Introduction
Neuroblastoma (NB) is one of the most common pediatric malignancies that derives from neural crest precursor cells. It is an extracranial solid tumor that most frequently occurs in adrenal gland [
1‐
2]. Tumor spontaneous regression or differentiation happens in patients with low-risk disease, and surgery with little or no adjunctive therapy is effective for these patients [
2‐
3]. While for patients with high-risk diseases, chemo-resistance and metastasis are the two main problems. Patients may initially response to chemotherapy, but chemo-resistance would develop soon. Wide metastasis usually occurs at diagnosis. Despite of multimodality chemotherapy and stem cell transplantation, satisfactory response still could not be achieved. The long-term survival rate of these patients is less than 40 % [
2‐
5]. So, more effective treatments are urgently needed.
Brain-derived neurotrophic factor (BDNF) is a member of neurotrophin family, and it is known as one of key factors for the sympathetic nervous system development [
2‐
3]. Previous reports show that patients with an unfavorable prognosis are more likely to express BDNF and its tyrosine kinase receptor TrkB [
6‐
8]. The association between BDNF/TrkB expression and chemo-resistance in NB has been studied. It was found that BDNF activation of TrkB induced chemo-resistance in NB, and that was mainly mediated by PI3K/Akt signaling pathway. Treatment targeting the PI3K/Akt pathway attenuated the BDNF/TrkB-induced chemo-resistance [
9‐
14]. Although TrkB expression with invasive capability in NB has been reported [
15‐
16], a systemic study of the role of BDNF/TrkB in the NB metastasis is still needed. In this study, we provide evidence supporting the hypothesis that BDNF/TrkB promotes the NB cell metastasis both in vitro and in vivo. We find that the metastasis potential stimulated by activation of the BDNF/TrkB pathway in NB cells is blocked by inhibiting activation of PI3K and MAPK pathways. Furthermore, we find that downstream targets of BDNF/TrkB-stimulated migration and invasion of NB cells may be Akt and mTOR. All of these results suggest that BDNF/TrkB and its downstream targets may be the new targets in the treatment of NB metastasis.
Discussion
In the present study, we indentified that BDNF/TrkB increased TB3 cell metastasis in vitro and in vivo. Inhibitors of PI3K pathway, MAPK pathway, Akt, and mTOR blocked the BDNF/TrkB-increased cell migration and invasion in vitro.
BDNF is needed for the development and maintenance of peripheral sympathetic and neural crest-derived sensory neurons [
17‐
19]. Binding of BDNF to its tyrosine kinase receptor TrkB activates PI3K, MAPK, and PLC-γ pathways [
9,
17‐
18]. BDNF/TrkB has been reported to be highly expressed in many malignancies and associated with poor prognosis [
20‐
24]. In NB, BDNF/TrkB expressions were found in tumors of patients with unfavorable prognosis [
6], and BDNF/TrkB promoted NB cell survival and proliferation, and induced resistance to chemo-therapy in vitro and in vivo [
25‐
27]. Our previous studies have indicated that BDNF/TrkB induced chemo-resistance via PI3K/Akt pathway and MAPK pathway [
9‐
11]; we and others have reported that inhibition of TrkB enhanced chemotherapeutic efficacy in NB in vitro and in vivo [
13,
28]. Studies also found that BDNF/TrkB increased metastasis in many malignancies, such as pancreatic ductal carcinoma [
29], prostate cancer [
30], lung cancer [
31], hepatocellular carcinoma [
21,
32], colorectal cancer [
33‐
34], gastric cancer [
35], choriocarcinoma [
36], ovarian cancer [
37], and multiple myeloma [
38]. Some of these reports showed that blocking TrkB or BDNF suppressed the BDNF/TrkB-induced metastasis [
21,
34,
36]. Studies have shown that BDNF/TrkB mediated NB cell invasiveness, and Douma S. and Geiger TR. et al. studied the mechanisms of BDNF/TrkB-induced cell invasiveness [
15‐
16,
39‐
40]. Douma S. et al. reported that TrkB increased rat intestinal epithelial (RIE) cell aggressiveness via suppression of anoikis (apoptosis resulting from loss of cell-matrix interactions), and their results showed that TrkB activated PI3K/PKB signaling, which contributed to anoikis resistance [
39]. Geiger TR et al. showed that TrkB increased NB cell aggressiveness via suppression of anoikis; further, they identified that kinase activity, but not adhesion domains, was required for TrkB-induced anoikis suppression in vitro and tumor metastatic capacity in vivo [
40]. But further mechanisms have not been shown in their reports. Hecht M. et al. focused their studies on HGF (hepatocyte growth factor) and its receptor c-Met, and their results showed that TrkB up-regulated HGF and c-Met expression to promote NB cell invasive capability [
15]. Cimmino F. et al. studied the role of galectin-1 in TrkB-mediated invasiveness in NB cells. They found that activation of TrkB up-regulated galectin-1 expression, and knockdown of galectin-1 mRNA expression or use galectin-1 inhibitor reduced the migratory and invasive capacity of NB cells [
16]. In our present study, we explored the role of BDNF/TrkB in NB cell metastasis in vitro and in vivo and further studied the pathways that mediated BDNF/TrkB effect on NB cell migration and invasion.
