HMGA2 alleviates ferroptosis by promoting GPX4 expression in pancreatic cancer cells

Abstract

Pancreatic cancer is one of the most malignant tumor types and is characterized by high metastasis ability and a low survival rate. As a chromatin-binding protein, HMGA2 is widely overexpressed and considered an oncogene with various undefined regulatory mechanisms. Herein, we demonstrated that HMGA2 is highly expressed in pancreatic cancer tissues, mainly distributed in epithelial cells, and represents a subtype of high epithelial-mesenchymal transition. Deletion of HMGA2 inhibits tumor malignancy through cell proliferation, metastasis, and xenograft tumor growth in vivo. Moreover, HMGA2 enhanced the cellular redox status by inhibiting reactive oxygen species and promoting glutathione production. Importantly, ferroptotic cell death was significantly ameliorated in cells overexpressing HMGA2. Conversely, HMGA2 deletion exacerbated ferroptosis. Mechanistically, HMGA2 activated GPX4 expression through transcriptional and translational regulation. HMGA2 binds and promotes cis-element modification in the promoter region of the GPX4 gene by enhancing enhancer activity through increased H3K4 methylation and H3K27 acetylation. Furthermore, HMGA2 stimulated GPX4 protein synthesis via the mTORC1-4EBP1 and -S6K signaling axes. The overexpression of HMGA2 alleviated the decrease in GPX4 protein levels resulting from the pharmacologic inhibition of mTORC1. Conversely, compared with the control, HMGA2 deletion more strongly reduced the phosphorylation of 4EBP1 and S6K. A strong positive correlation between HMGA2 and GPX4 expression was confirmed using immunohistochemical staining. We also demonstrated that HMGA2 mitigated the sensitivity of cancer cells to combination treatment with a ferroptosis inducer and mTORC1 inhibition or gemcitabine. In summary, our results revealed a regulatory mechanism by which HMGA2 coordinates GPX4 expression and underscores the potential value of targeting HMGA2 in cancer treatment.

Fig. 1. Features of HMGA2 expression in pancreatic cancer.

A The transcriptome expression of HMGA2 in RNA-seq datasets from 869 pancreatic cancer tissues and 417 adjacent normal tissues. B Proteomic expression of HMGA2 in PDAC samples from CPTAC patients. C The survival probability of patients with pancreatic cancer with high or low HMGA2 expression is shown as the median expression value of HMGA2. The data were collected from the TCGA database. D The UMAP plot shows the categorization of single cells from human pancreatic cancer cells into different cell types (left) and the single-cell expression levels of HMGA2 (right). E Reclustering of epithelial cells from D revealed that HMGA2 was highly expressed in the epithelial cluster 4 subtype. F Functional enrichment analysis of the marker genes of epithelial cluster 4 (log2FC > 3 and p value adjusted < 0.01) via the Metascape web server.

Fig. 2. HMGA2 is highly expressed in pancreatic cancer and promotes cancer cell malignancy.

A Representative images of IHC staining showing HMGA2 expression in pancreatic cancer tissue. The graph bar represents the results of the quantitative analysis. The intensity of HMGA2 staining was quantified using the integral optical density (IOD). The scale bar indicates 100 µm. B Representative images of HMGA2 and αSMA IHC double-stained pancreatic cancer tissue. Cell growth detection after HMGA2 overexpression in MIAPaCa-2 (C) and PANC-1 cells (D) or after HMGA2 deletion in PANC-1 cells (E). Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) (n = 4). The efficiency of overexpression or deletion was confirmed using Western blotting, as indicated. The band intensity was quantified by ImageJ software. Evaluation of the effect of HMGA2 overexpression on the clonal growth and migration of MIAPaCa-2 (F) and PANC-1 cells (G). H Xenograft tumor model investigating the role of PANC-1 sgNC and PANC-1 sgHMGA2 in SCID mice. The graph bar represents the average tumor weight. The data are presented as the means ± SDs for each group of mice (n = 7). * indicates p < 0.05, ** indicates p < 0.01.

Fig. 3. HMGA2 is correlated with ferroptosis signaling and influences the cellular redox status.

Cell signaling pathway analysis (A) and gene set enrichment analysis (GSEA) (B) based on HMGA2 expression levels. Datasets from TCGA and GTEx were collected and divided into two groups, HMGA2-low and HMGA2-high, based on the median expression value of HMGA2 to collect DEGs for functional pathway analysis. Evaluation of cellular ROS (C), lipid peroxidation (F), and GSH levels (I) in MIAPaCa-2 cells overexpressing HMGA2. Evaluation of cellular ROS (D, E), lipid peroxidation (G, H), and GSH levels (J, K) in HMGA2-depleted PANC-1 and BxPC3 cells (n = 3). Cellular ROS were assessed using DCFH staining, and lipid peroxidation was assessed using BODIPY™ 581/591 C11 staining followed by flocytometry analysis. GSH was assessed using GSH and GSSG Assay Kits. The p value was determined by Student’s t test and is indicated in the figure.

