Abstract Disease caused by plasticizers has received increasing attention. Di(2-ethylhexyl) phthalate (DEHP), one of the most widely exposed plasticizers, has been shown to be closely associated with the development of breast cancer (BRCA) in epidemiological studies, but the specific mechanistic targets and related pathways are still unclear. In this study, we aimed to elucidate the potential pathogenic targets and mechanisms of DEHP-induced BRCA through network toxicology and molecular docking. Databases including GeneCards, OMIM, ChEMBL, and SwissTargetPrediction were first used to identify DEHP-related targets and BRCA-related targets, and 691 potential targets were obtained from the intersection analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to clarify the biological functions and pathways of potential targets. Protein–protein interaction (PPI) analysis revealed the interactions between potential targets, and 14 hub targets of DEHP-induced BRCA were further screened. To verify the clinical significance of the hub targets, the expression of the target proteins was verified in the TCGA database, and the affinity between DEHP and 12 key targets (hub targets with p < 0.05) was determined via molecular docking. Our study provides a theoretical basis for DEHP-induced BRCA from the “Homo sapiens” perspective and reveals the potential risks caused by exposure to DEHP, thus providing new strategies for the prevention and treatment of DEHP-induced BRCA. Keywords: Breast cancer, Di(2-ethylhexyl) phthalate, Network toxicology, Molecular docking Subject terms: Breast cancer, Environmental sciences, Materials science Introduction Breast cancer (BRCA) is the second most prevalent cancer worldwide, and seriously affects women’s health ^[26]1. Among the potential pathogenic factors, the impact of environmental factors such as air pollution, chemical toxicity, and toxins in food on BRCA has attracted increasing attention. Plasticizers, as chemical additives to polymer materials, are ubiquitous from food packaging to household items in daily life ^[27]2. Although the development of plasticizers meets various people’s needs, their lipophilic nature and stability also brings potential health risks ^[28]3. For example, phthalate ester (PAE) plasticizers can act as estrogen-like molecules to promote the progression and development of BRCA ^[29]4. Di(2-ethylhexyl) phthalate (DEHP), as one of the PAEs, is a colourless and odourless liquid, and is added to mainly polyvinyl chloride (PVC) products to increase extensibility and flexibility. DEHP is physically bound to PVC moleculars in a lipophilic manner but not a chemically bound via covalent bonds. As a result, DEHP can be continuously released from PVC products, causing severe pollution to air, soil, food, etc. Studies have shown that humans are exposed to DEHP at doses of approximately 3–30 μg/kg per day through the gastrointestinal tract, respiratory tract and dermal contact ^[30]5,[31]6. DEHP and its metabolites were also detected in urine samples from all study subjects in Silva MJ’s research ^[32]7. Exposure to DEHP at low doses is generally considered safe, while its long-term accumulation can also cause health damage, including cancer. Growing evidence suggests a bi-directionally supportive relationship between DEHP and BRCA, with findings from epidemiologic and mechanistic studies forming complementary pillars. A large-scale cohort study has confirmed a strong association between DEHP exposure and increased BRCA risk in humans, providing population-level evidence for this link ^[33]8. On the other hand, in vitro studies revealed that DEHP promotes normal breast cells proliferation and BRCA progression via estrogen receptor-mediated pathways, offering a biological mechanism that could underlie the observed epidemiologic association ^[34]9,[35]10. In addition, DEHP and its metabolite mono(2-ethylhexyl) phthalate (MEHP) enhance BRCA cells proliferation by dysregulating progesterone receptors ^[36]11. These findings confirm the critical role of DEHP as an endocrine disruptor, and highlight the susceptibility of hormone receptor-positive BRCA to DEHP ^[37]12. However, these studies were limited to only the cell line level, and lacked a comprehensive and systematic investigation. In addition, the World Health Organization has defined DEHP as a class of 2B carcinogen, mainly considering the limited or insufficient evidence of carcinogenesis in humans ^[38]13. Thus, more evidence is required to elucidate the association between DEHP and BRCA. In this study, network toxicology combined with molecular docking provided novel evidence