The PFKFB3 Knockout A549 Cell Line is a CRISPR/Cas9-edited knockout cell line derived from A549 human lung adenocarcinoma cells, engineered to disrupt the gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3). This product provides a stable loss-of-function model for investigating the role of PFKFB3 in cancer metabolism, particularly in the context of non-small cell lung cancer (NSCLC). Disruption of PFKFB3 abrogates the production of fructose-2,6-bisphosphate, a key allosteric activator of phosphofructokinase-1 (PFK-1), thereby impairing glycolytic flux and enabling studies of metabolic reprogramming, hypoxia adaptation, and tumor cell survival.
The A549 host cell line is a well-characterized model of human lung adenocarcinoma, originally derived from the explanted tumor tissue of a 58-year-old Caucasian male. These cells exhibit epithelial morphology and retain characteristics of alveolar type II epithelial cells, including the expression of surfactant proteins. Importantly, A549 cells harbor a KRASG12S mutation, a common driver of lung adenocarcinoma, and display robust activation of the PI3K/AKT/mTOR signaling axis, making them particularly suitable for investigating oncogene-driven metabolic alterations and therapeutic resistance mechanisms.
PFKFB3 functions as a potent glycolytic activator and is transcriptionally upregulated by HIF-1??, c-Myc, and E2F1 in response to hypoxia, growth factors, and oncogenic signals. The kinase domain of PFKFB3 generates fructose-2,6-bisphosphate, which allosterically activates PFK-1 to accelerate glycolytic carbon flux. This activity is regulated by upstream kinases including AKT and AMPK, as well as by PTEN and PI3K-dependent pathways. PFKFB3 interacts with 14-3-3 proteins, the APC/C-Cdh1 ubiquitin ligase complex, and AMPK, integrating nutrient and energy status with cell cycle progression. Downstream, PFK-1 activation drives lactate production, ATP generation, and rerouting of glucose-6-phosphate into the pentose phosphate pathway for nucleotide biosynthesis. PFKFB3 also enhances VEGF expression and CDK1/cyclin B activity, linking glycolysis to angiogenesis and cell division.
In the A549 lung adenocarcinoma background, PFKFB3 knockout profoundly disrupts the metabolic flexibility required for proliferation under normoxic and hypoxic conditions. Loss of fructose-2,6-bisphosphate attenuates glycolytic flux, reduces lactate secretion, and diminishes ATP and biosynthetic intermediate production, thereby impairing cell proliferation and clonogenic survival. The knockout sensitizes cells to metabolic stress and apoptosis, and abrogates the HIF-1??-driven angiogenic program by downregulating VEGF. This model therefore recapitulates the critical dependency of KRAS-mutant NSCLC on PFKFB3-mediated glycolysis and provides a powerful tool for dissecting the intersection of oncogenic signaling, hypoxia responses, and metabolic adaptation.
This knockout cell line is ideally suited for a wide range of biomedical research applications, including cancer metabolism studies, investigation of glycolytic inhibition, hypoxia response analysis, and screening of antiglycolytic agents such as PFKFB3 inhibitors. Representative experimental approaches include metabolic flux analysis using Seahorse assays, lactate and glucose consumption measurements, Western blotting for PFKFB3 and downstream targets (e.g., PFK-1, GLUT1, LDH), RT-qPCR quantification of PFKFB3 mRNA, and cell-based functional assays (proliferation, apoptosis, colony formation, migration, and invasion). Additional applications encompass HIF-1?? reporter assays, co-immunoprecipitation of PFKFB3 interactors, flow cytometric cell cycle profiling, and drug sensitivity testing with compounds like 2-deoxyglucose or 3PO. For further information and technical support, please contact Ascent Research.
