O-GlcNAc-modified HOXA9 suppresses ferroptosis via promoting UBR5-mediated SIRT6 degradation in nasopharyngeal carcinoma

Abstract

Background: Nasopharyngeal carcinoma (NPC) is the most common malignancy of the nasopharynx. Ferroptosis induction shows anti-tumor activities in cancers including NPC. Elucidating the regulatory mechanism of ferroptosis is crucial for developing targeted therapeutic strategies for NPC.

Methods: The GEO dataset (GSE68799) was used to analyze HOXA9 expression in NPC. Cell viability, levels of MDA, total iron, Fe²⁺ and GSH, and lipid peroxidation were examined for ferroptosis evaluation. O-GlcNAcylation levels on HOXA9 and ubiquitination levels on SIRT6 were detected by immunoprecipitation. ChIP and luciferase assays were applied for determining the interaction of HOXA9 and UBR5. The interaction between UBR5 and SIRT6, OGT and HOXA9 were evaluated by Co-IP assays. A subcutaneous NPC mouse model was established to explore whether knockdown of HOXA9 or UBR5 regulates tumor growth in vivo.

Results: HOXA9 was highly expressed in NPC, and knockdown of HOXA9 elevated total iron, Fe²⁺ and lipid peroxidation and reduced GSH and NPC cell viability. O-GlcNAcylation stabilized HOXA9 and facilitated its nuclear translocation in NPC cells. HOXA9 directly bound to UBR5 promoter to increase its expression, thus accelerating ubiquitination and degradation of SIRT6. HOXA9 restrained ferroptosis via promoting UBR5 expression, and UBR5 suppressed ferroptosis through promotion of SIRT6 ubiquitination and degradation. Knockdown of HOXA9 or UBR5 promoted ferroptosis and inhibited NPC growth in mice.

Conclusion: O-GlcNAc-modified HOXA9 inhibits ferroptosis by enhancing UBR5 expression and ubiquitination and degradation of SIRT6 in NPC cells, thus accelerating NPC progression. Our study provides potential therapeutic targets for NPC treatment.

Keywords: Ferroptosis; HOXA9; Nasopharyngeal carcinoma; O-GlcNAcylation; SIRT6; UBR5.

Fig. 1

The expression of HOXA9 was increased in NPC. (A) GEO dataset (GSE68799) analysis of differentially expressed genes in NPC (normal, n = 4; NPC, n = 42). (B) qRT-PCR analysis of TMEM68, SUOX, SLC20A2, RAC1, NFE2L2, HOXA9, FBXO32, FAM110A, DNAJB6 and BAHCC1 in NP69, 5-8F, NPC/HK1, HNE3 and C666-1 cells. (C) GEO database (GSE68799) analysi of HOXA9 expression in NPC. (D) IHC detected HOXA9 level in tumor tissues of patients. (Scale bar, 100 and 50 µm). Western blotting (E) and qRT-PCR (F) analysis of HOXA9 in NP69, 5-8F, NPC/HK1, HNE3 and C666-1 cells. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Fig. 2

Knockdown of HOXA9 enhanced ferroptosis in NPC cells. NPC/HK1 and C666-1 cells were transfected with sh-NC, sh-HOXA9#1 or sh-HOXA9#2 and treated with Erastin at 0, 2.5, 5, 10, 20 or 40 µM or RSL3 at 0.1, 0.5, 1, 5 or 10 µM for 24 hours in the presence or absence of Fer-1 at 5 μM. DMSO was used as the solvent control. qRT-PCR (A) Cell viability was evaluated with CCK-8 after treatment with different concentrations of Erastin and RSL3. Relative MDA (B), total iron (C), Fe²⁺ (D), and GSH (E) in NPC/HK1 and C666-1 cells after Erastin (10 μM) or RSL3 (1 μM) treatment. (F and G) Measurement of lipid peroxidation by flow cytometry after Erastin (10 μM) or RSL3 (1 μM) treatment. (H and I) Western blotting analysis of ACSL4, GPX4, SIRT6 and GAPDH after Erastin (10 μM) or RSL3 (1 μM) treatment. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Fig. 3

O-GlcNAcylation stabilized HOXA9 and facilitated its nuclear translocation in NPC cells. (A) Two potential O-GlcNAc modification sites (Thr4 and Ser127) on HOXA9 was predicated through YinOYang-1.2. (B) HOXA9 was immunoprecipitated, and its O-GlcNAcylation was examined by Western blotting in NP69, NPC/HK1 and C666-1 cells. (C) Western blotting analysis of HOXA9 and GAPDH in NPC/HK1 and C666-1 cells treated with control, TMG and OSMI-4. (D) MYC-OGT and FLAG-HOXA9 were transfected into HEK293T cells, and FLAG-HOXA9 was immunoprecipitated. The abundance of MYC-OGT and FLAG-HOXA9 was determined with Western blotting. (E) HEK293T cells were transfected with FLAG-HOXA9, FLAG-HOXA9-T4A or FLAG-HOXA9-S127A. FLAG was immunoprecipitated, and its O-GlcNAcylation was examined by Western blotting. (F) Homology analysis of HOXA9 in different species. (G) Protein levels of HOXA9 in cells transfected with MYC-OGT, FLAG-HOXA9 or FLAG-S127A and treated with CHX for 0, 2, 4, 8 or 16 hours. (H) IF staining of HOXA9 in NPC/HK1 and C666-1 cells treated with control, TMG and OSMI-4. Scale bar, 25 µm * p < 0.05, ** p < 0.01 and *** p < 0.001.

