Environmental Lead Promotes Breast Cancer Migration and Invasion via the AKR1C3–NF-κB–MMP Axis

TL;DR

This study shows how environmental lead exposure increases breast cancer cell migration and invasion through a specific molecular pathway involving AKR1C3, NF-κB, and MMPs.

Contribution

The study identifies a novel Pb-induced AKR1C3–NF-κB–MMP axis in breast cancer progression.

Findings

Pb exposure increases breast cancer cell migration and invasion without affecting proliferation.

AKR1C3 activates NF-κB signaling, which upregulates MMP-2 and MMP-9 to promote cancer cell movement.

AKR1C3 is proposed as a potential therapeutic target for breast cancer linked to heavy metal exposure.

Abstract

Background/Objectives: Environmental exposure to heavy metals is an established risk factor for breast cancer development; however, the molecular mechanisms underlying the contribution of lead (Pb) to disease progression remain unclear. This study aimed to investigate the effects of Pb exposure on breast cancer cells and to delineate the associated mechanisms. Methods: We examined Pb-induced migration and invasion of breast cancer cells using wound-healing and Transwell assays; assessed cell proliferation by flow cytometry and MTT assay; identified potential target genes via RNA sequencing; and further elucidated the underlying mechanisms using integrated molecular biology approaches (including immunofluorescence, Western blotting, and ELISA), functional cellular assays, and bioinformatics analysis. Results: Pb exposure significantly enhanced the migratory and invasive capabilities of breast cancer cells by upregulating aldo-keto reductase family 1 member C3 (AKR1C3), without markedly affecting cell proliferation. Mechanistically, AKR1C3 promoted migration and invasion through activation of NF-κB signaling, leading to upregulated expression of MMP-2 and MMP-9. Conclusions: This study reveals a novel molecular axis—Pb exposure promotes breast cancer cell migration and invasion via the AKR1C3–NF-κB–MMP-2/MMP-9 pathway—and identifies AKR1C3 as a potential therapeutic target for breast cancer associated with environmental heavy metal exposure.

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Full text

1. Introduction

Breast cancer is the most prevalent malignancy affecting women’s health worldwide. According to the International Agency for Research on Cancer (IARC), breast cancer accounted for 23.8% of all new cancer cases in women globally in 2022. In the same year, approximately 660,000 women died from the disease, representing 15.4% of all cancer-related deaths in women [1]. Metastasis, a multistep and sequential process, is the leading cause of mortality among breast cancer patients [2]. This process is characterized by enhanced migratory and invasive capacities of cancer cells [3,4]. Tumor cells further facilitate dissemination by secreting matrix metalloproteinases (MMPs), which degrade the extracellular matrix, enabling entry into lymphatic and blood circulation and eventual colonization of distant organs. Among the MMP family, MMP-2 and MMP-9 play central roles in tumor invasion and metastasis [5,6], and their overexpression is strongly associated with aggressive breast cancer and poor prognosis [6]. For example, in lung adenocarcinoma, MMP-2 and MMP-9 are downstream targets of the NF-κB signaling, promoting epithelial–mesenchymal transition and bone metastasis [7]. Similarly, in breast cancer, the G-CSF/Stat3 pathway enhances migration and invasion by upregulating MMP-2 and MMP-9 expression [8]. Therefore, elucidating the mechanisms behind the dysregulated expression of MMP-2 and MMP-9 is essential to developing targeted anti-metastatic therapies.

Epidemiological studies indicate that environmental factors contribute significantly to breast cancer initiation and progression. Heavy metals, in particular, can act as metalloestrogens, promoting breast carcinogenesis [9]. Pb, a pervasive environmental toxicant, is classified as a Group 2A carcinogen by the IARC [10]. Following exposure, Pb can accumulate in various organs through protein binding or the formation of insoluble deposits, increasing cancer risk [11]. While some studies report positive correlations between Pb exposure and cancers of the lung, thyroid, and colorectum, associations with gastric or renal cancer are less clear [12,13]. Pb has also been linked to gastrointestinal cancers, including gastric, pancreatic, and bladder cancer [10,14].

Regarding breast cancer, evidence suggests that Pb exposure may elevate risk, often associated with oxidative stress [15,16], although other studies report no significant correlation [17]. Mechanistically, Pb can promote gastric cancer progression through oxidative stress, impaired DNA repair, and inflammation [12], and in breast cancer, Pb exposure enhances tumor aggressiveness via genomic instability and anti-apoptotic mechanisms [13]. Pb has also been shown to stimulate breast cancer cell proliferation through activation of the Erα pathway [18] and positively correlates with tumor volume, suggesting a role in tumor growth [19]. However, the specific mechanisms by which Pb contributes to breast cancer malignancy remain insufficiently understood. A systematic investigation into Pb’s role in breast cancer progression could improve environmental risk assessment and inform prevention and therapeutic strategies for exposed populations.

