4-Hydroxyestradiol Promotes Breast Carcinogenesis via Androgen Receptor: Evidence from Urinary Metabolite Profiling and Functional Studies

4-Hydroxyestradiol Promotes Breast Carcinogenesis via Androgen Receptor: Evidence from Urinary Metabolite Profiling and Functional Studies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 4-Hydroxyestradiol Promotes Breast Carcinogenesis via Androgen Receptor: Evidence from Urinary Metabolite Profiling and Functional Studies Ziquan Wang, Linzhu Zhang, Xiaofan Liu, Lin Fu, Rong Wu, Huanhuan Fei, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8136540/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective Breast cancer (BC) is a major global health threat with increasing incidence, and estrogen metabolites are implicated in its pathogenesis. To investigate alterations in urinary estrogen metabolites in BC patients and elucidate the oncogenic role and molecular mechanism of the metabolite 4-hydroxyestradiol (4-OH-E2) in BC progression. Methods A total of 126 treatment-naive BC patients and 103 healthy women were recruited to detect urinary estrogen metabolites using gas chromatography-tandem mass spectrometry (GC-MS/MS). MCF10A cells were treated with estradiol (E2) or 4-OH-E2 for 8 weeks (designated MCF10A-E and MCF10A-H, respectively), followed by assessments of cell morphology, migration (Transwell, scratch assay), invasion (Transwell), colony formation, and cell cycle (flow cytometry). Tumorigenicity assays were performed in BALB/c nude mice. Potential targets of 4-OH-E2 were predicted via SwissTargetPrediction and validated using GEPIA database. Loss-of-function experiments (AR silencing) were conducted to explore the role of androgen receptor (AR) in BC cells, with qRT-PCR, Western blot, and functional assays (proliferation, migration, invasion) used for verification. Results BC patients demonstrate dysregulated estrogen metabolism, marked by significantly elevated urinary 4-OH-E2. Chronic 4-OH-E2 exposure drives malignant transformation in mammary epithelial cells, enhancing tumorigenic phenotypes in vitro and in vivo. AR is a critical mediator of 4-OH-E2’s oncogenic effects, and its loss-of-function accelerates BC progression. Conclusion 4-OH-E2 is significantly elevated in BC patients and promotes breast carcinogenesis by enhancing cell malignant phenotypes and tumorigenicity, with AR as a critical target. These findings highlight 4-OH-E2 and AR as potential targets for BC prevention and treatment. Breast cancer Estrogen 4-Hydroxyestradiol Androgen Receptor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Breast cancer (BC), a significant global health concern affecting women, has emerged as the predominant malignancy among the female population worldwide. Recent epidemiological data indicate a continuous increase in its global incidence, with an estimated 2.3 million new cases diagnosed annually[ 1 , 2 ]. In China, the incidence of BC is exhibiting a similar upward trajectory, with a trend towards younger age of onset, imposing a substantial burden on affected families and the healthcare system. The pathogenesis of BC is characterized by its complexity, currently understood as a multifactorial, multistep process involving multiple genes[ 3 ]. A comprehensive elucidation of the underlying mechanisms of BC is paramount for the development of effective prevention, diagnostic, and therapeutic strategies. Accumulating evidence from clinical investigations reveals that approximately 70% of BC patients exhibit estrogen receptor (ER) positivity, highlighting the critical dependence of tumor growth on estrogen signaling, thereby classifying these tumors as hormone-dependent[ 4 ]. Clinical epidemiological studies further demonstrate that sustained elevation of endogenous estrogen levels or exogenous estrogen supplementation is significantly associated with increased BC incidence. Estrogen, a class of steroid hormones essential for the development and maintenance of the female reproductive system, exerts a complex and multifaceted role in both physiological and pathological processes within breast tissue. Its primary mechanism of action involves binding to ERs within breast cells, including the ERα and ERβ subtypes, with ERα considered a dominant driver in BC development and progression[ 5 ]. Upon ligand binding, ERs undergo a series of intricate molecular events, including conformational changes, dimerization, and translocation to the nucleus. Subsequently, the ER complex interacts with estrogen response elements (EREs) located within the promoter regions of target genes, modulating the transcription of numerous downstream genes involved in cellular proliferation, differentiation, apoptosis, and angiogenesis[ 6 – 8 ]. Under normal physiological conditions, estrogenic activity is subject to stringent regulatory control, ensuring a dynamic equilibrium between breast cell proliferation and programmed cell death. However, in pathological states, this delicate balance is frequently disrupted, and persistent, excessive estrogen stimulation may lead to aberrant proliferation of breast epithelial cells, ultimately promoting malignant transformation[ 9 , 10 ]. Estrogen is not a static entity in vivo , but rather undergoes complex metabolic transformations, resulting in the generation of diverse metabolites. An increasing body of research suggests that estrogen metabolites play a significant role in the initiation and progression of BC[ 11 ]. Numerous studies have posited that hydroxylated estrogens may exhibit important biological activities, thereby influencing BC development[ 12 , 13 ]. For example, a nested case-control study demonstrated that in postmenopausal women, the 2-hydroxylation pathway of estrone and estradiol was associated with an elevated risk of BC, independently of unbound estradiol levels[ 14 ]. A further nested case-control study within the Shanghai Women's Health Study cohort demonstrated that diminished urinary concentrations of maternal estrogens, coupled with increased 2-hydroxylation, were associated with reduced postmenopausal BC risk in a low-risk population[ 15 ]. Furthermore, the research by Wang et al. revealed that an elevated urinary ratio (> median) of 2-OHE1 to 16-OHE1 in BC patients was inversely correlated with overall mortality. This correlation was especially pronounced in patients receiving chemotherapy prior to urine collection[ 16 ]. Currently, the precise roles of several estrogen metabolites in BC remain incompletely characterized. Therefore, accurate quantification of changes in urinary estrogen metabolites, in conjunction with in-depth systematic investigation of their mechanisms of action in BC, will not only enhance our understanding of BC etiology but also provide a robust foundation for the development of novel targeted therapeutics and the optimization of personalized treatment regimens. This review will focus on key estrogen metabolites and their biological functions in BC, with the aim of providing a comprehensive and valuable reference for the scientific community engaged in BC research. 2. Materials and Methods 2.1 Detection of Urinary Estrogen and Its Metabolites in Breast Cancer Patients and Healthy Women From 2020 to 2021, a total of 126 patients with pathologically confirmed, treatment-naive breast cancer and 103 healthy women were recruited from Nanjing First Hospital. Exclusion criteria included smoking and alcohol abuse, pregnancy or lactation, reproductive and endocrine system disorders, impaired liver or kidney function, and a history of hormone therapy in both patients and healthy women. These factors were excluded as they can potentially alter endogenous estrogen levels and introduce errors in the detection results. Morning urine samples were collected from premenopausal women on days 13–15 of their menstrual cycle, while samples were collected from postmenopausal women on any random day. All samples were collected post-diagnosis but pre-surgery. Urine specimens from all participants were analyzed using gas chromatography-tandem mass spectrometry (GC-MS/MS). This study was approved by the Ethics Committee of Nanjing First Hospital (KY20251103-KS-02), and all the study procedures strictly adhered to the ethical principles outlined in the Declaration of Helsinki. Written informed consent was obtained from all participants with documentation maintained in secured study records. 2.2 Cells culture and treatment MCF10A human breast epithelial cells and MCF-7 human breast cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines had been authenticated by short tandem repeat (STR) profiling by ATCC and were confirmed to be free of Mycoplasma contamination prior to use. MCF10A cells were cultured in 1 mL of complete culture medium (DMEM/F12 (1:1) (C11330500BT, Gibco) supplemented with 5% horse serum (BL209A, biosharp), 10 µg/mL insulin (I189675, ALADDIN), 20 ng/mL epidermal growth factor (EGF, HYC020M01, Healthgen Biotechnology), 100 ng/mL cholera toxin (CTX, MX0931-1ml, MK), and 0.5 µg/mL hydrocortisone (IH0100, Solarbio). The cell suspension was mixed gently and then transferred to a 6 cm culture dish containing 2 mL of complete culture medium. The cells were incubated at 37°C in a humidified incubator with 5% CO 2 , and the medium was checked and replaced the following day. When the cell culture reached approximately 90% confluency, the supernatant was discarded, and the cells were washed twice with PBS. The cells were then detached by adding 0.25% trypsin, collected, and transferred to a 5 mL centrifuge tube. After centrifugation at 1200 rpm for 3 minutes, the supernatant was removed. The cell pellet was resuspended in 3 mL of complete culture medium, and the cells were passaged at a 1:3 ratio and cultured in a 37°C, 5% CO 2 incubator. MCF10A cells were treated with 10⁻⁹mol/L of E2 (HY-B0141, MCE) and 4-hydroxy-E2 (HY-N10403. MCE) for 8 weeks. These treated cells, along with the original MCF10A cells, were seeded onto 6-well plates at a density of 10⁴cells per well, with a volume of 2.5 mL per well. The plates were then incubated in a cell culture incubator at 37°C, 5% CO 2 , and saturated humidity for 48 hours. Cellular morphology was observed, and images were captured using a microscope (DMi1, Leica). The SiRNA transfection of AR in MCF-7 cells was performed using Lipofectamine 2000 (11668-027, Invitrogen) according to the operating instructions. The siRNA sequences of AR as follows: siRNA-1, 5’-GACUCAGCUGCCCCAUCCATT-3’; siRNA-2, 5’CACCAAUGUCAACUCCAGGAUTT-3’; siRNA-3, 5’-AAGACGCUUCUACCAGCUCACTT-3’. 2.3 Transwell Migration Assay MCF10A, MCF10A-E, and MCF10A-H cells in the logarithmic growth phase were harvested and the cell density adjusted to 5 × 10⁴cells/mL. For each cell line, 200 µL of the cell suspension was seeded into the upper chamber of a 24-well Transwell plate. The lower chamber was filled with 800 µL of culture medium supplemented with 20% serum. The plates were incubated for 24 h at 37°C in a humidified incubator with 5% CO 2 . After incubation, the inserts were removed, and the medium in the upper chamber was aspirated. Non-migrated cells on the upper surface of the membrane were carefully removed with a cotton swab. The membranes were washed twice with pre-warmed PBS (37°C) and fixed in ice-cold 4% paraformaldehyde (441244, Sigma) for 30 min, followed by staining with crystal violet (C0121, Beyotime) for 10 min. The polycarbonate membranes were then carefully excised from the bottom of the upper chamber, mounted onto microscope slides, and the cells that had migrated to the lower surface of the membrane were counted under a microscope. Images were captured from randomly selected fields under a light microscope. 2.4 Transwell Invasion Assay Matrigel (354234, BD), stored at -20°C, was thawed overnight on ice at 2–8°C. 100 µL of Matrigel was then mixed thoroughly with 100 µL of ice-cold culture medium using pre-chilled pipette tips. 50 µL of this diluted Matrigel solution was added to the upper chamber of each Transwell insert, covering the entire polycarbonate membrane. The plates were incubated at 37°C for 30 minutes to allow the Matrigel to polymerize and form a gel. MCF10A, MCF10A-E, and MCF10A-H cells in logarithmic growth phase were harvested and the cell density adjusted to 5 × 10⁴ cells/mL. For each cell line, 200 µL of the cell suspension was seeded into the upper chamber of a 24-well Transwell plate, coated with Matrigel as described above. The lower chamber was filled with 800 µL of culture medium supplemented with 20% serum. The plates were incubated for 24 h at 37°C in a humidified incubator with 5% CO 2 . After incubation, the inserts were removed, and the medium in the upper chamber was aspirated. The Matrigel gel and non-invading cells on the upper surface of the membrane were gently removed with a moistened cotton swab. The membranes were washed twice with pre-warmed PBS (37°C), fixed in ice-cold 4% paraformaldehyde for 30 min, and stained with crystal violet for 10 min. The polycarbonate membranes were carefully excised from the Transwell inserts, and the number of invading cells on the lower surface of the membrane was counted under a microscope. Images were captured from randomly selected fields under a light microscope. 