Targeting b3-adrenergic receptor signaling inhibits neuroblastoma cell growth via suppressing the mTOR pathway

Jing Deng b, 1, Ping Jiang b, 1, Tianyou Yang a, 1, Mao Huang b, Weiwei Qi b, Ti Zhou b, Zhonghan Yang b, Yan Zou a, ***, Guoquan Gao a, b, c, **, Xia Yang a, b, d, *

Keywords:
Neuroblastoma
b3-adrenergic receptor Proliferation
mTOR

A B S T R A C T

Neuroblastoma (NB), the most common extracranial solid tumor in childhood, always leads to an un- favorable prognosis. b3-adrenergic receptor (b3-AR) signaling plays an important role in lipid meta- bolism. Although previous studies have focused mainly on the role of b2-AR in tumor cells; there are few studies about the cancer-related function of b3-AR. Herein, we showed that b3-AR expression was significantly increased in clinical NB tissue compared with that in the less malignant ganglioneuroma (GN) and ganglioneuroblastoma (GNB) tissues. Further cellular assays demonstrated that treatment of NB cells with SR59230A (a specific b3-AR antagonist) suppressed NB cells growth and colony formation, and siRNA knockdown of b3-AR expression also inhibited NB cell proliferation. The mechanistic study revealed that b3-AR knockdown and SR59230A inhibited the phosphorylation and thereby the activation of the mTOR/p70S6K pathway. Activation of the mTOR pathway with the activator MHY1485 reversed the inhibitory effect of SR59230A on NB cell growth. Above all, our study clarifies a novel regulatory role of b3-AR in NB cell growth and provides a potent therapeutic strategy for this disease by specific tar- geting of the b3-AR pathway.

1. Introduction

Neuroblastoma (NB), which is derived from the primary neural crest, is the most common extracranial solid tumor in childhood. As a form of peripheral nervous system tumor, NB always leads to unfavorable outcomes in children, causing 9%-15% of cancer-related deaths in infancy [1e4]. The oncogene MYCN is an important prognostic marker for NB [5e8], where a high copy number often predicts a poor outcome for patients with this disease [9,10]. The clinical biological behavior of NB is significantly heterogeneous, which is characterized by spanning from spontaneous regression to high-risk aggressive progression [11,12]. Treatments for NB mainly include surgical resection, chemotherapy and pro-differentiation of the tumor cells. Although a series of therapeutic strategies have been used to cure NB, the disease recurrence rate remains very high, resulting in pain for the patients and enhanced rates of mortality. Thus, studies are needed to clarify the underlying mechanism about the tumorigenesis and aggressive progression of NB, and to find potent targets for its treatment. The b-adrenergic receptor(b-AR), which belongs to the family of G protein-coupled receptors, acts by binding with their catechol- amine hormone ligands, such as adrenaline and norepinephrine.

The b-AR signaling pathway plays an important role in physiolog- ical and pathological processes, including regulation of the car- diovascular system, lipid metabolism and glucose utilization [13e15]. b-ARs comprise three subtypes, namely, b1-AR, b2-AR and b3-AR. Among these receptors, b1-AR and b2-AR are predomi- nantly expressed in the heart of many mammalian species [16], whereas b3-AR is mainly expressed in adipose tissues and plays a pivotal role in lipolysis, the thermogenesis of adipose tissue and the mediation of glucose metabolism [17,18]. Recent studies have revealed that the b-AR pathways are closely related to the tumor- igenesis and aggressive progression of tumors, with b2-AR being the most widely studied in this regard. For instance, it was reported that b2-AR expression was obviously enhanced in hepatocellular carcinoma tissues, and activation of the b2-AR pathway promoted hepatocellular carcinoma cell growth in vivo and in vitro, whereas blocking b2-AR with a specific b2-AR antagonist ICI118,551 inhibited tumor growth in mice [19]. However, until now, the function of b3-AR in tumors has not been well clarified, and the role of b3-AR signaling in NB has not been reported. Thus, in this study, we used clinical NB tissues and cells to investigate the role of the b3-AR pathway in the tumorigenesis and progression of NB.

