TRIM7 modulates NCOA4-mediated ferritinophagy and ferroptosis in glioblastoma cells

Abstract

Objective: Glioblastoma is one of the most common intracranial malignant tumors with an unfavorable prognosis, and iron metabolism as well as ferroptosis are implicated in the pathogenesis of glioblastoma. The present study aims to decipher the role and mechanisms of tripartite motif-containing protein 7 (TRIM7) in ferroptosis and glioblastoma progression.

Methods: Stable TRIM7-deficient or overexpressing human glioblastoma cells were generated with lentiviral vectors, and cell survival, lipid peroxidation and iron metabolism were evaluated. Immunoprecipitation, protein degradation and ubiquitination assays were performed to demonstrate the regulation of TRIM7 on its candidate proteins.

Results: TRIM7 expression was elevated in human glioblastoma cells and tissues. TRIM7 silence suppressed growth and induced death, while TRIM7 overexpression facilitated growth and inhibited death of human glioblastoma cells. Meanwhile, TRIM7-silenced cells exhibited increased iron accumulation, lipid peroxidation and ferroptosis, which were significantly reduced by TRIM7 overexpression. Mechanistically, TRIM7 directly bound to and ubiquitinated nuclear receptor coactivator 4 (NCOA4) using K48-linked chains, thereby reducing NCOA4-mediated ferritinophagy and ferroptosis of human glioblastoma cells. Moreover, we found that TRIM7 deletion sensitized human glioblastoma cells to temozolomide therapy.

Conclusion: We for the first time demonstrate that TRIM7 modulates NCOA4-mediated ferritinophagy and ferroptosis in glioblastoma cells, and our findings provide a novel insight into the progression and treatment for human glioblastoma.

Keywords: Ferritin; Ferroptosis; Glioblastoma; NCOA4; TRIM7.

Fig. 1

TRIM7 expression is elevated in human glioblastoma cells and tissues. (A) Human normal brain astroglia cells (SVG and HA1800) and human glioblastoma cells (A172 and U87MG) were subjected to IB assay to detect TRIM7 expression. (B) The expression of TRIM7 protein in human glioblastoma tissues. N = 6 for each group. All values are presented as the mean ± S.D., *P < 0.05 is considered statistically significant.

Fig. 2

TRIM7 silence suppresses growth and induces death of human glioblastoma cells. (A) Stable TRIM7-deficient cells were subjected to IB assay to validate the efficiency. (B) Stable TRIM7-deficient or control cells were cultured and exposed to CCK-8 assay to evaluate cell viability. (C) Colony formation assay. (D) Cell proliferation was assessed by BrdU assay at 72 h. (E) LDH releases from human glioblastoma cells to the medium at 72 h. N = 6 for each group. All values are presented as the mean ± S.D., *P < 0.05 is considered statistically significant.

Fig. 3

TRIM7 overexpression accelerates growth and inhibits death of human glioblastoma cells. (A) Stable TRIM7-overexpressing cells were subjected to IB assay to validate the efficiency. (B) Stable TRIM7-overexpressing or control cells were cultured and exposed to CCK-8 assay to evaluate cell viability. (C) Colony formation assay. (D) Cell proliferation was assessed by BrdU assay at 72 h. (E) LDH releases from human glioblastoma cells to the medium at 72 h. N = 6 for each group. All values are presented as the mean ± S.D., *P < 0.05 is considered statistically significant.

Fig. 4

TRIM7 silence causes intracellular iron accumulation and ferroptosis of human glioblastoma cells. (A) Stable TRIM7-deficient cells were treated with inhibitors of ferroptosis (Fer-1 and Lip-1), necroptosis (NSA) and apoptosis (Z-VAD), and then cell viability was measured using CCK-8 assay at 72 h. (B) LDH releases from human glioblastoma cells to the medium at 72 h. (C) Relative LPO level. (D) Quantification of MDA content. (E–F) The levels of H₂O₂ and superoxide anion in human glioblastoma cells. (G–H) Intracellular ferrous iron and LIP levels. N = 6 for each group. All values are presented as the mean ± S.D., *P < 0.05 is considered statistically significant.