to clarify the potential correlations. The workflow of the investigation strategy is illustrated in Fig. [39]1. Network toxicology is an emerging interdisciplinary subject that integrates systems biology, chemistry, and bioinformatics. Different from traditional toxicology, network toxicology can explore biological mechanisms from molecules, cells and biological processes, thus providing systematical and comprehensive insights into the pathogenesis. Meanwhile, molecular docking can predict specific binding patterns as well as affinities between chemicals and target proteins, thus contributing to elucidating the underlying mechanisms of carcinogenesis ^[40]14. Owing to the advantages of both technologies, this study aimed to reveal the toxicity profiles of DEHP and the underlying molecular mechanism of DEHP-mediated BRCA. More importantly, our study comprehensively elucidates the targets and pathogenic pathways of DEHP-induced BRCA from the perspective of “Homo sapiens”, and numerous potential targets (691) reflect the complexity and profound influence of DEHP on BRCA, which provides new strategies for the prevention and intervention of BRCA. Fig. 1. [41]Fig. 1 [42]Open in a new tab The workflow of our study. Step one: database mining, using the public database to identify DEHP-related targets, BRCA-related targets, and potential targets of DEHP-induced BRCA. Step two: exploring the biological functions of potential targets, including PPI, Go analysis, and KEGG pathways enrichment analysis. Step three: TCGA database validation of hub targets and molecular docking analysis of key targets. Methods The data collected for this study were obtained from online databases, with links available in Table [43]S1. Preliminary toxicity analysis of DEHP The integration of molecular structure, functional groups, data mining, machine learning algorithms, etc., is used to evaluate the chemical toxicity in toxicity prediction tools. ProTox-3.0 and ADMETlab 3.0 were adopted to predict the toxicity of DEHP. The SMILES of DEHP (CCCCC(CC)COC(=O)C1=CC=CC=C1C(=O)OCC(CC)CCCC) obtained from the PubChem database was used for toxicity prediction retrieval. In ProTox‑3.0, the toxicity prediction results are classified into four categories: active with a probability > 0.7, active with a probability < 0.7, inactive with a probability > 0.7, and inactive with a probability < 0.7. In ADMETlab, the prediction probability values are transformed into three grades: excellent (0–0.3), medium (0.3–0.7), and poor (0.7–1.0). Collection of DEHP-related targets The keyword of “di(2-ethylhexyl) phthalate” was used to acquire the targets in the ChEMBL database, and the organism was limited to “Homo sapiens”. The SMILES of DEHP was used to acquire targets in the SwissTargetPrediction database, and targets with “Probability > 0” were collected for further analysis. Subsequently, the targets obtained from the two databases were merged and deduplicated as to obtain the final EDHP-related targets. Collection of BRCA-related targets and intersection analysis The GeneCards and OMIM databases were chosen to collect BRCA-related targets, and the keyword of “breast cancer” was used for searching. Targets with “Score > median” in the GeneCards database were selected for further analysis. Then, the targets from the two databases were merged and deduplicated as to obtain the final BRCA-related targets. After that, intersection analysis (Venn plot) was performed between DEHP-related targets and BRCA-related targets, and the intersection targets were considered as the potential targets of DEHP-induced BRCA. The target names were standardized using the UniProt database. Protein–protein interaction (PPI) network analysis and hub targets screening A PPI network was constructed via the STRING database. The standardized target names were entered into the multiple proteins’ module, and the organisms were limited to the “Homo sapiens”. The parameter “minimum required interaction score” was set as “high confidence (0.700)”. Cytoscape software (version 3.9.1) was used for visualization and analysis of the PPI network. The CytoNCA plugin in Cytoscape software was used to screen the hub targets according to the following the six critical topological parameter criteria: ① betweenness centrality (BC) > median, ② closeness centrality (CC) > median, ③ degree centrality (DC) > 6 × median, ④ eigenvector centrality (EC) > median, ⑤ local average connectivity-based method centrality (LAC) > median, and ⑥ network centrality (NC) > median. