Synergistic Activation of LEPR and ADRB2 Induced by Leptin Enhances Reactive Oxygen Specie Generation in Triple-Negative Breast Cancer Cells

1. Cell culture and treatment

Two murine-derived TNBC cell lines, 4T1 and EMT6 (purchased from ATCC), were cultured in RPMI-1640 medium (BI, Israel, South America) supplemented with 10% fetal bovine serum (Newzerum) under standard conditions of 37°C and 5% CO₂. Cells were seeded in six-well plates and allowed to reach approximately 60% confluence before being subjected to treatment.

For experimentation, cells were treated with varying concentrations (10, 50, 100, 200 ng/mL) of recombinant mouse leptin (R&D Systems) for a duration of 24 hours. Additionally, the LEPR antagonist, Allo-aca (100 nM for 24 hours, MCE), and the specific ADRB2 inhibitor, ICI118551 (10 μM for 24 hours, Sigma-Aldrich), were separately added to 4T1 and EMT6 cells at appropriate densities prior to further experimentation.

2. Quantitative Real-time PCR

Total RNA was extracted from the samples, followed by cDNA synthesis using the Hifair II 1st Strand cDNA Synthesis Kit (Yeasen). Subsequently, DNA amplification was carried out using the UltraSYBR Mixture (Cwbio). The relative expression levels of target genes were determined using the formula 2^-ΔΔCT, with normalization to β-actin.

The primer sequences utilized were as follows: LEPR: forward 5′-AGCTAGGTGTAAACTGGGACA-3′, reverse 5′-GCAGAGGCGAATCATCTATGAC-3′; ADRB2: forward 5′-ATGTCGGTTATCGTCCTGGC-3′, reverse 5′-GGTTTGTAGTCGCTCGAACTTG-3′; NADPH oxidase 4 (NOX4): forward 5′-TTTCTCAGGTGTGCATGTAGC-3′, reverse 5′-GCGTAGGTAGAAGCTGTAACCA-3′; β-actin: forward 5′-GGCTGTATTCCCCTCCATCG-3′, reverse 5′-CCAGTTGGTAACAATGCCATGT-3′.

3. Western blot

Total protein was extracted utilizing RIPA lysis buffer (Solarbio), followed by quantification using the BCA assay kit (Beyotime Biotechnology). Subsequently, protein samples were denatured in a metal bath at 95°C for 8 minutes. Electrophoresis was then performed using a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel, and proteins were transferred onto a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with 10% skimmed milk at room temperature for 1 hour, followed by overnight incubation at 4°C with primary antibodies. Anti-LEPR and anti-ADRB2 antibodies were sourced from Abcam (ab5593, ab182136), anti-NOX4 antibody from Boster (A00403), and anti-β-actin antibody from Santa Cruz Biotechnology (sc-81178, Santa Cruz Biotechnology). After washing thrice with PBST (phosphate buffered saline with Tween 20), membranes were incubated with secondary antibodies at room temperature for 1 hour. Protein bands were visualized using chemiluminescent horseradish peroxidase substrate (ECL, WBKlS0100, Millipore), and band densities were analyzed using Image J.

4. Cell viability assay

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Beyotime Biotechnology) following the manufacturer’s instructions. Briefly, cells were seeded in 96-well plates and allowed to adhere for 24 hours before treatment with varying concentrations of leptin (10, 50, 100, and 200 ng/mL) for an additional 24 hours. In a separate experiment, cells were treated with leptin (100 ng/mL) alone or in combination with Allo-aca (100 nM). Following treatment, CCK-8 solution was added to each well, and absorbance was measured at 450 nm to evaluate cell viability.

