Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, 67100 L'Aquila, Italy.
Int J Mol Sci. 2022 Sep 17;23(18):10895. doi: 10.3390/ijms231810895.
Patients with advanced neuroblastoma (NB) receive multimodal clinical therapy, including the potent anthracycline chemotherapy drug doxorubicin (Dox). The acquisition of Dox resistance, however, is a major barrier to a sustained response and leads to a poor prognosis in advanced disease states, reinforcing the need to identify and inhibit Dox resistance mechanisms. In this context, we report on the identification and inhibition of a novel Dox resistance mechanism. This mechanism is characterized by the Dox-induced activation of the oncogenic TrkAIII alternative splice variant, resulting in increased Dox resistance, and is blocked by lestaurtinib, entrectinib, and crizotinib tyrosine kinase and LY294002 IP3-K inhibitors. Using time lapse live cell imaging, conventional and co-immunoprecipitation Western blots, RT-PCR, and inhibitor studies, we report that the Dox-induced TrkAIII activation correlates with proliferation inhibition and is CDK1- and Ca-uniporter-independent. It is mediated by ryanodine receptors; involves Ca-dependent interactions between TrkAIII, calmodulin and Hsp90; requires oxygen and oxidation; occurs within assembled ERGICs; and does not occur with fully spliced TrkA. The inhibitory effects of lestaurtinib, entrectinib, crizotinib, and LY294002 on the Dox-induced TrkAIII and Akt phosphorylation and resistance confirm roles for TrkAIII and IP3-K consistent with Dox-induced, TrkAIII-mediated pro-survival IP3K/Akt signaling. This mechanism has the potential to select resistant dormant TrkAIII-expressing NB cells, supporting the use of Trk inhibitors during Dox therapy in TrkAIII-expressing NBs.
患有晚期神经母细胞瘤 (NB) 的患者接受多模式临床治疗，包括强效蒽环类化疗药物阿霉素 (Dox)。然而，获得 Dox 耐药性是持续反应的主要障碍，并导致晚期疾病状态预后不良，这就需要确定和抑制 Dox 耐药机制。在这种情况下，我们报告了一种新的 Dox 耐药机制的鉴定和抑制。这种机制的特征是 Dox 诱导的致癌 TrkAIII 选择性剪接变体的激活，导致 Dox 耐药性增加，并且可以被 lestaurtinib、entrectinib 和 crizotinib 酪氨酸激酶和 LY294002 IP3-K 抑制剂阻断。通过延时活细胞成像、常规和共免疫沉淀 Western blot、RT-PCR 和抑制剂研究，我们报告说 Dox 诱导的 TrkAIII 激活与增殖抑制相关，并且与 CDK1 和钙通道无关。它是由肌醇 1,4,5-三磷酸受体介导的；涉及 Dox 诱导的 TrkAIII、钙调蛋白和 HSP90 之间的 Ca 依赖性相互作用；需要氧气和氧化；发生在组装的 ERGICs 内；并且不会与完全剪接的 TrkA 一起发生。lestaurtinib、entrectinib、crizotinib 和 LY294002 对 Dox 诱导的 TrkAIII 和 Akt 磷酸化和耐药性的抑制作用证实了 TrkAIII 和 IP3-K 的作用，与 Dox 诱导的 TrkAIII 介导的生存 IP3K/Akt 信号一致。这种机制有可能选择耐药的休眠 TrkAIII 表达 NB 细胞，支持在 TrkAIII 表达的 NB 中在 Dox 治疗期间使用 Trk 抑制剂。
