PRDX4, as an oncogene in breast cancer, affects the proliferation, invasion, migration, and apoptosis of tumor cells. Figure [123]7J shows the oncogenic role of PRDX4 in breast cancer. Fig. 7. [124]Fig. 7 [125]Open in a new tab Impact of PRDX4 knockdown on the malignant features of breast cancer cells (A–D) The impact of PRDX4 on the proliferation of MDA-MB-231 cells detected using clone formation test and EdU uptake assays (Scale bar: 100 μm) (E–G) Detection of with/without PRDX4 on the invasive and migratory ability of MDA-MB-231 cells (Scale bar: 100 μm). (H,I) Effect of knockdown of PRDX4 on apoptosis in MDA-MB-231 cells. Each experimental group was set up with three replicates. Student’s t-test was used to compare groups, and the data were presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. EdU, 5-Ethynyl-2’-deoxyuridine. Candidate drug exploration and molecular docking of PRDX4 in breast cancer The CSCA web tool was used to screen out CTRP drugs, which showed that the IC[50] was strongly positive for the expression level of PRDX4. Subsequently, 23 drugs with a correlation coefficient > 0.2 and FDR < 0.05 were picked out. Their association with PRDX4 was visualized by a lollipop chart (Fig. [126]8A). Additionally, we found that neratinib, HER2 targeted inhibitor contained in 23 CTRP drugs, was sensitive to PRDX4 (Fig. [127]8C). Thus, the association between the expression level of PRDX4 and HER2 status in breast cancer was investigated using the BEST database. The results showed that the expression of PRDX4 in HER2-positive breast cancer was higher than that in HER2-negative breast cancer (Fig. [128]8B). In addition, molecular docking carried out using Autodock4 showed that the binding energy between neratinib and PRDX4 was − 6.8, and interactive forces, including H bonds and hydrophobic forces, helped maintain the stability of the compound (Fig. [129]8D). This finding indicates that PRDX4 has good affinity and sensitivity to neratinib. Fig. 8. [130]Fig. 8 [131]Open in a new tab Drug sensitivity analysis and molecular docking of PRDX4 with neratinib. (A) The graph illustrates the association between PRDX4 expression and the IC[50] of each CTRP drug. (B) Association between PRDX4 expression and HER2 in [132]GSE162228, which contains a total of 109 breast cancer samples and 24 adjacent normal breast samples. (C) Correlation between PRDX4 expression and IC[50] of neratinib in [133]GSE12093 (with 136 breast cancer samples) and [134]GSE17705 (with 298 breast cancer samples) datasets. (D) PDB 4RQX of PRDX4 as the molecular interacting with the ligand neratinib. Note: In the upper left two figures, the left one showes that PRDX4 has a linear structure docked with the small molecular neratinib and the right showes the pocket structure of PRDX4 wrapping the ligand. In the below left two figure below, the left one exhibited that a purple spherical ligand interacted with PRDX4 in the form of surface structure, and the right showed the details: the white band means the local part of PRDX4; the residues that interacted with the ligand were in the form of purple sticks; the solid red line was the hydrogen bond, the blue dashed line meant the hydrophobic force and the solid yellow line was the pi bond; the labels contained the name of each residue and the distance of each interacting bond. The right figure shows the power of the interaction between PRDX4 and the ligand. HER2, human epidermal growth factor receptor 2; IC[50], half maximal inhibitory concentration. Effects of PRDX4 knockdown on the sensitivity of breast cancer cells to neratinib To further investigate the impact of PRDX4 on the sensitivity of breast cancer cells to neratinib, we performed qRT-PCR to determine the mRNA expression levels of PRDX4 in patients with HER2-positive breast cancer. The results showed that PRDX4 was highly expressed in the breast cancer tissues compared with the adjacent normal tissues (Fig. [135]9A). Moreover, the expression of PRDX4 was significantly higher in HER2-positive breast cancer patients than in adjacent normal tissues (Fig. [136]9B). Furthermore, we examined the cell viability after altering PRDX4 expression in two cell lines: the triple-negative breast cancer cell line MDA-MB-231 and the HER2-positive cell line SK-BR-3. After drug treatment, the viability of the triple-negative breast cancer cell line decreased to 62.3% compared with the blank control group, while that of the HER2-positive cell line dropped to 49%. The viability of the HER2-positive cell line was 0.78 times that of the triple-negative cell line, indicating a more significant reduction in the former. When PRDX4 was knocked down simultaneously with drug treatment, the viability of the HER2-positive cell line decreased by 7.68-fold compared with the group treated with the drug alone. In