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis To explore the significant biological functions and pathways associated with potential targets of DEHP-induced BRCA, the Metascape online tool was applied for GO and KEGG pathway enrichment analysis. The GO analysis incorporates three modules: including biological processes (BP), cellular components (CC), molecular functions (MF). The parameters of GO analysis were set as follows: min overlap = 3, p-value cutoff = 0.05, min enrichment = 1.5. Correspondingly, the parameters of KEGG enrichment were set as: min overlap = 3, p-value cutoff = 0.05, min enrichment = 1.5. The species was selected as “Homo sapiens”. Afterward, visualization of the relevant results was accomplished via the Bioinformatics online platform. Hub targets expression analysis of BRCA in the TCGA database The clinical phenotype and hub targets transcription levels of BRCA patients were obtained from RNA-seq FPKM-UQ data in the TCGA database. The Wilcoxon rank-sum test was used to compare the expression levels of the hub targets in “primary tumor” and “solid normal tissue”. To visualize the results, the R package ggplot2 was applied to construct a box plot with available p-values. Molecular docking of DEHP with key targets Molecular docking was utilized to estimate the affinity between DEHP and the key targets. The 3D structure of DEHP (SDF format) was obtained from the PubChem database and converted to MOL format using OpenBabel (version 3.1.1). The crystal structures of the receptor protein were collected from the RCSB Protein Data Bank (PDB). Then, the water molecules were eliminated using PyMol (version 3.9.1), followed by the residual ligands’ separation. Hydrogenation was completed using the AutoDock (version 1.5.7), and the molecular docking processes were achieved through AutoDock Vina (version 1.2.5) with the grid boxes parameters (Table [44]S2). Finally, the visualization of the docking results was completed using PyMOL (version 3.9.1). Results Chemical information and toxicity prediction of DEHP The chemical information of the plasticizer DEHP was obtained from the Pubchem database, and the details are shown in Figure [45]S1. As one of the most typical plasticizers in daily life, epidemiology research has revealed a correlation between DEHP and BRCA. Here, we first performed a preliminary validation using the toxicity prediction tools, and the prediction outcomes indicated that DEHP has the potential for carcinogenicity (ProTox3.0 = 0.86 (active with a probability > 0.7), ADMETlab3.0 = 0.328 (medium)) (Figure [46]S2). These findings provide evidence and support for further study of DEHP-induced BRCA. Potential targets identification of DEHP-induced BRCA After merging and deduplication, a total of 1726 DEHP-related targets were collected from the ProTox3.0 and ADMETlab3.0 databases. Moreover, 6147 BRCA-related targets were acquired from GeneCards and OMIM databases. Furthermore, intersection analysis was performed between DEHP-related targets and BRCA-related targets, and the intersection targets were defined as potential targets. As shown in the Venn plot (Fig. [47]2), 691 proteins were identified as potential targets identification of DEHP-induced BR. It is conceivable that the numerous potential targets imply a significant impact on mammary gland, and further biological function investigations are required to explore the pivotal role in causing BRCA. Fig. 2. Fig. 2 [48]Open in a new tab Intersection analysis (Venn plot) between BRCA-related targets and DEHP-related targets. GO and KEGG pathway enrichment analysis GO analysis was performed to elucidate the biological functions of the potential targets (Fig. [49]3A). In total, the results revealed 3654 significant GO terms, including 3038 GO terms for BP, 169 GO terms for CC, and 447 GO terms for MF. In BP, notable enrichment terms were observed in phosphorylation, response to hormone, positive regulation of phosphorylation, response to xenobiotic stimulus, positive regulation of programmed cell death and others. For example, phosphorylation usually occurs extensively in the metabolic process of cellular metabolism, whereas metabolic reprogramming is a critical characteristic of tumor adaptation. These findings indicate that the genes under investigation are likely involved in these crucial biological activities. Regarding CC, the enriched components mainly located in the membrane raft, perinuclear region of cytoplasm, and receptor complex, suggesting the functions of cellular trafficking and signaling transduction within specific cellular structures. In MF, GO terms of phosphotransferase activity, alcohol group as acceptor, oxidoreductase activity, and protein tyrosine kinase activity were ranked