Aldo–keto reductase 1C3 (AKR1C3), also known as type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5), is a member of the human aldo-keto reductase superfamily [20]. It participates in steroid hormone metabolism and exhibits prostaglandin synthase activity [21]. AKR1C3 is highly expressed in multiple cancers, including prostate [22] and esophageal cancer [23], and its expression is significantly associated with tumor progression and prognosis. As a proto-oncogene, AKR1C3 regulates epithelial-mesenchymal transition (EMT) and angiogenesis, modulating proliferation, migration, and drug resistance in small-cell lung cancer [24]. In hepatocellular carcinoma, AKR1C3 activates NF-κB signaling to enhance tumor growth and metastasis [25], whereas silencing AKR1C3 in thyroid cancer suppresses autophagy-dependent glycolysis via ERK pathway inactivation [26]. Although AKR1C3 overexpression in breast cancer is well documented [27], prior studies have primarily focused on its role in chemoresistance [28,29], leaving its contribution to metastasis largely uncharacterized.

In this study, we demonstrated that Pb exposure markedly enhances the migratory and invasive capacities of breast cancer cells. Omics-based analyses identified AKR1C3 as a potential mediator of these pro-migratory and pro-invasive effects. Subsequent experiments confirmed that Pb upregulates AKR1C3 expression, which in turn modulates MMP-2 and MMP-9 levels, thereby promoting malignant progression in breast cancer cells. These findings provide a mechanistic link between environmental Pb exposure and breast cancer aggressiveness, highlighting AKR1C3 as a potential target for therapeutic intervention.

2. Materials and Methods

2.1. Cell Culture and PbAc Treatment

All cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). MCF-10A cells were grown in DMEM/F12 medium (PM150310, Procell, Wuhan, China) supplemented with 5% horse serum (164215, Procell), 20 ng/mL epidermal growth factor (E9644, Sigma-Aldrich, St. Louis, MO, USA), 0.5 mg/mL hydrocortisone (H0135, Sigma-Aldrich), 10 μg/mL insulin (PB180432, Procell), and 100 U/mL penicillin, 100 μg/mL streptomycin (P1400, Solarbio, Beijing, China). MCF-7 cells were maintained in RPMI-1640 medium (PM150113, Procell) supplemented with 10% fetal bovine serum (FBS, FSP500, ExCell Bio, Suzhou, China). Next, 293T cells were cultured in DMEM (PM150217, Procell) supplemented with 10% FBS and 100 U/mL penicillin, 100 μg/mL streptomycin. All cells were incubated at 37 °C in a humidified atmosphere of 5% CO_2_. Pb acetate (PbAc, 316512, Sigma-Aldrich) was first dissolved in ultrapure water to prepare a high-concentration stock solution (100 mM). Prior to use, this stock solution was further diluted with fresh complete RPMI-1640 or DMEM/F12 medium to the desired working concentrations (10, 20, 80, and 100 μM). All diluted working solutions were stored at 4 °C until use.

2.2. Cell Proliferation and Wound Healing Assays

MCF-10A and MCF-7 cells were seeded in 96-well plates (TCP011096, Biofil, Guangzhou, China) at a density of 5 × 10^3^ cells per well and treated with PbAc for 48 h. Cell proliferation was assessed using the MTT assay. Briefly, 10 μL of MTT solution (ST1537, Beyotime, Shanghai, China) was added to each well and incubated at 37 °C for 4 h, followed by the addition of 100 μL DMSO (CD4731, Coolaber, Beijing, China) and further incubation at 37 °C for 15 min to solubilize the formazan crystals. Absorbance was measured at 492 nm and recorded as the value at day 0, with measurements repeated at 24 h intervals for subsequent time points. The same experimental setup was used for wound healing-related analyses, with six replicates included for each treatment group. Cell viability was calculated using the formula [(As − Ab)/(Ac − Ab)] × 100%, where As, Ab, and Ac represent the absorbance values of the experimental, blank, and control groups, respectively.