5. Clone formation assay

EMT6 single-cell suspension were seeded in a six-well plate at a density of 500 cells per well. Once adhered, cells were subjected to treatment with either leptin or Allo-aca, and the culture medium was refreshed every three days. The experiment was concluded when the number of cells in most individual clones exceeded 50 cells, as determined under microscopic observation. Following this, cells were fixed with 4% paraformaldehyde and stained with 1 mL of crystal violet dye for 10 minutes per well. After rinsing with PBS, the cells were allowed to air-dry before being photographed.

6. Wound closure assay

EMT6 cells were seeded in a six-well plate at a density of 2×10⁵ cells and allowed to incubate overnight. Using a pipette tip, scratches were carefully made on the cell monolayer. Following this, the cells were gently washed three times with PBS and treated with either leptin or Allo-aca for 18 hours. The scratches were observed and photographed under a microscope and the rate of scratch closure was quantitatively analyzed using ImageJ software.

7. Transwell migration assay

After pre-culturing 4T1 cells in serum-free medium for 24 hours, a single-cell suspension was prepared at a density of 5×10⁵ cells/mL. Subsequently, 200 μL of the cell suspension was added to the upper chamber of a transwell chamber. In the lower chamber of the 24-well plate, 600 μL of medium containing 15% fetal bovine serum along with either leptin or Allo-aca was added. The cells were then incubated at 37°C for 24 hours. Then the transwell chamber was washed twice with PBS, fixed with methanol, and stained with 0.1% crystal violet. The upper layer of non-migrated cells was gently wiped off with a cotton swab. Migrated cells were then photographed and quantitatively analyzed using Image J.

8. ROS measurement

The levels of ROS were assessed using the ROS fluorescent probe DCFH-DA (Invitrogen) via flow cytometry. Following 24 hours of various treatments, cells were harvested, washed with PBS, and then incubated with serum-free medium containing 10 μM DCFH-DA for 20 minutes, protected from light. The levels of ROS were measured using flow cytometry (BD FACSCanto II).

9. Measurement of mitochondrial ROS

4T1 cells and EMT6 cells were treated with MitoSOX Red fluorescent dye (5 μM, Thermo Fisher, Waltham, MA) and incubated at 37°C for 30 minutes. Following the incubation period, cells were washed with PBS and examined under a confocal microscope (Olympus FV1000).

10. Double labeling immunofluorescence analysis

After treatment with leptin (100 ng/mL) alone or in combination with Allo-aca (100 nM) for 24 hours, cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature. The cells were blocked with 5% goat serum for 30 minutes and then incubated overnight at 4°C with a 1:100 dilution of anti-LEPR antibody (sc-8391, Santa Cruz Biotechnology) and a 1:200 dilution of anti-ADRB2 antibody (ab128136, Abcam). This was followed by incubation with the corresponding Alexa Fluor-conjugated secondary antibodies (1:200, Affinity) for 1 hour in the dark. Cell nuclei were stained with DAPI. Immunoreactivity was captured using a confocal microscope with 488 and 594 laser excitations (Olympus FV1000), and the fluorescence intensity was analyzed using the coloc2 plug-in (ver. 2.0, Fiji) of Image J v1.8.0.

11. In vivo tumorigenesis

Female BALB/c mice, aged 6-8 weeks, were procured from Vital River Lab Animal Technology Co., Ltd. All experimental procedures were ethically approved by the Medical Ethics Committee, Nankai University (2023-SYDWLL-000531). Following subcutaneous inoculation of 1×10⁵ 4T1 cells into the breast gland of the mice, the animals were randomly divided into four groups: control group, leptin group (intraperitoneal injection, 1 mg/kg), leptin (1 mg/kg) combined with Allo-aca (subcutaneous injection, 0.1 mg/kg), and leptin (1 mg/kg) combined with ICI118551 (intraperitoneal injection, 0.5 mg/kg). Each drug combination was administered once every two days. Throughout the experiment, the mice’s food intake and body weight were monitored and recorded every two days. Tumor volume was measured and calculated using the formula V=(length×width²)/2. Following 3 weeks of drug administration, the mice were euthanized, and blood serum from the eyeball, as well as tumors and lungs, were collected for subsequent experiments.