Fig. 4

HOXA9 directly bound to the promoter of UBR5 to promote its expression in NPC cells. (A) qRT-PCR analysis of SIRT6 in NPC/HK1 and C666-1 cells transfected with sh-NC, sh-HOXA9#1 or sh-HOXA9#2. (B) Multiple potential ubiquitination modification sites in SIRT6 was predicted through BioGRID database analysis. (C) qRT-PCR analysis of MDM2, RNF20, RNF40, STUB1, UBE3A, HECTD1, UBR5, HECTD4, HERC2, HUWE1, MYCBP2 and SKP2 in NPC/HK1 and C666-1 cells transfected with sh-NC, sh-HOXA9#1 or sh-HOXA9#2. (D) GEPIA2 database analysis of UBR5 expression in head and neck squamous cell carcinoma. qRT-PCR (E) and Western blotting (F and G) analysis of UBR5 in NP69, 5-8F, NPC/HK1, HNE3 and C666-1 cells. qRT-PCR (H) and Western blotting (I and J) analysis of UBR5 in NPC/HK1 and C666-1 cells transfected with sh-NC, sh-HOXA9#1 or sh-HOXA9#2. (K) Prediction of three binding sites for HOXA9 in the promoter of UBR5 through JASPAR. (L) The binding site for HOXA9 in the promoter of UBR5 was determined by ChIP assays. (M) The luciferase activity of UBR5 reporters in NPC/HK1 and C666-1 cells transfected with sh-NC, sh-HOXA9#1 or sh-HOXA9#2. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Fig. 5

UBR5 facilitated the ubiquitination and degradation of SIRT6 to reduce its expression in NPC cells. qRT-PCR (A) and Western blotting (B) analysis of UBR5 in NPC/HK1 and C666-1 cells transfected with empty vector or UBR5-overexpressing vector. (C) Protein levels of SIRT6 in cells transfected with empty vector or UBR5-overexpressing vector and UBR5-overexpressing cells treated with MG132. (D) Co-IP assays were applied to evaluate the interaction between UBR5 and SIRT6. (E) SIRT6 ubiquitination analysis in cells transfected with empty vector or UBR5-overexpressing vector. (F) Protein levels of UBR5 and SIRT6 in cells transfected with empty vector or UBR5-overexpressing vector and treated with CHX for 0, 2, 4 or 8 hours. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Fig. 6

O-GlcNAc-modified HOXA9 inhibited ferroptosis via targeting UBR5 in NPC cells. NPC/HK1 and C666-1 cells were transfected with Vector + sh-NC, HOXA9 + sh-NC or HOXA9 + sh-UBR5 and treated with Erastin at 10 μM and OSMI-4 at 20 μM for 24 hours. DMSO was used as the solvent control. (A) Cell viability was evaluated with CCK-8. Relative MDA (B), total iron (C), Fe²⁺ (D), and GSH (E) in NPC/HK1 and C666-1 cells. (F and G) Measurement of lipid peroxidation. (H and I) Western blotting analysis of UBR5, ACSL4, GPX4, SIRT6 and GAPDH. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Fig. 7

UBR5 suppressed ferroptosis through SIRT6 in NPC cells. NPC/HK1 and C666-1 cells transfected with sh-NC, sh-UBR5 or sh-UBR5 + sh-SIRT6 were treated with Erastin at 10 μM. DMSO was used as the solvent control. (A) Cell viability was evaluated with CCK-8 after Erastin (10 μM) treatment. Relative MDA (B), total iron (C), Fe²⁺ (D), and GSH (E) in NPC/HK1 and C666-1 cells after Erastin (10 μM) treatment. (F and G) Measurement of lipid peroxidation by flow cytometry after Erastin (10 μM) treatment. (H and I) Western blotting analysis of ACSL4, GPX4, SIRT6, NCOA4, FTH1 and GAPDH after Erastin (10 μM) treatment. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Fig. 8

Knockdown of HOXA9 or UBR5 promoted ferroptosis through SIRT6 and repressed NPC growth in mice. NPC/HK1 and C666-1cells were lentivirally transfected with sh-NC, sh-HOXA9 or sh-UBR5 and subsequently subcutaneously injected into mice. Mice were administrated with vehicle or Erastin. (A) Photos of excised subcutaneous tumors. (B) Tumor volume (n = 6 per group). (C) Tumor weight (n = 6 per group). (D and E) Protein levels of UBR5 and SIRT6 in tumor tissues. (F and G) Protein levels of ACSL4 and GPX4 in tumor tissues. (H) IHC staining of Ki-67 and GPX4 (Scale bar, 50 µm). * p < 0.05, ** p < 0.01 and *** p < 0.001.