contrast, the viability of the triple-negative cell line decreased by only 0.92-fold, showing almost no change. These results indicate that PRDX4 selectively modulates cell sensitivity, and HER2-positive cell lines are more sensitive to the resistance-reversing effect of neratinib (Fig. [137]9C, D). In addition, we used qRT-PCR to detect the expression of HER2 in the HER2-positive cell line SK-BR-3 after different treatments. The treatment groups were as follows: control group, PRDX4 knockdown group, drug treatment group, and drug treatment combined with PRDX4 knockdown group. The results showed that the expression of HER2 decreased sequentially in these groups. The lowest HER2 expression was observed in the group with combined drug treatment and PRDX4 knockdown (Fig. [138]9E). These findings suggest that PRDX4 can directly or indirectly affect the mRNA expression level of HER2 during neratinib intervention, and the PRDX4-HER2 axis may serve as a potential combined target for neratinib therapy. Fig. 9. [139]Fig. 9 [140]Open in a new tab Expression level of PRDX4 in breast cancer detected by qRT-PCR assay. (A) PRDX4 mRNA expression level in breast cancer tissues and matching normal tissues of 29 pairs of patients with breast cancer. Data analysis was performed using the paired t-test. (B) PRDX4 expression in breast cancer tissues and adjacent normal tissues of 12 pairs of patients with HER2-positive breast cancer. Data analysis was performed using the paired t-test. (C) Sensitivity of the HER2-positive cell line SK-BR-3 to neratinib after PRDX4 knockdown. (D) Sensitivity of the triple-negative breast cancer cell line MDA-MB-231 to neratinib after PRDX4 knockdown. (E) The expression levels of HER2 in four groups, namely the control group, PRDX4 knockdown group, drug treatment group, and drug treatment combined with PRDX4 knockdown group. (F) The schematic presents the role of PRDX4 in breast cancer. Each group was set up with three replicates. Student’s t-test was used for to compare groups, and the data were presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Discussion According to the latest global cancer statistics, breast cancer has surpassed lung cancer to become the most prevalent cancer among women^[141]31. Additionally, it is the second leading cause of death among females^[142]5. Therefore, there is an urgent need to identify effective potential therapeutic targets to facilitate the prevention and control of breast cancer^[143]6,[144]32. Numerous studies in recent years have shown that members of this family are highly expressed in various tumor tissues and can promote tumorigenesis and development through multiple pathways. For instance, highly expressed PRDX4 can metabolize hydrogen peroxide produced by nicotinamide adenine dinucleotide phosphate oxidase 4, thereby maintaining the growth and survival of pancreatic cancer cells^[145]33. However, the role of this gene family in breast cancer progression remains unclear and requires comprehensive analysis. Therefore, we employed bioinformatics analysis to comprehensively explore the prognostic value of PRDX family members in breast cancer and their potential as therapeutic targets. Tumorigenesis is closely related to dysregulation of gene expression^[146]34. Previous studies have also explored the expression of specific PRDX family members in breast cancer as well as the associated mechanisms of action. For example, Cha et al.^[147]35 demonstrated that PRDX1 was overexpressed in breast cancer and that its expression correlated with tumor grade. This suggests that it could be a potential prognostic biomarker. Based on this, we systematically explored the expression of each member of the PRDX family in breast cancer at the transcriptional and protein levels. We found that the mRNA and protein levels of PRDX1, PRDX2, PRDX4, and PRDX6 were much higher in breast cancer tissues than in normal tissues. PRDX3 was expressed in breast cancer tissues at much higher protein levels than in neighbouring normal tissues, but the mRNA level analysis showed different results. We speculate that this difference may be due to insufficient specimens. These results preliminarily reveal the potential roles of PRDX1, PRDX2, PRDX4, and PRDX6 as oncogenes in breast cancer. Further clinicopathological correlation analysis demonstrates that, except for PRDX3, the expression levels of the remaining members of the PRDX family are significantly associated with the pathological stage and lymph node metastasis of breast cancer. Among them, high mRNA expression of PRDX4 is significantly correlated with poor OS, DSS, and PFI in patients. Verified by the multivariate Cox regression model, PRDX4 has the potential to serve as an independent prognostic indicator. Additionally, high expression of PRDX1 and PRDX3 is also associated