2.5 Scratch Assay MCF10A, MCF10A-E, and MCF10A-H cells in logarithmic growth phase were seeded into 6 cm dishes to achieve approximately 80% confluence. Once the cells had formed a confluent monolayer, a scratch was created in each dish by dragging a 200 µL pipette tip across the cell layer, ensuring consistent scratch width across all dishes. The cell culture medium was then aspirated, and the dishes were washed three times with PBS to remove any cell debris generated during the scratching process. The cells were then incubated at 37°C in a humidified incubator with 5% CO 2 for 24 hours. After incubation, the supernatant was aspirated, and the cells were washed twice with PBS, followed by the addition of 2 mL of complete culture medium. Images of the same scratch location in each group were captured under a microscope (DMi1, Leica) at 0 hours and 24 hours. ImageJ software was used to quantify and analyze the migration distance of the cells in each group. 2.6 Colony Formation Assay MCF10A, MCF10A-E, and MCF10A-H cells in logarithmic growth phase were seeded into 6-well plates at a density of 300 cells per well. After allowing the cells to attach overnight (approximately 24 hours) at 37°C in a humidified incubator with 5% CO 2 (observed under an inverted microscope), MCF10A-E and MCF10A-H groups were supplemented with E2 and 4-OH-E2 at a concentration of 10⁻⁹mol/L. The cells were then cultured for an additional 10 days, with medium changes every 3 days. After 10 days, the supernatant was aspirated, and the cells were fixed with 4% paraformaldehyde for 20 minutes, washed twice with PBS, and stained with crystal violet for 10 minutes. Finally, the plates were rinsed with distilled water, and images were captured. 2.7 Cell Cycle Analysis by Flow Cytometry MCF10A, MCF10A-E, and MCF10A-H cells in logarithmic growth phase were seeded into 6-well cell culture plates at a density of 1 × 10⁵ cells/mL, with 2 mL per well and three replicates per group. After 24 hours of culture, the supernatant was removed, and the cells were washed twice with PBS. The cells were then trypsinized, collected, and centrifuged at 1500 rpm for 5 minutes, followed by removal of the supernatant. The cell pellet was washed once with 2 mL of PBS and centrifuged again to remove the PBS. The cells were then fixed by adding ice-cold 70% ethanol and incubating overnight at 4°C. After fixation, the cells were centrifuged at 1000 rpm for 3 minutes, the ethanol was discarded, and the cells were washed with PBS, followed by centrifugation at 1000 rpm for 3 minutes. The cell pellet was then resuspended in 0.5 mL of prepared propidium iodide (PI) staining solution (containing 0.5 mL staining buffer, 25 µL PI and 10 µL RNase A (C1052, Beyotime) and incubated at 37°C for 30 minutes. After staining, the cells were washed with ice-cold PBS solution and centrifuged at 1000 rpm for 3 minutes. Finally, 500 µL of the single-cell suspension was analyzed by flow cytometry (CytoFLEX, Beckman), detecting red fluorescence at an excitation wavelength of 488 nm, and the data were analyzed using appropriate software. 2.8 Animal Experiment The experiment was conducted in two independent runs, with each run involving two groups of five 6-week-old BALB/c nude mice (n = 5 per group), totaling four groups: A1, A2, B1, and B2. Prior to the experiment, mice were housed in a specific pathogen-free (SPF) environment for one week to allow for acclimatization. Logarithmically growing cells were trypsinized, suspended in PBS buffer at a concentration of 10⁷cells/75 µL PBS, and kept on ice. Immediately before injection, the cell suspension was mixed with an equal volume of Matrigel. Mice were held with their abdomen facing upwards. A 1 mL syringe with a needle was inserted into the left (or right) flank, slightly above the waist, ensuring that the distance from the injection site was less than the needle length. The needle was advanced towards the head, being careful not to puncture the skin outwards or the muscle layer inwards. Once the needle reached the injection site, the cell suspension was injected, and the needle was then withdrawn. MCF10A cells (left flank) and MCF10A-H cells (right flank) were injected into the same mouse. MCF10A-H cells (right flank) and MCF10A-E cells (left flank) were injected into the same mouse. Following cell injection, estradiol valerate tablets were ground into a powder, dissolved in a small amount of PBS, and 0.125 mg estradiol valerate was administered to each mouse via subcutaneous injection in the back once a week. Tumor size was measured every 7 days. On day 28 post-implantation, all nude mice were euthanized by cervical dislocation, and the tumors were excised. The study protocol was approved by the Animal Ethics Committee of Nanjing First Hospital (DWSY-25092724). All experiments were conducted in compliance with the relevant guidelines and regulations. 2.9 Target Prediction and Expression Analysis of 4-Hydroxyestradiol Potential targets of 4-hydroxyestradiol were predicted in silico using SwissTargetPrediction ( http://swisstargetprediction.ch/ ). Subsequent expression analysis of the predicted target genes was performed utilizing data from the GEPIA (Gene Expression Profiling Interactive Analysis, http://gepia.cancer-pku.cn/ ) database. 2.10 RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR Total RNA was isolated from cells using TRIzol reagent (BS259A, Biosharp) according to the manufacturer's protocol. First-strand cDNA was synthesized from the purified RNA using the cDNA Reverse Transcription Kit (KCD-M1003, Cronda) following the manufacturer's recommendations. Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green PCR Master Mix (KCD-M1004, Cronda) on a StepOne real-time PCR System (MA-600, Mmolarray). Quantitative PCR was performed with the following cycling parameters: initial denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 30 s and annealing/extension at 60°C for 1 min. A melting curve analysis was performed from 65°C to 97°C following the amplification. Relative gene expression was quantified using the ΔCT method, normalizing target gene CT values to the housekeeping gene β-actin using the formula: ΔCT = CTreference − CTtarget. The primer pairs were synthesized by and the sequences of the primers were as follows: AR, Forward: 5’-GACGACCAGATGGCTGTCATT-3’, Reverse: 5’-GGGCGAAGTAGAGCATCCT-3’;β-actin, Forward: 5’-ACGTGGACATCCGCAAAG-3’, Reverse༚5’-TGGAAGGTGGACAGCGAGGC-3’. 2.11 Western blot Cells were lysed in RIPA buffer (BL504A, Biosharp) supplemented with protease inhibitors (A32955, Thermo) and protein concentrations were determined using the BCA Protein Assay kit (BL521A, Biosharp) with absorbance measured at 750 nm on a multiwell spectrophotometer (DR-3518GL, Diatek). 20µg protein were separated by 10% SDS-PAGE and transferred to PVDF membranes (IPVH00010, Millipore). Membranes were blocked for 2 h at room temperature in TBS-T containing 5% non-fat milk and then incubated overnight at 4°C with primary antibodies: anti-AR (1:5000, 22089-1-AP, Proteintech) and anti-β-actin (1:2000, GB11001, Serivicebio). Following overnight incubation, membranes were washed and incubated with anti-mouse or anti-rabbit secondary antibodies (Cell Signaling Technology) at 1:10,000. Protein bands were visualized using enhanced chemiluminescence (WBKLS0100, Millipore). Band intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA). 2.12 Flow Cytometry Detection of EDU MCF-7 cells in the logarithmic growth phase were seeded in 6-well plates at adjusted densities and divided into three groups: Control, si-NC, and si-AR. Cells were transfected with si-NC or si-AR using Lipofectamine 2000, followed by replacement with fresh medium after 6 hours of incubation. After 48 hours of treatment, pre-configured 2× EDU working solution was added to achieve a final concentration of 10 µM, and cells were incubated for 2 hours. The supernatant was removed, and cells were washed with PBS, digested with trypsin, collected by centrifugation, and fixed with 4% paraformaldehyde for 15 minutes. After removing the fixative by centrifugation, cells were washed three times, permeabilized with PBS containing 0.3% Triton X-100 for 10–15 minutes, and washed 1–2 times. The Click reaction solution was prepared, and 0.5 ml was added to each well for a 30-minute incubation at room temperature in the dark. The Click reaction solution was removed by centrifugation, and cells were washed three times. A 500 µL single-cell suspension was analyzed using a flow cytometer (excitation wavelength: 488 nm) to detect green fluorescence, followed by software analysis. 2.13 Statistical Analysis Data are presented as mean ± SD from at least three independent experiments and analyzed using GraphPad Prism 6.0. Differences between two groups were assessed using unpaired t-tests (two-tailed), with Welch's correction applied when variances were unequal. Clinical data from lung cancer patients were analyzed using SPSS (version 15). Baseline characteristics were compared using the Chi-squared test. Statistical significance was defined as P < 0.05. 3. Results 3.1 The urinary concentration of 4-OH-E2 is significantly elevated in breast cancer patients A comparative analysis of demographic and lifestyle factors, including age, BMI, smoking status, alcohol consumption habits, and age at menarche, revealed no statistically significant differences between the breast cancer patient cohort and the healthy control group (Table 1 ). Table 1 Baseline Characteristics of Breast Cancer Patients and Healthy Women Characteristics Control (N = 103) Breast cancer (N = 126) P value Age 39.03 ± 3.48 39.94 ± 5.14 0.35 BMI 21.89 ± 1.74 22.25 ± 2.83 0.50 Smoking 0/37 0/54 1 Alcohol consumption 0/37 0/54 1 Age at menarche 14.40 ± 1.35 13.88 ± 1.45 0.27 Subsequent analysis, involving the quantification of E1, E2, and E3 estrogens and their corresponding metabolites, demonstrated a statistically significant reduction in urinary 2-OH-E1 concentrations in breast cancer patients relative to the control group. Conversely, concentrations of 2-Methoxy-E1, 2-Methoxy-E2, 4-OH-E1, 4-OH-E2, and 4-Methoxy-E1 were significantly increased in the breast cancer cohort. Notably, 4-OH-E2 exhibited the most substantial alteration in its metabolic profile concerning E2 (Table 2 , Fig. 1 A-C). In addition, the expression of SNCG and CYP1B1 which contribute to promote the shift of estrogen metabolism toward the production of the genotoxic metabolite 4-hydroxyestradiol was significantly elevated in the breast cancer compared with normal group (Fig. 1 D). Therefore, 4-OH-E2 was consequently chosen for further investigation. Table 2 Urinary Estrogen Metabolite Levels in Breast Cancer Patients and Healthy Controls Control (N = 103) Breast cancer (N = 126) Control (N = 103) OR (95% CI) * p* Estrone (E1) 3.09 ± 2.18 4.4 ± 3.44 0.05 1.89(0.97–3.66) 0.06 Estradiol (E2) 1.24 ± 0.81 1.47 ± 0.89 0.21 1.3(0.83–2.05) 0.26 Estriol (E3) 1.75 ± 1.14 2.45 ± 2.25 0.09 1.52(0.88–2.62) 0.13 2-OH-E1 0.89 ± 0.68 1.48 ± 1.04 0 2.18(1.25–3.82) 0.01 2-OH-E2 0.25 ± 0.22 0.22 ± 0.15 0.5 0.9(0.59–1.38) 0.63 2-Methoxy-E1 0.29 ± 0.19 0.92 ± 0.82 0 27.68(4.94-155.05) 0 2-Methoxy-E2 0.04 ± 0.03 0.08 ± 0.06 0 4.99(2.18–11.44) 0 4-OH-E1 0.18 ± 0.13 0.56 ± 0.38 0 20.47(4.94–84.87) 0 4-OH-E2 0.09 ± 0.04 0.15 ± 0.09 0 3.36(1.6–7.07) 0 4-Methoxy-E1 0.03 ± 0.02 0.05 ± 0.06 0.01 3.22(1.3–7.99) 0.01 4-Methoxy-E2 0.02 ± 0.02 0.03 ± 0.02 0.22 1.39(0.84–2.28) 0.2 16α-OH-E1 0.96 ± 0.58 0.89 ± 0.6 0.62 0.93(0.61–1.42) 0.74 3.2 4-OH-E2 Alters MCF10A Cell Morphology and Promotes Its Proliferation​, Invasion, Migration, Colony Formation, and Cell Cycle Progression Compared with MCF10A cells, MCF10A cells and MCF10A cells treated with E2 and 4-OH-E2 exhibited significant morphological changes. MCF10A cells showed a typical epithelial cell morphology, being polygonal or elliptical. MCF10A cells treated with E2 appeared polygonal or elongated. MCF10A-H cells treated with 4-OH-E2 were slender, with pseudopodia extending, enlarged nuclei, and an accelerated proliferation rate (Fig. 2 A). Transwell assay results for cell migration showed that compared with the MCF10A group, the number of migrating cells treated with E2 or 4-OH-E2 significantly increased, and the migration ability of MCF10A cells treated with 4-OH-E2 was stronger than that of MCF10A cells treated with E2. Transwell assay results for cell invasion indicated that compared with the MCF10A group, the number of invading cells treated with E2 and 4-OH-E2 dramatically increased, and the invasion ability of MCF10A cells treated with 4-OH-E2 was stronger than that of MCF10A cells treated with E2 (Fig. 2 B). Scratch test results demonstrated that compared with the MCF10A group, the scratch healing rate of cells treated with E2 or 4-OH-E2 was significantly increased, and the migration ability of MCF10A cells treated with 4-OH-E2 was superior to that of MCF10A cells treated with E2 (Fig. 2 C). Colony formation assay results revealed that compared with the MCF10A group, the colony formation rate of cells treated with E2 or 4-OH-E2 groups was significantly increased, and the colony-forming ability of MCF10A cells treated with 4-OH-E2 was stronger than that of MCF10A cells treated with E2 (Fig. 2 D). Flow cytometry analysis of the cell cycle showed that the number of MCF10A cells treated with E2 or 4-OH-E2 in the G0/G1 phase significantly decreased, while the number of cells in the S and G2/M phases significantly increased, indicating more cells in the proliferative state. Moreover, the proliferation ability of MCF10A cells treated 4-OH-E2 was greater than that of MCF10A cells treated with E2 (Fig. 2 E). 