Autocrine phosphatase PDP2 inhibits ferroptosis by dephosphorylating ACSL4 in the Luminal A Breast Cancer

Abstract

Phosphatases can dephosphorylate phosphorylated kinases, leading to their inactivation, and ferroptosis is a type of cell death. Therefore, our aim is to identify phosphatases associated with ferroptosis by analyzing the differentially expressed genes (DEGs) of the Luminal A Breast Cancer (LumABC) cohort from the Cancer Genome Atlas (TCGA). An analysis of 260 phosphatase genes from the GeneCard database revealed that out of the 28 DEGs with high expression, only the expression of pyruvate dehydrogenase phosphatase 2 (PDP2) had a significant correlation with patient survival. In addition, an analysis of DEGs using gene ontology, Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analysis revealed a significant variation in the expression of ferroptosis-related genes. To further investigate this, we analyzed 34 ferroptosis-related genes from the TCGA-LumABC cohort. The expression of long-chain acyl-CoA synthetase 4 (ACSL4) was found to have the highest correlation with the expression of PDP2, and its expression was also inversely proportional to the survival rate of patients. Western blot experiments using the MCF-7 cell line showed that the phosphorylation level of ACSL4 was significantly lower in cells transfected with the HA-PDP2 plasmid, and ferroptosis was correspondingly reduced (p < 0.001), as indicated by data from flow cytometry detection of membrane-permeability cell death stained with 7-aminoactinomycin, lipid peroxidation, and Fe2+. Immunoprecipitation experiments further revealed that the phosphorylation level of ACSL4 was only significantly reduced in cells where PDP2 and ACSL4 co-precipitated. These findings suggest that PDP2 may act as a phosphatase to dephosphorylate and inhibit the activity of ACSL4, which had been phosphorylated and activated in LumABC cells. Further experiments are needed to confirm the molecular mechanism of PDP2 inhibiting ferroptosis.

Fig 1. PDP2 is highly expressed in LumABC tissues, and its expression is inversely correlated with patient prognosis.

(A) Volcano plot showing the differentially expressed genes (DEGs) between LumABC and adjacent tissues based on data from the TCGA-LumABC cohort. (B) Forest plot showing the LASSO and univariate Cox regression analyses of five phosphatase-related genes based on data from the TCGA-LumABC cohort. (C) Risk diagnostic plot showing the diagnostic values of the five phosphatase genes. (D) Kaplan-Meier survival of the five phosphatases based on gene expression and clinical survival data from the TCGA-LumABC cohort.

Fig 2. GO and KEGG analysis reveal abnormalities in multiple pathways associated with ferroptosis.

(A-C) Results of the GO analysis, showing the first fifteen cellular compartments (A), biological processes (B), and molecular functions (C), respectively. (D) Results of the KEGG pathways analysis, showing the first ten pathways. (E) Visualization of the ferroptosis pathway in the KEGG analysis, showing the connections and interactions among various molecules related to ferroptosis.

Fig 3. GSEA analysis indicates the inhibition of ferroptosis in the TCGA-LumABC cohort.

(A) Thw results of the GSEA analysis show the first twenty pathways. (B) Out of the twenty pathways, three pathways related to ferroptosis are blocked.

Fig 4. Correlation between ACSL4 and PDP2 expression.

(A) ferroptosis-related genes with expression data in the TCGA-LumABC cohort. (B and C) Volcano plots (B) and heatmaps (C) showing differentially expressed genes (DEGs) among the ferroptosis-related genes in the TCGA-LumABC cohort. (D) Correlation analysis of the five phosphatases and the fifteen ferroptosis-related genes.

Fig 5. ACSL4 expression is correlated with the prognosis of LumABC patients.

(A) Forest plot showing the univariate Cox proportional hazards regression analysis of 16 ferroptosis-related genes based on data from the TCGA-LumABC cohort. (B) Kaplan-Meier survival of the three ferroptosis-related genes based on data from the TCGA-LumABC cohort.

Fig 6. PDP2 inhibits ferroptosis through dephosphorylating phosphorylated ACSL4.

MCF-7 cells were treated with the indicated plasmids and erastin. (A) Immunoblotting detection of PDP2, ACSL4, and phosphorylated ACSL4 (pACSL4), showing typical images in three replicates. (B) Flow cytometry detection of lipid peroxides using C11-BODIPY (n = 5). (C) Flow cytometry analysis of the proportion of 7-AAD-positive dead cells (n = 5). (D) Transmission electron microscopy observation of cell morphology, with white arrows indicating ferroptotic mitochondria and yellow arrows indicating normal mitochondria. (E) Flow cytometry detection of the fluorescence intensity of Fe²⁺ in cells stained with FerroOrange (n = 5). (F) Co-immunoprecipitation with anti-HA (PDP2) and immunoblotting detection of PDP2, ACSL4, and pACSL4, respectively. The flow cytometry data represent the mean ± SD from three independent experiments and were analyzed using two-way ANOVA followed by Tukey’s post-hoc multiple comparison analysis. Statistical significance is denoted as *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001.