Abstract
Purpose
Invasive ductal carcinoma (IDC) accounts for 90% of triple-negative breast cancer (TNBC). IDC is mainly derived from the breast ductal epithelium which is innervated by the 4th to 6th thoracic sympathetic nerves. However, little is known about the contribution of the interactions between sympathetic nerves and breast cancer cells to the malignant progression of TNBC.
Methods
The expression levels of the β2-adrenergic receptor (β2-AR, encoded by ADRB2 gene), nerve growth factor (NGF), and tropomyosin receptor kinase A (TrkA) were determined using immunohistochemistry (IHC). NGF expression levels in the serum were compared by enzyme-linked immunosorbent assay (ELISA). Cell proliferation was assessed using the Cell Counting Kit-8 assay. The β2-AR, NGF, p-ERK, and p-CERB expression levels were determined using western blotting. TNBC cells and neuronal cells of the dorsal root ganglion (DRG) in 2-day-old Sprague Dawley rats were co-cultured. Using norepinephrine (NE), NGF, and β2-AR, NGF/TrkA blocker pretreatments, the axon growth of each group of DRG neuron cells was detected by immunofluorescence analysis.
Results
The sympathetic adrenergic neurotransmitter NE activated the ERK signaling pathway in TNBC cells. NE/β2-AR signaling promotes NGF secretion. NGF further facilitates the malignant progression of TNBC by increasing sympathetic neurogenesis. In the co-culture assay, the sympathetic adrenergic NE/β2-AR signal pathway also enhanced NGF secretion. NGF binds TrkA in DRG neurons and promotes axonal growth.
Conclusion
These results suggest that NE/β2-AR pathway promotes cell proliferation and NGF production in triple-negative breast cancer.
Keywords: Nerve Growth Factors; Norepinephrine; Receptor, trkA; Receptors, Adrenergic, beta-2; Triple Negative Breast Neoplasms
INTRODUCTION
Breast cancer is the most frequently diagnosed malignant disease among women worldwide and the main cause of cancer-related deaths [1]. Triple-negative breast cancer (TNBC), which represents approximately 15%–20% of all diagnosed breast carcinomas, is characterized by an aggressive clinical course, lymph node metastasis, and dismal prognosis [2,3]. A total of 90% of the pathological types of TNBCs are invasive ductal carcinomas (IDCs). The IDC is primarily derived from breast ductal epithelium which is innervated by the 4th to 6th thoracic sympathetic nerves [4]. The sympathetic nervous system (SNS) is a primary component of the tumor microenvironment and plays an important role in tumorigenesis and tumor progression [5]. However, in the TNBC tumor microenvironment, the sympathetic nerve-breast cancer cell interaction and its role in the malignant progression of TNBC remain unclear.
Most patients with breast cancer experience distress such as severe depression and anxiety, which increases the density of sympathetic nerve fiber in the breast tissue releasing a large amount of norepinephrine (NE) [6,7]. NE primarily binds to the β2-adrenergic receptor (β2-AR) on the surface of tumor cells in the tumor microenvironment [8]. β2-ARs are associated with heterotrimeric guanine nucleotide-binding proteins (G proteins) and initiate various signaling pathways such as adenylyl cyclase and mitogen-activated protein kinases (MAPKs) [9,10]. β2-ARs are abnormally high in breast cancer cells and can regulate cell proliferation [11]. Clinical studies have shown that blocking β2-AR signaling improves the prognosis of patients with breast cancer [12]. However, there are few relevant studies on whether sympathetic nerve fibers are expressed in TNBC and whether they affect breast cancer cell growth.
Nerve growth factor (NGF) is highly expressed in breast cancer [13], ovarian cancer [14], pancreatic cancer [15], lung cancer [16], liver cancer [17], melanoma [18], thyroid cancer [19], and other types of cancer. NGF is an important neurotrophic factor that binds to tropomyosin receptor kinase A (TrkA) and p75NTR on nerve fiber endings and plays a key role in the differentiation, advancement, and regeneration of neurons [20]. Besides nerve nutrition, NGF plays an important role in breast cancer occurrence and development, including proliferation, apoptosis, invasion, metastasis, angiogenesis, and neurogenesis [13]. Renz et al. [21] demonstrated that pancreatic cancer cells secrete NGF to induce the axonal growth of dorsal root ganglion (DRG) neurons in vitro. Hayakawa et al. [22] also found that in a mouse model of gastric cancer, NGF secreted by gastric cancer cells binds to TrkA at the nerve fiber endings around gastric cancer cells and induces nerve fiber proliferation in tumor tissues. In the tumor microenvironment, NGF secreted by tumor cells can induce nerve fiber proliferation in tumor tissues.
Developing/progressing tumors are classified as a result of the mutual interactions between tumor cells and their surrounding microenvironment. Renz et al. [21] reported that β2-AR/NGF/TrkA feed-forward loop promotes pancreatic tumorigenesis. To investigate the role of the NE/β2-AR pathway in the malignant progression of TNBC, we performed experiments using human TNBC cell lines. In this study, we found high expression of NE and β2-AR in TNBC cells and report that the NE/β2-AR pathway promotes cell proliferation and the production of nerve growth factors in TNBC.
