Background: Arachidonate 5-lipoxygenase (ALOX5), a key enzyme in lipid metabolism and inflammation, has been linked to the progression of various cancer types. However, its specific function in thyroid cancer and the tumor immune microenvironment remains unclear.

Methods: ALOX5 expression in thyroid cancer tissues and cell lines was analyzed using The Cancer Genome Atlas (TCGA) and GTEx databases, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blotting. A series of cellular assays—namely cell counting kit-8 (CCK-8), 5-Ethynyl-2′-Deoxyuridine (EdU) incorporation, colony formation, wound closure, and Transwell invasion tests—were conducted to assess the impact of ALOX5 knockdown on cell proliferation, migration, and invasion. The expression levels of cytokines (C-C motif chemokine ligand 2 (CCL2) and colony-stimulating factor 1 (CSF1)) and M2 macrophage surface markers (Cluster of Differentiation 163 (CD163) and Cluster of Differentiation 206 (CD206)) were evaluated using RT-qPCR and Western blot analyses in both conditioned medium and co-culture models. Flow cytometry was performed to quantify the expression of CD163 and CD206 in THP-1–derived macrophages following exposure to conditioned medium from thyroid cancer cells or direct co-culture. Furthermore, expression levels of proteins associated with the Notch signaling pathway were evaluated, and rescue experiments with Jagged canonical Notch ligand 1 (Jagged1) overexpression were conducted to validate the specific role of this pathway.

Results: ALOX5 was significantly upregulated in thyroid cancer tissues and cell lines (p < 0.001). Silencing ALOX5 suppressed cell proliferation, migration, and invasion. Additionally, ALOX5 knockdown reduced CCL2 and CSF1 expression and inhibited M2 macrophage polarization. Mechanistically, ALOX5 positively regulated the Jagged1-Notch signaling pathway, as evidenced by decreased expression of Jagged1, Notch intracellular domain (NICD), Hairy/enhancer-of-split related with YRPW motif protein 1 (HEY1), and Hairy and enhancer of split-1 (HES1) upon ALOX5 silencing. Additionally, overexpression of Jagged1 reversed the inhibitory effects of ALOX5 knockdown on tumor cell behavior and the production of immunosuppressive cytokines.

Conclusion: ALOX5 promotes thyroid cancer progression and the polarization of immunosuppressive tumor-associated macrophage polarization through activation of the Jagged1-Notch signaling cascade. Inhibiting ALOX5 could represent a promising therapeutic avenue for managing thyroid cancer by concurrently restraining tumor progression and reshaping the immune landscape of the tumor microenvironment.