Fig. 5

TRIM7 overexpression reduces ferroptosis of human glioblastoma cells. (A) TRIM7-overexpressing or control cells were stimulated with erastin or RSL3 for 72 h, and relative LPO level was determined. (B) Quantification of MDA content. (C) Cell viability was measured using CCK-8 assay. (D) LDH releases from human glioblastoma cells to the medium. N = 6 for each group. All values are presented as the mean ± S.D., *P < 0.05 is considered statistically significant.

Fig. 6

TRIM7 silence promotes NCOA4-mediated ferritinophagy and ferroptosis of human glioblastoma cells. (A–C) Relative protein levels of TFR, TF, FPN1, IRP2, FTH1 and NCOA4 in human glioblastoma cells with or without TRIM7 deficiency. (D) TRIM7 and NCOA4 mRNA levels in human glioblastoma cells. (E) Stable TRIM7-deficient cells were treated with 3-MA or CQ to inhibit autophagy, and then subjected to IB assay to detect FTH1 protein. (F) Stable TRIM7-deficient cells were infected with or without shNCOA4, and then subjected to IB assay to detect FTH1 protein. (G–H) Ferrous iron and LIP levels in stable TRIM7-deficient cells with autophagic inhibition or NCOA4 silence. (I–J) Quantification of MDA content. (K–N) Cell viability and LDH releases. N = 6–10 for each group. All values are presented as the mean ± S.D., *P < 0.05 is considered statistically significant.

Fig. 7

TRIM7 directly binds to and ubiquitinates NCOA4 using K48-linked chains. (A) His-Ub and HA-NCOA4 plasmids were co-transfected into HEK293T cells with or without Flag-TRIM7 plasmid, and then cell lysates were prepared to IP assay with anti-HA antibody-conjugated agarose beads. (B) The Flag-TRIM7 and HA-NCOA4 proteins were mixed with ubiquitin conjugation mixture at 30 °C for 1 h, and then subjected to IP assay with anti-HA antibody-conjugated agarose beads. (C) HA-NCOA4 plasmid-transfected HEK293T cells with or without TRIM7 overexpression were transfected with different ubiquitin mutant plasmids, and then cell lysates were prepared to IP assay with anti-HA antibody-conjugated agarose beads. (D) Stable TRIM7-overexpressing A172 cells were treated with CHX to inhibit protein synthesis, and then NCOA4 protein levels were determined by IB assay. (E) Stable TRIM7-overexpressing A172 cells were treated with or without MG132, and then NCOA4 protein levels were determined. (F–G) HA-NCOA4 plasmid was co-transfected into HEK293T cells with different mutant TRIM7 plasmids, and then cell lysates were prepared to IB assay. (H) HA-NCOA4 plasmid was co-transfected into HEK293T cells with different truncated TRIM7 plasmids, and then cell lysates were prepared to IP assay with anti-Flag antibody-conjugated agarose beads. N = 6 for each group. All values are presented as the mean ± S.D., *P < 0.05 is considered statistically significant.

Fig. 8

TRIM7 deletion sensitizes human glioblastoma cells to TMZ therapy. (A) TRIM7-KO or WT A172 cells were treated with TMZ, and cell viability was detected using CCK-8 assay at indicating times. (B) Cell proliferation was assessed by BrdU assay at 36 h. (C) LDH releases from human glioblastoma cells to the medium at 36 h. (D) Tumor volume over time from mice inoculated with TRIM7-KO or WT A172 cells in the presence of TMZ treatment. (E) Tumor weight at 4 weeks from mice inoculated with TRIM7-KO or WT A172 cells in the presence of TMZ treatment. (F) Stable TRIM7-deficient or control GSCs were cultured and exposed to CCK-8 assay to evaluate cell viability. (G) Cell proliferation was assessed by BrdU assay at 72 h. (H) LDH releases from human GSCs to the medium at 72 h. (I) Tumor volume over time in PDX models with TMZ treatment. (J) Tumor weight at 4 weeks in PDX models with TMZ treatment. N = 6 for each group. All values are presented as the mean ± S.D., *P < 0.05 is considered statistically significant.

Fig. 9

A proposed molecular model of TRIM7 in regulating ferroptosis of human glioblastoma cells. TRIM7 directly binds to and ubiquitinates NCOA4 using K48-linked chains and subsequently reduces NCOA4 expression, thereby inhibiting the degradation of ferritin and decreasing intracellular iron levels, which then prevents lipid peroxidation and ferroptotic cell death of human glioblastoma cells.