3.3 4-OH-E2 Promotes Tumorigenesis of MCF10A Cells in Nude Mice To verify whether E2 or 4-OH-E2 has the same functional changes in vivo, MCF10A cells treated with E2 or 4-OH-E2 were subsequently subjected to tumorigenicity assays in nude mice. The results showed that MCF10A cells treated with E2 or 4-OH-E2 could form tumors in vivo, while untreated MCF10A cells could not (Fig. 3 A). Moreover, the tumorigenicity rate and tumor volume of MCF10A cells treated with 4-OH-E2 were greater than those of MCF10A cells treated with E2 (Fig. 3 B and C ). The above results indicate that 4-OH-E2 promotes tumorigenesis of MCF10A cells. 3.4 AR is a Key Target of 4-Hydroxyestradiol​ The targets of 4-hydroxyestradiol were predicted using SwissTargetPrediction ( http://swisstargetprediction.ch/ ), and a total of 48 valid targets were obtained ( Table 3 ). Expression analysis of the top 5 targets (including AR, ADCY10, ESR1, ESR2 and SHBG) using the TCGA database revealed that compared with adjacent tissues, the expression of AR and ESR1 in cancer tissues was significantly increased (Fig. 4 ), especially for AR with a probability value for 1.0, suggesting that AR may be a key target of 4-hydroxyestradiol, which requires further research. 3.5 Silencing AR Promotes Proliferation, Migration, Invasion, Colony Formation, and Cell Cycle Progression of Breast Cancer Cells​ To further investigate the role of AR in breast cancer, we performed loss-of-function experiments by knocking down AR expression in breast cancer cells. Quantitative real-time PCR (RT-qPCR) analysis of AR mRNA levels demonstrated a significant reduction in AR expression in the si-AR group compared to the si-NC (negative control) group for all three siRNAs tested (Fig. 5 A). The si-AR-2 siRNA exhibited the highest silencing efficiency (Fig. 5 A) and was thus selected for subsequent experiments. Consistently, Western blot analysis revealed a marked decrease in AR protein expression in the si-AR group compared to the si-NC group (Fig. 5 B). Transwell migration and invasion assays showed a significant increase in the number of migrating and invading cells following AR knockdown (Fig. 5 C). Similarly, wound healing assays demonstrated a significantly enhanced migratory capacity of cells upon AR silencing (Fig. 5 D). Furthermore, colony formation assays revealed a substantial increase in colony formation ability after AR knockdown (Fig. 5 E). Cell cycle analysis indicated a reduction in the proportion of cells in the G0/G1 phase and a concomitant increase in the proportion of cells in the S and G2/M phases following AR knockdown, suggesting an accelerated cell proliferation rate (Fig. 5 F). In line with these findings, EDU incorporation assays showed a significantly higher number of EDU-positive cells after AR knockdown (Fig. 5 G), indicating an increased proportion of cells undergoing active proliferation. 4. Discussion Breast cancer (BC), characterized by its intricate heterogeneity, presents a formidable challenge to women's health on a global scale. Among the multitude of factors implicated in its pathogenesis, the role of estrogens has remained a central focus of intense investigation. These steroid hormones are recognized to exert a critical influence on the proliferation of both normal and neoplastic mammary epithelial cells. Epidemiological evidence has consistently substantiated long-held hypotheses regarding the potential carcinogenic effects of estrogens, which they mediate through diverse mechanisms of action[ 17 ]. Historically, research efforts have primarily concentrated on the "cumulative estrogen exposure" paradigm, wherein elevated circulating concentrations of estradiol (E2) and estrone (E1) were postulated to promote mammary epithelial proliferation and facilitate the accumulation of somatic mutations via estrogen receptor (ER)-mediated transcriptional programs. Estrogens exert their effects through binding to both nuclear and membrane-associated ERs, initiating a cascade of intracellular signaling pathways that ultimately stimulate cellular proliferation. However, with the advancement of high-resolution mass spectrometry techniques and the emergence of single-cell spatial metabolomics platforms, the emphasis in BC research has gradually transitioned from quantifying individual hormone concentrations towards elucidating the complex "estrogen metabolic profile". In vivo , estrogens undergo a series of metabolic transformations, including Phase I (hydroxylation) and Phase II (methylation, glucuronidation, and sulfation) reactions, resulting in the generation of a diverse array of metabolites exhibiting distinct biological activities. These metabolites include 2-hydroxy, 4-hydroxy, and 16α-hydroxy derivatives. Certain metabolic products, such as 4-hydroxyestradiol-quinone, possess the capacity to directly form DNA adducts, thereby inducing genomic instability. Conversely, 16α-hydroxyestrone exhibits potent ER agonist activity. These observations have led to the development of dualistic carcinogenic models, encompassing both "receptor-driven" and "genotoxic" mechanisms, which provide a more nuanced theoretical framework for comprehending the molecular underpinnings of estrogen-promoted breast carcinogenesis[ 11 ]. For instance, studies have demonstrated significant associations between circulating concentrations of estradiol and estrone with elevated breast cancer risk. Furthermore, the 2-hydroxylation pathway of estrone and estradiol has been implicated in increased risk, independent of the concentration of non-conjugated estradiol[ 14 , 18 ]. Within the context of the present investigation, we sought to characterize the estrogen landscape through the targeted analysis of E1, E2, E3, and their corresponding metabolites. Our findings revealed a significant reduction in urinary 2-OH-E1 concentrations among breast cancer patients. Concomitantly, we observed a marked elevation in the levels of 2-Methoxy-E1, 2-Methoxy-E2, 4-OH-E1, 4-OH-E2, and 4-Methoxy-E1, suggesting that these estrogen metabolites may play a salient role in the pathogenesis and progression of BC. Estradiol (E2), the principal circulating estrogen, undergoes metabolic biotransformation primarily via the action of cytochrome P450 (CYP) enzymes. Among the resulting metabolites, 4-hydroxyestradiol (4-OH-E2) has garnered considerable attention within the realm of cancer research, owing to its classification as a putatively carcinogenic estrogen derivative. Evidence suggests that 4-OH-E2 elicits a diverse array of biological effects that may contribute to the development and progression of breast cancer. For example, 4-OH-E2 has been implicated in the induction of reactive oxygen species (ROS) generation, which are well-documented mediators of DNA damage and may promote neoplastic transformation in human mammary epithelial cells[ 19 , 20 ]. Furthermore, 4-OH-E2 exhibits inherent estrogenic activity, capable of binding to estrogen receptors (ERα and ERβ), thereby modulating downstream gene expression and influencing cellular function. Studies have also demonstrated that 4-OHE2 can augment the expression of heme oxygenase-1 (HO-1) in breast cells, with HO-1 serving as a critical component of the cellular oxidative stress response. Notably, 4-OHE2-mediated induction of HO-1 expression may be regulated by the Nrf2/Keap1 signaling axis, ultimately influencing cellular proliferation and malignant transformation[ 21 ]. In a relevant study, Wu et al. reported that long-term exposure of MCF-10A cells to 4-OHE2 resulted in the acquisition of malignant characteristics, including enhanced cellular proliferation, epithelial-mesenchymal transition (EMT), and augmented migratory and invasive capacity, accompanied by a significant upregulation of CYP1B1 expression. Intriguingly, pharmacological blockade of CYP1B1 attenuated the malignant phenotype observed in MCF-10A cells following chronic exposure to 4-OHE2[ 22 ]. Complementary investigations conducted by KIM et al. revealed that nordihydroguaiaretic acid (NDGA), functioning as both a substrate and inhibitor of catechol-O-methyltransferase (COMT), suppressed COMT-mediated formation of 4-methoxyestradiol (4-MeOE2), while concomitantly exacerbating 4-OHE2-induced DNA damage and cellular toxicity. These findings suggest that NDGA possesses the potential to modulate COMT activity within mammary tissues, potentially preventing the inactivation of mutagenic estradiol metabolites and thereby enhancing catechol estrogen-mediated genotoxicity[ 23 ]. In concordance with these cumulative findings, our current investigation revealed that 4-OH-E2 promotes the invasive, migratory, and clonogenic capabilities of MCF10A cells, while also accelerating cell cycle progression. Furthermore, our in vivo studies demonstrated that 4-OH-E2 facilitates the tumorigenicity of MCF10A cells and fosters subsequent tumor growth. To further elucidate the downstream targets of 4-OH-E2, we performed in silico prediction of its target genes, leading to the identification of the androgen receptor (AR). AR, a member of the nuclear receptor superfamily, functions as a transcription factor modulating eukaryotic gene expression. It plays a pivotal role in the development and maintenance of homeostasis in various physiological systems, including the reproductive, skeletal muscle, cardiovascular, neurological, immune, and hematopoietic systems[ 24 – 26 ]. In the oncological context, while AR was initially considered relevant primarily to male-related tissues, a growing body of evidence implicates aberrant AR signaling in the pathogenesis of breast cancer and other malignancies, such as prostate, bladder, kidney, and lung cancers[ 27 – 29 ]. Breast cancer is broadly classified into subtypes including estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-positive, and triple-negative breast cancer (TNBC) [ 30 ]. The androgen receptor (AR) is increasingly recognized to play a significant role in the pathogenesis of breast cancer (BC). AR is expressed in over 70% of breast cancer tumors, rendering it a potential biomarker and therapeutic target [ 31 ]. Studies have demonstrated a significant association between AR expression and histological grade, recurrence patterns, and molecular subtypes, with ER, PR, HER2, and tumor recurrence identified as independent factors influencing AR expression[ 32 ]. AR expression is typically elevated in ER-positive breast cancer[ 33 ], and AR activation may suppress the growth of ER-positive breast cancer cells, suggesting a potential interplay between AR and ER signaling pathways[ 34 , 35 ]. Concurrently, ER-positive breast cancer patients exhibiting high AR expression demonstrate a diminished response to neoadjuvant chemotherapy, yet display improved survival outcomes[ 36 ]. AR expression is observed in approximately 60%-70% of TNBC cases, which can be further categorized into distinct molecular subtypes, including the Luminal Androgen Receptor (LAR) subtype characterized by elevated AR expression[ 30 ]. Umay et al. utilized digital image analysis (AR-DIA) to objectively assess AR expression, demonstrating its superiority over subjective scoring methods, with a 10% AR-DIA cutoff value identified as the strongest negative prognostic threshold for distant metastasis. While AR-DIA did not confer additional prognostic value in the favorable-prognosis TNBC subgroup, it was significantly informative in the poor-prognosis subgroup, underscoring the importance of AR expression assessment for TNBC prognosis and AR-targeted therapeutic strategies [ 37 ]. A multitude of studies have indicated that AR functions as a tumor suppressor and a favorable prognostic marker in ER-positive breast cancer. Conversely, in ER-negative breast cancer, AR is implicated as a tumor promoter and a less favorable prognostic indicator[ 38 , 39 ]. In a study involving 931 patients, survival curve analysis revealed a significant association between the presence of AR in ER + tumors and superior disease-free survival (DFS) and overall survival (OS). A similar trend was observed in a study encompassing 1467 postmenopausal breast cancer patients[ 40 ]. Furthermore, an independent investigation revealed that elevated AR expression in ER + tumors correlates with reduced lymphocyte infiltration, indicative of a more favorable prognosis and prolonged survival [ 4 ]. Some studies have suggested that AR may contribute to