2.3. Transwell Migration and Invasion Assays

First, MCF-10A and MCF-7 cells (3 × 10^4^ cells) were resuspended in 200 μL of serum-free medium containing the indicated concentrations of PbAc and plated in the upper chambers of Transwell inserts (3422, Corning, Corning, NY, USA). The lower chambers were filled with 500 μL of complete medium. After 24 h of incubation, the inserts were removed. The cells on the lower surface of the membrane were fixed with 4% paraformaldehyde (P1110, Solarbio) for 15 min and stained with 0.1% crystal violet (ST1537, Beyotime) for 30 min. Non-migrated cells on the upper side of the membrane were gently removed using a cotton swab, and the membrane was rinsed with PBS. The stained cells were imaged under a microscope, with five random fields captured per membrane. The migratory and invasive abilities were quantified by counting the average number of stained cells from three randomly selected fields. And the data from each experimental group were normalized to their corresponding control group. The data are presented as the mean ± SD from three independent biological replicates. To conduct the invasion assay, GM6001 (HY-15768, MedChemExpress, Monmouth Junction, NJ, USA) was added to the cells at a concentration of 10 μmol/L. Recombinant MMP-9 protein (HY-P73300, MedChemExpress) was activated with APMA (HY-148905, MedChemExpress) and then added to the cell culture medium at a concentration of 20 μg/mL. Transwell chambers were pre-coated with Matrigel (356234, Corning); all other procedures were identical to those used for the migration assay.

2.4. CCK-8 Assay

Cells in the logarithmic growth phase were seeded into 96-well plates. After complete adherence, the cells were treated with the respective compounds for 48 h. Subsequently, 10 μL of CCK-8 solution (SC119-02, Seven, Beijing, China) was added to each well, and the plates were incubated at 37 °C for 30 min. Then, the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader. Cell viability was calculated using the formula Cell Viability = [(As − Ab)/(Ac − Ab)] × 100%, where As represents the absorbance of the experimental group, Ab represents the absorbance of the blank group, and Ac represents the absorbance of the control group.

2.5. Detection of Intracellular Reactive Oxygen Species (ROS)

Cells in the logarithmic growth phase were seeded into 96-well plates. After complete adherence, the cells were treated with the respective compounds for 48 h. Subsequently, an appropriate concentration of a fluorescent probe (S0033S, Beyotime) was added to each well, and the plates were incubated at 37 °C for 30 min. The cells were then washed with serum-free medium. Finally, the fluorescence intensity of each group was measured using a fluorescence microplate reader. Each group contained three replicate wells, and data from three independent biological replicate experiments were used for statistical analysis.

2.6. Enzyme-Linked Immunosorbent Assays

The concentrations of MMP-2 (MM-0069H1, Meimian, Yancheng, Jiangsu, China) and MMP-9 (MM-0149H2, ELISA) in cell culture supernatants were measured using ELISA kits. Standards and samples were added to a microplate pre-coated with capture antibodies, incubated at 37 °C, and washed. Biotin-labeled detection antibodies and horseradish peroxidase (HRP)-conjugated streptavidin were then added sequentially, with thorough washing after each incubation step. Next, the TMB substrate was added for color development in the dark, and the reaction was terminated with the stop solution at the appropriate time. Absorbance was measured at 450 nm, and protein concentrations were calculated based on a standard curve. Throughout the procedure, reaction time was strictly controlled, washing was performed thoroughly, and contamination was carefully avoided.

2.7. RNA Extraction and qRT-PCR

Total RNA was isolated from cells with RNAiso Plus (9108, Takara, Kyoto, Japan), and cDNA was reverse-transcribed using the PrimeScript™ RT Reagent Kit (AU341, TransGen Biotech, Beijing, China). Quantitative real-time PCR (qRT-PCR) was conducted using a TB Green reagent kit (RR420A, TaKaRa). The relative expression of the target genes was calculated by the 2–ΔΔCT method and normalized to that of the housekeeping gene GAPDH. All experiments were conducted with at least three biological replicates, and data are presented as the mean ± standard deviation (SD) of triplicate measurements. The primers used were as follows: AKR1C3 (forward, 5′-CGAGACAAACGATGGGTGGA-3′; reverse, 5′-GTCTGATGCGCTGCTCATTG-3′), AKR1C1 (forward, 5′-CCAGTGTCTGTAAAGCCAGGT-3′; reverse, 5′-GTTGAAGTTGGACACCCCGA-3′), SLC7A11 (forward, 5′-TGTGTGGGGTCCTGTCACTA-3′; reverse, 5′-CAGTAGCTGCAGGGCGTATT-3′), and GAPDH (forward, 5′-ATGACCCCTTCATTGACCTCA-3′; reverse, 5′-GAGATGATGACCCTTTTGGCT-3′). SNAIL1 (forward, 5′-GCTGCAGGACTCTAATCCAGA-3′; reverse, 5′-ATCTCCGGAGGTGGGATG-3′), ZEB1 (forward, 5′-GGGAGGAGCAGTGAAAGAGA-3′; reverse, 5′-TTTCTTGCCCTTCCTTTCTG-3′).