12. Glutathione assay

The measurement of glutathione (GSH) content in serum or tumor samples was conducted using a GSH kit (NjJcBio) following the manufacturer’s instructions. Absorbance readings were taken at a wavelength of 420 nm.

13. Lung metastasis in tumor-implanting mice

The mouse lung tissue sections were stained with hematoxylin and eosin (H&E) and imaged at Zhongke Guanghua Biotechnology Company.

14. Bioinformation analysis

Survival analysis data was obtained from the Kaplan-Meier plotter database (http://kmplot.com/analysis/index.php?p=service&cancer), using the ‘auto-select best cutoff’ option for cutoff determination. Correlation analysis was performed using bc-GenExMiner (http://bcgenex.ico.unicancer.f/BC-GEM/GEM-requete.php) and GEPIA (http://gepia.cancer-pku.cn/). Additionally, the prognostic significance in breast cancer patients was assessed by receiver operating characteristic (ROC) curve using the Plotter platform (http://rocplt.org).

15. Statistical analysis

The data was presented as mean±standard error of mean, and statistical analysis was performed using Prism 9.0 (GraphPad). Student’s t test was utilized for comparisons between two groups, while one-way ANOVA followed by Tukey’s test was employed for comparisons among multiple groups. Statistical significance was set at p < 0.05.

1. Leptin upregulates LEPR expression in TNBC cells

Using the publicly accessible Kaplan-Meier plotter database, we identified that elevated LEPR expression in TNBC tissues correlated with poorer overall survival (OS) and distant metastasis-free survival (p < 0.05) (Fig. 1A and B). We further investigated the impact of leptin on LEPR expression in murine TNBC cell lines, 4T1 and EMT6. Both cell types showed increased LEPR expression in response to leptin treatment (0, 10, 50, 100, and 200 ng/mL) for 24 hours, with peak expression at 100 ng/mL (Fig. 1C and D). Therefore, we used 100 ng/mL leptin for subsequent experiments. To counteract leptin’s effects, we used the LEPR antagonist peptide Allo-aca (Allo). Allo alone did not significantly alter LEPR mRNA and protein levels in 4T1 or EMT6 cells. However, it effectively inhibited leptin-induced LEPR upregulation (p < 0.05) (Fig. 1E and F). These findings suggest that LEPR in murine TNBC cell lines can be activated by leptin, and Allo may suppress this effect.

Fig. 1.

Leptin upregulates LEPR expression in TNBC cells. (A, B) Bioinformatics data from Kaplan-Meier database (https://kmplot.com/analysis/index.php?p=service) showed OS curve (A) and DMFS curve (B) among TNBC patients stratified by high and low LEPR expression levels. Higher LEPR expression was associated with poor prognosis. (C, D) Quantitative PCR and western blot were performed to analyze the expression of LEPR mRNA (C) and LEPR protein (D) in 4T1 and EMT6 cells following stimulation with increasing concentrations of leptin (0, 10, 50, 100, 200 ng/mL) for 24 hours, respectively. n=3-4. (E, F) Quantitative LEPR mRNA (E) and LEPR protein (F) expression in 4T1 and EMT6 cells following 24 hours of leptin (100 ng/mL) and/or Allo-aca (Allo, 100 nM) treatment. n=3. DMFS, distant metastases-free survival; HR, hazard ratio; LEPR, leptin receptor; OS, overall survival; PCR, polymerase chain reaction; SEM, standard error of mean; TNBC, triple-negative breast cancer. All data are presented as the mean±SEM. ns, no significance; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

2. LEPR activation promotes TNBC cell proliferation and migration

To investigate the role of LEPR in breast cancer progression, we examined cell proliferation using CCK-8 and colony formation assays. Treatment of 4T1 and EMT6 cells with varying concentrations of leptin for 24 hours increased cell viability, peaking at 100 ng/mL for 4T1 cells and 50 ng/mL for EMT6 cells (p < 0.05) (Fig. 2A). While Allo alone did not significantly affect cell proliferation, it counteracted the proliferative effects of 100 ng/ml leptin (p < 0.05) (Fig. 2B). Colony formation assays showed similar results in EMT6 cells (p < 0.05) (Fig. 2C).

