Decoding BTN3A3: A potential biomarker and therapeutic target to promote glycolytic metabolism reprogramming and nasopharyngeal carcinoma progression.

Abstract

e15193 Background: Butyrophilin subfamily 3 member A3 (BTN3A3) is expressed in various tumors, where it modulates glycolysis, proliferation, invasion, and migration to influence progression. However, its molecular mechanisms in nasopharyngeal carcinoma (NPC) remain unclear. This study aims to elucidate BTN3A3's role in NPC progression, identify biomarkers for invasion/metastasis risk stratification, and provide foundations for novel therapeutic targets. Methods: Immunohistochemistry (IHC) profiled BTN3A3 expression in nasopharyngeal tissues and correlated it with clinicopathological parameters and prognosis. BTN3A3 was overexpressed or silenced in NPC cell lines. Proliferation and tumorigenicity were assessed via CCK-8, colony-formation, and subcutaneous xenograft assays. Cell motility was quantified by wound-healing and transwell assays. RNA-sequencing and KEGG pathway enrichment were used to identify BTN3A3-regulated pathways. Western blotting evaluated its impact on glycolytic proteins. Integrated analysis of mass-spectrometry interactome data and the GeneCards glycolysis gene set identified PKM2 as a candidate binding partner. The BTN3A3–PKM2 interaction was predicted by molecular docking and validated by co-immunoprecipitation and immunofluorescence co-localization. Functional rescue experiments were conducted using PKM2 knockdown. Results: BTN3A3 mRNA and protein levels were significantly elevated in NPC versus chronic inflammation tissues (P < 0.001), and further increased in metastatic primary tumors (P = 0.003). Multivariate Cox analysis identified BTN3A3 as an independent predictor of poorer overall and progression-free survival. KEGG enrichment of RNA-seq data identified glycolysis as the top pathway regulated by BTN3A3, and western blotting confirmed BTN3A3 manipulation altered key glycolytic enzymes (GLUT1, HK2, LDHA; P < 0.01). Integrated interactome analysis nominated PKM2 as a candidate partner, and molecular docking predicted a high-affinity BTN3A3–PKM2 interaction, which was validated by co-immunoprecipitation and co-localization. BTN3A3 increased total and phospho-PKM2-Ser37 levels. Functionally, PKM2 knockdown attenuated BTN3A3-driven increases in proliferation, invasion, migration, and glycolytic flux (P < 0.05). Furthermore, BTN3A3 promoted PKM2 nuclear translocation, and enhanced the association between ERK1/2 and PKM2, indicating it facilitates ERK1/2-mediated phosphorylation and nuclear shuttling of PKM2 to drive glycolysis and NPC progression. Conclusions: BTN3A3 is upregulated in NPC and associated with poor prognosis and distant metastasis. Functionally, it promotes glycolytic reprogramming and tumor progression by enhancing ERK1/2-dependent phosphorylation of PKM2 at Ser37 and PKM2 nuclear translocation.