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

目的：Lenvatinib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)无反应性进展性甲状腺癌(TC)。对Lenvatinib的客观有效率为65%，是非持久的，大多数患者对Lenvatinib治疗产生抵抗力。鉴于RAI无反应性甲状腺癌的临床特征是氟脱氧葡萄糖正电子发射断层成像证实的高葡萄糖摄取率，我们推测葡萄糖代谢可能在TKI抵抗中起作用。 方法：采用两株对Lenvatinib敏感的人TC细胞系THJ29T和TPC1，通过不断增加药物浓度诱导其耐药。阻力系数为2(这是什么，定义？)在LR细胞中也有表达。通过分析细胞周期和凋亡率来评价LR细胞的表型，并用流式细胞仪对其进行定量。免疫印迹法检测线粒体呼吸链各成分的表达。有氧糖酵解通过SeaHorse XF Cell Mito Stress Kit进行测量，该试剂盒在富含葡萄糖的条件下定量测定线粒体氧化磷酸化(OXPHOS)。P值为0.05被认为具有统计学意义。 结果： 与LS细胞相比，LR TC细胞具有相似的细胞周期模式或增强的G2-M期，表现出促进有丝分裂和增殖的模式。LR细胞较LS细胞早期凋亡率低(THJ29T：0.410.03vs0.80.1，p=0.001；TPC1：0.400.02vs0.810.12，p=0.003)。与LS-TC相比，LR细胞线粒体呼吸标志物：复合体I NDUFB8、复合体II SDHB、复合体III UQCRC2、复合体IV COX II和复合体V ATP5A的蛋白表达显著上调。与LS细胞相比，LR细胞的线粒体最大耗氧率显著增加(THJ29T-LR4013306vsTHJ29T-LS2293334，p&lt；0.001；TPC1-LR3999358 vsTPC1-LS1774208，p&lt；0.0001)。 结论：线粒体呼吸增强是导致列瓦替尼耐药的原因之一。包括OXPHOS抑制剂在内的联合疗法应作为克服代谢耐药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.