METHODS
Antibodies
Anti-tyrosine hydroxylase (TH) Ab (75875), anti-S100 Ab (112), and anti-β2-AR Ab (182136) were purchased from Sigma (St. Louis, USA); anti-CREB Ab (12208-1-AP), phosphorylated anti-CREB Ab (ser133), anti-ERK Ab (16443-1-AP), Phosphorylated anti-ERK Ab (T202/Y204), and anti-NGF Ab (52918) were purchased from Cell Signaling Technology (Danvers, USA); anti-beta-actin Ab was purchased from Beijing Zhongshan Jinqiao Company (Beijing, China); anti-TrkA BAS-bs (10210R) was purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China); horseradish peroxidase (HRP)-conjugated Goat Anti-Rabbit IgG (H+L) (SA00001-2) was purchased from Proteintech Group (Rosemont, USA).
Cell culture
MCF-10A, MDA-MB-231, BT549, MDA-MB-468, HS-578T, and MCF-7 cells were purchased from the American Type Culture Collection (ATCC, Manassas, USA). MCF-10A cells were cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS, v/v), penicillin (100 U/mL) and streptomycin (100 mg/mL). MDA-MB-231 cells were cultured in DMEM (Gibco, Grand Island, USA), supplemented with 10% FBS(Gibco) (v/v), penicillin (100 U/mL) and streptomycin (100 mg/mL). BT549, MDA-MB-468, HS-578T, and MCF-7 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS (v/v), penicillin (100 U/mL), and streptomycin (100 mg/mL). The cells were maintained at 37°C in a humidified incubator with 5% CO2.
Cell proliferation assay
Cell proliferation was evaluated using Cell Counting Kit (CCK) assay (Gibco). MCF-10A, MDA-MB-231, BT549, MDA-MB-468, HS-578T, and MCF-7 cells were seeded at a density of 5 × 103 cells in 96-well culture plates. The cells were stimulated with 0, 0.1, 1, 10, and 20 μM of NE (Sigma). Then isoprenaline (ISO), propranolol (Pro), atenolol (Ate), and ICI (Sigma) were added into the culture medium at final concentrations of 10 μM. After 6 hours, 10 μL of CCK-8 solution was added to each well, and cells were further incubated in a CO2 incubator for 2 hours. The optical density (OD) value of each well was measured using a microplate reader (Thermo, Vantaa, Finland) at 450 nm.
Western blot
Protein samples (20 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a constant current of 20 mA using the Mini Protean 3 Cell (Bio-Rad, Hercules, USA). Proteins separated on the gels were transferred onto polyvinylidene fluoride membranes (Merck Millipore, Burlington, USA). Membranes were blocked with 5% fat-free milk or 5% bovine serum albumin in Tris buffered saline with Tween 20 (10 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20) for 1 hour at room temperature and incubated with the indicated primary antibodies. After washing three times, the membrane was probed with a 1:1,000 diluted HRP-conjugated secondary antibody for 1 hour. The membranes were visualized using an ECL system (Amersham, UK). A Chemi DocTM XRS + gel imaging system was used to scan the Images, and analyze the gray matter density.
Clinical samples
Clinical samples were collected between September 1, 2017 and December 31, 2020 at The First Affiliated Hospital of Dalian Medical University from 120 patients with TNBC who underwent radical surgical treatment and served as the observation group and 45 patients with primary breast cancer who underwent surgery and patients with breast hyperplasia who served as the control group. The mean age of the observation group was 47.65 ± 5.67 years, and that of the control group was 50.37 ± 5.81 years.
The pathological diagnosis of TNBC was based on the World Health Organization classification of breast tumor histology [23]. Clinical annotation included histological type, histological grade, tumor size, lymph node status, and regional lymph node metastasis status. Immunohistochemistry (IHC) tests included estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2, Ki-67, TH, β2-AR, NGF, and TrkA. Tumor size was measured based on the largest diameter of the invasive component in the histological sections. When the largest diameter of the metastatic lesion > 0.2 mm in the axillary lymph nodes, it indicated regional lymph node metastasis. ER, PR, and AR were defined according to the American Society of Clinical Oncology (ASCO), the College of American Pathologists (CAP) and the 2013 ASCO/CAP Guidelines for the Detection of HER-2 in Breast Cancer [24]. Breast cancer molecular typing was performed according to the 2013 St. Gallen Conference [25]. TNM staging was based on the 8th edition of the American Joint Committee on Cancer 2017 breast cancer staging criteria [26]. The First Affiliated Hospital of Dalian Medical University granted the ethical approval to conduct this study within its facilities (application ref.: YJ-KY-FB-2021-16). Breast cancer tissue and serum samples were processed anonymously per ethical and health laws and regulations. Informed consent was obtained from all patients.