the growth and metastasis of HER2-positive breast cancer cells Targeting Androgen Receptor in Estrogen Receptor-Negative Breast Cancer. In the present study, we observed that AR knockdown promoted the proliferation, migration, invasion, colony formation, and cell cycle progression of MCF-7 breast cancer cells. AR has been established as a tumor suppressor in ER + breast cancer, an observation that highlights the intricate interplay between AR and the ERα signaling pathway in this malignancy. Studies have demonstrated that, in ER-positive breast cancer, AR agonists can impede tumor cell proliferation through interactions with the ER signaling cascade[ 41 ]. Furthermore, research indicates that AR activation can trigger the dissociation of ERα from chromatin, with AR occupying over 40% of ERα binding sites (ERBS), consequently suppressing the binding of ERα to estrogen response elements (EREs). Concomitantly, investigations have revealed that ERα can acquire novel binding targets by translocating to a subset of AR binding sites (ARBS), subsequently modulating the expression of AR target genes, including the tumor suppressors SEC14L2, EAF2, and ZBTB16, ultimately resulting in inhibited cell growth. In addition, AR can compete with ERα for binding to the shared co-activator p300, a factor critical for ERα activity. Given that ERα relies on the co-regulatory protein SRC-3 to facilitate p300 recruitment, whereas AR can bind directly to p300, AR may possess a competitive advantage, thereby attenuating ER signaling pathway activation[ 42 ]. AR may also exert indirect inhibitory effects on ERα activity through specific mediator proteins. Estrogen receptor beta (ERβ), a known inhibitor of ERα, is subject to regulation by activated AR. Through binding to androgen response elements (AREs) located in the promoter region of ERβ, AR can upregulate ERβ gene expression, consequently dampening the ER signaling pathway. Collectively, AR activation can suppress ERα activity via multiple distinct mechanisms. Considering the pivotal role of ERα as a major driver of tumor growth in ER-positive breast cancer, the inhibition of ERα activity represents a potentially effective strategy for slowing disease progression[ 43 ]. Furthermore, AR and ER may exert reciprocal influences on their respective functions through modulation of intracellular signaling pathways, such as the PI3K/AKT and MAPK cascades[ 31 ]. Spatial genomics analyses suggest that the molecular characteristics of AR-expressing breast cancer cells within the tumor microenvironment correlate with improved overall survival in patients, providing clinical validation for the tumor-suppressive role of AR. In ER-positive breast cancer xenografts, ligand-mediated AR activation reprograms cistromes, attenuates oncogenic signaling pathways, and promotes cellular resilience toward a more differentiated phenotype. However, sustained AR activation can induce cistrome rearrangement, favoring the transcription factor PROP paired-like homeobox 1, thereby transforming AR into an oncogene and activating the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, culminating in lineage plasticity and a transition to a resistant, invasive subtype[ 44 ]. However, these observations warrant further investigation. 5. Conclusion This study establishes 4-hydroxyestradiol (4-OH-E2) as a critical metabolite linked to breast cancer pathogenesis through multifaceted evidence. Mechanistically, 4-OH-E2 likely promotes breast cancer development, at least in part, by antagonizing or downregulating the tumor-suppressive function of AR, provides a novel mechanistic link for the metabolite's carcinogenic effects and points to the AR pathway as a potential therapeutic avenue. Abbreviations BC, breast cancer 4-OH-E2, 4-hydroxyestradiol E2, estradiol AR, androgen receptor ER, estrogen receptor ERα, estrogen receptor alpha ERβ, estrogen receptor beta PR, progesterone receptor HER2, human epidermal growth factor receptor 2 GC-MS/MS, gas chromatography–tandem mass spectrometry GEPIA, Gene Expression Profiling Interactive Analysis SPF, specific pathogen-free FBS, fetal bovine serum EGF, epidermal growth factor CTX, cholera toxin PBS, phosphate-buffered saline DMSO, dimethyl sulfoxide PI, propidium iodide EDU, 5-ethynyl-2´-deoxyuridine siRNA, small interfering RNA qRT-PCR, quantitative real-time polymerase chain reaction WB, Western blot HO-1, heme oxygenase-1 NDGA, nordihydroguaiaretic acid COMT, catechol-O-methyltransferase EMT, epithelial-mesenchymal transition ARE, androgen response element ERE, estrogen response element ARBS, androgen receptor binding site ERBS, estrogen receptor binding site SD, standard deviation HR, hazard ratio CI, confidence interval Declarations Ethics approval and Consent form This study was reviewed and approved by the Ethics Committee of Nanjing First Hospital (KY20251103-KS-02), and all the study procedures strictly adhered to the ethical principles outlined in the World Medical Association Declaration of Helsinki. Written informed consent was obtained from all participants with documentation maintained in secured study records. The animal experiment was approved by the Animal Ethics Committee of Nanjing First Hospital (DWSY-25092724). All experiments were conducted in compliance with the relevant guidelines and regulations. Availability of data and materials All data generated or analysed during this study are included in this published article. The original data can be available from the corresponding author on reasonable request. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This study was supported by Nanjing Medical Science and Technology Development Fund (YKK23129), National Natural Science Foundation of China (82203182), China Postdoctoral Science Foundation (2024M763658), and the Project of Future Technology Star of Nanjing First Hospital. Author contributions Wei Liang, Fei Fei and Xiaowei Wei developed the study concept and design. Ziquan Wang, Linzhu Zhang, and Xiaofan Liu completed the acquisition of data. Fei Fei and Linzhu Zhang made contributions to data analysis. Lin Fu, Rong Wu, and Huanhuan Fei made contributions to data interpretation. Fei Fei and Ziquan Wang drafted the manuscript. All authors critically revised, read and approved the final version of the manuscript, and agreed to be accountable for the study. 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Breast cancer research: BCR. 2014;16(1):R21. Asemota S, Effah W, Holt J, Johnson D, Cripe L, Ponnusamy S, Thiyagarajan T, Khosrosereshki Y, Hwang DJ, He Y, et al. A molecular switch from tumor suppressor to oncogene in ER + ve breast cancer: Role of androgen receptor, JAK-STAT, and lineage plasticity. Proc Natl Acad Sci USA. 2024;121(40):e2406837121. Table 3 Table 3 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table3.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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17:02:52","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":167355,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8136540/v1/ca83f287423afcd84e0d55cb.html"},{"id":98439740,"identity":"1697dc89-b142-4e28-a073-d386d91e7b41","added_by":"auto","created_at":"2025-12-17 17:02:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1045265,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe urinary concentration of 4-OH-E2 is significantly elevated in breast cancer patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-C. The urinary concentration of 2-OH, 4-OH and 16α-OH- in breast cancer patients；D, The expression of SNCG and CYP1B1. **, p\u0026lt;0.01; ***, p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8136540/v1/1e5100263e62fee3e355b744.png"},{"id":98377197,"identity":"2da44a29-e889-4729-9c5c-5de57f60aa08","added_by":"auto","created_at":"2025-12-17 07:11:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1427342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e4-OH-E2 alters MCF10A cell morphology and enhances proliferation, invasion, migration, colony formation, and cell cycle progression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA．Representative micrographs of cell morphology for MCF10A；B. Transwell assay was used to detect the migration and invasion of MCF10A treated with E2 or 4-OH-E2; C.Wound-healing assay was used to detect the wound closure of MCF10A treated with E2 or 4-OH-E2 at 0 h and 24 h; D. Colony formation assay was used to detect the colony formation rate of MCF10A treated with E2 or 4-OH-E2; E. Flow cytometry was used to detect cell cycle of MCF10A treated with E2 or 4-OH-E2; *, p\u0026lt;0.05; **, p\u0026lt;0.01; ***, p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8136540/v1/4b0b547b2d63a9f47cc9720f.png"},{"id":98377196,"identity":"76e53bc7-54de-4748-980c-fbbf7605257e","added_by":"auto","created_at":"2025-12-17 07:11:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":223402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e4-OH-E2 promotes tumorigenesis of MCF10A cells in nude mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Representative images of tumor formation in nude mice injection with MCF10A cells treated with E2 or 4-OH-E2; B. Tumor volume growth curve from MCF10A-E cells treated with E2 or 4-OH-E2 over 28 days; C. Quantitative analysis of tumorigenicity rate;\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8136540/v1/54b54c08c4b1608569ca3c27.png"},{"id":98440140,"identity":"80343c51-def4-4f9c-b195-8182eda7b77f","added_by":"auto","created_at":"2025-12-17 17:03:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1531451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAR is a key target of 4-hydroxyestradiol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, Top 5 potential targets of 4-OH-E2 predicted by SwissTargetPrediction; B, Expression analysis using TCGA database shows significantly higher AR expression in breast cancer tissues compared to adjacent normal tissues; *, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8136540/v1/0387ca185cdf0022895bc98b.png"},{"id":98377200,"identity":"5c18f6b9-1d0d-4ef9-8bce-74cb8512f922","added_by":"auto","created_at":"2025-12-17 07:11:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":482962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAR silencing enhances proliferation, migration, invasion, colony formation, and cell cycle progression in breast cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. RT-qPCR analysis of AR mRNA expression after transfection with three siRNA of AR dependently; B, Western blot analysis of AR protein expression; C: Transwell assay was used to detect migration and invasion of MCF-7 cells with AR silence; D. Wound-healing assay was used to detect the migratory capacity of MCF-7 cells with AR silence; E. The colony-forming ability of MCF-7 cells affected by AR was detected by colony formation assay; F. Flow cytometry was used to detect cell cycle of MCF-7 cells affected by AR; G. EdU staining was used to detect the proliferation of MCF-7 cells affected by AR. ns, no significance; **, p\u0026lt;0.01; ***, p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8136540/v1/e0258d95550b82a710567a3e.png"},{"id":109759243,"identity":"c65f9c65-bd31-407c-b7ee-5acd248a1c67","added_by":"auto","created_at":"2026-05-22 07:26:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6435539,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8136540/v1/098039e2-2c24-48f6-a302-9086330f3fcb.pdf"},{"id":98440652,"identity":"dc38f04b-ce2d-4098-943f-dbb363414010","added_by":"auto","created_at":"2025-12-17 17:04:08","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16051,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8136540/v1/e10bd93e05c326776a644027.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"4-Hydroxyestradiol Promotes Breast Carcinogenesis via Androgen Receptor: Evidence from Urinary Metabolite Profiling and Functional Studies","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBreast cancer (BC), a significant global health concern affecting women, has emerged as the predominant malignancy among the female population worldwide. Recent epidemiological data indicate a continuous increase in its global incidence, with an estimated 2.3\u0026nbsp;million new cases diagnosed annually[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In China, the incidence of BC is exhibiting a similar upward trajectory, with a trend towards younger age of onset, imposing a substantial burden on affected families and the healthcare system. The pathogenesis of BC is characterized by its complexity, currently understood as a multifactorial, multistep process involving multiple genes[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A comprehensive elucidation of the underlying mechanisms of BC is paramount for the development of effective prevention, diagnostic, and therapeutic strategies.