Effective treatment options for patients with advanced thyroid cancer remain limited, underscoring the urgent need for novel therapeutic strategies. One promising approach is to target the reprogrammed metabolism that supports oncogenic transformation and rapid tumor growth. In particular, cancer cells frequently rely on glutamine for their bioenergetic and biosynthetic needs. This study investigated whether interfering with glutamine metabolism could serve as a viable therapeutic strategy for thyroid cancer.

To explore this possibility, researchers evaluated the expression of key enzymes involved in glutamine metabolism, namely glutaminase (GLS) and glutamate dehydrogenase (GDH), in thyroid cancer tissues using immunohistochemistry. In parallel, the expression of glutamine metabolism-related genes was assessed by real-time qPCR and western blotting. The functional impact of inhibiting glutamine metabolism was examined using 6-diazo-5-oxo-L-norleucine (DON) in thyroid cancer cells, with effects on cell proliferation measured by CCK-8, colony formation, and Edu incorporation assays. Additional analyses, including flow cytometry and Transwell assays, evaluated cell cycle progression and the migratory and invasive capabilities of the cancer cells. Mechanistic insights were gained through further real-time qPCR, western blotting, Seahorse metabolic assays, and gas chromatography-mass spectrometry. Finally, the therapeutic potential of a DON prodrug, JHU-083, was assessed in vivo using xenograft tumor models in BALB/c nude mice.

The results revealed that both GLS and GDH were overexpressed in thyroid cancer tissues, with GLS expression correlating positively with lymph-node metastasis and advanced TNM stage. Thyroid cancer cells demonstrated a marked dependence on glutamine, as evidenced by significant growth inhibition when cultured in glutamine-free medium. Treatment with DON inhibited cell proliferation without promoting apoptosis; instead, it increased the proportion of cells in the S phase of the cell cycle, accompanied by a decrease in cyclin-dependent kinase 2 and cyclin A expression. Moreover, DON treatment significantly impaired the migration and invasion of thyroid cancer cells, which was associated with reduced expression of key proteins such as N-cadherin, Vimentin, matrix metalloproteinase-2, and matrix metalloproteinase-9. DON also led to reduced levels of non-essential amino acids—including proline, alanine, aspartate, asparagine, and glycine—providing a potential explanation for the observed decrease in proteins that support migration, invasion, and cell cycle progression. In vivo, the DON prodrug JHU-083 not only suppressed tumor proliferation and metastatic potential but also enhanced the innate immune response in xenograft tumors, as reflected by decreased expression of the immune checkpoint molecules CD47 and programmed cell death ligand 1 (PD-L1).

In conclusion, thyroid cancer cells exhibit enhanced glutamine metabolism, relying on the high expression of multiple glutamine metabolism-related genes for growth and survival. Targeting this metabolic dependency with DON, particularly through the use of its prodrug JHU-083, emerges as a promising therapeutic option for advanced thyroid cancer. These findings pave the way for further clinical investigations to assess the efficacy of glutamine metabolism inhibitors in overcoming treatment resistance and improving patient outcomes.