Aveic Sanja, Pantile Marcella, Seydel Anke, Esposito Maria Rosaria, Zanon Carlo, Li Gary, Tonini Gian Paolo
Neuroblastoma Laboratory, Pediatric Research Institute, Città della Speranza, Padua, Italy.
Ignyta Inc., San Diego, California, USA.
Oncotarget. 2016 Feb 2;7(5):5646-63. doi: 10.18632/oncotarget.6778.
Neuroblastoma (NB) is a threatening childhood malignancy. Its prognosis is affected by several morphological, and biological characteristics, including the constitutive expression of ALK tyrosine kinase. In this study we examined the therapeutic potential of a novel ALK inhibitor, entrectinib, in obliterating NB tumor cells. Entrectinib showed the growth-inhibitory effects on NB cells with a 50% inhibitory concentration range of 0.03-5 μM. In the ALK-dependent cells, entrectinib mediated G1-arrest, which was associated with modified expression of multiple cell-cycle regulators. Down-regulation of Ki-67, and attenuated phosphorylation of ERK1/2, and STAT3, correlated with observed antiproliferative capacity of entrectinib. Initial cytostatic activity of entrectinib was followed by concentration-dependent apoptotic cell death, and Caspase-3 activation. However, we delineated a reduced sensitivity of ALK mutated NB cells to entrectinib, and demonstrated strong activation of autophagy in SH-SY5YF1174L NB cell line. Abrogation of autophagy by chloroquine increased significantly the toxicity of entrectinib, as confirmed by enhanced death rate, and PARP protein cleavage in SH-SY5YF1174L cells. In aggregate, our data show that entrectinib inhibits proliferation, and induces G1-arrest, and apoptosis in NB cells. We propose entrectinib for further consideration in treatment of NB, and recommend pharmacological inhibition of autophagy to be explored for a combined therapeutic approach in NB patients that might develop resistance to entrectinib.
神经母细胞瘤（NB）是一种威胁儿童生命的恶性肿瘤。其预后受多种形态学和生物学特征影响，包括ALK酪氨酸激酶的组成性表达。在本研究中，我们检测了新型ALK抑制剂恩曲替尼在清除NB肿瘤细胞方面的治疗潜力。恩曲替尼对NB细胞显示出生长抑制作用，其50%抑制浓度范围为0.03 - 5 μM。在ALK依赖的细胞中，恩曲替尼介导G1期阻滞，这与多种细胞周期调节因子的表达改变有关。Ki-67的下调以及ERK1/2和STAT3磷酸化的减弱，与恩曲替尼观察到的抗增殖能力相关。恩曲替尼最初的细胞生长抑制活性之后是浓度依赖性的凋亡细胞死亡和半胱天冬酶-3激活。然而，我们发现ALK突变的NB细胞对恩曲替尼的敏感性降低，并在SH-SY5YF1174L NB细胞系中证明了自噬的强烈激活。氯喹对自噬的消除显著增加了恩曲替尼的毒性，这在SH-SY5YF1174L细胞中死亡率增加和PARP蛋白裂解得到证实。总体而言，我们的数据表明恩曲替尼抑制NB细胞增殖，诱导G1期阻滞和凋亡。我们建议进一步考虑将恩曲替尼用于NB的治疗，并推荐探索对自噬的药理学抑制，以用于可能对恩曲替尼产生耐药性的NB患者的联合治疗方法。
