Abstract 6652: PDCD4/mTORC2 axis regulates tumorigenesis through PFKFB3 in NSCLC

Abstract Programmed cell death 4 (PDCD4) is a tumor suppressor whose expression is frequently downregulated in various cancers, including lung cancer. PDCD4 has been shown to suppress tumor cell proliferation, migration, invasion, and metastasis in cultured cells and in mouse models. It has also been suggested to play a role in regulating glucose metabolism; however, the detailed mechanisms remain unclear. Using reverse phase protein array, we found that knockdown of PDCD4 upregulated 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), a critical enzyme in regulation of glycolysis. PFKFB3 converts fructose-6-phosphate to fructose-2, 6-bisphosphate to activate the rate-limiting enzyme, 6-phosphofructo-1-kinase, in glycolysis. PFKFB3 expression is upregulated in lung cancer and correlates with tumor stage progression. Inhibition or down-regulation of PFKFB3 in lung cancer cells induces cell apoptosis and inhibits tumor growth in nude mice. Given that PDCD4 inhibits mTORC2 activity, we next examined how mTORC2 affects PFKFB3 expression. We found that mTORC2 interacted with PFKFB3 in NSCLC cells. Using In vitro kinase assays demonstrated that can phosphorylate PFKFB3. Further analysis pinpointed a Ser residue as the target site, leading to the generation of phosphorylation-deficient (S to A) and phospho-mimetic (S to D) mutants. Stability assays revealed that phosphorylation at this specific Ser is essential for maintaining PFKFB3 protein stability; as the phosphorylation-deficient mutant underwent rapidly degradation, whereas the phospho-mimetic mutant showed increased it’s half-life. Functional assays showed that WT PFKFB3 promoted high proliferation rates, whereas the phosphorylation-deficient mutant impaired colony formation and proliferation. Taken together, these findings uncover a key mechanism of metabolic reprogramming in NSCLC, in which mTORC2 drives glycolysis by directly phosphorylating PFKFB3, highlighting the therapeutic potential of targeting the PDCD4/mTORC2/PFKFB3 axis in NSCLC treatment. Citation Format: Elham Zokaei, Qing Wang, Hsin-Sheng Yang. PDCD4/mTORC2 axis regulates tumorigenesis through PFKFB3 in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6652.