IHC
Clinical specimens were collected from breast cancer tissues of patients with TNBC (n = 120). Among the specimens, we selected 30 cases of sympathetic nerve fibers positive (TH+) tissues for quantification of IHC for S100, TH, β2-AR, NGF, and TrkA. Sections were deparaffinized twice in xylene and hydrated using a graded series of ethanol to phosphate-buffered saline (PBS). Endogenous peroxidase activity was blocked with 3% H2O2 for 5 minutes. After washing with PBS containing 0.1% Tween 20 (PBS-T), the slides were blocked with a blocking kit and incubated anti-β2-AR, anti-NGF, and anti-TrkA Ab. Slides were washed again and covered with HRP-labeled Abs for 30 minutes. Finally, the slides were visualized with 3, 3’-diaminobenzidine and counterstained with hematoxylin and eosin (HE) (Beijing Solarbio Co. Ltd., Beijing, China.). IHC was performed using the ImmPRESS detection kit (Vector Laboratories, Newark, USA).
Enzyme-linked immunosorbent assay (ELISA)
Serum samples were collected from patients with breast hyperplasia and TNBC. NGF expression levels were compared by ELISA. ELISA was performed using an NGF ELISA kit (L200729898; Cloud-Clone Corp., Katy, USA) (Shanghai Langton Biotechnology Co., Ltd., Shanghai, China).
MDA-MB-231 cells and DRG co-culture
Primary cultures of DRG were established according to a published protocol [21]. Two days old Sprague Dawley rats were obtained from the Experimental Animal Center of Dalian medical university (permit number SCXK 2008-0002). Briefly, DRG was dissected from newborn Sprague Dawley (2 days old) and pooled in an L-15 medium (Gibco) on ice. The DRG was then transferred to the lower chamber of the Transwell which was covered with rat tail collagen Type I (Invitrogen, USA). After 24 h, the DRG neurons were purified with 5 μg/ml cytarabine (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). MDA-MB-231 cells were cultured in the upper chambers of the 6-well plates at a density of 1 × 104 cells/cm2. The control group contained only the DRG (referred to as the DRG group). The cells were cultured at 37°C in a 5% CO2 incubator for 7 days and the medium was changed every 2 days. Using NE, NGF, and β2-AR/TrkA blocker (Shandong Topscience, Rizhao, China) pretreatment, the axon growth of each group of DRG neurons was detected by immunofluorescence staining.
Statistical analysis
SPSS 20.0 (IBM Corp., Armonk, USA) was used for statistical analyses. The measurement data were compared between two groups using an unpaired t-text, and among three group using a one-way analysis of variance and least significant difference. The χ2 test was used to compare differences in categorical data. Statistical significance was set at p < 0.5.
RESULTS
The sympathetic nerve is closely related to the malignant progression of TNBC
To explore the correlation between sympathetic nerves and the malignant progression of TNBC, clinical samples of TNBC and its adjacent tissues (as controls) were used and stained with H&E. In the adjacent TNBC tissue, sympathetic nerves, arteries, veins, and lymphatic vessels were located in the fibrous interstitium surrounding the breast lobules, whereas sympathetic nerve fibers were tightly surrounded by breast cancer cells and were significantly increased in TNBC tissues (Figure 1A). S100 and TH are biomarkers that recognize nerves and sympathetic nerve fibers, respectively [27,28]. Next, to characterize the proliferation of nerve fibers in TNBC tissues, the distribution of nerve fibers in TNBC tissues was determined by IHC staining with an anti-S100 Ab for Schwann cell markers of peripheral nerve fibers (Figure 1B). To further confirm the presence of sympathetic nerve fibers, we tested sympathetic nerves by staining with anti-TH Ab. The results showed that TNBC tissues expressed TH+ sympathetic nerve fibers (Figure 1C). We also observed that TNBC cells are tightly wrapped around the sympathetic nerve fibers (represented by the arrow) (Figure 1A).
Figure 1. Detection of sympathetic nerve fibers in triple-negative breast cancer.
(A) Representative images of breast HE slides (scale bar: 100 μm). The breast tissues were collected from mammary gland hyperplasia (n = 30) and TNBC (n = 30) patients. Arrows point to nerve trunks (composed of many nerve fibers). Bar graphs showing quantification of neural numbers (nerve trunk numbers/mm2 tissue section). (B) IHC for the neuronal marker S100 (scale bar: 100 μm). (C) IHC for the sympathetic nervous marker TH (scale bar: 100 μm).
HE = hematoxylin and eosin; TNBC = triple-negative breast cancer; IHC = immunohistochemistry; TH = tyrosine hydroxylase; ctrl = control.
*p < 0.001.
To test for an association between sympathetic fibers and clinicopathological parameters in TNBC, we classified 120 patients with TNBC into sympathetic nerve fiber positive and negative groups (Table 1). Among all patients with TNBC, 32% (38/120) were positive for sympathetic nerve fibers. Among the 120 TNBC samples, 108 were of the IDC pathological type, and the proportion of sympathetic nerve fiber-positive samples was 34.3% (37/108) in all cases, indicating that sympathetic nerve fibers are associated with TNBC tumorigenesis. In patients with TNBC, 45.5% (30/66) of sympathetic nerve fibers reaction were positive for lymph node metastasis and 14.8% (8/54) were negative. This difference was significant between the two groups. These data suggest that sympathetic nerves are associated with lymph node metastasis in TNBC. Regarding histological grade, the positive rate of TH in grade 3 was 36.3%, and the positive rate of TH was 6.7% in grade 2. The difference between the two groups was significant. The data showed that sympathetic nerves were associated with the malignant progression of TNBC. Taken together, these data suggest that sympathetic nerve fibers are distributed in the TNBC tumor microenvironment and are closely related to the malignant progression of TNBC.
