Objective

Innate and acquired resistance is the principle factor limiting the efficacy of tyrosine kinase inhibitors in lung cancer. We have observed a dramatic upregulation of the cell surface co-receptor neuropilin-2b in lung cancers clinically treated with tyrosine kinase inhibitors correlating with acquired resistance. We hypothesize that neuropilin-2b plays a functional role in acquired tyrosine kinase inhibitor resistance.

Methods

Non–small cell lung cancer proliferation and survival were determined during chronic tyrosine kinase inhibitor exposure in the presence or absence of neuropilin-2b knock-down. Interactions of neuropilin-2a and neuropilin-2b isoforms with PTEN and GSK3β were assessed by immunoprecipitation. Neuropilin-2a and neuropilin-2b mutants deleted for their cytoplasmic domains were used to identify regions responsible for neuropilin-2b–GSK3β interaction. Because GSK3β is known to phosphorylate and degrade PTEN, phospho-PTEN and total PTEN levels were assessed after transfection of neuropilin-2a and neuropilin-2b wild-type and mutant constructs.

Results

Non–small cell lung cancer cells chronically treated with gefitinib or osimertinib developed drug resistance and exhibited logarithmic growth in the presence of endothelial growth factor receptor tyrosine kinase inhibitors. However, neuropilin-2b knockdown cells remained sensitive to gefitinib. Likewise, neuropilin-2b knockdown suppressed and neuropilin-2a knockdown enhanced cellular migration. Acquired drug resistance and cell migration correlated with neuropilin-2b-dependent AKT activation with the intermediate step of GSK3β-dependent PTEN degradation. A specific binding site for GSK3β on the cytoplasmic domain of neuropilin-2b was identified with truncated protein constructs and computer modeling.

Conclusions

Neuropilin-2b facilitates non–small cell lung cancer resistance to tyrosine kinase inhibitors, and this biological effect relates to AKT activation. Neuropilin-2b GSK3β interactions appear to be essential for PTEN degradation and AKT activation in lung cancer cells. Disruption of the neuropilin-2b GSK3β interaction may represent a novel treatment strategy to preserve sensitivity to tyrosine kinase inhibitors in non–small cell lung cancer.

中文翻译：

---

Neuropilin-2b 促进非小细胞肺癌对酪氨酸激酶抑制剂的耐药性

客观的

先天和获得性耐药是限制酪氨酸激酶抑制剂在肺癌中疗效的主要因素。我们观察到在临床上用与获得性耐药相关的酪氨酸激酶抑制剂治疗的肺癌中，细胞表面共受体神经纤维蛋白 2b 显着上调。我们假设neuropilin-2b 在获得性酪氨酸激酶抑制剂抗性中起功能性作用。

方法

在存在或不存在neuropilin-2b敲低的情况下，在慢性酪氨酸激酶抑制剂暴露期间确定非小细胞肺癌的增殖和存活率。通过免疫沉淀评估neuropilin-2a 和neuropilin-2b 亚型与PTEN 和GSK3β 的相互作用。Neuropilin-2a 和 neuropilin-2b 的细胞质结构域缺失突变体被用来识别负责 neuropilin-2b-GSK3β 相互作用的区域。因为已知 GSK3β 磷酸化和降解 PTEN，所以在转染神经纤维蛋白 2a 和神经纤维蛋白 2b 野生型和突变体构建体后评估磷酸化 PTEN 和总 PTEN 水平。

结果

用吉非替尼或奥希替尼长期治疗的非小细胞肺癌细胞产生耐药性，并在内皮生长因子受体酪氨酸激酶抑制剂存在下表现出对数增长。然而，neuropilin-2b 敲低细胞仍然对吉非替尼敏感。同样，neuropilin-2b 组合式抑制和neuropilin-2a 组合式增强细胞迁移。获得性耐药性和细胞迁移与依赖于 GSK3β 的 PTEN 降解的中间步骤的神经纤毛蛋白-2b 依赖性 AKT 活化相关。用截短的蛋白质构建体和计算机建模鉴定了神经纤毛蛋白-2b 细胞质结构域上 GSK3β 的特异性结合位点。

结论

Neuropilin-2b 促进非小细胞肺癌对酪氨酸激酶抑制剂的耐药性，这种生物学效应与 AKT 激活有关。Neuropilin-2b GSK3β 相互作用似乎对肺癌细胞中的 PTEN 降解和 AKT 激活至关重要。破坏neuropilin-2b GSK3β相互作用可能代表一种新的治疗策略，以保持对非小细胞肺癌对酪氨酸激酶抑制剂的敏感性。