The mammalian target of rapamycin (mTOR) is a downstream protein kinase of the PI3K/AKT signaling pathway, and plays a pivotal role in physiological and pathological processes, such as cell proliferation, cell differentiation, autophagy, protein synthesis and cancer growth [20]. mTOR signaling can be activated by growth factors, and mTOR regulates protein translation and synthesis by activating the downstream regulator kinase p70S6K [20]. The aberrant regulation of mTOR signaling is closely associated with tumor cell growth and mTOR is usually considered as a target for tumor treatments. It was reported that mTOR signaling has a cen- tral role in lipid homeostasis and lipid metabolism [21], which led us to speculate that there may be some crosstalk between the b3- AR and mTOR signaling pathways. Since there are no reports about the interaction between b3-AR and mTOR signaling in NB, we aimed to conduct such investigation on role of b3-AR in NB and whether it regulates the mTOR pathway in NB cells.

2. Materials and methods

2.1. Reagents and antibodies

Epinephrine (E4642) and SR59230A (S8688) were from Sigma- Aldrich. MHY1485 (S7811) was from Selleck. The b3-AR (ab76249)antibody for the Western blot assay, and the b3-AR (ab140713) antibody for the immunohistochemical (IHC) assay were from Abcam. Antibodies for p-mTOR (2974), mTOR (2983), p- p70S6K (9205), and p70S6K (2708) were from Cell Signaling Technology, -and the antibody for b-actin (A2228) was from Sigma- Aldrich.

2.2. Clinical tumor sample collection

To test the expression of b3-AR in clinical NB tissues, we collected three types of clinical tissue samples from the Guangzhou Women and Children’s Medical Center; namely, ganglioneuroma (GN, n 35), ganglioneuroblastoma (GNB, nodular and intermixed, n 34), and NB (n 71). These clinical samples were used to create a tissue microarray (TMA) performed by the Shanghai Outdo Biotech Company.

2.3. Immunohistochemistry

For the IHC assay, the clinical tumor samples were fixed, embedded in paraffin, and finally assembled into a tissue microarray on slides. The sample slides were deparaffinized and rehydrated according to standard methods and then subjected to IHC staining. In brief, the slides were pretreated with 0.01 mol/Lcitrate buffer (pH 6.0) for 2 min at 100 ◦C in an autoclave. After the tissue sections had been naturally cooled, they were immersed in 3% H2O2 and 100% methanol for 20 min to quench the endogenous peroxidases. Then the sections were treated with normal goat serum to block non-specific staining, following which the slides were incubated with anti-b3-AR (1:200) antibody at 4 ◦C overnight. After the incubation of primary antibody, the slides were incubated with horseradish peroxidase-labeled anti-rabbit or mouse sec- ondary antibody for 30 min at room temperature and then stained by substrate diaminobenzidine (DAB) from Dako for 30s, and then counterstained with hematoxylin for 20s. The staining scores were evaluated according to the positive area plus positive intensity of the clinical neuroblastoma samples.

2.4. Cell culture

The NB cells, which were a kind gift from Professor Li Bo, Sun Yat-sen Medical School, were cultured in high glucose dulbecco’s modified eagle medium（DMEM, Corning)，supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) under 5% CO2 in a humidified incubator at 37 ◦C.

2.5. Cell Counting Kit-8 assay

CCK-8 reagent was from Dojindo (Japan). For the Cell Counting Kit-8 (CCK-8) assay, SK-N-AS and SHEP cells was seeded in 96-well plates at a density of 3 103 cells per well, respectively. After culturing for 12 h, the cells were treated with different concentra- tions of the selective b3-AR antagonist SR59230A for 24 h. Then, 10 mL of CCK-8 reagents were added and the cells were incubated for 1e4 h. Finally, the absorbance at 450 nm was measured ac- cording to the manufacturer’s instructions.

2.6. Western blot assay

Western blot assays were used to detect the expression of pro- teins, and we have tissue and cell protein to test the level of pro- teins. For tissue protein extraction, tissues were separated and washed with cold PBS to exclude the influence of blood cells et al. Then tissue homogenate was obtained and sonicated to get cell lysates and tissue cell lysates were centrifuged at 12000 rpm for 15min in 4 ◦C and then boiled for 10min followed by the testing of protein concentration. For the collection of cell protein, cells were harvested and lysed for total protein extraction using SDS sample buffer (glycerol, 0.5MTris-HCL, 10%SDS, ddH2O). Protein concen- trations were measured using a BCA protein assay kit (KeyGEN Biotech, China) according to the manufacturer’s protocol. The protein expression was then detected by SDS-PAGE gels and then transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% defatted milk powder for 1 h at room tem- perature and incubated overnight with primary antibodies, b3-AR (1:1000), mTOR (1:1000), p-mTOR(1:1000), p70S6K(1:1000), p- p70S6K(1:1000), b-actin ((1:6000). Then the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody for 2 h at room temperature before detection.