2.8. Western Blotting Analysis

Before conducting the Western blotting analysis, the MCF-7 and MCF-10A cells were treated with various concentrations of PbAc. The cells were subsequently lysed and collected in RIPA buffer (C190419, YangGuangBio, Beijing, China) containing a cocktail of protease inhibitors (H221661, YangGuangBio). Protein extracts were then resolved by electrophoresis on SDS-PAGE gels using the Bio-Rad Protean II system. Once the proteins were transferred to a PVDF membrane, the membrane was blocked with a 5% non-fat dry milk solution in TBS-T for 60 min at room temperature. The membrane was subsequently incubated with the primary antibodies overnight at 4 °C (Table S1). The following day, the membrane was washed three times with TBS-T (15 min in total), followed by incubation with a fluorescently labeled secondary antibody at a dilution of 1:5000 for 50 min at room temperature. After further washing with TBS-T three times (15 min in total), the immunoreactive bands were visualized using the Tanon 4800 automated chemiluminescence imaging system.

2.9. RNA Sequencing, Functional Enrichment, and Hub Gene Analysis

After MCF-7 cells were treated with 10 or 20 μM PbAc for 48 h, total RNA was extracted using RNAiso Plus reagent, and RNA samples from three biological replicates per condition were packaged, snap-frozen on dry ice, and sent to GeneWiz (Suzhou, China) for RNA sequencing (RNA-seq). The quality control criteria include using fastp (0.22.0) to remove sequences with 3′-end adapters and discarding reads with an average quality score below Q20. All subsequent analyses were performed based on the clean data, using a fold change threshold of |Log2FC| > 2 and a p-value < 0.05 for identifying differentially expressed genes (DEGs). DEGs were subjected to functional enrichment analyses, including Gene Ontology (GO) term enrichment using the TopGO package and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using the clusterProfiler package (see Figure S1 for details). Hub genes were subsequently selected and analyzed using Gene Expression Profiling Interactive Analysis (GEPIA), a web-based platform integrating RNA-seq data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project. GEPIA was applied to evaluate the differential expression of hub genes between breast cancer tissues and corresponding normal tissues using the Expression DIY module with thresholds of |log2 fold change| ≥ 1 and p < 0.05. Furthermore, the Kaplan–Meier plotter was employed to assess the associations between hub gene expression levels and overall survival in breast cancer patients [30].

2.10. Construction of Stable Cell Lines Expressing Wild-Type and Enzymatically Inactive Mutant AKR1C3

The Myc-DDK-tagged AKR1C3 overexpression plasmid (RC200210, pCMV6-Entry) was provided by Professor Li Zhao (China Pharmaceutical University). The pWPXLd-AKR1C3 plasmid was generated by synthesizing cDNA from the Myc-DDK-tagged AKR1C3 template, followed by cloning it into the lentiviral expression vector pWPXLd (12258, Addgene, Cambridge, MA, USA). To produce lentiviral particles, 293T cells were co-transfected with pWPXLd-AKR1C3 and the lentiviral packaging plasmids psPAX2 and pMD2.G using the Lipo6000 transfection reagent (C0526, Beyotime Biotechnology, Shanghai, China). After 48 h, the lentivirus-containing supernatant was collected, filtered through a 0.22 μm syringe filter, and used to infect MCF-10A and MCF-7 cells. The enzyme-dead mutant plasmid (AKR1C3▲) was constructed by mutating Tyr 55 and His 117 to Ala in the pWPXLd-AKR1C3 plasmid using a site-directed mutagenesis kit (D0206S; Beyotime Biotechnology). The subsequent steps were the same as those used for generating AKR1C3-overexpressing cells.

2.11. Gene Silencing, Immunofluorescence, and Transcription Factor Prediction

Small interfering RNA (siRNA) targeting AKR1C3 (si-AKR1C3) and a non-targeting negative control (siRNA-NC) were designed and synthesized by Xi’an Calgary Biotechnology Co., Ltd. (Xi’an, Shaanxi, China) (Table S2). Cells were seeded in six-well plates and transfected at approximately 60% confluence using the Lipo6000 transfection reagent according to the manufacturer’s instructions. After 48 h, RNA or protein was extracted to evaluate transfection efficiency, and three independent experiments were used for subsequent analyses. For immunofluorescence (IF) assays, cells were seeded in 24-well plates at a density of 1 × 10^4^ cells per well, fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, and blocked with 4% BSA [31]. Cells were then incubated with primary antibodies at 4 °C overnight, followed by incubation with the corresponding secondary antibodies for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI-containing anti-fade mounting medium, and images were acquired using a Leica confocal fluorescence microscope.