Table 1. Association between the presence of sympathetic nerve fibers and clinicopathological parameters in triple-negative breast cancer.
| Parameter | Sympathetic nerve fibers positive | Sympathetic nerve fibers negative | p-value | |
|---|---|---|---|---|
| All cases (n = 120) | 38 (32) | 82 (68) | ||
| Pathological subtype | 0.132 | |||
| All cases (n = 120) | 38 (32) | 82 (68) | ||
| Pathological subtype | 0.132 | |||
| IDC (n = 108) | 37 (34.3) | 71 (65.7) | ||
| Other subtypes (n = 12) | 1 (8.3) | 11 (91.7) | ||
| Patient age | 1.000 | |||
| ≤ 35 (n = 11) | 3 (27.3) | 8 (72.7) | ||
| > 35 (n = 109) | 35 (32.1) | 74 (67.9) | ||
| Menopausal status | 0.393 | |||
| Premenopausal (n = 69) | 24 (34.8) | 45 (65.2) | ||
| Postmenopausal (n = 51) | 14 (27.5) | 37 (72.5) | ||
| Tumor values (T) | 0.074 | |||
| 1 (n = 23) | 3 (13) | 20 (87) | ||
| 2 (n = 88) | 32 (36.4) | 56 (63.6) | ||
| 3 (n = 9) | 3 (33.3) | 6 (66.7) | ||
| Lymphovascular space involvement | 0.110 | |||
| Positive (n = 112) | 38 (33.9) | 74 (66.1) | ||
| Negative (n = 8) | 0 (0) | 8 (100) | ||
| Lymph nodes status | < 0.001* | |||
| Positive (n = 66) | 30 (45.5) | 36 (54.5) | ||
| Negative (n = 54) | 8 (14.8) | 46 (85.2) | ||
| TNM stage | 0.080 | |||
| Stage I (n = 9) | 0 (0) | 9 (100) | ||
| Stage II+III (n = 111) | 38 (34.2) | 73 (65.8) | ||
| Histologic grade | 0.032* | |||
| 1 (n = 3) | 0 (0) | 3 (100) | ||
| 2 (n = 15) | 1 (6.7) | 14 (93.3) | ||
| 3 (n = 102) | 37 (36.3) | 65 (63.7) | ||
| Proliferative fraction (Ki-67) | 0.110 | |||
| < 14 (n = 8) | 0 (0) | 8 (100) | ||
| ≥ 14 (n = 112) | 38 (33.9) | 74 (66.1) | ||
| ADRB2 receptor | < 0.001* | |||
| ADRB2 positive (n = 78) | 35 (44.9) | 43 (55.1) | ||
| ADRB2 negative (n = 42) | 3 (7.1) | 39 (92.9) | ||
| Nerve growth factor | < 0.001* | |||
| NGF positive (n = 64) | 33 (51.6) | 31 (48.4) | ||
| NGF negative (n = 56) | 5 (8.9) | 51 (91.1) | ||
Values are presented as number (%).
IDC = invasive ductal carcinomas; NGF = nerve growth factor; TNM = tumor, node, metastasis.
*Statistically significant p-values (p < 0.05 using χ2 test).
Sympathetic adrenergic NE promotes the proliferation of TNBC cells
Breast cancer patients have severe depression and anxiety resulting from the fact that the SNS is in a state of excessive stress, which promotes sympathetic nerve fiber in the breast tissue, releasing large amounts of NE [6,7,8]. To study the effect of sympathetic adrenergic neurotransmitter NE on TNBC cells, we stimulated several breast cancer cell lines, such as the normal breast epithelial cell line (MCF10A), TNBC cell lines (MDA-MB-231, MDA-MB-468, BT-549, and HS-578T), and non-TNBC cell line (MCF7) with different NE concentrations. Cell proliferation was determined using a CCK-8 assay. As shown in Figure 2, 10 μM of NE significantly enhanced the proliferation of MDA-MB-231, MDA-MB-468, and BT-549, but not of MCF10A, HS-578T, and MCF7 cells. These results suggest that the sympathetic adrenergic neurotransmitter NE promotes TNBC cell proliferation.
Figure 2. The sympathetic adrenergic neurotransmitter norepinephrine promotes the proliferation of triple-negative breast cancer cells. Normal breast epithelial cell line MCF10A and human breast cancer cell lines, MDA-MB-231, MDA-MB-468, BT-549, HS-578T, and MCF7, were starved overnight and then treated with 0, 0.1, 1, 10, and 20 μM of norepinephrine for 24 hours. Cell viability was determined by Cell Counting Kit-8 assay. Data are shown as means ± standard deviation from triplicate experiments.
*p < 0.01, †p < 0.001.