\u003c/p\u003e \u003cp\u003eAccumulating evidence from clinical investigations reveals that approximately 70% of BC patients exhibit estrogen receptor (ER) positivity, highlighting the critical dependence of tumor growth on estrogen signaling, thereby classifying these tumors as hormone-dependent[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Clinical epidemiological studies further demonstrate that sustained elevation of endogenous estrogen levels or exogenous estrogen supplementation is significantly associated with increased BC incidence. Estrogen, a class of steroid hormones essential for the development and maintenance of the female reproductive system, exerts a complex and multifaceted role in both physiological and pathological processes within breast tissue. Its primary mechanism of action involves binding to ERs within breast cells, including the ERα and ERβ subtypes, with ERα considered a dominant driver in BC development and progression[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Upon ligand binding, ERs undergo a series of intricate molecular events, including conformational changes, dimerization, and translocation to the nucleus. Subsequently, the ER complex interacts with estrogen response elements (EREs) located within the promoter regions of target genes, modulating the transcription of numerous downstream genes involved in cellular proliferation, differentiation, apoptosis, and angiogenesis[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Under normal physiological conditions, estrogenic activity is subject to stringent regulatory control, ensuring a dynamic equilibrium between breast cell proliferation and programmed cell death. However, in pathological states, this delicate balance is frequently disrupted, and persistent, excessive estrogen stimulation may lead to aberrant proliferation of breast epithelial cells, ultimately promoting malignant transformation[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Estrogen is not a static entity \u003cem\u003ein vivo\u003c/em\u003e, but rather undergoes complex metabolic transformations, resulting in the generation of diverse metabolites. An increasing body of research suggests that estrogen metabolites play a significant role in the initiation and progression of BC[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Numerous studies have posited that hydroxylated estrogens may exhibit important biological activities, thereby influencing BC development[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. For example, a nested case-control study demonstrated that in postmenopausal women, the 2-hydroxylation pathway of estrone and estradiol was associated with an elevated risk of BC, independently of unbound estradiol levels[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. A further nested case-control study within the Shanghai Women's Health Study cohort demonstrated that diminished urinary concentrations of maternal estrogens, coupled with increased 2-hydroxylation, were associated with reduced postmenopausal BC risk in a low-risk population[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, the research by Wang et al. revealed that an elevated urinary ratio (\u0026gt;\u0026thinsp;median) of 2-OHE1 to 16-OHE1 in BC patients was inversely correlated with overall mortality. This correlation was especially pronounced in patients receiving chemotherapy prior to urine collection[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, the precise roles of several estrogen metabolites in BC remain incompletely characterized. Therefore, accurate quantification of changes in urinary estrogen metabolites, in conjunction with in-depth systematic investigation of their mechanisms of action in BC, will not only enhance our understanding of BC etiology but also provide a robust foundation for the development of novel targeted therapeutics and the optimization of personalized treatment regimens. This review will focus on key estrogen metabolites and their biological functions in BC, with the aim of providing a comprehensive and valuable reference for the scientific community engaged in BC research.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Detection of Urinary Estrogen and Its Metabolites in Breast Cancer Patients and Healthy Women\u003c/h2\u003e \u003cp\u003eFrom 2020 to 2021, a total of 126 patients with pathologically confirmed, treatment-naive breast cancer and 103 healthy women were recruited from Nanjing First Hospital. Exclusion criteria included smoking and alcohol abuse, pregnancy or lactation, reproductive and endocrine system disorders, impaired liver or kidney function, and a history of hormone therapy in both patients and healthy women. These factors were excluded as they can potentially alter endogenous estrogen levels and introduce errors in the detection results. Morning urine samples were collected from premenopausal women on days 13\u0026ndash;15 of their menstrual cycle, while samples were collected from postmenopausal women on any random day. All samples were collected post-diagnosis but pre-surgery. Urine specimens from all participants were analyzed using gas chromatography-tandem mass spectrometry (GC-MS/MS). This study was approved by the Ethics Committee of Nanjing First Hospital (KY20251103-KS-02), and all the study procedures strictly adhered to the ethical principles outlined in the Declaration of Helsinki. Written informed consent was obtained from all participants with documentation maintained in secured study records.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cells culture and treatment\u003c/h2\u003e \u003cp\u003eMCF10A human breast epithelial cells and MCF-7 human breast cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines had been authenticated by short tandem repeat (STR) profiling by ATCC and were confirmed to be free of Mycoplasma contamination prior to use. MCF10A cells were cultured in 1 mL of complete culture medium (DMEM/F12 (1:1) (C11330500BT, Gibco) supplemented with 5% horse serum (BL209A, biosharp), 10 \u0026micro;g/mL insulin (I189675, ALADDIN), 20 ng/mL epidermal growth factor (EGF, HYC020M01, Healthgen Biotechnology), 100 ng/mL cholera toxin (CTX, MX0931-1ml, MK), and 0.5 \u0026micro;g/mL hydrocortisone (IH0100, Solarbio). The cell suspension was mixed gently and then transferred to a 6 cm culture dish containing 2 mL of complete culture medium. The cells were incubated at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e, and the medium was checked and replaced the following day. When the cell culture reached approximately 90% confluency, the supernatant was discarded, and the cells were washed twice with PBS. The cells were then detached by adding 0.25% trypsin, collected, and transferred to a 5 mL centrifuge tube. After centrifugation at 1200 rpm for 3 minutes, the supernatant was removed. The cell pellet was resuspended in 3 mL of complete culture medium, and the cells were passaged at a 1:3 ratio and cultured in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. MCF10A cells were treated with 10⁻⁹mol/L of E2 (HY-B0141, MCE) and 4-hydroxy-E2 (HY-N10403. MCE) for 8 weeks. These treated cells, along with the original MCF10A cells, were seeded onto 6-well plates at a density of 10⁴cells per well, with a volume of 2.5 mL per well. The plates were then incubated in a cell culture incubator at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, and saturated humidity for 48 hours. Cellular morphology was observed, and images were captured using a microscope (DMi1, Leica). The SiRNA transfection of AR in MCF-7 cells was performed using Lipofectamine 2000 (11668-027, Invitrogen) according to the operating instructions. The siRNA sequences of AR as follows: siRNA-1, 5\u0026rsquo;-GACUCAGCUGCCCCAUCCATT-3\u0026rsquo;; siRNA-2, 5\u0026rsquo;CACCAAUGUCAACUCCAGGAUTT-3\u0026rsquo;; siRNA-3, 5\u0026rsquo;-AAGACGCUUCUACCAGCUCACTT-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Transwell Migration Assay\u003c/h2\u003e \u003cp\u003eMCF10A, MCF10A-E, and MCF10A-H cells in the logarithmic growth phase were harvested and the cell density adjusted to 5 \u0026times; 10⁴cells/mL. For each cell line, 200 \u0026micro;L of the cell suspension was seeded into the upper chamber of a 24-well Transwell plate. The lower chamber was filled with 800 \u0026micro;L of culture medium supplemented with 20% serum. The plates were incubated for 24 h at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. After incubation, the inserts were removed, and the medium in the upper chamber was aspirated. Non-migrated cells on the upper surface of the membrane were carefully removed with a cotton swab. The membranes were washed twice with pre-warmed PBS (37\u0026deg;C) and fixed in ice-cold 4% paraformaldehyde (441244, Sigma) for 30 min, followed by staining with crystal violet (C0121, Beyotime) for 10 min. The polycarbonate membranes were then carefully excised from the bottom of the upper chamber, mounted onto microscope slides, and the cells that had migrated to the lower surface of the membrane were counted under a microscope. Images were captured from randomly selected fields under a light microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Transwell Invasion Assay\u003c/h2\u003e \u003cp\u003eMatrigel (354234, BD), stored at -20\u0026deg;C, was thawed overnight on ice at 2\u0026ndash;8\u0026deg;C. 100 \u0026micro;L of Matrigel was then mixed thoroughly with 100 \u0026micro;L of ice-cold culture medium using pre-chilled pipette tips. 50 \u0026micro;L of this diluted Matrigel solution was added to the upper chamber of each Transwell insert, covering the entire polycarbonate membrane. The plates were incubated at 37\u0026deg;C for 30 minutes to allow the Matrigel to polymerize and form a gel. MCF10A, MCF10A-E, and MCF10A-H cells in logarithmic growth phase were harvested and the cell density adjusted to 5 \u0026times; 10⁴ cells/mL. For each cell line, 200 \u0026micro;L of the cell suspension was seeded into the upper chamber of a 24-well Transwell plate, coated with Matrigel as described above. The lower chamber was filled with 800 \u0026micro;L of culture medium supplemented with 20% serum. The plates were incubated for 24 h at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. After incubation, the inserts were removed, and the medium in the upper chamber was aspirated. The Matrigel gel and non-invading cells on the upper surface of the membrane were gently removed with a moistened cotton swab. The membranes were washed twice with pre-warmed PBS (37\u0026deg;C), fixed in ice-cold 4% paraformaldehyde for 30 min, and stained with crystal violet for 10 min. The polycarbonate membranes were carefully excised from the Transwell inserts, and the number of invading cells on the lower surface of the membrane was counted under a microscope. Images were captured from randomly selected fields under a light microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Scratch Assay\u003c/h2\u003e \u003cp\u003eMCF10A, MCF10A-E, and MCF10A-H cells in logarithmic growth phase were seeded into 6 cm dishes to achieve approximately 80% confluence. Once the cells had formed a confluent monolayer, a scratch was created in each dish by dragging a 200 \u0026micro;L pipette tip across the cell layer, ensuring consistent scratch width across all dishes. The cell culture medium was then aspirated, and the dishes were washed three times with PBS to remove any cell debris generated during the scratching process. The cells were then incubated at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours. After incubation, the supernatant was aspirated, and the cells were washed twice with PBS, followed by the addition of 2 mL of complete culture medium. Images of the same scratch location in each group were captured under a microscope (DMi1, Leica) at 0 hours and 24 hours. ImageJ software was used to quantify and analyze the migration distance of the cells in each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Colony Formation Assay\u003c/h2\u003e \u003cp\u003eMCF10A, MCF10A-E, and MCF10A-H cells in logarithmic growth phase were seeded into 6-well plates at a density of 300 cells per well. After allowing the cells to attach overnight (approximately 24 hours) at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e (observed under an inverted microscope), MCF10A-E and MCF10A-H groups were supplemented with E2 and 4-OH-E2 at a concentration of 10⁻⁹mol/L. The cells were then cultured for an additional 10 days, with medium changes every 3 days. After 10 days, the supernatant was aspirated, and the cells were fixed with 4% paraformaldehyde for 20 minutes, washed twice with PBS, and stained with crystal violet for 10 minutes. Finally, the plates were rinsed with distilled water, and images were captured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell Cycle Analysis by Flow Cytometry\u003c/h2\u003e \u003cp\u003eMCF10A, MCF10A-E, and MCF10A-H cells in logarithmic growth phase were seeded into 6-well cell culture plates at a density of 1 \u0026times; 10⁵ cells/mL, with 2 mL per well and three replicates per group. After 24 hours of culture, the supernatant was removed, and the cells were washed twice with PBS. The cells were then trypsinized, collected, and centrifuged at 1500 rpm for 5 minutes, followed by removal of the supernatant. The cell