Transcription factors potentially regulating AKR1C3 were predicted using the online TF Target Finder tool [32]. Candidate transcription factors were identified by intersecting results obtained from hTFtarget, TCGA, GTEx, and KnockTF databases, and subsequently filtered using the JASPAR database (https://jaspar.elixir.no, 9 December 2025). To predict transcription factor-binding sites, the core promoter region of AKR1C3 (Homo sapiens chromosome 10, GRCh38.p14 primary assembly: 5046780–5048780) was analyzed using JASPAR database. Statistical analyses were performed using GraphPad Prism (version 8.0), with data presented as the mean ± SD. Student’s t-test was used for comparisons between two groups, and one-way ANOVA was used for multiple-group comparisons, with p < 0.05 considered statistically significant.

3. Results

3.1. Pb Exposure Markedly Enhanced the Migratory and Invasive Capacities of Breast Cancer Cells Without Significantly Affecting Cell Proliferation

We first investigated the effects of Pb exposure on the migratory and invasive capacities of breast cancer cells. Wound healing assays (Figure 1A,B) revealed that Pb treatment promoted cell migration. This finding was confirmed by Transwell assays (Figure 1C,D), which also revealed enhanced migration. Moreover, Pb exposure significantly increased the invasiveness of MCF-7 cells (Figure 1E). Pb exposure promoted the migration and invasion of breast cancer cells. Importantly, this effect was not attributable to changes in cell viability or intracellular ROS levels at the concentrations used (Figure S2). Together, these observations indicate that Pb specifically activates a pro-metastatic program, rather than inducing non-specific cellular stress responses. As EMT is a key driver of tumor cell migration and invasion, we examined EMT markers. We found that Pb treatment induced the downregulation of the epithelial marker E-cadherin and upregulation of the mesenchymal marker N-cadherin (Figure S3A). We also found that the gene expression of Snail and ZEB1, negative regulators of E-cadherin, was upregulated upon Pb treatment (Figure S3B,C). To assess the effect of Pb on cell proliferation, we analyzed the cell cycle distribution by conducting flow cytometry assays. The results of the analysis revealed no significant difference in the percentages of cells in the G1, S, or G2 phase between the control and Pb-treated groups (Figure 1F,G). Consistent with these findings, MTT assays confirmed that the proliferative capacity of the cells remained unchanged following exposure to Pb (Figure 1H,I). Taken together, our data suggest that Pb enhances the migration and invasion but does not affect the proliferation of breast cancer cells.

3.2. Transcriptomic Analysis Revealed Potential Molecular Targets Underlying Pb-Induced Migration and Invasion in Breast Cancer Cells

To elucidate the mechanism by which Pb promotes the migration and invasion of breast cancer cells, MCF-7 cells were treated with 10 μM and 20 μM Pb for 48 h, and gene expression changes after Pb exposure were analyzed by RNA-seq (Files S1 and S2). The heatmap revealed that Pb treatment significantly altered the expression of multiple genes (Figure 2A). To identify potential gene targets involved in Pb-induced migration and invasion, DEGs were analyzed between each Pb-treated group and the control group. Compared to those in the control group, 29 DEGs were identified in the 10 μM Pb-treated group, among which 15 genes were significantly upregulated, and 14 genes were downregulated. In the 20 μM Pb-treated group, 18 DEGs were detected, including 11 upregulated genes and seven downregulated genes (Figure 2B,C). A Venn diagram analysis was performed to identify common DEGs across the two treatment groups, revealing six overlapping genes (Figure 2D). Based on evidence from other studies linking these genes to cancer progression, three genes—AKR1C1, AKR1C3, and SLC7A11—were selected for further validation. The results of the qRT-PCR assays confirmed that the expression of AKR1C1, AKR1C3, and SLC7A11 was significantly upregulated in Pb-exposed breast cancer cells (Figure 2E), which was consistent with the RNA-seq data. The results of the Western blotting analysis revealed that only AKR1C3 was significantly upregulated at the protein level (Figure 2F). Taken together, our data suggest that AKR1C3 may be a key target gene through which Pb enhances the migratory and invasive abilities of breast cancer cells.