2.7. EdU cell proliferation assay

For EdU assay, a density of 1 105 cells per well of SK-N-AS and SHEP cells were mounted onto coverslips in 6-well plates. After culturing for 12 h, cells were treated with SR59230A and epinephrine for another 24 h. Then cells were incubated with EdU reagents (KeyGEN Biotech, China) for 2 h, followed by subsequent staining according to the manufacturer’s instructions. The images were captured by fluorescence microscope Olympus BX63 (Japan).

2.8. siRNA transfection

We used siRNA transfection to knockdown the expression of b3- AR. The siRNA was synthesized from RiboBio (Guangzhou, china). For b3-AR silencing, SK-N-AS cells were seeded in 6-well plate at a density of 3 × 105 cells per well, and then transfected with 100 nM b3-AR and negative control siRNA using transfection reagent .RNAiMAX (Invitrogen) according to the manufacturer’s in- structions. After incubating for 48 h, the silence efficiency was verified by western blotting assay. Once the silence efficiency was verified, cells were subjected to the following experiments based on the above conditions.

2.9. Colony formation assay

For colony formation assay, SK-N-AS and SHEP cells were seeded in 6-well plates at a density of 1000 cells/well, and treated with different concentration of drugs. After incubating for 1 week, the colonies were stained with 0.1% crystal violet, and the images were captured with GS-800 software.

2.10. Statistical analysis

Student’s t-test and one – way ANOVA was used to assess the difference of two or more than three groups by GraphPad Prism5, and p-value<0.05 was considered as statistically significant.

3. Results

3.1. The expression of b3-AR is significantly increased in clinical NB tissues

To study the role of the b3-AR pathway in NB, we detected the expression of b3-AR in three subtypes of childhood peripheral neuroblastic tumors, namely, GN, GNB and NB. Of these three subtypes, GN and GNB are less malignant and thus served as control groups. These clinical tissues were assembled into a tissue micro- array for detection of b3-AR level. The IHC assay was applied to test the expression of b3-AR (Fig. 1A) and the b3-AR levels in GN, GNB and NB were shown in Fig.1B. We analyzed the differences in b3-AR expression among these tumor subtypes. The results indicated that the expression of b3-AR was significantly higher in the NB clinical tissues than in the GN and GNB tissues (Fig. 1C). Additionally, we extracted the protein from fresh clinical tumor tissues and further evaluated the expression of b3-AR in NB. Western blot results indicated that the level of b3-AR was higher in NB than in GNB (Fig. 1D). Our findings suggested that the b3-AR pathway is hyperactivated in NB and may play an important role in the tumorigenesis of NB. Since b3-AR is known to be expressed mainly in adipose tissue where it has a crucial role in mediating lipid metabolism and thermogenesis, we questioned why it would be so highly expressed in NB and sought to further examine its connec- tion with this disease.

3.2. Blocking b3-AR suppresses NB cell proliferation and colony formation

Based on the of IHC staining results in Fig. 1, we speculated that the activation of the b3-AR pathway is associated with NB cell growth. In order to study the role of b3-AR signaling in NB, we chose two NB cell lines that express higher levels of b3-AR (data not shown) as cell models. We stimulated these cells with the b-AR activation ligand epinephrine and the specific b3-AR antagonist SR59230A. The cell viability assay results revealed that the epinephrine treatment had improved NB cell viability, whereas b3- AR antagonist SR59230A inhibited cell growth in a dose-dependent manner (Fig. 2A; 2B). EdU assays were used to further verify the proliferation of NB cells under these treatment conditions. The results again indicated that epinephrine treatment promoted cell proliferation, whereas SR59230A suppressed cell proliferation (Figure.2C; 2D). The colony formation assay also demonstrated that SR59230A could inhibit NB cell colony formation (Fig. 2E). Taken together, these results demonstrated that blocking the b3-AR pathway could suppress the NB cell proliferation and colony for- mation abilities.

