HMGA1 stimulates MYH9-dependent ubiquitination of GSK-3β via PI3K/Akt/c-Jun signaling to promote malignant progression and chemoresistance in gliomas

Abstract

Myosin heavy chain 9 (MYH9) plays an essential role in human diseases, including multiple cancers; however, little is known about its role in gliomas. In the present study, we revealed that HMGA1 and MYH9 were upregulated in gliomas and their expression correlated with WHO grade, and HMGA1 promoted the acquisition of malignant phenotypes and chemoresistance of glioma cells by regulating the expression of MYH9 through c-Jun-mediated transcription. Moreover, MYH9 interacted with GSK-3β to inhibit the expression of GSK-3β protein by promoting its ubiquitination; the downregulation of GSK-3β subsequently promoted the nuclear translocation of β-catenin, enhancing growth, invasion, migration, and temozolomide resistance in glioma cells. Expression levels of HMGA1 and MYH9 were significantly correlated with patient survival and should be considered as independent prognostic factors. Our findings provide new insights into the role of HMGA1 and MYH9 in gliomagenesis and suggest the potential application of HMGA1 and MYH9 in cancer therapy in the future.

Fig. 1. Upregulation of HMGA1 and MYH9 was correlated with the progression of gliomas.

A, B HMGA1 (A) and MYH9 (B) mRNA expression were upregulated in glioma tissues compared to normal brain tissues, and overexpressed levels of HMGA1 and MYH9 in glioma patients were positively correlated with the status of pathological classification. C The expression of HMGA1 was positively related to MYH9 expression. D Immunohistochemistry assay identified HMGA1 was overexpressed in glioma sample tissues compared to normal brain tissues. E MYH9 was overexpressed in glioma sample tissues. Strong expression of MYH9 was mainly observed in cytoplasmic (B, D, and E) and partly found in the nucleus (C and F) in glioma tissues. F, G Analysis of Chinese Glioma Genome Atlas (CCGA) database suggested HMGA1 (F) and MYH9 (G) mRNA level was significantly increased with the increase of WHO grade. H The expression of HMGA1 was positively related to MYH9 expression in CCGA database. I Dose–response curves of TMZ treatment were examined for HMGA1-overexpression groups and the control groups. J Western blot assay confirmed the change of the downstream pathway of HMGA1.

Fig. 2. HMGA1 increased the expression of MYH9 through c-Jun-mediated transcription.

A Knockdown HMGA1 leads to the decreasing of MYH9 mRNA expression. B Schematic diagram of putative c-Jun binding sites in MYH9 promotor and the primers of each binding site. C, D Knockdown c-Jun induced inhibition of MYH9 mRNA (C) and protein (D) expression. E c-Jun bound to all the predicted sites inside the transcription regulatory region of MYH9, and knockdown of c-Jun could inhibit the protein–DNA interactions in all three binding sites. F Dual-luciferase reporter assay showed that the reporter plasmid with the promotor region of MYH9 resulted in a significant decrease in luciferase activity after transfection with si-c-Jun, whereas the plasmid without MYH9 promotor had no change in luciferase activity.

Fig. 3. MYH9 promoted proliferation, invasion, migration, and TMZ resistance in gliomas.

A, B MTT assays showed MYH9 downregulation observably inhibited the cell growth. C Edu incorporation assays showed knockdown of MYH9 decreased the cell proliferation by blocking G1/S phase transition in glioma cells. D Intracranial orthotopic transplantation suggested knockdown of MYH9 inhibited the tumorigenesis in gliomas. E, F Transwell (E) and Boyden (F) chamber assays confirmed knockdown of MYH9 inhibited the ability of cell invasion and migration. G, H Dose–response curves were delineated for TMZ treatment in shMYH9 cells and the control groups to identify the role of MYH9 in chemoresistance. I Western blot assay confirmed the change of the downstream pathway of MYH9.

Fig. 4. HMGA1 promoted malignant phenotypes and chemoresistance of glioma cells via MYH9.

A–C MTT (A and B) and Edu incorporation (C) assays showed that MYH9 upregulation reversed the shHMGA1-mediated reduction of growth ability in glioma cells. D, E Transwell (D) and Boyden (E) chamber assays verified that the ability of cell invasion and migration was recovered in shHMGA1+ MYH9 plasmid-treated cells group compared to shHMGA1-treated cells group. F, G Dose–response curves for TMZ showed the TMZ concentration causing IC₅₀ was rescued in shHMGA1+MYH9 plasmid-treated cells groups compared to shHMGA1-treated cells groups. H Western blot assay confirmed MYH9 upregulation rescued shHMGA1-mediated reduction of the EMT-related proteins, the cell cycle-related factors, and the stemness-related factors, and MYH9-overexpression could reverse the shHMGA1-mediated increase of GSK-3β.

Fig. 5. MYH9 interacted with GSK-3β and promotes its ubiquitination.

A Knockdown of MYH9 had no effect on the GSK-3β mRNA level in glioma cells. B Overexpression of MYH9 could downregulate GSK-3β protein level and vice versa. C GSK-3β and MYH9 proteins bind to each other, and overexpression of MYH9 downregulated GSK-3β protein level and remarkedly increased Ubiquitin activity. D Mutation of all three potential ubiquitination sites could induce the reduction of ubiquitination of GSK-3β. E Mutation of two potential interaction domains of MYH9 could both upregulate the GSK-3β protein level and inhibit Ubiquitin activity. F Immunofluorescence staining showed MYH9 and GSK-3β both mainly located in the cytoplasm, and overexpression of MYH9 could reduce the cytoplasmic expression of GSK-3β.

Fig. 6. Elevated HMGA1 and MYH9 expression as an unfavorable factor in glioblastoma.

A A brief illustration of the oncogenic HMGA1/MYH9 signaling pathway. B, C Survival analysis from CCGA database suggested that mRNA expression level of HMGA1 (B) and MYH9 (C) had a positive relationship with glioma patients’ survival time. D, E The results of TCGA database also suggested that patients with low expression of HMGA1 or low expression MYH9 had a longer survival time compared to patients with high expression. F Survival analysis from TCGA database showed patients with both high expressions of HMGA1 and MYH9 had significantly worse survival times compared to patients with both low expressions. G, H Survival analysis about primary glioma cases in our department showed HMGA1 expression and MYH9 expression were found to be positively associated with the overall survival. I. Patients with both high expression of HMGA1 and MYH9 had the shortest survival time, whereas patients with both low expressions of HMGA1 and MYH9 had the best survival time.