pellet was washed once with 2 mL of PBS and centrifuged again to remove the PBS. The cells were then fixed by adding ice-cold 70% ethanol and incubating overnight at 4\u0026deg;C. After fixation, the cells were centrifuged at 1000 rpm for 3 minutes, the ethanol was discarded, and the cells were washed with PBS, followed by centrifugation at 1000 rpm for 3 minutes. The cell pellet was then resuspended in 0.5 mL of prepared propidium iodide (PI) staining solution (containing 0.5 mL staining buffer, 25 \u0026micro;L PI and 10 \u0026micro;L RNase A (C1052, Beyotime) and incubated at 37\u0026deg;C for 30 minutes. After staining, the cells were washed with ice-cold PBS solution and centrifuged at 1000 rpm for 3 minutes. Finally, 500 \u0026micro;L of the single-cell suspension was analyzed by flow cytometry (CytoFLEX, Beckman), detecting red fluorescence at an excitation wavelength of 488 nm, and the data were analyzed using appropriate software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Animal Experiment\u003c/h2\u003e \u003cp\u003eThe experiment was conducted in two independent runs, with each run involving two groups of five 6-week-old BALB/c nude mice (n\u0026thinsp;=\u0026thinsp;5 per group), totaling four groups: A1, A2, B1, and B2. Prior to the experiment, mice were housed in a specific pathogen-free (SPF) environment for one week to allow for acclimatization. Logarithmically growing cells were trypsinized, suspended in PBS buffer at a concentration of 10⁷cells/75 \u0026micro;L PBS, and kept on ice. Immediately before injection, the cell suspension was mixed with an equal volume of Matrigel. Mice were held with their abdomen facing upwards. A 1 mL syringe with a needle was inserted into the left (or right) flank, slightly above the waist, ensuring that the distance from the injection site was less than the needle length. The needle was advanced towards the head, being careful not to puncture the skin outwards or the muscle layer inwards. Once the needle reached the injection site, the cell suspension was injected, and the needle was then withdrawn. MCF10A cells (left flank) and MCF10A-H cells (right flank) were injected into the same mouse. MCF10A-H cells (right flank) and MCF10A-E cells (left flank) were injected into the same mouse. Following cell injection, estradiol valerate tablets were ground into a powder, dissolved in a small amount of PBS, and 0.125 mg estradiol valerate was administered to each mouse via subcutaneous injection in the back once a week. Tumor size was measured every 7 days. On day 28 post-implantation, all nude mice were euthanized by cervical dislocation, and the tumors were excised. The study protocol was approved by the Animal Ethics Committee of Nanjing First Hospital (DWSY-25092724). All experiments were conducted in compliance with the relevant guidelines and regulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Target Prediction and Expression Analysis of 4-Hydroxyestradiol\u003c/h2\u003e \u003cp\u003ePotential targets of 4-hydroxyestradiol were predicted in silico using SwissTargetPrediction (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Subsequent expression analysis of the predicted target genes was performed utilizing data from the GEPIA (Gene Expression Profiling Interactive Analysis, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from cells using TRIzol reagent (BS259A, Biosharp) according to the manufacturer's protocol. First-strand cDNA was synthesized from the purified RNA using the cDNA Reverse Transcription Kit (KCD-M1003, Cronda) following the manufacturer's recommendations. Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green PCR Master Mix (KCD-M1004, Cronda) on a StepOne real-time PCR System (MA-600, Mmolarray). Quantitative PCR was performed with the following cycling parameters: initial denaturation at 95\u0026deg;C for 15 min, followed by 40 cycles of denaturation at 95\u0026deg;C for 30 s and annealing/extension at 60\u0026deg;C for 1 min. A melting curve analysis was performed from 65\u0026deg;C to 97\u0026deg;C following the amplification. Relative gene expression was quantified using the ΔCT method, normalizing target gene CT values to the housekeeping gene β-actin using the formula: ΔCT\u0026thinsp;=\u0026thinsp;CTreference\u0026thinsp;\u0026minus;\u0026thinsp;CTtarget. The primer pairs were synthesized by and the sequences of the primers were as follows: AR, Forward: 5\u0026rsquo;-GACGACCAGATGGCTGTCATT-3\u0026rsquo;, Reverse: 5\u0026rsquo;-GGGCGAAGTAGAGCATCCT-3\u0026rsquo;;β-actin, Forward: 5\u0026rsquo;-ACGTGGACATCCGCAAAG-3\u0026rsquo;, Reverse༚5\u0026rsquo;-TGGAAGGTGGACAGCGAGGC-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Western blot\u003c/h2\u003e \u003cp\u003eCells were lysed in RIPA buffer (BL504A, Biosharp) supplemented with protease inhibitors (A32955, Thermo) and protein concentrations were determined using the BCA Protein Assay kit (BL521A, Biosharp) with absorbance measured at 750 nm on a multiwell spectrophotometer (DR-3518GL, Diatek). 20\u0026micro;g protein were separated by 10% SDS-PAGE and transferred to PVDF membranes (IPVH00010, Millipore). Membranes were blocked for 2 h at room temperature in TBS-T containing 5% non-fat milk and then incubated overnight at 4\u0026deg;C with primary antibodies: anti-AR (1:5000, 22089-1-AP, Proteintech) and anti-β-actin (1:2000, GB11001, Serivicebio). Following overnight incubation, membranes were washed and incubated with anti-mouse or anti-rabbit secondary antibodies (Cell Signaling Technology) at 1:10,000. Protein bands were visualized using enhanced chemiluminescence (WBKLS0100, Millipore). Band intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Flow Cytometry Detection of EDU\u003c/h2\u003e \u003cp\u003eMCF-7 cells in the logarithmic growth phase were seeded in 6-well plates at adjusted densities and divided into three groups: Control, si-NC, and si-AR. Cells were transfected with si-NC or si-AR using Lipofectamine 2000, followed by replacement with fresh medium after 6 hours of incubation. After 48 hours of treatment, pre-configured 2\u0026times; EDU working solution was added to achieve a final concentration of 10 \u0026micro;M, and cells were incubated for 2 hours. The supernatant was removed, and cells were washed with PBS, digested with trypsin, collected by centrifugation, and fixed with 4% paraformaldehyde for 15 minutes. After removing the fixative by centrifugation, cells were washed three times, permeabilized with PBS containing 0.3% Triton X-100 for 10\u0026ndash;15 minutes, and washed 1\u0026ndash;2 times. The Click reaction solution was prepared, and 0.5 ml was added to each well for a 30-minute incubation at room temperature in the dark. The Click reaction solution was removed by centrifugation, and cells were washed three times. A 500 \u0026micro;L single-cell suspension was analyzed using a flow cytometer (excitation wavelength: 488 nm) to detect green fluorescence, followed by software analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Statistical Analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from at least three independent experiments and analyzed using GraphPad Prism 6.0. Differences between two groups were assessed using unpaired t-tests (two-tailed), with Welch's correction applied when variances were unequal. Clinical data from lung cancer patients were analyzed using SPSS (version 15). Baseline characteristics were compared using the Chi-squared test. Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The urinary concentration of 4-OH-E2 is significantly elevated in breast cancer patients\u003c/h2\u003e \u003cp\u003eA comparative analysis of demographic and lifestyle factors, including age, BMI, smoking status, alcohol consumption habits, and age at menarche, revealed no statistically significant differences between the breast cancer patient cohort and the healthy control group (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline Characteristics of Breast Cancer Patients and Healthy Women\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (N\u0026thinsp;=\u0026thinsp;103)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBreast cancer (N\u0026thinsp;=\u0026thinsp;126)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.94\u0026thinsp;\u0026plusmn;\u0026thinsp;5.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlcohol consumption\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge at menarche\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSubsequent analysis, involving the quantification of E1, E2, and E3 estrogens and their corresponding metabolites, demonstrated a statistically significant reduction in urinary 2-OH-E1 concentrations in breast cancer patients relative to the control group. Conversely, concentrations of 2-Methoxy-E1, 2-Methoxy-E2, 4-OH-E1, 4-OH-E2, and 4-Methoxy-E1 were significantly increased in the breast cancer cohort. Notably, 4-OH-E2 exhibited the most substantial alteration in its metabolic profile concerning E2 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). In addition, the expression of SNCG and CYP1B1 which contribute to promote the shift of estrogen metabolism toward the production of the genotoxic metabolite 4-hydroxyestradiol was significantly elevated in the breast cancer compared with normal group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Therefore, 4-OH-E2 was consequently chosen for further investigation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUrinary Estrogen Metabolite Levels in Breast Cancer Patients and Healthy Controls\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (N\u0026thinsp;=\u0026thinsp;103)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBreast cancer (N\u0026thinsp;=\u0026thinsp;126)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl (N\u0026thinsp;=\u0026thinsp;103)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOR (95% CI) *\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEstrone (E1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.09\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.89(0.97\u0026ndash;3.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEstradiol (E2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.3(0.83\u0026ndash;2.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEstriol (E3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.52(0.88\u0026ndash;2.62)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-OH-E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.18(1.25\u0026ndash;3.82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-OH-E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.9(0.59\u0026ndash;1.38)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-Methoxy-E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27.68(4.94-155.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-Methoxy-E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.99(2.18\u0026ndash;11.44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-OH-E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20.47(4.94\u0026ndash;84.87)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-OH-E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.36(1.6\u0026ndash;7.