Our results showed that BDNF activation of TrkB increased the TB3 cell migration and invasion in vitro, and metastasis was significantly increased in TrkB-expressing TB3 tumors compared to that in non-TrkB-expressing TB3 tumors in vivo. These results were similar to Mineyoshi Aoyama and others’ reports in which BDNF/TrkB promoted NB cell invasive activity [
6,
15‐
16,
40]. Although there are studies that focus on the mechanisms of BDNF/TrkB-mediated NB cell metastasis [
15‐
16,
40], the signal transduction pathways that mediated the invasive activity were not fully investigated. Douma S. et al. have reported that TrkB activated PI3K/PKB signaling, which contributed to anoikis resistance in RIE cells [
39]. In our present study, we found that suppression of PI3K and MAPK pathways inhibited BDNF/TrkB-increased migration or invasion in TB3 cells, and blocked BDNF/TrkB-induced increase of P-Akt(Ser473) and P-Erk(Thr202/Tyr204) (Fig.
3a–d). Furthermore, inhibitors for Akt and mTOR, two downstream targets in PI3K pathway [
26], could also block BDNF/TrkB-increased cell migration and invasion, and blocked BDNF/TrkB-induced increase of P-Akt(Ser473) and P-mTOR(Ser2481) (Fig.
4a–d). Our results showed that inhibitor of mTOR (rapamycin) suppressed the BDNF/TrkB-increased migration and invasion in TB3 cells. This result is differed from Douma S.’s study in which rapamycin did not contribute to TrkB-mediated anoikis resistance in RIE cells [
39]. These may suggest that although mTOR is not involved in TrkB-mediated anoikis resistance in RIE cells, it may involve in other key processes during NB cell migration and invasion. Further studies are needed to explain this.
Epithelial mesenchymal transition (EMT) is the conversion of epithelial cells to mesenchymal cells, which is considered to be associated with an increase of cell migration and invasion capacity, as well as resistance to anoikis/apoptosis. EMT was reported to be accompanied by an increase of N-cadherin and Slug, or by a decrease of E-cadherin [
41]. In our present study, we investigated the expressions of N-cadherin, E-cadherin, and Slug under the BDNF treatment. We found that the BDNF treatment of TrkB-expressing TB3 cells increased E-cadherin expression, decreased Slug expression, but had no effect on N-cadherin expression within the time points we tested (Suppl. Fig.
1). Inhibitors for PI3K, MAPK, Akt, and mTOR could individually attenuate the BDNF/TrkB-induced increase of E-cadherin or decrease of Slug. These inhibitors had no effect on N-cadherin expression (Suppl. Fig.
2). As our results are different from some reports [
41], so further studies are needed to investigate whether or not E-cadherin and Slug are involved in the BDNF/TrkB-induced migration or invasion, and if they are, how do they affect the migration and invasion? Recent reports show that EMT is not required for metastasis in breast cancer and pancreatic cancer [
42,
43], so the role of EMT during metastasis in different types of cancers may vary.
Our study provided evidence that BDNF/TrkB increased migration and invasion of TB3 cells through PI3K/Akt and MAPK pathways; together with previous studies that BDNF/TrkB mediated chemo-resistance through PI3K/Akt pathway in the treatment of NB patients [
9‐
14], we believe that BDNF/TrkB and its downstream targets will play very important roles in the treatment of high-risk NB patients, who are suffering from metastasis or/and chemo-resistance. Also, there are some limitations in our present study, such as no validation of the results in clinical tumor samples, only one cell line used, and no more inhibitors targeting BDNF/TrkB signaling pathways. In the future, we will try to finish these studies to fully elucidate the effect of BDNF/TrkB on NB metastasis.
Above all, our study showed that BDNF/TrkB increased TB3 cell migration and invasion through PI3K/Akt/mTOR and MAPK pathways. Based on these findings, we may provide new potential molecular targets for the treatment of NB metastasis.