PFKL-mediated PLIN2 S159 phosphorylation promotes the survival and proliferation of HCC cells and tumor growth.a, Huh7 cells (2 × 104 cells) with depleted endogenous PLIN2 or PFKL and reconstituted expression of the indicated Flag-rPLIN2 (left) or Flag-rPFKL (right) were treated with or without 2-DG (25 mM) or Etomoxir (Eto, 40 μM) for 48 h. The cells were counted. Data are presented as means ± SD of three biologically independent replicates, analyzed by one-way ANOVA; n = 5. b-e, Huh7 cells with depleted endogenous PLIN2 or PFKL and reconstituted expression of the indicated Flag-rPLIN2 or Flag-rPFKL were subcutaneously injected into six-week-old male athymic BALB/c nude mice (n = 12 per group). Ten days after tumor cell injection, 0.2 mL of 2-DG (500 mg/kg) or PBS control was intraperitoneally injected daily for 18 days (n = 6). The tumor volumes (b) and weights (c) were determined. Data are presented as means ± SD of 6 biologically independent replicates, analyzed by one-way ANOVA. The indicated tumor tissues were analyzed by TUNEL assay. Representative images from three independent experiments are shown. Apoptotic cells in 10 microscope fields were quantified. Data were analyzed by two-sided unpaired Student’s t-test; n = 10 (d, e).Credit:Glycolytic enzyme PFKL governs lipolysis by promoting lipid droplet-mitochondria tethering to enhance β-oxidation and tumor cell proliferation.

Cancer cells, especially those with high glycolytic rates, face significant energy stress due to their rapid proliferation. To survive and thrive under these conditions, they must find alternative energy sources. One such source is the oxidation of fatty acids stored in lipid droplets, a process known as β-oxidation. However, for β-oxidation to occur efficiently, lipid droplets must be in close proximity to mitochondria, where fatty acids are transported and oxidized. This spatial arrangement is crucial for the efficient transfer of fatty acids across the mitochondrial membrane.

The mechanisms governing the interaction between lipid droplets and mitochondria, and how this interaction is regulated, remain poorly understood. Previous studies have suggested that the perilipin (PLIN) proteins, which coat lipid droplets, play a role in this process. Specifically, PLIN2 and PLIN3 have been implicated in mediating the association of lipid droplets with autophagosomes, which are involved in the degradation of lipid droplets. Additionally, PLIN2 and PLIN3 have been shown to interact with other proteins, such as adipose triglyceride lipase (ATGL), which hydrolyzes triglycerides into fatty acids for β-oxidation.

In this context, the glycolytic enzyme PFKL (phosphofructokinase liver type) has emerged as a potential player. PFKL is a key enzyme in the glycolytic pathway, catalyzing the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. However, PFKL also possesses non-canonical functions, including its ability to act as a protein kinase. Previous studies have shown that PFKL can phosphorylate other proteins, such as histones and PFKP, and regulate their activity.

Building on these observations, a team of researchers led by Ying Meng from Zhejiang University conducted a comprehensive study to investigate the role of PFKL and PLIN2 in mediating the tethering of lipid droplets to mitochondria and promoting β-oxidation in tumor cells. Their findings, published in Nature Metabolism, provide new insights into the metabolic adaptations of cancer cells and identify potential targets for anti-cancer therapy.

The study began by examining the interaction between lipid droplets and mitochondria under glucose deprivation, a common energy stress condition in tumor cells. The researchers found that glucose deprivation enhanced the binding of the lipid droplet-associated protein PLIN2 to the mitochondrial protein CPT1A, which was essential for the tethering of lipid droplets to mitochondria.

To understand the molecular mechanism underlying this interaction, the authors focused on PFKL, an enzyme that catalyzes the rate-limiting step of glycolysis. They observe that glucose deprivation enhanced the binding of PFKL to PLIN2 and its phosphorylation by the p38 MAP kinase. This phosphorylation event converted PFKL from a tetrameric form, which is active in glycolysis, to a monomeric form, which loses its glycolytic activity.

Importantly, the phosphorylated PFKL acted as a protein kinase and phosphorylated PLIN2 at Ser159. This phosphorylation event enhanced the interaction between PLIN2 and CPT1A, promoting the tethering of lipid droplets to mitochondria and the subsequent mobilization of fatty acids for β-oxidation.

To further validate the role of PFKL and PLIN2 in this process, the researchers performed various experiments. They showed that depletion of PFKL or PLIN2 prevented the tethering of lipid droplets to mitochondria and the reduction in total lipid droplets induced by glucose deprivation.

In addition, the mutation of PFKL at Thr331, the site of p38-mediated phosphorylation, or PLIN2 at Ser159 abrogated the interaction between PLIN2 and CPT1A, the tethering of lipid droplets to mitochondria, and the subsequent β-oxidation.

They also found that the expression of PFKL mutants or PLIN2 mutants that lacked the ability to be phosphorylated at the relevant sites inhibited tumor cell proliferation and promoted apoptosis in vitro and in vivo.

Finally, the study explored the clinical significance of these findings. The authors performed immunohistochemical staining of human HCC samples and found that the phosphorylation levels of PFKL at Thr331 and PLIN2 at Ser159 were significantly higher in HCC tissues compared to normal tissues. Furthermore, these phosphorylation levels correlated with poor survival in HCC patients.

In conclusion, this study reveals a novel moonlighting function of PFKL as a protein kinase that promotes β-oxidation and tumor cell proliferation by tethering lipid droplets to mitochondria. This discovery sheds light on the complex interplay between glycolysis and lipid metabolism in cancer cells and provides a potential target for the development of novel anti-cancer therapies.

1. Meng Y, Guo D, Lin L, Zhao H, Xu W, Luo S, Jiang X, Li S, He X, Zhu R, Shi R, Xiao L, Wu Q, He H, Tao J, Jiang H, Wang Z, Yao P, Xu D, Lu Z. Glycolytic enzyme PFKL governs lipolysis by promoting lipid droplet-mitochondria tethering to enhance β-oxidation and tumor cell proliferation. Nat Metab. 2024 Jun;6(6):1092-1107.