Abstract Programmed cell death 4 (PDCD4) is a tumor suppressor frequently downregulated in non-small cell lung cancer (NSCLC), yet its role in metabolic regulation remains poorly understood. Using reverse-phase protein array analysis, we found that loss of PDCD4 significantly upregulates the glycolytic enzyme PFKFB3, which produces fructose-2,6-bisphosphate to activate the rate-limiting enzyme PFK1. PFKFB3 is highly expressed in NSCLC and correlates with advanced tumor stage. Because PDCD4 suppresses mTORC2 activity, we investigated whether mTORC2 regulates PFKFB3. Co-immunoprecipitation and in vitro kinase assays showed that mTORC2 directly interacts with and phosphorylates PFKFB3, and mapping analysis identified Ser461 as the targeted phosphorylation site. To determine its functional significance, we generated phosphorylation-deficient (S461A) and phospho-mimetic (S461D) PFKFB3 mutants. Protein stability assays revealed that S461A undergoes rapid proteasomal degradation, whereas S461D exhibits enhanced stability compared with wild-type (WT) PFKFB3. Seahorse XF glycolysis stress tests demonstrated that WT and S461D significantly increased basal and maximal glycolytic activity, while S461A markedly impaired glycolysis and metabolic capacity. Consistent with these effects, WT and S461D promoted proliferation and colony formation, whereas S461A suppressed cell growth. These findings identify mTORC2-mediated phosphorylation of PFKFB3 at Ser461 as a critical regulator of PFKFB3 stability and glycolytic function and show that PDCD4 loss enhances mTORC2 activity to drive glycolytic reprogramming in NSCLC. Targeting the PDCD4-mTORC2-PFKFB3 axis may offer a promising therapeutic approach to disrupt tumor metabolism in lung cancer. Citation Format: Elham Zokaei, Hsin-Sheng Yang, Qing Wang, Yanquan Zhang. Pdcd4/mTORC2 axis regulates glycolysis via PFKFB3 in NSCLC abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 586.
Zokaei et al. (2026) studied this question.