at the top three, providing insights into the specific activities and capabilities of the genes at the molecular level. Fig. 3. [50]Fig. 3 [51]Open in a new tab (A) GO analysis (top 10) of the potential targets, including BP, CC, and MF three parts. (B) KEGG pathway enrichment analysis (top 20) of the potential targets. KEGG pathway enrichment analysis was conducted to identify the potential signaling pathways associated with potential targets, and the analysis uncovered several significant pathways ^[52]15 (Fig. [53]3B). Among the enriched pathways, ‘pathways in cancer’ stood out, which reflected a tight correlation between DEHP and BRCA. Overall, the identification of these GO terms and KEGG pathways provides a framework for understanding the molecular mechanisms underlying the biological processes investigated in this study. PPI network construction and hub targets screening To gain further insights into the interactions among the potential targets of DEHP-induced BRCA, the STRING database combined with Cytoscape software are applied for the construction and visualization of PPI network. As shown in Fig. [54]4A, the PPI network is exhibited in a concentric circles’ layout, consisting of 615 nodes and 2749 edges with an average neighbor number of 9.018. The nodes represent potential targets (genes), and the edges represent PPI. The nodes in concentric circles are ranked on the basis of their degree values, while the size and color shading also indicate their degree values. Fig. 4. [55]Fig. 4 [56]Open in a new tab (A) PPI analysis of the 691 potential targets. (B) PPI analysis of the 14 hub targets. (C) KEGG pathway enrichment analysis (top 10) of the 14 hub targets. Hub targets screening was performed using the CytoNCA plugin with strict criteria. Topological parameters including BC > 317.015, CC > 0.105, DC > 36, EC > 0.008, LAC > 1.714, and NC > 2.429 were used as screening criteria for hub targets, and 14 genes (nodes of the inner circle in the PPI network) including HSP90AA1, TNF, IL6, MAPK3, MAPK1, BCL2, EP300, CASP3, KRAS, ERBB2, BCL2L1, GRB2, SHC1, and CYP3A4 were identified as hub targets of DEHP-induced BRCA. The detailed topological parameters of the hub targets are listed in Table [57]1. Furthermore, PPI network among hub targets, composed of 14 nodes and 78 edges, was also constructed (Fig. [58]4B), and a KEGG pathway analysis of hub targets is shown in Fig. [59]4C. Interestingly, many of the hub targets have been reported to be involved in cell proliferation, tumorigenicity, and other biological functions, and we will explore these further in the following discussion section. Table 1. Detailed topological parameters of the hub targets. Hub targets DC EC LAC BC CC NC HSP90AA1 660.197 0.197 6.606 49,572.473 0.120 23.673 TNF 66 0.213 9.212 30,668.100 0.118 33.769 IL6 61 0.218 9.410 20,419.432 0.117 29.642 MAPK3 56 0.216 8.679 16,958.135 0.118 24.628 MAPK1 55 0.220 9.709 15,442.079 0.118 26.553 BCL2 54 0.203 9.407 21,391.152 0.118 26.424 EP300 51 0.099 4.745 35,135.530 0.118 18.872 CASP3 47 0.174 8.085 19,491.512 0.117 21.100 KRAS 45 0.199 10.222 15,298.309 0.118 20.553 ERBB2 41 0.148 8.000 14,112.325 0.116 17.768 BCL2L1 40 0.166 9.100 6947.265 0.116 19.571 GRB2 39 0.131 7.949 7777.711 0.114 18.409 SHC1 36 0.128 7.944 4579.432 0.113 16.720 CYP3A4 36 0.019 12.167 3974.472 0.106 27.356 [60]Open in a new tab Hub targets expression analysis of BRCA in the TCGA database To further investigate the clinical value of the hub targets, BRCA data from the TCGA database were used for validation. The expression analysis of the hub targets revealed that 12 targets (HSP90AA1, IL6, MAPK1, BCL2, EP300, CASP3, KRAS, ERBB2, BCL2L1, GRB2, SHC1, and CYP3A4) exhibited significant differences (p < 0.05) in BRCA and solid normal tissues, and were defined as key targets (Fig. [61]5). Among these, 7 key targets (HSP90AA1, CASP3, KRAS, ERBB2, BCL2L1, GRB2, and SHC1) were up-regulated in BRCA, and the remaining 5 key targets were down-regulated. The expression of the hub targets of TNF and MAPK3 exhibited no difference. The altered expression of key targets revealed a potential association with BRCA, which were consistent with previous studies. For example, HSP90AA1 protein is increased in BRCA tissues compared with normal tissues, and is associated with poor overall survival (OS) ^[62]16. ERBB2, also named as HER2, is involved in the carcinogenesis of BRCA through several signaling pathways ^[63]17, such as the PI3K-AKT, MAPK pathways. These findings correlate the altered expression of key targets directly with potential pathogenic mechanisms and therapeutic targets of BRCA. Fig. 