Cross-talk between oncogene-driven signaling pathways and thyroid cancer metabolism

目的：乐伐替尼是FDA批准的酪氨酸激酶抑制剂（TKI），用于治疗转移性放射性碘（RAI）无反应的进行性甲状腺癌（TC）。乐伐替尼的客观缓解率为65%，是不持久的，大多数患者对乐伐替尼治疗产生耐药性。鉴于RAI无反应性甲状腺癌的临床现象已得到证实，其特征是氟脱氧葡萄糖正电子发射断层扫描成像证实的葡萄糖摄取较高，我们假设葡萄糖代谢可能在TKI耐药中发挥作用。 研究方法：我们使用了两种人Lenvatinib敏感（LS）TC细胞系THJ 29 T和TPC 1，并通过连续暴露于浓度增加的药物诱导Lenvatinib耐药性（LR）。阻力系数为2（这是什么，定义？）在LR细胞中获得。通过流式细胞仪定量细胞周期和凋亡率的分析来评估LR细胞的表型。进行免疫印迹以分析线粒体呼吸链组分的表达。通过Seahorse XF Cell Mito Stress Kit测量有氧糖酵解，该试剂盒定量富含葡萄糖条件下的线粒体氧化磷酸化（OXPHOS）。认为p值0.05具有统计学显著性。 结果如下： LR TC细胞的特征在于与LS细胞相似的细胞周期模式或细胞周期的G2-M期增强，揭示了促进有丝分裂和增殖的模式。LR细胞的早期凋亡率低于LS细胞（THJ 29 T：0.41 ± 0.03 vs 0.8 ± 0.1，p=0.001，TPC 1：0.40 ± 0.02 vs 0.81 ± 0.12，p=0.003）。与LS-TC相比，LR细胞中线粒体呼吸标记物：复合物I NDUFB 8、复合物II SDHB、复合物III UQCRC 2、复合物IV考克斯II和复合物V ATP 5A的蛋白表达显著上调。与LS细胞相比，LR细胞的特征在于显著增加的最大线粒体耗氧速率（THJ 29 T-LR 4013 306 vs THJ 29 T-LS 2293 334，p<0.001; TPC 1-LR 3999 358 vs TPC 1-LS 1774 208，p<0.0001）。 结论：线粒体呼吸增强是导致乐伐替尼耐药的因素之一。应将包括OXPHOS抑制剂在内的联合治疗作为克服代谢抗性机制的潜在途径进行测试。

Objective: Lenvatinib is an FDA-approved tyrosine kinase inhibitor (TKI) used for the treatment of metastatic radioactive iodine (RAI) non-responsive progressive thyroid cancer (TC). The objective response rate to Lenvatinib of 65% is non-durable, and most patients develop resistance to therapy with Lenvatinib. Given a well-established clinical phenomenon of RAI-non-responsive thyroid cancer being characterized by a higher glucose uptake evidenced by fluorodeoxyglucose positron emission tomography imaging, we hypothesized that glucose metabolism might play a role in resistance to TKIs. Methods: We used two human Lenvatinib sensitive (LS) TC cell lines THJ29T and TPC1 and induced Lenvatinib resistance (LR) through continuous exposure to increasing concentrations of the drug. A resistance factor of 2 (what is this, definition?) was attained in the LR cells. The LR cells phenotype was assessed through analysis of the cell cycle and apoptosis rate, that were quantified with flow cytometry. Immunoblot was performed to analyze the expression of the components of the mitochondrial respiratory chain. Aerobic glycolysis was measured via Seahorse XF Cell Mito Stress Kit, that quantifies mitochondrial oxidative phosphorylation (OXPHOS) in glucose-rich conditions. A p-value of 0.05 was considered statistically significant. Results: LR TC cells are characterized by either a similar cell cycle pattern or enhanced G2-M phase of cell cycle as compared with LS cells, revealing a pattern promoting mitosis and proliferation. There was a lower early apoptosis rate in LR vs LS cells (THJ29T: 0.41 0.03 vs 0.8 0.1, p=0.001, TPC1: 0.40 0.02 vs 0.81 0.12, p=0.003). There was a significant upregulation of the protein expression of mitochondrial respiration markers: complex I NDUFB8, complex II SDHB, complex III UQCRC2, complex IV COX II, and complex V ATP5A in LR cells as compared with LS-TC. Consistently, LR cells were characterized by a significantly increased maximal mitochondrial oxygen consumption rate as compared with LS cells (THJ29T-LR 4013 306 vs THJ29T-LS 2293 334, p<0.001; TPC1-LR 3999 358 vs TPC1-LS 1774 208, p<0.0001). Conclusions: Enhanced mitochondrial respiration is one of the contributing factors towards resistance to Lenvatinib. Combination therapies including OXPHOS inhibitors should be tested as potential avenues to overcome metabolic resistance mechanisms.