3.3. Knockdown of b3-AR expression inhibits NB cell proliferation

Besides using the specific b3-AR antagonist to treat NB cells, we performed a b3-AR siRNA knockdown assay to further verify the role of the b3-AR pathway in NB cell proliferation. The efficiency of the siRNA in knocking down b3-AR was first verified (Fig. 3A), where we tested three b3-AR siRNA fragments to choose the best one. The kinase pathway plays a crucial role in regulating cell growth, and mTOR signaling has an important role in modulating cell growth and proliferation. The deregulation of mTOR pathway is closely associated with a variety of tumors including solid and hematologic tumors. However, there are no reports about the crosstalk between the b3-AR and mTOR pathways in NB cells. The Western blot results indicated that knockdown of b3-AR inhibited the activation of mTOR/p70S6K signaling in NB cells (Fig. 3B). Then, we used the EdU assay to determine the effect of b3-AR siRNA knockdown on NB cell proliferation. As shown in Fig. 3C, silencing b3-AR expression inhibited the proliferation of NB cells, which suggested that the b3-AR pathway plays an important role in NB cell proliferation.

3.4. SR59230A inhibits mTOR pathway activation and activating mTOR signaling promotes NB cell proliferation

Besides using b3-AR siRNA knockdown assay, we also treated NB cells with SR59230A to determine the change of mTOR/p70S6K signaling. The results showed that SR59230A treatment suppressed the phosphorylation and thus the activation of mTOR/p70S6K signaling (Fig. 4A). Next, we used the mTOR activator MHY1485 to treat NB cells and tested the proliferation of NB cells. The results showed that MHY1485 promoted the proliferation of NB cells and reversed the inhibitory effect of SR59230A on NB cell proliferation (Fig. 4B). The results illustrated that inhibition of the b3-AR pathway suppressed NB cell proliferation via inactivating the mTOR signaling pathway.

4. Discussion

An increasing number of studies have reported that stress is closely related to tumorigenesis and tumor progression. Under stressful conditions, the secretion of catecholamine hormones such as adrenaline and norepinephrine are increased. These hormones bind with b-AR to activate downstream signaling pathways. Acti- vation of the b-AR can result in the modulation of tumor cell growth, proliferation and metastasis, where b2-AR has been the most widely studied in this regard [22,23]. However, of the three b- AR subtypes, b3-AR has not been well investigated in terms of its effect on NB. In our study, we demonstrated that the level of b3-AR was significantly increased in the clinical NB tissues compared with that in the less malignant tumors GN and GNB, suggesting that the b3-AR pathway is deregulated and over-activated in NB. Further cellular function assays revealed that blocking the b3-AR pathway inhibited NB cell proliferation and colony formation, indicating that b3-AR pathway inhibition suppressed NB cell growth in vitro. There are some limitations in our research. We performed the experi- ments using cell lines only and not in tumor-bearing mouse models; therefore, animal studies are needed to further investigate the role of the b3-AR pathway in NB. The b-AR pathway not only has a role in tumor growth, but is also associated with tumor metastasis. We analyzed the expression of b3-AR in different clin- ical stages of NB, and found the level to be upregulated in high- grade clinical stages (data not shown), which implies that the b3- AR pathway may regulate NB cell metastasis. Thus, the role of the b3-AR pathway in NB cell metastasis needs further investigation.

The mTOR signaling pathway has a very important role in modulating cell growth and proliferation, and is always dysregu- lated in different types of tumors. mTOR signaling is a vital thera- peutic target for many tumors [24]. It was reported that the dual mTORC1 and mTORC2 inhibitor AZD8055 was an effective reagent for inhibiting cancer cell growth [25]. Recent studies also found a critical role of the mTOR pathway in mediating adipose tissue function, including lipid metabolism, adipogenesis and thermogenesis, revealing that deregulated mTOR signaling is involved in obesity and related metabolic diseases [21]. In our study, we uncovered that the targeting of b3-AR inhibited NB cell growth by suppressing mTOR signaling, which clarifies a novel regulatory axis of b3-AR and mTOR in NB cells. In summary, we have demonstrated that the expression of b3-
AR is significantly increased in clinical NB tissues and blocking the b3-AR pathway inhibited NB cell growth. Further mechanistic study revealed that targeting b3-AR suppressed NB cell growth by repressing the mTOR/p70S6K signaling pathway. Thus, our study clarifies a novel regulatory axis of the b3-AR and mTOR/p70S6K signaling pathway in NB and provides a potent therapeutic target for the treatment of this devastating childhood tumor.

Conflicts of interest
No Conflict Of Interest.

Acknowledgements

We thank professor Bo Li for providing neuroblastoma cell lines to us. This study was funded by the National Nature Science Foundation of China, grant numbers: 81572342, 81770808.

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