5. [64]Fig. 5 [65]Open in a new tab Expression analysis of hub targets between BRCA and normal tissues in the TCGA database. Molecular docking results Finally, the molecular docking analysis was carried out to elucidate the affinities of DEHP for the key targets utilizing AutoDock vina. With phenyl and alkyl groups, DEHP has high lipophilicity, which was able to be inserted into the hydrophobic pocket of the target proteins. As presented in Table [66]S2, except for the GRB2 protein (− 4.8 kcal/mol), almost all of the key targets exhibited strong binding energy with DEHP (usually < − 5.0 kcal/mol). Further visualization of potential binding modes between DEHP and key proteins were accomplished with PyMOL software (Fig. [67]6). Among these, four key targets (HSP90AA1, MAPK1, BCL2, and CYP3A4) exhibited relatively high affinity (energy < − 6.0 kcal/mol). Notably, hydrogen bonds, which account for the high affinity, are formed between DEHP and proteins due to the four hydrogen bond receptors of the carboxyl groups, including ASN-51 of HSP90AA1, ARG-24 of IL6, LYS-54 of MAPK1, GLN-20 of BCL2, GLN-20 of EP300, ARG-207 of CASP3, GLN-68 and THR-45 of ERBB2, LYS-87 of BCL2L1, and ARG-112 of GRB2. These findings indicated an extensive affinity between DEHP and disease targets, providing evidence for DEHP-induced BRCA from the molecular docking perspective. Fig. 6. [68]Fig. 6 [69]Open in a new tab Molecular docking analysis results of key targets with DEHP. The molecular docking was achieved through AutoDock Vina (version 1.2.5, [70]https://vina.scripps.edu/), and the visualization of the docking results was completed using PyMOL (version 3.9.1, [71]https://www.pymol.org/). Discussion The role of environmental factors in health has attracted increasing attention, and the wide application of plasticizers such as DEHP makes long-term exposure unavoidable, causing potential harms including BRCA. Humans are exposed to the plasticizer DEHP through various routes, of which low-dose dietary intake is a common and widespread approach, especially through packaged foods and beverages. In addition, DEHP can enter the body through the respiration and skin contact. Unfortunately, a previous study has reported that even at low-dose of long-term exposure to DEHP can accumulate in the body, increasing the risk of BRCA ^[72]18. On the one hand, cell and animal experiments have revealed potential links between DEHP and BRCA. The exposure of mice to a high dose of DEHP is accompanied by the abnormal alterations of breast tissue ^[73]19,[74]20, including mammary hyperplasia and tumor formation. Cell experiments have shown that DEHP can promote the proliferation and drug resistance of BRCA cells ^[75]11,[76]21. These findings provide important clues for further investigations of the DEHP effects on human health, while cell and animal studies are not directly equivalent to the situation in humans. On the other hand, several studies have attempted to reveal potential links between DEHP and BRCA from an epidemiological perspective. However, epidemiological studies have indicated that DEHP-induced BRCA is controversial, as some studies found that exposure to DEHP increases BRCA risk ^[77]8,[78]22, whereas others not ^[79]23,[80]24. But undeniably, those results were influenced by numerous factors, including variations in study methodologies, sample size, exposure assessment accuracy, and pathogenic complexity. The current viewpoint about DEHP-induced BRCA is mainly based on the endocrine disrupting characteristic of DEHP. Estrogen plays a crucial role in the growth and development of breast tissue, whereas DEHP is able to emulate the natural estrogenic activities, thus interfering with the normal function of the endocrine system ^[81]25. Studies have shown that exposure to DEHP may affect estrogen metabolism and signal transduction ^[82]20,[83]26, and that abnormal estrogen levels or disordered estrogen signaling pathways increase the risk of BRCA ^[84]27. In addition, the occurrence of BRCA is a complex process caused by multiple factors working together. Potential factors such as genetic predispositions, lifestyles and environments, may contribute to the development of BRCA. Thus, the endocrine disrupting characteristic of DEHP may be one of many pathogenic mechanisms, and comprehensive of systematic studies are required to further investigate the potential mechanism of DEHP-induced BRCA. In this context, the present study was conducted to systematically investigate potential targets and pathogenic pathways of DEHP-induced BRCA specific to “Homo sapiens” via network toxicology and molecular docking