Sympathetic adrenergic NE/β2-AR signaling promotes TNBC cell growth via the ERK pathway
Given that NE binds to β2-AR on the cancer cells and triggers various signaling pathways in the breast cancer microenvironment [29], TNBC and its adjacent tissues (as a control) were used to investigate β2-AR distribution and expression by IHC. The results show that β2-AR was expressed in both groups and that β2-AR expression was significantly high in breast cancer cells compared with that in the control group (Figure 3A). Interestingly, β2-AR expression was obvious in breast cancer cells that closely surround the nerves (Figure 3B). Among the 120 clinical samples analyzed, β2-AR expression was high in 78 TNBC patients, and the positive rate of TH was a 44.9%. In contrast, the other 42 cases of TNBC patients showed low β2-AR expression with a 7.1% (p < 0.001) positive rate of TH (Table 1). These indicate that high β2-AR expression in TNBC cells could be associated with the distribution of sympathetic nerves. Furthermore, β2-AR expression in breast cancer cells was determined using the Cancer Cell Line Encyclopedia (CCLE) gene database. The results show that β2-AR expression was high in TNBC cells than in non-TNBC cells (Supplementary Figure 1).
Figure 3. β2-adrenergic receptor expression was upregulated in the triple-negative breast cancer cells.
(A) The breast tissues were collected from mammary gland hyperplasia (n = 30) and TNBC (n = 30) patients that expressed TH. The tissues were immunostained with anti-β2-AR (scale bar: 100 μm). Positive β2-AR expression was most obvious in TNBC cells tightly surrounding the nerve. The graph shows the AOD measurements. (B) TNBC cells infiltrated into the surrounding nerve trunks, and β2-AR expression was high in the TNBC cells (scale bar: 100 μm).
TNBC = triple-negative breast cancer; TH = tyrosine hydroxylase; β2-AR = β2-adrenergic receptor; AOD = average optical density; ctrl = control.
*p < 0.001.
Next, to further demonstrate that NE targets breast cancer via the NE/β2-AR pathway, β2-AR expression in MCF10A, MDA-MB-231, MDA-MB-468, BT-549, HS-578T, and MCF7 cells was also determined. β2-AR expression was high in MCF10A, MDA-MB-231, MDA-MB-468, and BT-549 cells, but low in HS-578T cells (Figure 4A). Next, the β2-AR-expressed MCF10A, MDA-MB-231 and BT-549 cells were used to check β2-AR expression after being treated with NE, β1-β2-AR activator (ISO), β1-AR inhibitor (Ate), β1-2-AR inhibitor (Pro), and β2-AR inhibitor (ICI). NE/ISO significantly enhanced the proliferation of MDA-MB-231 and BT-549 cells, but not MCF10A cells (Figure 4B). NE and ISO significantly increased β2-AR expression in MDA-MB-231 and BT-549 cells (Figure 4C and D). The treatments with Pro or ICI significantly reversed the increased proliferation induced by NE, but not under Ate treatment (Figure 4B), indicating that NE enhances cell proliferation via β2-AR but not β1-AR.
Figure 4. Norepinephrine binds to β2-adrenergic receptor on triple-negative breast cancer cells.
(A) Expression of β2-AR protein. Cell lysates were subjected to immunoblotting with antibodies against β2-AR (1:500) and β-actin (1:1,000). Quantitation of the western blotting results is shown in the bar graph. Data from at least three independent experiments are expressed as means ± SD. (B) Cell proliferation by CCK-8 assay. MCF-10A, MD-MB231, and BT549 cells were treated with 10 µM NE/ISO/Ate/Pro/ICI for 24 hours to measure the cell viability. Data are shown as means ± SD from triplicate experiments, β2-AR expression after several treatments in MD-MB231 (C) and BT549 cells (D). After starvation with serum-free medium for 24 hours, the cells were treated with 10 µM NE/ISO/Ate/Pro/ICI for 6 hours. Cell lysates were subjected to immunoblotting with antibodies against β2-AR (1:500) and β-actin (1:1,000). Quantitation of the western blotting results is shown in the bar graph. Data from three independent experiments are expressed as means ± SD.
β2-AR = β2-adrenergic receptor; SD = standard deviation; CCK = Cell Counting Kit; NE = norepinephrine; ISO = isoprenaline; Ate = atenolol; Pro = propranolol.
*p < 0.01, †p < 0.001.
NE binds to β2-AR and activates extracellular signal-regulated kinase (ERK and CREB downstream) [10]. To investigate the effect of NE/β2-AR downstream signal on the proliferation of TNBC cells, the ERK and CREB phosphorylation levels were investigated in MD-MB-231 and BT549 cells with NE or ISO which are treated with Pro, ICI, and Ate. ERK phosphorylation was significantly higher following NE or ISO treatment in MD-MB-231 (Figure 5A) and BT549 cells (Figure 5B). Moreover, ERK phosphorylation was significantly attenuated by pretreatment with either Pro or ICI, whereas CREB remained the same. Cell proliferation was also suppressed by pretreatment with an ERK kinase-specific inhibitor (U0126), but not suppressed by a PKA-specific inhibitor (KT-5720), in MD-MB-231 and BT549 cells (Figure 5C). Collectively, these results suggest that NE binds to the β2-AR on TNBC cells and induces ERK activation, which is required for cell proliferation.
Figure 5. Norepinephrine/β2-adrenergic receptor signal promotes the growth of triple-negative breast cancer cells via ERK activation.
