The Role of PFKFB3 in EGFR-TKIs Tolerance in Non-Small Cell Lung Cancer

Background Introduction

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths worldwide. Epidermal growth factor receptor (EGFR) mutations are relatively common in NSCLC, accounting for approximately 15-30% of cases. Although EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib and osimertinib have shown significant efficacy in treating NSCLC patients with EGFR mutations, many patients eventually develop drug resistance, leading to tumor recurrence. The development of resistance is not only related to genetic mutations but also closely associated with non-genetic mechanisms such as metabolic reprogramming. Metabolic reprogramming enables cancer cells to survive under continuous drug treatment, eventually developing into drug-resistant cell populations.

In recent years, studies have found that the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) plays a critical role in maintaining cancer cell metabolism and redox balance. By regulating glycolytic flux and redox homeostasis, PFKFB3 helps cancer cells survive under drug pressure. However, the mechanisms by which PFKFB3 functions during EGFR-TKIs treatment remain unclear. Therefore, this study aims to explore the metabolic and redox regulatory roles of PFKFB3 in EGFR-TKIs treatment, providing new therapeutic strategies to overcome NSCLC resistance.

Source of the Paper

This paper was co-authored by Nadiia Lypova, Susan M. Dougherty, Brian F. Clem, and other researchers from the University of Louisville School of Medicine and the Brown Cancer Center in the United States. The paper was published in 2024 in the journal Cancer & Metabolism, titled PFKFB3-dependent redox homeostasis and DNA repair support cell survival under EGFR-TKIs in non-small cell lung carcinoma.

Research Process and Results

1. Research Design and Experimental Process

This study employed various experimental methods to systematically investigate the role of PFKFB3 in EGFR-TKIs treatment. The research process included the following main steps:

a) Metabolomics Analysis

The researchers first conducted metabolomics analysis to track changes in glucose metabolism in lung cancer cells under PFKFB3 inhibition and EGFR-TKIs treatment. Stable isotope-labeled glucose ([u-13C]-glucose) was used for metabolic tracing, and changes in metabolites in glycolysis, the tricarboxylic acid (TCA) cycle, and the polyol pathway were analyzed.

b) Oxidative Stress and DNA Damage Assessment

Through live-cell imaging and DCFDA oxidation experiments, the researchers quantified oxidative stress levels in cells under different treatment conditions. Additionally, immunocytochemistry and neutral comet assays were used to assess DNA integrity, particularly the damage caused by oxidative stress.

c) Impact of PFKFB3 Inhibition on Cell Survival

The researchers used PFKFB3 inhibitors PFK-158 and Kan0438757, combined with EGFR-TKIs (erlotinib and osimertinib), to evaluate the impact of PFKFB3 inhibition on lung cancer cell survival. Cell viability assays and ATP level measurements were used to analyze the effects of PFKFB3 inhibition on cell metabolism and energy balance.

d) DNA Repair Mechanism Study

Through immunoblotting and immunofluorescence microscopy, the researchers assessed the impact of PFKFB3 inhibition on the expression of DNA repair enzymes (such as MPG, NTHL1, and UNG2) and explored the role of PFKFB3 in base excision repair (BER).

2. Key Research Findings

a) PFKFB3 Maintains Glycolytic Flux

The study found that PFKFB3 inhibition significantly reduced glucose uptake and glycolytic flux in lung cancer cells under EGFR-TKIs treatment. PFKFB3 inhibition also led to a decrease in ATP levels, indicating that PFKFB3 plays a critical role in maintaining cellular energy balance.

b) PFKFB3 Inhibition Exacerbates Oxidative Stress

PFKFB3 inhibition significantly increased reactive oxygen species (ROS) levels in lung cancer cells and reduced the expression of glutathione peroxidase 4 (GPX4). GPX4 is a key component of the cellular antioxidant defense system, and its decreased expression further exacerbated oxidative stress.

c) PFKFB3 Regulates DNA Repair

The study found that PFKFB3 inhibition significantly reduced the expression of DNA glycosylases (such as MPG, NTHL1, and UNG2), leading to increased DNA oxidative damage. Additionally, PFKFB3 inhibition reduced the expression of ATM (ataxia-telangiectasia mutated), further impairing DNA damage repair capacity.

d) PFKFB3 Inhibition Enhances EGFR-TKIs Cytotoxicity

PFKFB3 inhibition significantly enhanced the cytotoxic effects of EGFR-TKIs on lung cancer cells, indicating that PFKFB3 plays an important role in maintaining cancer cell tolerance to EGFR-TKIs.

3. Research Conclusions

This study demonstrates that PFKFB3 helps lung cancer cells survive under EGFR-TKIs treatment by regulating glycolysis, redox balance, and DNA repair. PFKFB3 inhibition effectively weakens the metabolic adaptability and DNA repair capacity of cancer cells, thereby enhancing the cytotoxicity of EGFR-TKIs. This finding provides a theoretical basis for developing novel anticancer therapies targeting PFKFB3, potentially helping to overcome NSCLC resistance.

Research Highlights

1. Multifaceted Role of PFKFB3: This study is the first to systematically reveal the multifaceted role of PFKFB3 in EGFR-TKIs treatment, including the regulation of glycolysis, redox balance, and DNA repair.
2. Metabolic Reprogramming and Drug Resistance: The research highlights the importance of metabolic reprogramming in EGFR-TKIs resistance, providing new insights into how cancer cells survive under drug pressure.
3. Novel Therapeutic Strategies: PFKFB3 inhibition, as a potential therapeutic strategy, may help eliminate cancer cells before they develop resistance, thereby improving the long-term efficacy of EGFR-TKIs.

Research Significance and Value

This study not only deepens our understanding of the role of PFKFB3 in lung cancer metabolism and drug resistance but also provides important theoretical support for developing novel anticancer therapies targeting PFKFB3. By inhibiting PFKFB3, it may be possible to effectively enhance the efficacy of EGFR-TKIs, reduce tumor recurrence, and offer new hope for NSCLC patients.