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-Methoxy-E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.22(1.3\u0026ndash;7.99)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-Methoxy-E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.39(0.84\u0026ndash;2.28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16α-OH-E1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.93(0.61\u0026ndash;1.42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 4-OH-E2 Alters MCF10A Cell Morphology and Promotes Its Proliferation​, Invasion, Migration, Colony Formation, and Cell Cycle Progression\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCompared with MCF10A cells, MCF10A cells and MCF10A cells treated with E2 and 4-OH-E2 exhibited significant morphological changes. MCF10A cells showed a typical epithelial cell morphology, being polygonal or elliptical. MCF10A cells treated with E2 appeared polygonal or elongated. MCF10A-H cells treated with 4-OH-E2 were slender, with pseudopodia extending, enlarged nuclei, and an accelerated proliferation rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Transwell assay results for cell migration showed that compared with the MCF10A group, the number of migrating cells treated with E2 or 4-OH-E2 significantly increased, and the migration ability of MCF10A cells treated with 4-OH-E2 was stronger than that of MCF10A cells treated with E2. Transwell assay results for cell invasion indicated that compared with the MCF10A group, the number of invading cells treated with E2 and 4-OH-E2 dramatically increased, and the invasion ability of MCF10A cells treated with 4-OH-E2 was stronger than that of MCF10A cells treated with E2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Scratch test results demonstrated that compared with the MCF10A group, the scratch healing rate of cells treated with E2 or 4-OH-E2 was significantly increased, and the migration ability of MCF10A cells treated with 4-OH-E2 was superior to that of MCF10A cells treated with E2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Colony formation assay results revealed that compared with the MCF10A group, the colony formation rate of cells treated with E2 or 4-OH-E2 groups was significantly increased, and the colony-forming ability of MCF10A cells treated with 4-OH-E2 was stronger than that of MCF10A cells treated with E2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Flow cytometry analysis of the cell cycle showed that the number of MCF10A cells treated with E2 or 4-OH-E2 in the G0/G1 phase significantly decreased, while the number of cells in the S and G2/M phases significantly increased, indicating more cells in the proliferative state. Moreover, the proliferation ability of MCF10A cells treated 4-OH-E2 was greater than that of MCF10A cells treated with E2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 4-OH-E2 Promotes Tumorigenesis of MCF10A Cells in Nude Mice\u003c/h2\u003e \u003cp\u003eTo verify whether E2 or 4-OH-E2 has the same functional changes in vivo, MCF10A cells treated with E2 or 4-OH-E2 were subsequently subjected to tumorigenicity assays in nude mice. The results showed that MCF10A cells treated with E2 or 4-OH-E2 could form tumors in vivo, while untreated MCF10A cells could not (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Moreover, the tumorigenicity rate and tumor volume of MCF10A cells treated with 4-OH-E2 were greater than those of MCF10A cells treated with E2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u003cb\u003eand C\u003c/b\u003e). The above results indicate that 4-OH-E2 promotes tumorigenesis of MCF10A cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 AR is a Key Target of 4-Hydroxyestradiol​\u003c/h2\u003e \u003cp\u003eThe targets of 4-hydroxyestradiol were predicted using SwissTargetPrediction (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and a total of 48 valid targets were obtained (\u003cb\u003eTable\u0026nbsp;3\u003c/b\u003e). Expression analysis of the top 5 targets (including AR, ADCY10, ESR1, ESR2 and SHBG) using the TCGA database revealed that compared with adjacent tissues, the expression of AR and ESR1 in cancer tissues was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), especially for AR with a probability value for 1.0, suggesting that AR may be a key target of 4-hydroxyestradiol, which requires further research.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5 Silencing AR Promotes Proliferation, Migration, Invasion, Colony Formation, and Cell Cycle Progression of Breast Cancer Cells​\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the role of AR in breast cancer, we performed loss-of-function experiments by knocking down AR expression in breast cancer cells. Quantitative real-time PCR (RT-qPCR) analysis of AR mRNA levels demonstrated a significant reduction in AR expression in the si-AR group compared to the si-NC (negative control) group for all three siRNAs tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The si-AR-2 siRNA exhibited the highest silencing efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and was thus selected for subsequent experiments. Consistently, Western blot analysis revealed a marked decrease in AR protein expression in the si-AR group compared to the si-NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Transwell migration and invasion assays showed a significant increase in the number of migrating and invading cells following AR knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Similarly, wound healing assays demonstrated a significantly enhanced migratory capacity of cells upon AR silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Furthermore, colony formation assays revealed a substantial increase in colony formation ability after AR knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Cell cycle analysis indicated a reduction in the proportion of cells in the G0/G1 phase and a concomitant increase in the proportion of cells in the S and G2/M phases following AR knockdown, suggesting an accelerated cell proliferation rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). In line with these findings, EDU incorporation assays showed a significantly higher number of EDU-positive cells after AR knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), indicating an increased proportion of cells undergoing active proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eBreast cancer (BC), characterized by its intricate heterogeneity, presents a formidable challenge to women's health on a global scale. Among the multitude of factors implicated in its pathogenesis, the role of estrogens has remained a central focus of intense investigation. These steroid hormones are recognized to exert a critical influence on the proliferation of both normal and neoplastic mammary epithelial cells. Epidemiological evidence has consistently substantiated long-held hypotheses regarding the potential carcinogenic effects of estrogens, which they mediate through diverse mechanisms of action[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Historically, research efforts have primarily concentrated on the \"cumulative estrogen exposure\" paradigm, wherein elevated circulating concentrations of estradiol (E2) and estrone (E1) were postulated to promote mammary epithelial proliferation and facilitate the accumulation of somatic mutations via estrogen receptor (ER)-mediated transcriptional programs. Estrogens exert their effects through binding to both nuclear and membrane-associated ERs, initiating a cascade of intracellular signaling pathways that ultimately stimulate cellular proliferation. However, with the advancement of high-resolution mass spectrometry techniques and the emergence of single-cell spatial metabolomics platforms, the emphasis in BC research has gradually transitioned from quantifying individual hormone concentrations towards elucidating the complex \"estrogen metabolic profile\". \u003cem\u003eIn vivo\u003c/em\u003e, estrogens undergo a series of metabolic transformations, including Phase I (hydroxylation) and Phase II (methylation, glucuronidation, and sulfation) reactions, resulting in the generation of a diverse array of metabolites exhibiting distinct biological activities. These metabolites include 2-hydroxy, 4-hydroxy, and 16α-hydroxy derivatives. Certain metabolic products, such as 4-hydroxyestradiol-quinone, possess the capacity to directly form DNA adducts, thereby inducing genomic instability. Conversely, 16α-hydroxyestrone exhibits potent ER agonist activity. These observations have led to the development of dualistic carcinogenic models, encompassing both \"receptor-driven\" and \"genotoxic\" mechanisms, which provide a more nuanced theoretical framework for comprehending the molecular underpinnings of estrogen-promoted breast carcinogenesis[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For instance, studies have demonstrated significant associations between circulating concentrations of estradiol and estrone with elevated breast cancer risk. Furthermore, the 2-hydroxylation pathway of estrone and estradiol has been implicated in increased risk, independent of the concentration of non-conjugated estradiol[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Within the context of the present investigation, we sought to characterize the estrogen landscape through the targeted analysis of E1, E2, E3, and their corresponding metabolites. Our findings revealed a significant reduction in urinary 2-OH-E1 concentrations among breast cancer patients. Concomitantly, we observed a marked elevation in the levels of 2-Methoxy-E1, 2-Methoxy-E2, 4-OH-E1, 4-OH-E2, and 4-Methoxy-E1, suggesting that these estrogen metabolites may play a salient role in the pathogenesis and progression of BC.\u003c/p\u003e \u003cp\u003eEstradiol (E2), the principal circulating estrogen, undergoes metabolic biotransformation primarily via the action of cytochrome P450 (CYP) enzymes. Among the resulting metabolites, 4-hydroxyestradiol (4-OH-E2) has garnered considerable attention within the realm of cancer research, owing to its classification as a putatively carcinogenic estrogen derivative. Evidence suggests that 4-OH-E2 elicits a diverse array of biological effects that may contribute to the development and progression of breast cancer. For example, 4-OH-E2 has been implicated in the induction of reactive oxygen species (ROS) generation, which are well-documented mediators of DNA damage and may promote neoplastic transformation in human mammary epithelial cells[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, 4-OH-E2 exhibits inherent estrogenic activity, capable of binding to estrogen receptors (ERα and ERβ), thereby modulating downstream gene expression and influencing cellular function. Studies have also demonstrated that 4-OHE2 can augment the expression of heme oxygenase-1 (HO-1) in breast cells, with HO-1 serving as a critical component of the cellular oxidative stress response. Notably, 4-OHE2-mediated induction of HO-1 expression may be regulated by the Nrf2/Keap1 signaling axis, ultimately influencing cellular proliferation and malignant transformation[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In a relevant study, Wu et al. reported that long-term exposure of MCF-10A cells to 4-OHE2 resulted in the acquisition of malignant characteristics, including enhanced cellular proliferation, epithelial-mesenchymal transition (EMT), and augmented migratory and invasive capacity, accompanied by a significant upregulation of CYP1B1 expression. Intriguingly, pharmacological blockade of CYP1B1 attenuated the malignant phenotype observed in MCF-10A cells following chronic exposure to 4-OHE2[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Complementary investigations conducted by KIM et al. revealed that nordihydroguaiaretic acid (NDGA), functioning as both a substrate and inhibitor of catechol-O-methyltransferase (COMT), suppressed COMT-mediated formation of 4-methoxyestradiol (4-MeOE2), while concomitantly exacerbating 4-OHE2-induced DNA damage and cellular toxicity. These findings suggest that NDGA possesses the potential to modulate COMT activity within mammary tissues, potentially preventing the inactivation of mutagenic estradiol metabolites and thereby enhancing catechol estrogen-mediated genotoxicity[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In concordance with these cumulative findings, our current investigation revealed that 4-OH-E2 promotes the invasive, migratory, and clonogenic capabilities of MCF10A cells, while also accelerating cell cycle progression. Furthermore, our in vivo studies demonstrated that 4-OH-E2 facilitates the tumorigenicity of MCF10A cells and fosters subsequent tumor growth.\u003c/p\u003e \u003cp\u003eTo further elucidate the downstream targets of 4-OH-E2, we performed in silico prediction of its target genes, leading to the identification of the androgen receptor (AR). AR, a member of the nuclear receptor superfamily, functions as a transcription factor modulating eukaryotic gene expression. It plays a pivotal role in the development and maintenance of homeostasis in various physiological systems, including the reproductive, skeletal muscle, cardiovascular, neurological, immune, and hematopoietic systems[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the oncological context, while AR was initially considered relevant primarily to male-related tissues, a growing body of evidence implicates aberrant AR signaling in the pathogenesis of breast cancer and other malignancies, such as prostate, bladder, kidney, and lung cancers[\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Breast cancer is broadly classified into subtypes including estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-positive, and triple-negative breast cancer (TNBC) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The androgen receptor (AR) is increasingly recognized to play a significant role in the pathogenesis of breast cancer (BC). AR is expressed in over 70% of breast cancer tumors, rendering it a potential biomarker and therapeutic target [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Studies have demonstrated a significant association between AR expression and histological grade, recurrence patterns, and molecular subtypes, with ER, PR, HER2, and tumor recurrence identified as independent factors influencing AR expression[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. AR expression is typically elevated in ER-positive breast cancer[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and AR activation may suppress the growth of ER-positive breast cancer cells, suggesting a potential interplay between AR and ER signaling pathways[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Concurrently, ER-positive breast cancer patients exhibiting high AR expression demonstrate a diminished response to neoadjuvant chemotherapy, yet display improved survival outcomes[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. AR expression