Fig. 2.

LEPR activation promotes the proliferation and migration of TNBC cells. (A) CCK-8 assay was employed to assess the proliferative capacity of 4T1 and EMT6 cells upon exposure to leptin (0, 10, 50, 100, 200 ng/mL). n=3. Scale bar=200 μm. (B, C) CCK-8 assay (B) and colony formation assay (C) were conducted to evaluate the proliferative ability of 4T1 and EMT6 cells treated with leptin (100 ng/mL) and/or Allo (100 nM). n=3. (D, E) Wound closure assay and transwell migration assay were employed to investigate the migratory ability of EMT6 (scale bar=500 μm) and 4T1 (scale bar=200 μm) cells following treatment with leptin and/or Allo, respectively. n=3. CCK-8, Cell Counting Kit-8; LEPR, leptin receptor; SEM, standard error of mean; TNBC, triple-negative breast cancer. All data are presented as the mean±SEM. ns, no significance; ^*p < 0.05, ^**p < 0.01.

We also explored the influence of the leptin/LEPR axis on TNBC cell migration. In both the wound closure assay for EMT6 cells and the transwell migration assay for 4T1 cells, leptin increased cell migration, while Allo significantly reduced the stimulatory effects of leptin (p < 0.05) (Fig. 2D and E).

3. Leptin induces NOX4-derived ROS and mitochondrial ROS production in TNBC cells

ROS production is crucial in both obesity and cancer. Leptin has been shown to induce ROS in various tissues, suggesting a potential signaling pathway via LEPR. In mammary gland cells, leptin induces ROS through NOX5 [18], but its effects on ROS in TNBC cells 4T1 and EMT6 were unexplored. We evaluated ROS production using the DCFH-DA probe, finding significantly elevated ROS levels in both cell types after leptin stimulation, although not in a dose-dependent manner (Fig. 3A). Allo alone did not affect ROS levels but inhibited leptin-induced ROS production (p < 0.05) (Fig. 3B).

Fig. 3.

Leptin induces NOX4-derived ROS production and mtROS generation. (A) Flow cytometry analysis of ROS levels in 4T1 and EMT6 cells treated with varying concentrations of leptin (0, 10, 50, 100, and 200 ng/mL), assessed using the fluorescent probe DCFH-DA. n=3. (B) Flow cytometry analysis of ROS levels in 4T1 and EMT6 cells treated with leptin (100 ng/mL) and/or Allo (100 nM), utilizing the fluorescent probe DCFH-DA. n=3. (C) Schematic diagram of the two main sources of ROS generation. (D, E) Quantitative PCR (D) and western blotting (E) were employed to evaluate the expression of NOX4 in 4T1 and EMT6 cells following stimulation with increasing concentrations of leptin (0, 10, 50, 100, and 200 ng/mL) for 24 hours. n=3. (F, G) Quantitative PCR (F) and western blotting (G) were employed to assess the expression of NOX4 in 4T1 and EMT6 cells treated with leptin (100 ng/mL) and/or Allo (100 nM). n=3. (H, I) MitoSOX staining assay to detect mtROS levels in 4T1 and EMT6 cells treated with leptin (100 ng/mL) and/or Allo (100 nM). n=3. mtROS, mitochondrial ROS; NOX4, NADPH oxidase 4; PCR, polymerase chain reaction; ROS, reactive oxygen species; SEM, standard error of mean. All data are presented as the mean±SEM. ns, no significance; ^*p < 0.05, ^**p < 0.01, ^*** p < 0.001.

