Ho Ruth, Eggert Angelika, Hishiki Tomoro, Minturn Jane E, Ikegaki Naohiko, Foster Patricia, Camoratto Anna Marie, Evans Audrey E, Brodeur Garrett M
Division of Oncology, the Children's Hospital of Philadelphia and the University of Pennsylvania, Philadelphia, Pennsylvania 19104-4318, USA.
Cancer Res. 2002 Nov 15;62(22):6462-6.
Neuroblastoma is a common childhood tumor derived from the peripheral nervous system. Favorable neuroblastomas usually express TrkA, the receptor for nerve growth factor (NGF), whereas unfavorable, MYCN-amplified neuroblastomas usually express TrkB and its ligand, brain-derived neurotrophic factor (BDNF). Here, we provide evidence that the TrkB-BDNF pathway is associated with enhanced survival and resistance to chemotherapy in neuroblastoma. We transfected the neuroblastoma line SH-SY5Y, which has endogenous expression of BDNF, with a full-length TrkB expression vector, and obtained clones with moderate or high levels of expression. Cells were exposed in vitro to chemotherapy agents used to treat neuroblastomas: doxorubicin, etoposide (VP16), and cisplatin. Chemoresistance was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell survival and by ELISA for cell death. In all cases, the TrkB-expressing subclones were more resistant to treatment than the parent line. Furthermore, when the TrkB tyrosine kinase was blocked with the Trk-specific inhibitor CEP-2563, or by neutralizing antibody to BDNF, sensitivity to chemotherapy was significantly increased. We also found constitutive phosphorylation of AKT at the Ser-473 site in TrkB transfectants, whereas there was only a minimal level of constitutive phosphorylation of AKT in SY5Y cells. These results show that the TrkB-BDNF pathway provides a survival advantage when exposed to DNA-damaging reagents, and, therefore, this autocrine pathway may play an important role in mediating the drug-resistant phenotype associated with TrkB-expressing neuroblastomas. Activation of PI3K/AKT survival pathway may contribute to the increased drug resistance in TrkB-expressing neuroblastomas.
神经母细胞瘤是一种源自外周神经系统的常见儿童肿瘤。预后良好的神经母细胞瘤通常表达TrkA，即神经生长因子（NGF）的受体，而预后不良、MYCN扩增的神经母细胞瘤通常表达TrkB及其配体脑源性神经营养因子（BDNF）。在此，我们提供证据表明TrkB - BDNF信号通路与神经母细胞瘤的生存增强及化疗耐药相关。我们用全长TrkB表达载体转染内源性表达BDNF的神经母细胞瘤细胞系SH - SY5Y，并获得了中度或高水平表达的克隆。将细胞在体外暴露于用于治疗神经母细胞瘤的化疗药物：阿霉素、依托泊苷（VP16）和顺铂。通过3 -（4,5 - 二甲基噻唑 - 2 - 基）- 2,5 - 二苯基四氮唑溴盐（MTT）法检测细胞存活情况以测定化疗耐药性，通过ELISA检测细胞死亡情况。在所有情况下，表达TrkB的亚克隆比亲代细胞系对治疗更具耐药性。此外，当用Trk特异性抑制剂CEP - 2563或抗BDNF中和抗体阻断TrkB酪氨酸激酶时，化疗敏感性显著增加。我们还发现TrkB转染细胞中AKT在Ser - 473位点存在组成型磷酸化，而在SY5Y细胞中AKT的组成型磷酸化水平极低。这些结果表明，TrkB - BDNF信号通路在暴露于DNA损伤试剂时提供了生存优势，因此，这种自分泌通路可能在介导与表达TrkB的神经母细胞瘤相关的耐药表型中起重要作用。PI3K / AKT生存信号通路的激活可能有助于表达TrkB的神经母细胞瘤耐药性增加。