with poor OS in patients, suggesting that the PRDX family is of great value in the prognostic assessment of breast cancer. In particular, PRDX4 warrants an in-depth investigation. In cancer treatment, immunotherapy has emerged as a crucial pillar. However, the lack of biomarkers significantly restricts its clinical efficacy^[148]36,[149]37. In breast cancer, the suppressive TME severely impairs the effectiveness of anti-tumor therapy by facilitating tumor immune evasion^[150]38,[151]39. Studies on liver cancer have demonstrated that the small-molecule compound parvifoline AA can significantly enhance the recognition and killing of tumor cells by natural killer (NK) cells by targeting PRDX1 and PRDX2, providing a theoretical basis for PRDX1 and PRDX2 as potential sensitizing targets for immunotherapy^[152]40. Inspired by these findings, our study analyzed the correlation between the mRNA expression of PRDX family members and immune cell infiltration in breast cancer. The results showed that PRDX4 had the strongest correlation with immune infiltration, while other family members exhibited weaker associations. Combining this with previous findings that PRDX4 overexpression can regulate the recruitment of neutrophils and macrophages in the wound microenvironment^[153]41we hypothesized that PRDX4 may serve as a diagnostic biomarker for the efficacy of breast cancer immunotherapy. From the perspective of molecular mechanisms, previous studies have demonstrated that members of the PRDX family can maintain the survival microenvironment of tumor cells by regulating intracellular redox homeostasis, thereby promoting tumor cell proliferation and survival^[154]42. Our pathway enrichment analysis of co-expressed genes also indicates that PRDX family members are associated with the invasion, progression, and drug resistance of breast cancer, suggesting their potential as therapeutic targets and prognostic markers. In past research, the roles of PRDX1 and PRDX6 in tumor development have been relatively well-defined. PRDX1 is mainly involved in the regulation of tumor cell proliferation, metastasis, and cell death^[155]31,[156]43,[157]44while PRDX6 regulates the membrane function and signal transduction of tumor cells through its phospholipase activity^[158]45. Existing evidence shows that PRDX4 is highly expressed in tissues and cell lines of endometrial cancer, CRC, and liver cancer, where it promotes tumor cell proliferation, migration, and inhibits apoptosis^[159]17,[160]46,[161]47. However, the oncogenic role of PRDX4 in breast cancer has not been reported. Our bioinformatics analysis revealed that in breast cancer, PRDX4 is primarily associated with biological functions crucial for tumor invasion and progression, such as invasion, EMT, and hypoxia. Subsequently, we investigated the expression of PRDX4 in various breast cancer cell lines and found that its expression pattern in vitro was consistent with that in vivo, both showing significantly higher expression levels compared to normal tissues or cells. We then conducted a series of experiments to explore the effects of PRDX4 on the proliferation, invasion, migration, and apoptosis of breast cancer cells. The results showed that knocking down PRDX4 reduced the invasive ability to 57% of the original level, the migratory ability to 29.6% of the baseline, and the proliferative ability of breast cancer cells to 20% of the control. However, the apoptosis rate increased to 1.5 times the original level after PRDX4 knockdown, which was notably disproportionate to the degree of proliferation inhibition. We hypothesized that PRDX4 may regulate other forms of cell death, such as ferroptosis and cuproptosis, contributing to the nearly 30-fold difference between the decrease in proliferation rate and the increase in apoptosis rate. Ferroptosis, a novel form of programmed cell death characterized by iron-dependent lipid peroxidation, has been reported to inhibit cell proliferation in lung cancer and osteosarcoma^[162]48,[163]49. Moreover, the inhibition of ferroptosis promotes the proliferation of MDA-MB-231 cells^[164]50. As a member of the antioxidant enzyme gene family, supressing PRDX4 expression can upregulate intracellular ROS levels and induce ferroptosis^[165]51potentially explaining the observed disparity between proliferation and apoptosis in our experiments. Additionally, studies on liver and gastric cancer have reported the role of regulating cuproptosis in inhibiting tumor proliferation^[166]52,[167]53. These findings suggest that knocking down PRDX4 may affect multiple cell death pathways, including ferroptosis and cuproptosis, in breast cancer cells, warranting further investigation. Resistance to targeted therapy and chemotherapy severely compromises the prognosis and quality of life of patients with breast