The primary drivers of cellular ROS production in physiological and pathological processes are NADPH oxidases (NOXs), with mitochondrial ROS serving as another significant source (Fig. 3C). Among NOX family members, NOX4 exhibited high expression in both 4T1 and EMT6 cells (data not shown). Leptin treatment increased NOX4 mRNA (p < 0.05) (Fig. 3D) and protein expression (p < 0.05) (Fig. 3E) in both cell lines, which was significantly reduced by Allo (p < 0.05) (Fig. 3F and G).Using MitoSOX Red, we found that leptin-induced mitochondrial ROS (mtROS) production was also significantly reduced by Allo treatment (Fig. 3H and I). Leptin treatment induced ROS generation, which contributed to the proliferation of EMT6 cells as shown by cell viability analysis, and promoted 4T1 cell migration as evidenced by wound closure assays. However, the ROS scavengers N-acetylcysteine or superoxide dismutase were able to eliminate the leptin-induced ROS effect (S1 Fig.). These findings suggest that the leptin/LEPR axis facilitates TNBC cell proliferation and migration by increasing NOX4-derived ROS and mtROS production, while Allo suppresses this effect.

4. Leptin upregulates ADRB2 expression in TNBC cells

To probe into the potential association between leptin and ADRB2 in breast cancer cells, we used quantitative real-time PCR and protein blotting to measure ADRB2 expression in the presence of leptin. Our results showed a significant increase in ADRB2 mRNA and protein levels in leptin-stimulated 4T1 and EMT6 cells (p < 0.05) (Fig. 4A and B). Furthermore, Allo, an inhibitor of LEPR, effectively suppressed leptin-induced upregulation of ADRB2 expression (p < 0.05) (Fig. 4C and D). Immunofluorescence double staining confirmed the presence of both LEPR, conjugated with Alexa Fluor 488, and ADRB2, conjugated with Alexa Fluor 594, on the cell membrane of EMT6 cells (Fig. 4E). Statistical analysis indicated that leptin treatment promoted the co-localization of LEPR and ADRB2, whereas Allo mitigated this effect. Additionally, the ADRB2 antagonist ICI118551 inhibited the co-localization of these receptors (Fig. 4F-J). The Pearson correlation coefficient statistics plot reconfirmed the fluorescence intensity changes of LEPR and ADRB2 in EMT6 cells under different treatments (Fig. 4K). These findings suggest a potential link between LEPR and ADRB2 in TNBC, like conditions observed in obesity. Moreover, they indicate that leptin not only activates LEPR but also induces the upregulation of ADRB2.

Fig. 4.

Leptin upregulates ADRB2 expression in TNBC cells. (A) Quantitative PCR was utilized to assess the expression levels of ADRB2 in 4T1 (n=3) and EMT6 (n=6) cells following stimulation with 50 ng/mL or 100 ng/mL leptin. (B) Western blotting was utilized to assess the expression levels of ADRB2 in 4T1 and EMT6 cells following stimulation with 50 ng/mL or 100 ng/mL leptin. n=4. (C, D) Quantitative PCR (C) and western blotting (D) were employed to analyze the expression of ADRB2 in 4T1 and EMT6 cells treated with leptin (100 ng/mL) and/or Allo (100 nM). n=3. (E) Representative images of immunofluorescence double staining showing the expression and co-localization of LEPR (Alexa Fluor 488 Conjugated) and ADRB2 (Alexa Fluor 594-conjugated) in the presence of leptin and/or Allo, or the ADRB2 inhibitor ICI118551 (ICI). (F-J) Statistical curves depicting the fluorescence intensity of LEPR and ADRB2 in the co-localization images for different experimental groups. (K) Quantitative analysis of LEPR-ADRB2 co-localization on cell membranes in each group, evaluated using Pearson’s correlation coefficient. Data are presented as mean±SEM, n=3. ADRB2, adrenergic receptor β2; LEPR, leptin receptor; PCR, polymerase chain reaction; SEM, standard error of mean; TNBC, triple-negative breast cancer. All data are presented as the mean±SEM. ns, no significance; ^*p < 0.05, ^**p < 0.01.