3.3. AKR1C3 Promotes the Migration and Invasion of Breast Cancer Cells

To assess the biological role of AKR1C3 in breast cancer, we generated stable cell lines overexpressing either wild-type AKR1C3 or an enzyme-dead mutant (AKR1C3▲, Figure S4). Wound healing (Figure 3A,B) and Transwell migration assays (Figure 3C,D) revealed that the overexpression of AKR1C3 enhanced the migration of MCF-10A and MCF-7 cells in an enzyme activity-dependent manner. Similarly, the overexpression of AKR1C3 significantly promoted the invasion of MCF-7 cells (Figure 3E). Additionally, overexpression of AKR1C3 leads to corresponding changes in EMT markers, whereas the enzyme activity mutant fails to induce such alterations (Figure S5). To assess the functional necessity of AKR1C3, we knocked down the expression of AKR1C3 using siRNA. The knockdown efficiency was determined by qRT-PCR and Western blotting assays (Figure S6), and two effective siRNA constructs (#1 and #2) were selected for subsequent functional assays. Both wound healing (Figure 3F,G) and Transwell migration assays (Figure 3H,I) revealed that knocking down AKR1C3 markedly impaired cell migration. Consistent results were observed in Transwell invasion assays using MCF-7 cells (Figure 3J). We sought to exclude contributions from non-enzymatic functions of AKR1C3. To this end, we verified that its catalytic mutant retained wild-type subcellular localization (Figure 3K) and performed structural analyses showing that the mutation caused no major conformational perturbation, suggesting protein interactions remained intact (Figure 3L). These findings collectively argue that AKR1C3 promotes breast cancer cell migration and invasion through its catalytic activity.

3.4. Pb Promotes the Migration and Invasion of Breast Cancer Cells by Upregulating AKR1C3

Having established that Pb exposure and AKR1C3 overexpression independently enhance breast cancer cell migration and invasion and that Pb upregulates the transcription of AKR1C3, we next investigated whether AKR1C3 is functionally required for Pb-induced malignant progression. Knocking down AKR1C3 via siRNA in Pb-treated cells effectively rescued the pro-migratory phenotype, as indicated by wound healing (Figure 4A,B) and Transwell migration (Figure 4C,D) assays. Similarly, knocking down AKR1C3 abolished Pb-induced invasion in MCF-7 cells (Figure 4E). Collectively, these findings demonstrate that AKR1C3 upregulation is a key mechanistic event through which Pb promotes the migratory and invasive abilities of breast cancer cells.

3.5. Pb Activates NF-κB Signaling via AKR1C3 in Breast Cancer Cells

Based on previous findings that AKR1C3 promotes malignant progression in liver cancer via activation of the NF-κB signaling [25], we investigated whether a similar mechanism operates in Pb-induced migration and invasion of breast cancer cells. Western blotting analysis revealed that both Pb exposure and AKR1C3 overexpression markedly increased the phosphorylation levels of IκBα and P65 (Figure 5A,B). Additionally, immunofluorescence assays showed that either Pb treatment or AKR1C3 overexpression facilitated the nuclear translocation of P65 (Figure 5C), consistent with NF-κB signaling activation. To determine whether AKR1C3 mediates Pb-induced NF-κB signaling, we knocked down AKR1C3 in Pb-treated cells and observed that AKR1C3 depletion attenuated Pb-triggered NF-κB activation (Figure 5D). These observations support the conclusion that Pb enhances the NF-κB signaling pathway through the upregulation of AKR1C3.