(A) Phosphorylation of ERK and CREB after NE treatment of MD-MB231 cells. After starvation, the cells were treated with 10 µM NE/ISO/Ate/Pro/ICI for 6 hours. Cell lysates were subjected to immunoblotting with antibodies against p-ERK (1:1,000), ERK (1:1,000), p-CREB (1:800), CREB (1:800), and β-actin (1:1,000). Quantitation of the western blotting results is shown in the bar graph. Data from three independent experiments are expressed as means ± SD. (B) Phosphorylation of ERK and CREB after NE treatment of BT549 cells. Data from three independent experiments are expressed as means ± SD. (C) CCK-8 assay of the MD-MB231 and BT549 cells. After starvation, MD-MB231 and BT549 cells were treated with 10 µM NE/ISO/KT5720/U0126 for 24 hours to measure the cell viability. Data are shown as means ± SD from triplicate experiments.
NE = norepinephrine; ISO = isoprenaline; Ate = atenolol; Pro = propranolol; SD = standard deviation; CCK = Cell Counting Kit; β2-AR = β2-adrenergic receptor.
*p < 0.01, †p < 0.001.
Sympathetic adrenergic NE/β2-AR pathway promotes NGF secretion in the TNBC cells
NE/β2-AR pathway upregulates NGF expression and secretion [21]. Data from the CCLE gene database also showed that NGF expression was significantly upregulated in TNBC cells compared with that in non-TNBC cells (Supplementary Figure 1). NGF levels in serum samples of patients with breast hyperplasia and TNBC was determined by ELISA. As shown in Figure 6A, NGF levels were significantly upregulated in the serum of patients with TNBC compared with those in the control group. To study the correlation between sympathetic nerves and NGF secretion in the tumor microenvironment of TNBC cells, clinical samples of TNBC and its adjacent tissues (as a control) were collected and subjected to IHC. Both normal breast and TNBC cells express NGF. However, NGF expression in TNBC cells was significantly higher than that in the control group (Figure 6B). Interestingly, NGF was co-expressed in TNBC cells and nerve fibers but not in the control tissue (Figure 6C), indicating that TNBC cells secrete NGF and interact with peripheral nerve fibers. NGF expression was particularly high in 64 patients with TNBC with a TH-positivity rate of 51.6%. However, NGF expression was low in the other 56 patients with TNBC who showed a TH-positivity rate of 8.9% (Table 1). These data suggest that NGF secretion by breast cancer cells may be associated with sympathetic nerves.
Figure 6. Norepinephrine/β2-adrenergic receptor signal promotes nerve growth factor expression via the ERK pathway in triple-negative breast cancer cells.
(A) The level of NGF detected by ELISA. The sera were obtained from the patients with mammary gland hyperplasia and TNBC (n = 120, mean ± SD). (B) IHC assay. The breast tissues were collected from mammary gland hyperplasia (n = 30) and TNBC (n = 30) patients that expressed TH. We stained samples with the anti-NGF Ab (scale bar: 100 μm) and calculated the AOD. (C) IHC against NGF in TNBC cells adjacent to nerve trunks (scale bar: 100 μm). Graph showing the AOD. Arrows point to nerve trunks. (D) NGF expression in human breast cancer cell lines. Cell lysates were subjected to immunoblotting with anti-NGF Ab (1:500) and anti-β-actin Ab (1:1,000). Quantitation of the western blotting results is shown in the bar graph. Data from three independent experiments are expressed as means ± SD. (E) NGF expression after NE treatment of MD-MB231 cells. Cells were treated with 10 µM NE for 0, 3, and 6 hours. Cell lysates were subjected to immunoblotting with antibodies against NGF (1:500) and β-actin (1:1,000). Data from three independent experiments are expressed as means ± SD. (F, G) NGF expression after NE treatment of MD-MB231 and the BT549 cells. After starvation, the cells were treated with 10 µM NE/ISO/Ate/Pro/ICI for the 6 hours. Cell lysates were subjected to immunoblotting with anti-NGF Ab (1:500) and anti-β-actin Ab (1:1,000). Quantitation of the western blotting results is shown in bar graph. Data from three independent experiments are expressed as means ± SD. (H) NGF secretion measured by ELISA. After starvation for 24 hours, cells were stimulated with 10 µM NE/ISO/Ate/Pro/ICI for 6 hours, and the media were collected. Data are shown as means ± SD from triplicate experiments. (I) NGF secretion was measured by ELISA. After starvation, cells were stimulated with 10 µM NE/ISO/KT5720/U0126 for 6 hours. Data are shown as means ± SD from triplicate experiments.
NGF = nerve growth factor; ELISA = enzyme-linked immunosorbent assay; TNBC = triple-negative breast cancer; SD = standard deviation; IHC = immunohistochemistry; TH = tyrosine hydroxylase; AOD = average optical density; NE = norepinephrine; ISO = isoprenaline; Ate = atenolol; Pro = propranolol; CCK = Cell Counting Kit; β2-AR = β2-adrenergic receptor.
*p < 0.01, †p < 0.001.
