Cancer Res Treat.  2020 Jul;52(3):830-847. 10.4143/crt.2019.510.

Activation of β₂-Adrenergic Receptor Promotes Growth and Angiogenesis in Breast Cancer by Down-regulating PPARγ

• Zhou J
• Liu Z
• Zhang L
• Hu X
• Wang Z
• Ni H
• Wang Y
• Qin J

• KMID: 2504463
• DOI: http://doi.org/10.4143/crt.2019.510

Abstract

Purpose
Chronic stress and related hormones are key in cancer progression. Peroxisome proliferator-activated receptor γ (PPARγ) and its agonists was reported that inducing anti-tumor effect. However, the function of PPARγ in pro-tumorigenic effects induced by chronic stress in breast cancer remains unknown. Herein, we have characterized a novel role of PPARγ and vascular endothelial growth factor (VEGF)/fibroblast growth factor 2 (FGF2) signals in breast cancer promoted by chronic stress.
Materials and Methods
We performed experiments in vivo and in vitro and used bioinformatics data to evaluate the therapeutic potential of PPARγ in breast cancer promoted by stress.
Results
Chronic stress significantly inhibited the PPARγ expression and promoted breast cancer in vivo. VEGF/FGF2-mediated angiogenesis increased in the chronic stress group compared to the control group. PPARγ agonist pioglitazone (PioG) injection offset the pro-tumorigenic effect of chronic stress. Moreover, specific β₂-adrenergic receptor (β₂R) antagonist ICI11-8551 inhibited the effect of chronic stress. In vitro, norepinephrine (NE) treatment had a similar tendency to chronic stress. The effect of NE was mediated by the β₂R/adenylate cyclase signaling pathway and suppressed by PioG. PPARγ suppressed VEGF/FGF2 through reactive oxygen species inhibition. Bioinformatics data confirmed that therewas a lowPPARγ expression in breast invasive carcinoma. Lower PPARγ was associated with a significantly worse survival.
Conclusion
β₂R activation induced by chronic stress and related hormones promotes growth and VEGF/FGF2-mediated angiogenesis of breast cancer by down-regulating PPARγ. Our findings hint that β receptor and PPARγ as two target molecules and the novel role for their agonists or antagonists as clinical medicine in breast cancer therapy

Keyword

β receptor; Chronic stress; PPARγ; Norepinephrine; Breast neoplasms

Figure

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  Fig. 1. Effect of chronic stress alone or in combination with pioglitazone (PioG) on tumor progression in a mouse model. (A) To establish the animal model, 4T1 cells were injected into the mice. On the same day, chronic stress was induced for 4 weeks. After 7 days, mice were treated with PioG for 3 weeks. (B) Tumor representative images from mice in control, chronic stress, PioG and chronic stress+PioG group (4-5 mice per group). Tumor growth curves (C) and average tumor weight (D) was measured on day 28 (4-5 tumors per group). (E) Representative images and calculated graph of lung nodules on day 28 (n=4 lungs per group). (F) H&E staining images of lung issues section from difference groups. Values are presented as mean±standard error of mean. #p < 0.05 vs. control group, *p < 0.05 vs. chronic stress+PioG group on day 28.

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  Fig. 2. Effect of chronic stress alone or in combination with pioglitazone (PioG) on angiogenesis in vivo. (A, B) Peroxisome proliferator-activated receptor γ (PPARγ) mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) in the tumor tissue from different groups. (C) PPARγ protein bands (left panel) and bar graph (right panel) in the tumor tissue from different groups. (D) Representative images of Matrigel plugs and newly formed blood vessels in the Matrigel (left panel) and hemoglobin (Hgb) content in the Matrigel matrix (right panel) in which we excised the Matrigel plugs and used them for quantification of angiogenesis. (E) Microvascular density was measured (left panel) and images of tumor section CD31 immunohistochemistry staining (right panel). (F) Quantitative vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) mRNA expression in tumor tissues detected by qPCR. (G) VEGF and FGF2 protein levels in tumor tissues were detected by western blot (WB). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Values are presented as mean±standard error of mean. *p < 0.05.

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  Fig. 3. Peroxisome proliferator-activated receptor γ (PPARγ) inhibition induced by norepinephrine (NE) improves viability and proliferation of 4T1 cells. (A-C) PPARγ mRNA expression in 4T1 cells treated with different concentrations of NE (0, 1, 10, and 100 μM) for different times (0 minute, 10 minutes, 1 hour, 3 hours, and 12 hours) by reverse transcription polymerase chain reaction (A) and quantitative real-time polymerase chain reaction. (B, C). *p < 0.05 vs. control group. (D) PPARγ expression in 4T1 cells detected by cell fluorescence staining. PE, phycoerythrin. (E) Cell viability measured by Cell Counting Kit-8 assay in 4T1 cells. PioG, pioglitazone. (F) Proliferation ability evaluated by soft agar colony formation assay in 4T1 cells. Values are presented as mean±standard error of mean. #p < 0.05 vs. control, *p < 0.05 vs. NE+PioG group.