cancer^[168]54. According to molecular classification, breast cancer can be categorized into several subtypes: hormone receptor-positive (Luminal A and Luminal B), HER-2-positive, and triple-negative^[169]55. Approximately 20% of breast cancers exhibit HER2 gene amplification or HER2 protein overexpression. Compared with hormone receptor-positive breast cancer, HER2-positive breast cancer is more aggressive, typically associated with a poorer prognosis, and prone to developing drug resistance^[170]56,[171]57. Studies have indicated that PRDX4 is closely associated with tumor resistance. It can upregulate the suppression of ROS generated during chemotherapy or radiotherapy, thereby conferring resistance of tumor cells to antitumor treatments^[172]58. By analyzing the top 36 drugs correlated with PRDX4 in the CTRP database, we found that PRDX4 expression was associated with resistance to neratinib, a HER2-targeted drug. Similarly, neratinib, a kinase inhibitor, selectively binds to the ATP-binding site of the HER2 tyrosine kinase, blocking its kinase activity and inhibiting tumor cell proliferation. It is primarily used for the enhanced adjuvant treatment of HER2-positive breast cancer^[173]59,[174]60. Further analysis of the BEST database revealed that PRDX4 expression was significantly higher in patients with HER2-positive breast cancer than in patients with HER2-negative breast cancer. Molecular docking results showed that PRDX4 has a strong affinity for neratinib and can form a stable complex. Based on the above analysis, we hypothesized that PRDX4 could serve as a target molecule to enhance the therapeutic efficacy of HER2-targeted drugs in HER2-overexpressing breast cancer. To validate this hypothesis, we collected paraffin-embedded tissue sections from patients with HER2-positive and triple-negative breast cancer and extracted RNA from these samples. qPCR results demonstrated that PRDX4 expression was significantly higher in patients with HER2-positive breast cancer. Furthermore, we investigated the impact of modulating PRDX4 expression on cell viability using two cell lines: the triple-negative breast cancer cell line MDA-MB-231 and the HER2-positive cell line SK-BR-3. After treatment with the drug, the viability of MDA-MB-231 cells decreased to 62.3% compared with the blank control group, whereas that of SK-BR-3 cells dropped to 49%. The viability of HER2-positive cells was 0.78 times that of triple-negative cells, indicating a more significant reduction in the former. Notably, when PRDX4 was knocked down simultaneously with drug treatment, the viability of SK-BR-3 cells decreased by 7.68-fold compared with the drug treatment group alone. Conversely, the viability of MDA-MB-231 cells decreased by only 0.92-fold, showing minimal change. These data suggest that PRDX4 selectively modulates cell sensitivity, rendering HER2-positive cell lines more susceptible to neratinib resistance. Subsequent qRT-PCR experiments revealed that neratinib treatment reduced HER2 expression in HER2-positive cells, and the combined treatment of neratinib and PRDX4 knockdown led to the lowest HER2 expression. This finding indicates that PRDX4 can directly or indirectly influence HER2 mRNA expression during neratinib intervention. Therefore, the PRDX4-HER2 axis may serve as a potential combined target for neratinib therapy, and targeting PRDX4 could represent a novel strategy to improve the efficacy of targeted therapy for patients with HER2-positive breast cancer. Conclusions Through a series of bioinformatics validations, we found that PRDX4, one of the members of the PRDX family, has the potential to serve as a biomarker and therapeutic target for breast cancer. Further research showed that PRDX4 is closely associated with biological processes such as invasion and EMT in breast cancer. It contributes to the proliferation, invasion, and metastasis of breast cancer cells while inhibiting apoptosis. These findings suggest that PRDX4 could potentially be used as a diagnostic and prognostic marker for patients with breast cancer, and targeting PRDX4 might represent a novel therapeutic strategy for breast cancer treatment. In addition, PRDX4 was also found to influence the efficacy of HER2-targeted therapy. Our study has preliminarily explored the role of the PRDX4-HER2 axis in regulating the sensitivity of breast cancer cells to neratinib treatment. However, the detailed mechanisms underlying this regulation remain unclear and require further investigation. Therefore, our study implies that PRDX4 has the potential to act as a prognostic marker and therapeutic target for breast cancer. Supplementary Information Below is the link to the electronic supplementary material. [175]Supplementary Material 1^ (635.8KB, docx) [176]Supplementary Material 2^ (25.1KB, xlsx) Acknowledgements