The transcription factor NF-κB can induce the expression of MMP2 and MMP9, which are known for their extracellular matrix-degrading capacity and contribute to breast cancer malignancy [33]. Our findings indicated that both Pb exposure and AKR1C3 overexpression upregulated MMP2 and MMP9 expression compared to the control (Figure 5E,F). Under these conditions, we also detected an increase in the secretion of MMP2/9 (Figure S7). Furthermore, we found that the addition of a broad-spectrum MMP inhibitor (GM6001, 10 umol/L [33,34]) to the cells effectively suppressed the enhancement of cell invasiveness induced by Pb exposure and AKR1C3 overexpression (Figure 5G,H). Moreover, knocking down AKR1C3 under Pb treatment considerably reduced MMP2 and MMP9 levels (Figure 5I), suggesting that Pb increases their expression via AKR1C3. To examine whether AKR1C3 acts through NF-κB signaling to promote the expression of MMP, we inhibited this pathway using JSH-23. The results of Western blotting analysis revealed that JSH-23 abolished the AKR1C3-mediated upregulation of MMP2 and MMP9 (Figure 5J). Consistent with these findings, immunofluorescence assays revealed that JSH-23 blocked AKR1C3-induced NF-κB activation (Figure 5K), indicating that AKR1C3 requires NF-κB signaling to stimulate the expression of MMP. Moreover, we found that adding recombinant active MMP9 to the cells effectively reversed the reduction in cell invasiveness caused by AKR1C3 knockdown and NF-κB signaling inhibition (Figure 5L,M), indicating that the changes in invasiveness are dependent on MMPs. We next assessed whether NF-κB signaling is essential for AKR1C3-driven migration and invasion. Transwell migration (Figure 5N) and wound healing assays (Figure 5O) demonstrated that NF-κB inhibition nullified the enhancing effects of AKR1C3 on cell migration and invasion, a finding confirmed by Transwell invasion experiments (Figure 5P). These results indicated that AKR1C3 promotes breast cancer cell migration and invasion by activating NF-κB signaling, thereby increasing the expression of MMP2 and MMP9. To summarize, Pb facilitates the migration and invasion of breast cancer cells through a signaling cascade involving AKR1C3-mediated NF-κB activation and the subsequent upregulation of MMP2 and MMP9.

3.6. AKR1C3 Promotes MMP2/9 Expression and Breast Cancer Cell Migration and Invasion via NF-κB

The transcription factor NF-κB can induce the expression of MMP2 and MMP9, which degrade the extracellular matrix and contribute to breast cancer malignancy [35]. Our results showed that both Pb exposure and AKR1C3 overexpression upregulated MMP2 and MMP9 expression compared to the control (Figure 5E,F). Knocking down AKR1C3 in Pb-treated cells considerably reduced MMP2 and MMP9 levels (Figure 5I), suggesting that Pb increases their expression via AKR1C3. Inhibition of NF-κB signaling using JSH-23 abolished AKR1C3-mediated upregulation of MMP2 and MMP9 (Figure 5J), and immunofluorescence assays confirmed that JSH-23 blocked AKR1C3-induced NF-κB activation (Figure 5K). Transwell migration (Figure 5N, wound healing (Figure 5O), and invasion assays (Figure 5P) demonstrated that NF-κB inhibition nullified the enhancing effects of AKR1C3 on cell migration and invasion. Together, these results indicate that AKR1C3 promotes breast cancer cell migration and invasion by activating NF-κB signaling, thereby upregulating MMP2 and MMP9. In summary, Pb facilitates breast cancer cell migration and invasion via an AKR1C3–NF-κB–MMP2/9 signaling cascade (Figure 6).

4. Discussion

Pb, a widespread environmental toxicant, is commonly present in air, water, soil, and food sources [36]. Chronic exposure to Pb has long been recognized as a contributor to pathological alterations across multiple organ systems, underscoring its broad toxicological impact [37,38,39]. Given its ubiquitous presence, the impact of Pb and related heavy metals on cellular functions has drawn considerable attention in biological research. In vitro studies on heavy metals typically employ either low-dose long-term or high-dose short-term exposure models [40,41]. Cytotoxicity studies commonly use Pb concentrations of 10 μM or higher [42]. In this study, we applied a high-dose, short-term exposure model by treating breast cancer cells with 10 μM or 20 μM Pb acetate for 48 h. Although the Pb concentrations used in this study exceed typical environmental exposure levels, they are commonly applied in short-term in vitro models to elucidate molecular mechanisms underlying metal-induced cellular responses. Previous reports suggest that Pb can modulate immune responses in mice through Th17- and NF-κB-related pathways [43], and alter lipid and small-molecule metabolite profiles in breast cancer patients, indicating a potential link to cancer progression [40]. However, the specific molecular mechanisms by which Pb influences breast cancer remain largely unexplored. Here, we demonstrated that Pb exposure enhances the migratory and invasive capabilities of breast cancer cells in a concentration-dependent manner, and these effects are associated with genes previously linked to poor prognosis [44,45]. Notably, we identified AKR1C3 as a key mediator of Pb-induced malignant progression in breast cancer cells. EMT is a critical process through which cells acquire migratory and invasive capabilities. Our study demonstrated that both Pb exposure and AKR1C3 overexpression induced alterations in EMT markers (Figures S3A and S5). Furthermore, we detected upregulated gene expression of the EMT-regulating transcription factors Snail and ZEB1 following Pb treatment. Together with previous findings, these results suggest that Pb exposure may ultimately promote changes in cellular invasion and migration by triggering the EMT process.

