PKP1 promotes lung cancer by modulating energy metabolism through stabilization of PFKP

Abstract

Lung cancer is the leading cause of cancer-related deaths worldwide, with lung squamous cell carcinoma (LUSC) lacking effective targeted therapies. Recent studies have identified Plakophilin-1 (PKP1) as one of the most differentially overexpressed genes in LUSC. This is particularly intriguing given that PKP1 is primarily known as a desmosomal component involved in cell adhesion, typically regarded as a tumor suppressor. To elucidate its biological role, we performed a genome-wide CRISPR knockout screening in PKP1-deficient models, revealing a strong dependence on mitochondrial metabolism. Metabolic assays further demonstrated that PKP1 loss significantly disrupts both mitochondrial function and glycolytic activity. In contrast, cells expressing PKP1 display a metabolically hyperactive phenotype, characterized by elevated oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Building on these findings, we found that PKP1 depletion selectively reduces platelet-type phosphofructokinase (PFKP) levels, a key rate-limiting enzyme in glycolysis, by enhancing its ubiquitination and subsequent degradation. Functional rescue experiments confirmed that PFKP mediates the proliferative role of PKP1. These findings suggest that PKP1 overexpression in LUSC promotes a hyperactive metabolic state binding to TRIM21 and preventing PFKP degradation, facilitating tumor progression. These effects were consistently observed across multiple LUSC cell lines, underscoring the robustness of the mechanism. These findings highlight a potential therapeutic vulnerability in LUSC metabolic regulation.

Graphical Abstract:

Supplementary Information: The online version contains supplementary material available at 10.1186/s40364-025-00815-w.

Keywords: Glycolysis and OXPHOS; Phosphofructokinase; Plakophilin-1; TRIM21.

Fig. 1

(A) Dot plot represents the gene essentiality score of all genes. Red labelled dots correspond to the top 20 genes within the lowest KO gene essentiality score (y-axis), and a neutral control gene essentiality score (x-axis) between − 0.5 and + 0.5. (B) Selection of GO (Biological Process - BP), REACTOME and CORUM terms overrepresented among the top 100 KO genes. The diameter of the circle is proportional to the number of genes, the colormap refers to the -log₁₀ FDR. X-axis specifies the enrichment score. (C) Left panel: OCR profile at baseline and in response to oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and antimycin A plus rotenone (Rot/AA) of SK-MES-1 KD model. Right panel: Bar plots show the quantification of basal respiration, maximal respiration and ATP production in the KD model. Results were normalized to total concentration of protein in each well. All experiments were conducted in triplicates and P values were calculated using an unpaired, two-tailed t-test. (D) Energy Map of basal OCR and basal ECAR in SK-MES-1 cells in KO and KD models, using the Seahorse Bioscience XF96 analyzer. Values represent mean ± SD (n = 3). MANOVA-test *P < 0.05; **P < 0.01; ***P < 0.001, solid line refers to the KO model comparison and dashed line to the silenced model. (E) GlycoPER profile at baseline and in response to Rot/AA and 2-deoxy-D-glucose (2-DG) of SK-MES-1 lacking PKP1 (left). Bar plots show quantification of basal and compensatory glycolysis in the KD model (right). Results were normalized to total concentration of protein of each well. All experiments were conducted in triplicates and P values were calculated using an unpaired, two-tailed t-test

Fig. 2

(A) Simplified glycolysis diagram highlighting the rate-limiting enzymes: hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PKM). (B) Representative immunoblot assay of the glycolytic rate-limiting enzymes: HK1, PFKP, PFKL, PFKM, and PKM2. ß-actin was used as loading control. (C) mRNA levels of PFKP in PKP1 KO and KD models. (D) Viability assays performed on silenced model of SK-MES-1 cell lines transfected with pCDNA3.1 + EV or pCDNA3.1 + PFKP. Growth was normalized to the EV. P values were calculated using an unpaired, two-tailed t-test (*t-test P < 0.05; **P < 0.01). (E) Immunoblot of PFKP and PKP1 in NCI-H520 overexpression PKP1 model. ß-actin was used as loading control. (F) Representative immunoblot of PFKP protein level in the PKP1 KD model upon MG132 treatment. (G) PFKP ubiquitination detected by anti-Ub immunoblotting in PKP1 KD model. (H) Ubiquitin-related protein interactors identify by IP-MS/MS. (I) Immunoblot monitoring co-IP of PKP1 and TRIM21 in SK-MES-1 cell line. ß-actin was used as negative control of the IP. (J). Representative image of immunofluorescence of PKP1 and TRIM21 and correlation between Overlap Coefficient and Pearson R (n = 117)