strategies. Toxicity analysis of chemicals is critical for the exploration of pathogenic mechanisms, which can provide the toxicity endpoints prediction from multiple aspects such as molecular similarity, fragment propensities, and machine learning ^[85]28. Prediction results indicate the carcinogenicity of DEHP, providing convincing evidence for DEHP-induced BRCA. Furthermore, DEHP also has toxicity on other endpoints, such as mutagenicity, immunotoxicity, cytotoxicity, and hepatotoxicity, may related to long-term chronic accumulation, whereas respiratory toxicity, skin sensitization and eye corrosion are caused mainly by short-term high-dose exposure. In addition, a total of 691 potential targets were identified from multiple disease-target databases, and these numerous potential targets manifest the complexity and profound influence on DEHP-induced BRCA. GO analysis of potential targets supports the association with BRCA. Phosphorylation and positive regulation of the phosphorylation process are usually involved in cell growth, apoptosis and signaling pathways through post-translational modification and cellular metabolism, and abnormal alteration in phosphorylation contribute to diseases, including cancers ^[86]29. AMP-activated protein kinase promotes BRCA metastasis by regulating the phosphorylation of PDHA ^[87]30. For hormone receptor-positive BRCA, the development of cancer cells is largely stimulated by a hormonal response. The activation of estrogen receptor leads to increased expression of cyclins, and prompts cancer cells to transition from the quiescent phase to the proliferative phase ^[88]31, leading to tumor growth and development. The CC terms related to membrane raft, cytoplasm, and the receptor complex suggested that potential targets may play the function of signaling transduction and transport, and MF related to receptor activity, enzyme activity and binding ability may be involved in signal transduction and molecular regulation of DEHP-induced BRCA. Furthermore, “Pathways in cancer” in KEGG enrichment analysis directly revealed the participation of potential targets in tumor signaling pathways. The “TNF signaling pathway” also plays an important role in promoting cell proliferation, inducing migration and metastasis, and regulating apoptosis in BRCA ^[89]31,[90]32. In addition, the pathways “Lipid and atherosclerosis” and “Fluid shear stress and atherosclerosis” may be involved in lipid metabolic disease. Fourteen hub targets of DEHP-induced BRCA were identified from numerous potential targets, and further validation was performed in the TCGA database. At present, many studies have reported the role of these hub targets in tumors. The HSP90AA1 protein, as a molecular chaperone, can bind to the HER2 protein to play an indirect role in BRCA pathogenesis. A stable structure that binding to HER2 proteins inhibits HER2 degradation, leading to sustained activation of the PI3K-AKT and RAS-MAPK signaling pathways, which contribute to BRCA progression ^[91]33. IL6 can directly act on BRCA cells to promote proliferation by activating the JAK-STAT3 signaling pathway ^[92]34. Meanwhile, IL6 can promote migration and invasion by inducing the expression of matrix metalloproteinases (MMPs) and activating epithelial-mesenchymal transformation ^[93]35. Regarding MAPK1 and MAPK3, studies have reported that they are involved in BRCA through several approaches, including the activation of cell cycle-related proteins and inhibition of antiapoptotic proteins by MAPK1/MAPK3 phosphorylation, and regulation of cytoskeletal proteins to promote migration ^[94]36. BCL2L1 and BCL2 are anti-apoptotic proteins belonging to the BCL2 family that function by preventing the oligomerization of the BAX and BAK proteins and maintaining the integrity of mitochondrial membrane, thus inhibiting cell apoptosis at high expression ^[95]37. GRB2, a HER2 adaptor protein, binds BECN1 to inhibit autophagy by suppressing VPS34 activity ^[96]38. This regulation promotes BRCA progression by enhancing cell proliferation through cross-talk with signaling pathways like RAS-RAF-MEK and PI3K-AKT, independent of mTORC1. EP300 usually regulates transcription factors mainly through acetylation and further activates cell cycle-related genes, thus affecting the progression of cancers ^[97]39. As for CASP3, studies have shown a correlation between abnormal expression and BRCA patient prognosis, and an abnormal CASP3-mediated apoptosis pathway is a potential mechanism of chemoresistance in BRCA ^[98]40,[99]41. KRAS can activate downstream factors through phosphorylation reactions, and ultimately promote cell