Background

Effective treatment for patients with advanced thyroid cancer is lacking. Metabolism reprogramming is required for cancer to undergo oncogenic transformation and rapid tumorigenic growth. Glutamine is frequently used by cancer cells for active bioenergetic and biosynthetic needs. This study aims to investigate whether targeting glutamine metabolism is a promising therapeutic strategy for thyroid cancer.

Methods

The expression of glutaminase (GLS) and glutamate dehydrogenase (GDH) in thyroid cancer tissues was evaluated by immunohistochemistry, and glutamine metabolism-related genes were assessed using real time-qPCR and western blotting. The effects of glutamine metabolism inhibitor 6-diazo-5-oxo-l-norleucine (DON) on thyroid cancer cells were determined by CCK-8, clone formation assay, Edu incorporation assay, flow cytometry, and Transwell assay. The mechanistic study was performed by real time-qPCR, western blotting, Seahorse assay, and gas chromatography–mass spectrometer assay. The effect of DON prodrug (JHU-083) on thyroid cancer in vivo was assessed using xenograft tumor models in BALB/c nude mice.

Results

GLS and GDH were over-expressed in thyroid cancer tissues, and GLS expression was positively associated with lymph-node metastasis and TNM stage. The growth of thyroid cancer cells was significantly inhibited when cultured in glutamine-free medium. Targeting glutamine metabolism with DON inhibited the proliferation of thyroid cancer cells. DON treatment did not promote apoptosis, but increased the proportion of cells in the S phase, accompanied by the decreased expression of cyclin-dependent kinase 2 and cyclin A. DON treatment also significantly inhibited the migration and invasion of thyroid cancer cells by reducing the expression of N-cadherin, Vimentin, matrix metalloproteinase-2, and matrix metalloproteinase-9. Non-essential amino acids, including proline, alanine, aspartate, asparagine, and glycine, were reduced in thyroid cancer cells treated with DON, which could explain the decrease of proteins involved in migration, invasion, and cell cycle. The efficacy and safety of DON prodrug (JHU-083) for thyroid cancer treatment were verified in a mouse model. In addition to suppressing the proliferation and metastasis potential of thyroid cancer in vivo, enhanced innate immune response was also observed in JHU-083-treated xenograft tumors as a result of decreased expression of cluster of differentiation 47 and programmed cell death ligand 1.

Conclusions

Thyroid cancer exhibited enhanced glutamine metabolism, as evidenced by the glutamine dependence of thyroid cancer cells and high expression of multiple glutamine metabolism-related genes. Targeting glutamine metabolism with DON prodrug could be a promising therapeutic option for advanced thyroid cancer.

中文翻译：

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靶向谷氨酰胺代谢在甲状腺癌中表现出抗肿瘤作用

背景

晚期甲状腺癌患者缺乏有效的治疗方法。癌症发生致癌转化和快速致瘤生长需要代谢重编程。癌细胞经常利用谷氨酰胺来满足活跃的生物能量和生物合成需求。本研究旨在调查靶向谷氨酰胺代谢是否是甲状腺癌的一种有前景的治疗策略。

方法

通过免疫组织化学评估甲状腺癌组织中谷氨酰胺酶（GLS）和谷氨酸脱氢酶（GDH）的表达，并使用实时qPCR和蛋白质印迹评估谷氨酰胺代谢相关基因。通过CCK-8、克隆形成实验、Edu掺入实验、流式细胞术和Transwell实验测定谷氨酰胺代谢抑制剂6-重氮-5-氧代-l-正亮氨酸（DON）对甲状腺癌细胞的影响。通过实时 qPCR、蛋白质印迹、海马分析和气相色谱-质谱分析进行了机理研究。使用 BALB/c 裸鼠的异种移植肿瘤模型评估了 DON 前药 (JHU-083) 对体内甲状腺癌的作用。

结果

GLS和GDH在甲状腺癌组织中过表达，且GLS表达与淋巴结转移和TNM分期呈正相关。在无谷氨酰胺培养基中培养时，甲状腺癌细胞的生长受到显着抑制。用 DON 靶向谷氨酰胺代谢可抑制甲状腺癌细胞的增殖。DON处理并不促进细胞凋亡，但S期细胞比例增加，并伴有细胞周期蛋白依赖性激酶2和细胞周期蛋白A表达下降。DON处理还通过降低细胞周期蛋白依赖性激酶2和细胞周期蛋白A的表达，显着抑制甲状腺癌细胞的迁移和侵袭。 N-钙粘蛋白、波形蛋白、基质金属蛋白酶-2 和基质金属蛋白酶-9。在用 DON 处理的甲状腺癌细胞中，非必需氨基酸（包括脯氨酸、丙氨酸、天冬氨酸、天冬酰胺和甘氨酸）减少，这可以解释参与迁移、侵袭和细胞周期的蛋白质的减少。DON 前药 (JHU-083) 用于治疗甲状腺癌的有效性和安全性在小鼠模型中得到验证。除了在体内抑制甲状腺癌的增殖和转移潜力之外，在 JHU-083 处理的异种移植肿瘤中还观察到由于分化簇 47 和程序性细胞死亡配体 1 的表达降低而增强的先天免疫反应。

结论

甲状腺癌表现出谷氨酰胺代谢增强，甲状腺癌细胞的谷氨酰胺依赖性和多个谷氨酰胺代谢相关基因的高表达证明了这一点。使用 DON 前药靶向谷氨酰胺代谢可能是晚期甲状腺癌的一种有前景的治疗选择。