As a prominent member of the AKR1C family, AKR1C3 performs diverse functions in hormone-related cancers. In prostate cancer, it promotes proliferation and invasion by catalyzing testosterone conversion to dihydrotestosterone (DHT) [46]. In cervical cancer, AKR1C3 enhances cell migration via suppression of lipocalin-2 (LCN2) [25]. In breast cancer, AKR1C3 overexpression correlates with tumor aggressiveness and chemotherapy resistance [47,48], and its role in hormone conversion can directly stimulate cancer cell proliferation [49]. Interestingly, in endometrial cancer, high AKR1C3 expression is associated with improved survival [50], indicating context-dependent functions. To examine its role in Pb-induced migration and invasion, we constructed an enzyme-dead mutant (Tyr55Ala/His117Ala) disrupting the NADP^+^ binding site [51]. Our results showed that Pb upregulates AKR1C3, activates NF-κB signaling, increases MMP2 and MMP9 expression, and promotes breast cancer cell migration and invasion, consistent with its tumor-promoting role in other cancers. The enzyme-dead mutant failed to activate NF-κB or upregulate MMP2/MMP9, confirming that AKR1C3’s enzymatic activity is required for its pro-migratory and pro-invasive functions. Although our data demonstrate that AKR1C3 enzymatic activity is required for NF-κB activation, the precise biochemical intermediates linking AKR1C3 catalysis to NF-κB signaling remain to be elucidated.

Transcription factors mediate gene expression by binding promoter or enhancer elements, thereby regulating RNA polymerase activity. To explore how Pb upregulates AKR1C3, we hypothesized that specific transcription factors might be involved. Bioinformatic analysis identified two potential TP63 binding sites within the AKR1C3 promoter, suggesting a role for TP63 in transcriptional regulation (Figure S8). TP63, a p53 family member, participates in multiple cancer-related processes, including cell growth, differentiation, and tumor suppression. For instance, TP63 regulates malignant phenotypes in esophageal cancer via SREBF1 [52] and coordinates with DLX5 and SOX2 in squamous cell carcinoma signaling [53]. Although bioinformatic evidence implies that TP63 may influence invasion and metastasis in invasive lobular carcinoma [54], functional validation is required. Whether TP63 mediates Pb-induced AKR1C3 upregulation remains an open question. Our bioinformatic analysis suggests TP63 as a potential upstream regulator of AKR1C3; however, functional validation will be required to confirm this regulatory relationship. Additionally, we observed that MCF-10A cells exhibited higher baseline migratory activity compared to MCF-7 cells. This difference may be attributed to the intrinsic biological characteristics of the two cell models: as an immortalized mammary epithelial cell line, MCF-10A often displays active baseline motility to facilitate anchorage and proliferation in vitro, whereas MCF-7, a luminal breast cancer cell line, is classically characterized by hormone-dependent proliferation and may maintain relatively stable epithelial colony morphology in standard two-dimensional migration assays. Importantly, this observation does not weaken but rather reinforces the core conclusion of our study. Our focus was to elucidate how Pb exposure enhances the migratory and invasive potential across different breast cancer cell models. The data demonstrate that Pb treatment significantly increased the baseline migration levels in both cell types. More critically, Pb exposure induced robust matrix-invasive capability exclusively in the malignant MCF-7 cells, a response directly relevant to the pathological process of cancer metastasis. In addition, a limitation of this study is that the functional connection to NF-κB was primarily established using a pharmacological inhibitor (JSH-23). We acknowledge that future studies should include rescue experiments involving forced activation of p65 to ultimately confirm its necessity within the entire research framework.

In summary, our study delineates a coherent signaling cascade in which Pb exposure promotes breast cancer cell migration and invasion by upregulating AKR1C3, activating NF-κB signaling, and inducing MMP2/MMP9 expression. Disruption of this AKR1C3–NF-κB–MMP2/MMP9 axis attenuates the malignant phenotype, highlighting its functional significance and potential as a therapeutic target for Pb-associated breast cancer progression.

5. Conclusions

This study demonstrates that environmental Pb exposure promotes the migration and invasion of breast cancer cells via an AKR1C3–NF-κB–MMP2/MMP9 signaling axis. Pb upregulates AKR1C3, which enzymatically activates NF-κB, leading to increased expression of MMP2 and MMP9 and enhanced malignant behavior. These findings provide novel mechanistic insights into how Pb contributes to breast cancer progression and identify the AKR1C3–NF-κB–MMP2/MMP9 axis as a potential therapeutic target for mitigating the effects of environmental heavy metal exposure on breast cancer.