proliferation by regulating signaling pathways such as the RAS-RAF-MEK-ERK and PI3K-AKT pathways ^[100]42. As a key tyrosine kinase adaptor, SHC1 promotes BRCA immune suppression by balancing STAT1 and STAT3 signaling. Phosphorylation at Y239/Y240 site enhances STAT3-driven immunosuppressive pathways (e.g., PD-L1 expression) while repressing STAT1-mediated immune surveillance, reducing CD8+T cell infiltration ^[101]43. CYP3A4 is mainly involved in the metabolism of chemotherapy drugs to interfere with tumor progression. Estrogen metabolism is also regulated by CYP3A4, thereby influencing the estrogen signaling pathway ^[102]44. Actually, the prediction results from the ADMETlab 3.0 indicate that DEHP is also a substrate and inhibitor of CYP3A4. HER2 is currently a clear therapeutic target for BRCA ^[103]45. The abnormally expression of HER2 can form homodimers or heterodimers with family proteins to activate the tyrosine kinase domain, and further activate the phosphorylation cascade of downstream signaling pathways ^[104]46. Notably, there was no difference in the TNF protein expression of between BRCA and normal tissues in TCGA database, which possibly due to the abnormal expression of TNF in physiological or pathological states such as the inflammatory response and immune regulation ^[105]47. BRCA is a systemic and complex disease, and what we briefly discuss here may involve many pathogenic factors. Molecular docking is a powerful tool in drug discovery and understanding protein–ligand interactions. In our study, molecular docking provides direct evidence for the interaction between DEHP and key targets at the atomic level, offering insights into DEHP-mediated BRCA. This approach allowed us to predict the most favourable binding poses and estimate binding energies. However, it should be noted that docking results are only approximations and need to be validated experimentally. Future work will focus on refining the docking protocol and conducting in vitro and in vivo experiments to confirm the predicted interactions. Conclusion Although the current evidence on DEHP-induced BRCA is limited mainly to epidemiological studies and cell/animal experiments, it is undeniable that DEHP, as a plasticizer widely accessible in daily life, has a non-negligible impact on human health. Conducting in-depth research on the relationship between DEHP and BRCA is of great significance for comprehensive understanding the BRCA pathogenesis, formulating effective preventive strategies and protecting public health. In this study, we preliminarily explored the potential targets and mechanisms via network toxicology and molecular docking techniques, thus providing potential evidence for DEHP-induced BRCA. On the other hand, our study also highlights the need to evaluate the safety of commonly used plasticizers. It must be acknowledged that our work is limited to computational predictions and lacks experimental validation, thereby limiting their translational relevance to human BRCA at this stage. To address this gap, the following in vitro/vivo validation strategies are proposed here. Functional assays including cell viability and migration/invasion assays (with DEHP-treated vs without DEHP-treated) should be conducted in BRCA cell lines such as MCF-7 and MDA-MB-231, and further investigate the dose–effect of DEHP. Western blotting (WB), immunohistochemistry (IHC) and Immunofluorescence (IF) are adopted to investigate the hub targets expression and signaling pathway activation (e.g., PI3K-AKT pathway, estrogen receptor pathway). As for in vivo validation, with/without DEHP-treated BRCA cell lines could be implanted into mice to assess their tumorigenicity. Correspondingly, WB, IHC and IF techniques could be used for analyzing hub targets expression and signaling pathways in mice. In addition, DEHP-exposed animal models could be constructed to evaluate the carcinogenicity of DEHP. Experimental validation is essential to bridge the gap between computational predictions and clinical relevance, which will strengthen the understanding of DEHP’s role in BRCA pathogenesis and inform evidence-based risk assessment of plasticizers. Supplementary Information [106]Supplementary Information.^ (73.5KB, doc) Author contributions Z.W. conceived the study and generated the hypotheses. Z.W. and Y.W. performed the experiments and analyzed the data. Z.W. wrote the manuscript. All authors contributed to the manuscript review, revision, and finalization. Data availability The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. Declarations Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-025-13201-1. References