5. ADRB2 mediates leptin-induced ROS generation in TNBC cells

To determine if ADRB2 plays a role in leptin-induced ROS generation, we conducted flow cytometry analysis. Our findings revealed that ICI118551 effectively inhibited leptin-induced ROS generation in both 4T1 and EMT6 cells (Fig. 5A). We then examined changes in ADRB2 and NOX4 expression in leptin-stimulated 4T1 cells treated with ICI118551 using immunoblotting. The results showed that ICI118551 treatment led to a downregulation of both ADRB2 and NOX4 expression, which were enhanced by leptin (p < 0.05) (Fig. 5B). We also assessed mtROS levels through confocal microscopy. The analysis demonstrated that mtROS levels elevated in the presence of leptin compared to the control group, and this increase was effectively inhibited by ICI118551 treatment (Fig. 5C). Collectively, our data indicate that leptin increases ROS levels by concurrently activating both LEPR and ADRB2 receptors, thereby fostering the progression of TNBC.

Fig. 5.

ADRB2 mediates the leptin-induced ROS generation. (A) Flow cytometry analysis of ROS levels in 4T1 and EMT6 cells treated with leptin and/or the ADRB2 inhibitor ICI, assessed using the fluorescent probe DCFH-DA. (B) Western blotting analyzed the expression of ADRB2 and NOX4 in 4T1 cells treated with leptin (100 ng/mL) and/or ICI (10 μM). n=3. (C) MitoSOX staining assay was utilized to detect mtROS levels in EMT6 cells treated with leptin (100 ng/mL) and/or ICI (10 μM). ADRB2, adrenergic receptor β2; mtROS, mitochondrial ROS; NOX4, NADPH oxidase 4; ROS, reactive oxygen species; SEM, standard error of mean. All data are presented as the mean±SEM. ns, no significance; ^*p < 0.05.

6. LEPR and ADRB2 in leptin-promoted breast cancer progression in vivo

To further validate our findings, we employed a transplanted tumor-bearing mouse model by inoculating 4T1 cells for in vivo experiments. Seven days post-cell inoculation, BALB/c mice were randomly assigned to receive one of the following treatments: normal saline, leptin, leptin combined with Allo, and leptin combined with ICI118551. Drug injections continued for 3 weeks, during which we monitored diet consumption, body weight, and tumor volume (Fig. 6A). Compared to the control group, the leptin group showed significantly larger tumor volumes and heavier tumor weights (p < 0.05) (Fig. 6B and C). However, Co-administration with the LEPR antagonist Allo or the ADRB2 inhibitor ICI118551 inhibited tumor growth compared to the leptin-alone group (p < 0.05) (Fig. 6B and C).

Fig. 6.

LEPR and ADRB2 in leptin-induced breast cancer progression in vivo. (A) Female BALB/c mice were subcutaneously inoculated with 1×10⁵ 4T1 cells into the mammary fat pad to establish the breast tumor animal model. Drug administration commenced 7 days postinoculation and continued for 21 days. Tumors, blood samples, and lungs were collected for analysis. (B) Representative image of tumors and tumor growth curves in different experimental groups. n=5. (C) Tumor weights were measured on day 28. n=5. (D, E) Food intake (D) and body weight curves (E) in different groups. n=5. (F) Metastatic lung nodules were visualized using H&E staining in different groups. (G) GSH content was assessed in both serum and tumor samples collected from different groups. n=3-5. (H, I) Real-time qPCR (H) and western blotting (I) were used to analyze the expression of LEPR, ADRB2, and NOX4 in tumors from different groups. n=3. ADRB2, adrenergic receptor β2; GSH, glutathione; LEPR, leptin receptor; NOX4, NADPH oxidase 4; qPCR, quantitative real-time polymerase chain reacion; SEM, standard error of mean. All data are presented as the mean±SEM. ns, no significance; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