Fig. 4. HMGA2 confers resistance to ferroptosis cell death.

Cell viability analysis of the effects of HMGA2 alteration in MIAPaCa-2 (A) and PANC-1 (B) cells with HMGA2 overexpression and PANC-1 cells with HMGA2 deletion (C) after RSL3 treatment (n = 4). Representative images of cell death indicated by propidium iodide (PI) staining after HMGA2 overexpression in MIAPaCa-2 cells (D) and HMGA2 deletion in PANC-1 cells (E) treated with RSL3. The scale bar indicates 100 µm. Lipid peroxidation analysis of HMGA2-overexpressing MIAPaCa-2 cells (F) and HMGA2-deleted PANC-1 cells (G) after RSL3 treatment was performed using a Lipid Peroxidation MDA Assay Kit (n = 3). The evaluation of cell survival of cells with HMGA2 overexpression in MIAPaCa-2 (H) and PANC-1 cells (I), and HMGA2 deletion PANC-1 (J) and BXPC3 cells (K) treated with RSL3 and ferrostatin-1 (Ferr), a ferroptosis inhibitor (n = 4). The p value was determined using Student’s t test. * indicates p < 0.05; ** indicates p < 0.01.

Fig. 5. HMGA2 enhances GPX4 transcription.

Detection of GPX4 protein expression in HMGA2-overexpressing MIAPaCa-2 (A) and PANC-1 cells (B) and HMGA2-depleted PANC-1 (C) and BXPC3 cells (D) using western blotting. The band intensity was quantified by ImageJ software. E Immunohistochemical staining analysis of HMGA2 and GPX4 expression in pancreatic cancer tissue. The scale bar indicates 100 µm. The correlation between HMGA2 and GPX4 expression was determined by statistical analysis. Detection of ferroptosis-related gene expression using RT‒qPCR in HMGA2-overexpressing MIAPaCa-2 (F) and PANC-1 cells (G) and HMGA2-deleted PANC-1 (H) and BXPC3 cells (I) (n = 3). J Enrichment analysis of the enhancer markers H3K4me1 and H3K27ac and of FLAG-HMGA2 fused to the cis-elements of the GPX4 promoter region using ChIP‒qPCR. K Luciferase activity assay demonstrating the transcriptional activation of GPX4 enhancer 3 and its promoter by HMGA2. The p value was determined by Student’s t-test.

Fig. 6. HMGA2 activates mTORC1 signaling.

A Demonstration of alterations in gene expression based on RNA sequence in HMGA2-overexpressing MIAPaCa-2 cells compared to control cells. B The 20 most notably enriched KEGG pathways significantly changed the expression of genes identified from the RNA sequences after HMGA2 overexpression. Western blot detection of the mTORC1 downstream genes p70S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and their phosphorylation status in HMGA2-overexpressing MIAPaCa-2 (C) and PANC-1 cells (D) and in HMGA2-deleted PANC-1 (E) and BXPC3 cells (F). The band intensity was quantified by ImageJ software.

Fig. 7. HMGA2 promotes GPX4 protein synthesis through mTORC1 signaling.

Western blot detection of the mTORC1 downstream genes p70S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and their phosphorylation status in PANC-1 (A) and MIAPaCa-2 (B) cells overexpressing HMGA2 and in PANC-1 cells with HMGA2 deletion (C) treated with the mTORC1 inhibitor rapamycin (Rap) and Torin1 for 2 h. Concentrations of rapamycin and Torin1 are indicated. Detection of S6K and 4EBP1 and their phosphorylation status in HMGA2-overexpressing MIAPaCa-2 (D) and HMGA2-deleted BXPC-3 (E) cells treated with AZD8055, an mTORC1 and mTPRC2 inhibitor. The band intensity was quantified by ImageJ software.

Fig. 8. HMGA2 alleviates sensitivity to a combination treatment with a ferroptosis inducer and mTORC1 inhibition.

Cell viability analysis of the effects of combination treatment with RSL3 and the mTORC1/2 inhibitors rapamycin (Rap), Torin1, and AZD8055 (AZD) in HMGA2-overexpressing MIAPaCa-2 (A), HMGA2-deleted PANC-1 (B) and BXPC-3 cells (C) (n = 4). Cell viability analysis of the effects of combination treatment with gemcitabine (GEM) and the mTORC1/2 inhibitors Rap, Torin1, and AZD on HMGA2-overexpressing MIAPaCa-2 (D) and HMGA2-deleted PANC-1 (E) and BXPC-3 cells (F) (n = 4). Cell viability analysis of the effects of the combination treatment of RSL3, GEM, and ferrostatin-1 (Ferr) and the mTORC1/2 inhibitors Rap, Torin1, and AZD on HMGA2 overexpression in MIAPaCa-2 (G) and HMGA2-deleted PANC-1 (H) and BXPC-3 cells (I) (n = 4). The p value was determined using a two-tailed unpaired Student’s t-test and is indicated.