PFKFB3 Knockout Cell Lines

Investigate the function of PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3) in glycolytic reprogramming and its role in protecting cancer cells from cisplatin-induced apoptosis.

1. Glycolysis and the Warburg Effect

Cancer cells often rely on enhanced glycolysis for energy production, even in the presence of oxygen—a phenomenon known as the Warburg effect. PFKFB3 is a key enzyme that catalyzes the production of fructose-2,6-bisphosphate (F2,6BP), a potent activator of glycolysis. Unlike other PFKFB family members, PFKFB3 is primarily localized in the nucleus, though the biological significance of this localization remains unclear .

2. PFKFB3 in Cancer Therapy

PFKFB3 has been linked to tumor growth and angiogenesis, but its role in chemotherapy resistance is poorly understood. Cisplatin, a widely used chemotherapeutic agent, induces DNA damage and apoptosis, but cancer cells often develop resistance through metabolic reprogramming .

3. Gaps in Knowledge

How does PFKFB3 regulate glycolysis in response to DNA damage?

Can PFKFB3 knockout (KO) cell lines serve as a model to study chemotherapy sensitization?

1. Generation of PFKFB3 KO Cell Lines

Method: CRISPR-Cas9 genome editing was used to disrupt the PFKFB3 gene in HeLa, A549, and HCT116 cancer cell lines. Two independent sgRNAs targeted the coding sequence, and KO was confirmed by immunoblotting and DNA sequencing .

Validation: PFKFB3 KO cells showed abolished protein expression and reduced basal glycolytic activity, as measured by extracellular acidification rate (ECAR) and lactate secretion .

2. Functional Assays in PFKFB3 KO Cells

Glycolytic Impairment: Cisplatin treatment normally stimulates glycolysis in wild-type cells, but this effect was completely blocked in PFKFB3 KO cells, as shown by ECAR and lactate assays .

Chemosensitization: PFKFB3 KO cells showed significantly increased sensitivity to cisplatin-induced apoptosis, with a >2-fold increase in Annexin V/PI-positive cells compared to wild-type cells .

Mechanistic Studies: Cisplatin induced acetylation of PFKFB3 at lysine 472 (K472), leading to cytoplasmic accumulation and enhanced phosphorylation by AMPK at serine 461 (S461), which activates glycolysis .

3. Therapeutic Implications

Xenograft Models: PFKFB3 KO cells showed reduced tumor growth in nude mice, and combination treatment with cisplatin and the PFKFB3 inhibitor PFK15 further suppressed tumor growth compared to single-agent therapy .

Acetylation-Mediated Regulation: Mutations in PFKFB3 (e.g., K472Q) that mimic acetylation promoted cytoplasmic localization, enhanced glycolysis, and protected cells from cisplatin, while nuclear-localized mutants lost this protective effect .

1. Mechanistic Insights

PFKFB3 regulates glycolysis and chemotherapy resistance through a dynamic acetylation-phosphorylation cascade:

DNA Damage Response: Cisplatin induces PFKFB3 acetylation at K472, disrupting its nuclear localization signal (NLS) and promoting cytoplasmic accumulation .

Glycolytic Activation: Cytoplasmic PFKFB3 is phosphorylated by AMPK at S461, increasing F2,6BP production and glycolytic flux, which supports cell survival under genotoxic stress .