To ensure leptin’s effects on breast cancer progression were not indirectly due to changes in diet and energy metabolism, we monitored food consumption and body weight. Drug administration had little effect on appetite (Fig. 6D). Mice in the leptin group lost a significant weight compared to the control group, with weight reduction peaking around the 10th day post-leptin injection. This weight loss stabilized over time and was alleviated by combined treatment with Allo or ICI118551 (Fig. 6E). H&E staining of lung tissue sections showed significantly larger metastatic nodules in the leptin group, while combined treatment with Allo or ICI118551 reduced nodule size (Fig. 6F).

GSH, a crucial antioxidant in redox homeostasis of tumor cells, was significantly reduced in the leptin group compared to controls, but this reduction was reversed by combination treatment with Allo or ICI118551 (p < 0.05) (Fig. 6G). Lastly, we quantified mRNA and protein levels in mouse tumor tissues. Consistent with our in vitro results, mRNA and protein expression of LEPR, ADRB2, and NOX4 were significantly upregulated in the leptin group compared to controls. This upregulation was reversed by combined treatment with Allo or ICI118551 (p < 0.05) (Fig. 6H and I).

7. The clinical significance of LEPR and ADRB2 in breast cancer patients

ROC curve analysis is a method used to evaluate the performance of diagnostic classification methods, with the area under the curve (AUC) serving as a key metric. In our study, both LEPR and ADRB2 had an AUC value of 0.629, indicating some diagnostic predictive ability (p < 0.001) (Fig. 7A and B), thus suggesting their potential as diagnostic markers of breast cancer. We further analyzed the correlation between LEPR and ADRB2 using the bc-GenExMiner and GEPIA databases. The data revealed a positive correlation between LEPR and both ADRB2 and NOX4, with ADRB2 showing a stronger positive trend (Fig. 7C-E). In contrast, glutathione peroxidase 4, a ROS scavenging enzyme, exhibited a negative correlation with LEPR (Fig. 7C). Kaplan-Meier plotter analysis demonstrated that high expression of LEPR and ADRB2 was associated with poor relapse-free survival in TNBC patients (p=0.007) (Fig. 7F).

Fig. 7.

The clinical significance of LEPR and ADRB2. (A, B) Receiver operating characteristic curve analysis was conducted to evaluate the prognostic significance of LEPR (A) and ADRB2 (B) in breast cancer patients. p < 0.01. (C-E) The correlation between LEPR, ADRB2, NOX4, and GPX4 was analyzed using the bc-GenExMiner database (C) and GEPIA database (D, E). (F) Kaplan-Meier plotter was utilized to illustrate RFS in TNBC patients with high expression of LEPR and ADRB2 combined. p=0.0071. (G) The schematic diagram of the study work. ADRB2, adrenergic receptor β2; AUC, area under the curve; GPX4, glutathione peroxidase 4; HR, hazard ratio; LEPR, leptin receptor; mtROS, mitochondrial reactive oxygen species; NOX4, NADPH oxidase 4; RFS, relapse-free survival; TNR, true-negative ratio; TPM, transcripts per million; TPR, true-positive ratio; TNBC, triple-negative breast cancer.

In summary, our study, conducted through both in vitro and in vivo experiments, demonstrates that concurrent activation of the membrane receptors LEPR and ADRB2 on TNBC cells stimulates NOX4-mediated cellular ROS generation and mitochondrial ROS production, fostering the proliferation and metastasis of TNBC (Fig. 7G). These findings suggest that dual targeting of LEPR and ADRB2 could be a promising predictive and therapeutic strategy in the clinical management of TNBC patients.