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  Fig. 4. Effect of norepinephrine (NE) alone or in combination with pioglitazone (PioG) on proangiogenic factors expression in vitro. (A) Peroxisome proliferator-activated receptor γ (PPARγ), vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF2) mRNA expression in 4T1 cells treated with NE (10 μM) alone or in combination with PioG (50 μM) by quantitative real-time polymerase chain reaction analysis. #p < 0.05 vs. control, *p < 0.05 vs. NE+PioG group. (B) PPARγ, VEGF, and FGF2 protein expression by western blot in the same condition. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (C) PPARγ, VEGF, and FGF2 mRNA expression in 4T1 cells treated with NE (10 μM) alone or in combination with αR inhibitor phent (1 μM) or βR inhibitor propranolol (PPL, 1 μM). *p < 0.05. (D) PPARγ, VEGF, and FGF2 mRNA expression in 4T1 cells treated with NE (10 μM) alone or in combination with β1R inhibitor metoprolol (1 μM) or β2R inhibitor ICI118551 (1 μM). *p < 0.05.

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  Fig. 5. β2-adrenergic receptor (β2R) is involved in peroxisome proliferator-activated receptor γ (PPARγ) inhibition and angiogenesis induced by norepinephrine (NE) in vitro. (A) PPARγ expression in 4T1 cells after β2R inhibitor treatment by flow cytometry analysis. (B) PPARγ expression detected by western blot in 4T1 cells transfected with siCtrl or siβ2R(1). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (C) PPARγ expression detected by cell fluorescence staining in 4T1 cells transfected with siCtrl or siβ2R(1). (D) PPARγ, vascular endothelial growth factor (VEGF), or fibroblast growth factor 2 (FGF2) mRNA expression analyzed by quantitative real-time polymerase chain reaction in 4T1 cells treated by adenylyl cyclase activator forskolin (10 μM). *p < 0.05; NS, no significance. (E) VEGF expression detected by western blot in 4T1 cells transfected with siPPARγ. (F) Reactive oxygen species (ROS) levels determined by flow cytometry in 4T1 cells with different treatment. (G, H) VEGF expression detected by western blot in 4T1 cells with different treatments, H2O2 (250 μmol/L) used to induce ROS (G), N-acetylcysteine (NAC, 5 mmol/L) used to eliminate ROS (H). *p < 0.05; NS, no significance.

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  Fig. 6. β2 receptor activation and peroxisome proliferator-activated receptor γ (PPARγ) inhibition is involved in chronic stress-induced tumor angiogenesis in vivo. (A) Representative tumor images from mice in control (n=4), ICI118551 (n=4) and GW9662 group (n=5). (B) Tumor growth curves. (C) Average tumor weight was measured on day 28. *p < 0.05 vs. control, #p < 0.05 vs. chronic stress. (D, E) PPARγ, vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF2) mRNA expression in tumor tissues by reverse transcription polymerase chain reaction (D) and quantitative real-time polymerase chain reaction (E). *p < 0.05. (F) PPARγ, VEGF, and FGF2 protein expression in tumor tissues by western blot. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (G) Representative images (left panel) and bar graph (right panel) of PPARγ expression detected in tumor tissue paraffin sections by immunohistochemistry staining. *p < 0.05. (H) Images of tumor section CD31 immunohistochemistry staining (left panel) and microvascular density was measured (right panel). *p < 0.05 vs. control, #p < 0.05 vs. chronic stress.

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  Fig. 7. Bioinformatics analysis of peroxisome proliferator-activated receptor γ (PPARγ) in breast invasive carcinoma (BRCA). (A, B) Higher PPARγ expression in normal tissue and lower PPARγ expression in BRCA tissue. Data from StarBase database (A) and data from GEPIA database (B). *p < 0.05. (C) High PPARγ expression is associated with longer overall survival (OS) compared to low PPARγ expression, according to StarBase v3.0 data (p=0.039). (D) High PPARγ expression is associated with longer relapse-free survival compared to low PPARγ expression, according to online database data (http://kmplot.com/analysis/index.php?p=service&cancer=breast) (p < 0.001). HR, hazard ratio. (E) Study model schematic. β2 receptor activation induced by chronic stress enables breast cancer progression by inactivating PPARγ and enhancing reactive oxygen species (ROS)–VEGF/fibroblast growth factor 2 (FGF2)–mediated angiogenesis.

Reference

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