is observed in approximately 60%-70% of TNBC cases, which can be further categorized into distinct molecular subtypes, including the Luminal Androgen Receptor (LAR) subtype characterized by elevated AR expression[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Umay et al. utilized digital image analysis (AR-DIA) to objectively assess AR expression, demonstrating its superiority over subjective scoring methods, with a 10% AR-DIA cutoff value identified as the strongest negative prognostic threshold for distant metastasis. While AR-DIA did not confer additional prognostic value in the favorable-prognosis TNBC subgroup, it was significantly informative in the poor-prognosis subgroup, underscoring the importance of AR expression assessment for TNBC prognosis and AR-targeted therapeutic strategies [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. A multitude of studies have indicated that AR functions as a tumor suppressor and a favorable prognostic marker in ER-positive breast cancer. Conversely, in ER-negative breast cancer, AR is implicated as a tumor promoter and a less favorable prognostic indicator[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In a study involving 931 patients, survival curve analysis revealed a significant association between the presence of AR in ER\u0026thinsp;+\u0026thinsp;tumors and superior disease-free survival (DFS) and overall survival (OS). A similar trend was observed in a study encompassing 1467 postmenopausal breast cancer patients[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Furthermore, an independent investigation revealed that elevated AR expression in ER\u0026thinsp;+\u0026thinsp;tumors correlates with reduced lymphocyte infiltration, indicative of a more favorable prognosis and prolonged survival [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Some studies have suggested that AR may contribute to the growth and metastasis of HER2-positive breast cancer cells Targeting Androgen Receptor in Estrogen Receptor-Negative Breast Cancer. In the present study, we observed that AR knockdown promoted the proliferation, migration, invasion, colony formation, and cell cycle progression of MCF-7 breast cancer cells.\u003c/p\u003e \u003cp\u003eAR has been established as a tumor suppressor in ER\u0026thinsp;+\u0026thinsp;breast cancer, an observation that highlights the intricate interplay between AR and the ERα signaling pathway in this malignancy. Studies have demonstrated that, in ER-positive breast cancer, AR agonists can impede tumor cell proliferation through interactions with the ER signaling cascade[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Furthermore, research indicates that AR activation can trigger the dissociation of ERα from chromatin, with AR occupying over 40% of ERα binding sites (ERBS), consequently suppressing the binding of ERα to estrogen response elements (EREs). Concomitantly, investigations have revealed that ERα can acquire novel binding targets by translocating to a subset of AR binding sites (ARBS), subsequently modulating the expression of AR target genes, including the tumor suppressors SEC14L2, EAF2, and ZBTB16, ultimately resulting in inhibited cell growth. In addition, AR can compete with ERα for binding to the shared co-activator p300, a factor critical for ERα activity. Given that ERα relies on the co-regulatory protein SRC-3 to facilitate p300 recruitment, whereas AR can bind directly to p300, AR may possess a competitive advantage, thereby attenuating ER signaling pathway activation[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. AR may also exert indirect inhibitory effects on ERα activity through specific mediator proteins. Estrogen receptor beta (ERβ), a known inhibitor of ERα, is subject to regulation by activated AR. Through binding to androgen response elements (AREs) located in the promoter region of ERβ, AR can upregulate ERβ gene expression, consequently dampening the ER signaling pathway. Collectively, AR activation can suppress ERα activity via multiple distinct mechanisms. Considering the pivotal role of ERα as a major driver of tumor growth in ER-positive breast cancer, the inhibition of ERα activity represents a potentially effective strategy for slowing disease progression[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Furthermore, AR and ER may exert reciprocal influences on their respective functions through modulation of intracellular signaling pathways, such as the PI3K/AKT and MAPK cascades[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Spatial genomics analyses suggest that the molecular characteristics of AR-expressing breast cancer cells within the tumor microenvironment correlate with improved overall survival in patients, providing clinical validation for the tumor-suppressive role of AR. In ER-positive breast cancer xenografts, ligand-mediated AR activation reprograms cistromes, attenuates oncogenic signaling pathways, and promotes cellular resilience toward a more differentiated phenotype. However, sustained AR activation can induce cistrome rearrangement, favoring the transcription factor PROP paired-like homeobox 1, thereby transforming AR into an oncogene and activating the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, culminating in lineage plasticity and a transition to a resistant, invasive subtype[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, these observations warrant further investigation.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study establishes 4-hydroxyestradiol (4-OH-E2) as a critical metabolite linked to breast cancer pathogenesis through multifaceted evidence. Mechanistically, 4-OH-E2 likely promotes breast cancer development, at least in part, by antagonizing or downregulating the tumor-suppressive function of AR, provides a novel mechanistic link for the metabolite's carcinogenic effects and points to the AR pathway as a potential therapeutic avenue.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBC, breast cancer\u003c/p\u003e \u003cp\u003e4-OH-E2, 4-hydroxyestradiol\u003c/p\u003e \u003cp\u003eE2, estradiol\u003c/p\u003e \u003cp\u003eAR, androgen receptor\u003c/p\u003e \u003cp\u003eER, estrogen receptor\u003c/p\u003e \u003cp\u003eERα, estrogen receptor alpha\u003c/p\u003e \u003cp\u003eERβ, estrogen receptor beta\u003c/p\u003e \u003cp\u003ePR, progesterone receptor\u003c/p\u003e \u003cp\u003eHER2, human epidermal growth factor receptor 2\u003c/p\u003e \u003cp\u003eGC-MS/MS, gas chromatography\u0026ndash;tandem mass spectrometry\u003c/p\u003e \u003cp\u003eGEPIA, Gene Expression Profiling Interactive Analysis\u003c/p\u003e \u003cp\u003eSPF, specific pathogen-free\u003c/p\u003e \u003cp\u003eFBS, fetal bovine serum\u003c/p\u003e \u003cp\u003eEGF, epidermal growth factor\u003c/p\u003e \u003cp\u003eCTX, cholera toxin\u003c/p\u003e \u003cp\u003ePBS, phosphate-buffered saline\u003c/p\u003e \u003cp\u003eDMSO, dimethyl sulfoxide\u003c/p\u003e \u003cp\u003ePI, propidium iodide\u003c/p\u003e \u003cp\u003eEDU, 5-ethynyl-2\u0026acute;-deoxyuridine\u003c/p\u003e \u003cp\u003esiRNA, small interfering RNA\u003c/p\u003e \u003cp\u003eqRT-PCR, quantitative real-time polymerase chain reaction\u003c/p\u003e \u003cp\u003eWB, Western blot\u003c/p\u003e \u003cp\u003eHO-1, heme oxygenase-1\u003c/p\u003e \u003cp\u003eNDGA, nordihydroguaiaretic acid\u003c/p\u003e \u003cp\u003eCOMT, catechol-O-methyltransferase\u003c/p\u003e \u003cp\u003eEMT, epithelial-mesenchymal transition\u003c/p\u003e \u003cp\u003eARE, androgen response element\u003c/p\u003e \u003cp\u003eERE, estrogen response element\u003c/p\u003e \u003cp\u003eARBS, androgen receptor binding site\u003c/p\u003e \u003cp\u003eERBS, estrogen receptor binding site\u003c/p\u003e \u003cp\u003eSD, standard deviation\u003c/p\u003e \u003cp\u003eHR, hazard ratio\u003c/p\u003e \u003cp\u003eCI, confidence interval\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and Consent form\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was reviewed and approved by the Ethics Committee of Nanjing First Hospital (KY20251103-KS-02), and all the study procedures strictly adhered to the ethical principles outlined in the World Medical Association Declaration of Helsinki. Written informed consent was obtained from all participants with documentation maintained in secured study records. The animal experiment was approved by the Animal Ethics Committee of Nanjing First Hospital (DWSY-25092724). All experiments were conducted in compliance with the relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article. The original data can be available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Nanjing Medical Science and Technology Development Fund (YKK23129), National Natural Science Foundation of China (82203182), China Postdoctoral Science Foundation (2024M763658), and the Project of\u0026nbsp;Future Technology Star of Nanjing First Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWei Liang, Fei Fei and Xiaowei Wei developed the study concept and design. Ziquan Wang, Linzhu Zhang, and Xiaofan Liu completed the acquisition of data. Fei Fei and Linzhu Zhang made contributions to data analysis. Lin Fu, Rong Wu, and Huanhuan Fei made contributions to data interpretation. Fei Fei and Ziquan Wang drafted the manuscript. All authors critically revised, read and approved the final version of the manuscript, and agreed to be accountable for the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful for the data support provided by the Information Department of Nanjing First Hospital, and we also express our sincere gratitude to Professor Wei Shi for providing professional English editing revision on the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGiaquinto AN, Sung H, Newman LA, Freedman RA, Smith RA, Star J, Jemal A, Siegel RL. Breast cancer statistics 2024. 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A molecular switch from tumor suppressor to oncogene in ER\u0026thinsp;+\u0026thinsp;ve breast cancer: Role of androgen receptor, JAK-STAT, and lineage plasticity. Proc Natl Acad Sci USA. 2024;121(40):e2406837121.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 3","content":"\u003cp\u003eTable 3 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, Estrogen, 4-Hydroxyestradiol, Androgen Receptor","lastPublishedDoi":"10.21203/rs.3.rs-8136540/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8136540/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eBreast cancer (BC) is a major global health threat with increasing incidence, and estrogen metabolites are implicated in its pathogenesis. To investigate alterations in urinary estrogen metabolites in BC patients and elucidate the oncogenic role and molecular mechanism of the metabolite 4-hydroxyestradiol (4-OH-E2) in BC progression.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 126 treatment-naive BC patients and 103 healthy women were recruited to detect urinary estrogen metabolites using gas chromatography-tandem mass spectrometry (GC-MS/MS). MCF10A cells were treated with estradiol (E2) or 4-OH-E2 for 8 weeks (designated MCF10A-E and MCF10A-H, respectively), followed by assessments of cell morphology, migration (Transwell, scratch assay), invasion (Transwell), colony formation, and cell cycle (flow cytometry). Tumorigenicity assays were performed in BALB/c nude mice. Potential targets of 4-OH-E2 were predicted via SwissTargetPrediction and validated using GEPIA database. Loss-of-function experiments (AR silencing) were conducted to explore the role of androgen receptor (AR) in BC cells, with qRT-PCR, Western blot, and functional assays (proliferation, migration, invasion) used for verification.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBC patients demonstrate dysregulated estrogen metabolism, marked by significantly elevated urinary 4-OH-E2. Chronic 4-OH-E2 exposure drives malignant transformation in mammary epithelial cells, enhancing tumorigenic phenotypes in vitro and in vivo. AR is a critical mediator of 4-OH-E2\u0026rsquo;s oncogenic effects, and its loss-of-function accelerates BC progression.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003e4-OH-E2 is significantly elevated in BC patients and promotes breast carcinogenesis by enhancing cell malignant phenotypes and tumorigenicity, with AR as a critical target. These findings highlight 4-OH-E2 and AR as potential targets for BC prevention and treatment.\u003c/p\u003e","manuscriptTitle":"4-Hydroxyestradiol Promotes Breast Carcinogenesis via Androgen Receptor: Evidence from Urinary Metabolite Profiling and Functional Studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-17 07:11:14","doi":"10.21203/rs.3.rs-8136540/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"38b2b698-b015-4cc5-8353-be28dc164c53","owner":[],"postedDate":"December 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T14:55:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-17 07:11:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8136540","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8136540","identity":"rs-8136540","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}