We examined NGF expression in MCF10A, MDA-MB-231, MDA-MB-468, BT-549, HS-578T, and MCF-7 cells. Compared with that in MCF10A cells, NGF expression was significantly upregulated in MDA-MB-231, HS-578T, and BT-549 cells (Figure 6D). The NGF expression peaked 6 hours after NE treatment in MD-MB-231 cells (Figure 6E). NE treatment enhanced NGF expression in the MD-MB-231 (Figure 6F) and BT549 cells (Figure 6G). In addition, the effect of NE/β2-AR signaling on NGF secretion in TNBC cells was determined. NE/ISO significantly enhanced NGF secretion in MDA-MB-231 and BT-549 cells. Treatment with Pro or ICI, but not with Ate treatment, significantly suppressed NE/ISO-induced NGF secretion (Figure 6H). To further investigate the effect of NE/β2-AR downstream signaling pathway on NGF secretion in the TNBC cells, MDA-MB-231 and BT549 cells were treated with either U0126 or KT-5720 and NGF secretion was measured. Treatment with U0126 significantly suppressed NGF secretion in MDA-MB-231 and BT549 cells, but not by treatment with KT-5720 (Figure 6I). The results indicate that NE/β2-AR signaling enhanced NGF secretion through activation of the ERK pathway in the TNBC cells.
Sympathetic adrenergic NE/β2-AR pathway induces the axon growth of DRG neuron cells by increasing NGF secretion in TNBC cells
NGF binds to TrkA, a highly specific receptor in peripheral nerve fibers, to promote neuronal axonal growth [20]. To investigate the correlation between NGF secreted by TNBC cells and peripheral sympathetic nerve fibers, IHC was performed to detect TrkA expression in peripheral nerve fibers. Compared to that in normal breast tissue, the TrkA expression level in nerve fibers was significantly higher in TNBC tissues (Figure 7A). These results suggest that, in the TNBC tumor microenvironment, NGF secreted by breast cancer cells may bind to TrkA at peripheral sympathetic nerve fiber endings and induce sympathetic nerve fiber hyperplasia.
Figure 7. Norepinephrine/β2-adrenergic receptor induces axon growth of dorsal root ganglion neuron cells by promoting nerve growth factor secretion.
(A) IHC assay. Breast tissues were collected from mammary gland hyperplasia (n = 30) and TNBC (n = 30) patients that expressed TH. The specimens were immunostained with anti-TrkA Ab (scale bar: 100 μm) and the AOD was calculated. Arrows point to nerve trunks. (B) Neurite length determination by fluorescence intensity quantitative analysis. Co-culture experiments were performed in Transwell Boyden chambers with the DRG neuron cells in the lower part and the breast cancer cells in the upper part. A negative control (with no breast cancer cells) and a positive control (addition of 50 ng/mL NGF) were set. ICI (10 μM) and TrkA blocker GW 441756 (10 μM) significantly inhibited the axonal growth of DRG neurons in L-15 medium.
IHC = immunohistochemistry; TNBC = triple-negative breast cancer; TH = tyrosine hydroxylase; TrkA = tropomyosin receptor kinase A; AOD = average optical density; DRG = dorsal root ganglion; NGF = nerve growth factor; ctrl = control; NE = norepinephrine.
*p < 0.05, †p < 0.01, ‡p < 0.001.
To further explore the mechanism of NE/β2-AR signal transduction on the axon growth of DRG neurons, the MDA-MB-231 cells were pretreated with NE or NGF and co-cultured with DRG neurons for 7 days. Immunofluorescence staining showed that pretreating MDA-MB-231 cells with NE or NGF significantly promoted axonal growth of DRG neurons. Moreover, the axonal growth of DRG neurons was suppressed by ICI treatment. Treatment with 10 μM of TrkA blocker (GW 441756) also inhibited the axonal growth of DRG neurons (Figure 7B). These results suggest that NGF binds to TrkA in DRG neurons and induces the axonal growth in the TNBC microenvironment.
DISCUSSION
The contribution of SNS to tumorigenesis has emerged as an important tumor microenvironment component [30]. Cancer activates nerve-dependent regenerative processes that promote survival and growth [11,31]. However, the mammary tumor sympathetic innervation and its capacity to regulate TNBC progression have not yet been described. In this study, we found that the sympathetic nerve fibers were significantly increased in TNBC tissues, and stimulation with NE promoted TNBC cell proliferation and NGF release.
Sympathetic nerves interact with tumor cells and promote the initiation and progression of malignancies, such as those of breast, prostate, and pancreatic cancer [15]. Magnon et al. [32] reported that many nerve fibers were positive for sympathetic nerve markers (TH and GAP-43) in prostate cancer tissue. Pundavela et al. [31] showed that neuronal marker (PGP9.5)-positive nerve fibers were closely associated with lymph node metastasis in breast cancer. The breast is abundantly innervated by the sympathetic and parasympathetic branches of the autonomic nervous system, which control breast growth and maintenance. In TNBC tissues, sympathetic nerve fibers proliferated significantly (Figure 1A). Sympathetic nerve fibers were mainly distributed in the tissues with grade 3 histological classification and lymph node metastasis. These results suggest that sympathetic nerve hyperplasia in TNBC tissues is closely associated with the malignant progression of TNBC.
