MEK5-ERK5 pathway mediates mitophagy by regulating Nur77 to promote tumorigenesis of osteosarcoma cells

Abstract

Objectives: To investigate the influence of MEK5/ERK5 pathway on mitophagy in osteosarcoma (OS), as well as the involved molecular mechanisms.

Methods: The overlapped genes of mitophagy-related genes from MSigDB database and DEGs between metastatic and primary OS groups from GSE32981 were identified. GSVA of mitophagy-related pathways between the metastatic and primary groups were analyzed. The relationships between Nur77 and mitophagy-related pathways, prognosis, immune infiltrating cells, immune response gene sets were investigated. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blotting were utilized to assess the expression levels of MEK5, ERK5, Nur77, PINK1, and Parkin. Cellular behaviors and mitochondrial potential were evaluated via CCK-8, Transwell assay and JC-1 staining.

Results: Total 4 overlapped genes were obtained as mitophagy-related DEGs, including GABARAPL1, HIF1A, PINK1, and RB1CC1. The activity scores of 3 mitophagy-related pathways exhibited significant differences between metastatic and primary groups. Importantly, Nur77 was significantly negatively correlated with a mitophagy-related pathway (GOBP MITOPHAGY: R = - 0.48, P = 0.02). The Nur77 expression in metastatic group was remarkedly higher than that in the primary group (P < 0.001). Patients with high Nur77 expression had poor prognosis, with AUC values all above 0.615 in predicting 1-, 3-, and 5-year survival. In addition, Nur77 was closely related to numerous immune cells, including activated dendritic cells, activated mast cells and M0 macrophages, and immune response gene sets chemokines and cytokines (all P < 0.05). In addition, MEK5/ERK5 pathway is activated in OS, and Nur77 is overexpressed in OS, and MEK5/ERK pathway promotes Nur77 expression, tumorigenesis and mitochondrial function in U2OS cells. Cytosporone B implement significantly increased the tumorigenesis of U2OS cells in sh-MEK5 group, and inhibited the weaken in mitochondrial membrane potential caused by MEK5 downregulation, and reversed the protein levels of mitophagy markers PINK1 and Parkin in sh-MEK5 group.

Conclusions: MEK5-ERK5 pathway mediates mitophagy by regulating Nur77 to promote tumorigenesis of OS cells. These findings offered promising therapeutic targets for OS.

Keywords: MEK5–ERK5 pathway; Mitophagy; Nur77; Osteosarcoma.

Fig. 1

Nur77 is significantly overexpressed and closely related to mitophagy in osteosarcoma (OS). A Expression level of Nur77 in the metastatic and primary groups. B Kaplan–Meier (KM) curve (left) and receiver operating characteristic (ROC) curve (right) of Nur77. C Volcano map (left) and heatmap (right) of differentially expressed genes (DEGs). D Venn diagram of the intersection of DEGs and mitophagy-related genes. E Expression level of 4 mitophagy-related DEGs in metastatic and primary groups. F Gene set variation analysis (GSVA) scores of three mitophagy-related pathways in the metastatic and the primary group. G Scatter plot of the correlation between Nur77 and the mitophagy-related pathways. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Relationship between Nur77 and immune in osteosarcoma (OS). A Immune cell content in each OS sample. B Differences of fraction of immune cells between metastatic and primary OS samples. C Correlation between Nur77 and immune cells. D Differences in immune response gene set activity between primary and metastatic OS samples. E Correlation between Nur77 and immune response gene sets. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

MEK5, ERK5, and Nur77 expression levels in human normal osteoblast cell line hFOB1.19 and osteosarcoma (OS) cell lines U2OS and MG63. A mRNA expression levels. B Protein expression levels. *P < 0.05, **P < 0.01

Fig. 4

MEK5/ERK pathway promotes Nur77 expression, tumorigenesis and mitochondrial function in U2OS cells. A Transfection efficiency measured by immunofluorescence; scale bar = 50 μm. B Western blotting detected MEK5, p-ERK5/ERK5, and Nur77 protein levels in U2OS cells. C Cell migration capability of U2OS cells was detected by Transwell assay; scale bar = 50 μm. D Cell invasive capability of U2OS cells was detected by Transwell assay; scale bar = 50 μm. E Cell viability of U2OS cells was detected by CCK-8 assay. F Mitochondrial membrane potential levels of U2OS cells were detected by JC-1 assay. *P < 0.05, **P < 0.01

Fig. 5

MEK5–ERK5 pathway mediates mitophagy by regulating Nur77 to promote tumorigenesis of U2OS cells. A Western blotting detected Nur77 protein level in U2OS cells. B Cell viability of U2OS cells was detected by CCK-8 assay. C Cell migration capability of U2OS cells was detected by Transwell assay; scale bar = 50 μm. D Cell invasive capability of U2OS cells was detected by Transwell assay; scale bar = 50 μm. E Mitochondrial membrane potential levels of U2OS cells were detected by JC-1 assay. F Western blotting detected the protein levels of PINK1 and Parkin in U2OS cells. **P < 0.01