Indeed, chronic psychological stress induces SNS over-tension and promotes NE release by sympathetic nerve fibers [33,34]. NE levels in serum and tumor tissues were abnormally elevated under the chronic confinement stress which promoted tumor growth [35,36,37]. NE mainly binds to β2-AR and promotes the proliferation of cancers, such as prostate cancer [35], lung cancer [36], colon cancer [38], etc. NE expression was significantly increased in the breast cancer tissues of patients with TNBC. In the IHC analysis, 65% of the TNBC cells expressed β2-AR, and 44.9% of them were positive for sympathetic nerve fibers. Interestingly, β2-AR expression was tightly linked with the sympathetic nerve fibers in breast cancer cells, indicating that ADRB2 may have had a certain relationship with the sympathetic nerve. In addition, TNBC cells expressed more β2-AR than other non-TNBC cells. Thaker et al. [37] have reported that NE recognizes β2-AR on the surface of ovarian cancer cells and induces CREB phosphorylation through the cAMP/PKA pathway of tumor cells [10]. In this study, the NE/β2-AR pathway induced ERK phosphorylation and enhanced proliferation of TNBC cells, but did not affect the proliferation of normal mammary epithelial cells (Figure 4B), indicating that the NE/β2-AR signaling was tumor-specific in the TNBC tumor microenvironment. The β2-AR blocker is expected to become a new adjuvant drug in the clinical treatment of TNBC patients [39].
Tumorigenesis involves nerve fiber hyperplasia of tumor tissue. NGF is produced by breast cancer and tumor-associated stromal cells such as macrophages [40], fibroblasts [41], and Schwann cells [42]. It has been reported that NE/β2-AR signaling up-regulates NGF expression in pancreatic cancer cells [21]. We first found that NE/β2-AR signal is the main factor for upregulating NGF expression and secretion in TNBC cells. NGF mediates its biological effects by binding to and activating tropomyosin receptor kinase neurotrophin receptors at the nerve terminals [28]. TrkA is expressed in the peripheral sympathetic and parasympathetic nerves as well as in sensory nerve fibers [43]. Hayakawa et al. [22] reported that NGF secreted by gastric cancer cells combines with the surrounding parasympathetic nerve fiber TrkA and enhances the proliferation of many parasympathetic nerve fibers in cancer tissues. Katebi et al. [44] reported that NGF/TrkA signaling promotes axonal growth in PC12 cells (derived from pheochromocytoma in the adrenal medulla of rats). In the present study, compared with that in normal breast tissue, TrkA was highly expressed in nerve fibers of TNBC tissue. NE/β2-AR signal promoted NGF secretion in TNBC cells, which combines with the TrkA and induces axonal growth of DRG neurons. Although normal breast epithelial cells (MCF10A) expressed β2-AR (Figure 4A) and NGF (Figure 6B), NE did not affect NGF secretion and cell proliferation (Figure 6E). In the TNBC microenvironment, NGF secreted by breast cancer cells binds to the surrounding sympathetic nerve fiber TrkA, which can induce sympathetic hyperplasia in breast cancer tissues. The NGF/TrkA pathway not only enhances sympathetic nerve hyperplasia but also sensory or parasympathetic nerve hyperplasia, and promotes the malignant progression of TNBC.
In summary, our research revealed that NE/β2-AR pathway promoted cell proliferation and NGF production in TNBC. Although further studies will be required to dissect the molecular events linking tumor “neurogenesis” to cancer progression, our data highlight the significant possibility that drugs targeting the SNS may be a useful therapy for breast cancer.
ACKNOWLEDGMENTS
We thank Prof. Xue Gao Department of Pathology, the first affiliated hospital of Dalian medical university, for providing the breast tissue sample and the Laboratory of Pathology for assisting with TNBC detection.
Footnotes
Funding: This work was supported by the National Nature Science Foundation of China (No. 81273923 to QPW).
Conflict of interest: The authors declare that they have no competing interests.
- Data curation: Zhou T.
- Formal analysis: Zhou T.
- Funding acquisition: Wen Q.
- Project administration: Li W, Wen Q.
- Writing - original draft: Jin M, Wang Y.
- Writing - review & editing: Jin M, Wang Y, Li W, Wen Q.
SUPPLEMENTARY MATERIAL
Heat maps showing distinct gene expression patterns associated with each breast cell line. The related information of β2-adrenergic receptor (encoded by ADRB2) gene expression of breast cancer cells in the Cancer Cell Line Encyclopedia gene bank. Data are presented in a matrix format: each row represents a breast cell, and each column a single gene. Red squares, the gene expression patterns of nerve growth factor and β2-adrenergic receptor were later found to be associated with triple-negative breast cancer.
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Associated Data
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Supplementary Materials
Heat maps showing distinct gene expression patterns associated with each breast cell line. The related information of β2-adrenergic receptor (encoded by ADRB2) gene expression of breast cancer cells in the Cancer Cell Line Encyclopedia gene bank. Data are presented in a matrix format: each row represents a breast cell, and each column a single gene. Red squares, the gene expression patterns of nerve growth factor and β2-adrenergic receptor were later found to be associated with triple-negative breast cancer.