TRIM21-SERPINB5 aids GMPS repression to protect nasopharyngeal carcinoma cells from radiation-induced apoptosis

Abstract

Background: The main strategy against nasopharyngeal carcinoma (NPC) is radiotherapy. However, radioresistance mediated recurrence is a leading clinical bottleneck in NPC. Revealing the mechanism of NPC radioresistance will help improve the therapeutic effect.

Methods: In this study, the role of TRIM21 (tripartite motif-containing 21) in NPC receiving ionizing radiation was firstly examined both in vivo and in vitro. Mass spectrometry analysis was performed to identify the downstream targets of TRIM21. NPC cells with TRIM21 or SERPINB5 (serpin family B member 5) overexpression or knockout were used to determine the epistatic relationship among SERPINB5, GMPS (guanine monophosphate synthase) and TRIM21. Flow cytometry, co-immunoprecipitation, western blot and immunofluorescence were employed to strengthen the results. Finally, immunohistochemistry using 4 radiosensitive and 8 radioresistent NPC patient samples was perform to examine the association between SERPINB5 or GMPS expression and patient radio-sensitivity.

Results: As an E3 ligase, TRIM21 was highly expressed in NPC. After ionizing radiation, TRIM21 repressed TP53 expression by mediating GMPS ubiquitination and degradation. Overexpression of TRIM21 protected NPC cells from radiation mediated cell apoptosis in vitro and in vivo. Further analysis revealed that TRIM21 mediated GMPS repression was dependent on SERPINB5, and SERPINB5 served as an adaptor which prevented GMPS from entering into the nucleus and introduced TRIM21 for GMPS ubiquitination. Moreover, the in vitro and in vivo results validated the finding that SERPINB5 promoted NPC cell radioresistance, and the radioresistant patients had higher SERPINB5 expression.

Conclusions: Overall, our data showed that TRIM21-SERPINB5-mediated GMPS degradation facilitated TP53 repression, which promoted the radioresistance of NPC cells. This novel working model related to TP53 suppression provided new insight into NPC radioresistence clinically.

Keywords: Apoptosis; GMPS; Nasopharyngeal carcinoma; SERPINB5; TP53; TRIM21.

Fig. 1

TRIM21 serves as an oncogene in NPC. a Western blot detection of TRIM21 expression in normal NP69 cell line and in NPC cell lines. b TRIM21 expression in healthy controls and patients with NPC in the Gene Expression Omnibus (GEO) dataset (81672252). c CCK-8 assay of HONE1 cells with TRIM21 GOF or LOF. d, e Colony formation assay and the quantification analysis of NPC cells with TRIM21 GOF or LOF. *P < 0.05, **P < 0.01, ***P < 0.001. Mu, mutant; ns, not significant

Fig. 2

TRIM21 protects NPC cells from radiation-induced apoptosis by manipulating the GMPS–TP53 cascade. a Western blot detection of TP53 expression in HONE1 cells after radiation. b Co-immunoprecipitation using anti-FLAG antibody following western blot detection of USP7 and TP53 expression in GMPS–FLAG-overexpressing NPC cells with or without X-ray radiation. c GMPS expression in TRIM21-overexpressing NPC cells with or without X-ray radiation. d Co-immunoprecipitation using anti-MYC antibody following western blot detection of GMPS expression in TRIM21–MYC-overexpressing NPC cells with or without X-ray radiation. e Flow cytometry analysis of Annexin V and PI staining in HONE1 cells with TRIM21 GOF or LOF after X-ray radiation. Annexin⁺PI⁻ cells, which were in the early apoptotic stage, were evaluated and quantified. f, g Quantification of the apoptotic HONE1 f and 5-8F g cells. h, i Clonogenic survival assay of HONE1 cells with TRIM21 GOF h or LOF i. j Absorbance intensity of TRIM21 GOF and LOF tumor cells and their counterpart controls in mice (n = 5 for each group). The tumors were evaluated 2 and 3 weeks, respectively, after implantation, and the mice received radiotherapy (2 Gy daily and a total of 12 Gy) after 2 weeks. k, l The absorbance intensity analysis of tumors in mice. For the co-immunoprecipitation assay, the cells were pre-treated with MG132 to avoid GMPS degradation before harvesting. *P < 0.05, **P < 0.01, ***P < 0.001. Mu, mutant; ns, not significant; IP, immunoprecipitation

Fig. 3

SERPINB5 is indispensable for TRIM21-mediated GMPS–TP53 repression after radiation. a Co-immunoprecipitation following western blotting in NPC cells with SERPINB5–HA and TRIM21–MYC overexpression. b Immunofluorescence staining analysis of SERPINB5 and TRIM21 in NPC cells. c Western blot detection of SERPINB5 expression in normal NP69 cell line and in NPC cell lines. d Western blot detection of GMPS and TP53 in NPC cells with TRIM21 GOF or SERPINB5 LOF. e Co-immunoprecipitation using anti-HA antibody in NPC cells with SERPINB5–HA and GMPS–FLAG overexpression. f Immunofluorescence staining to detect the co-localization of GMPS and SERPINB5 in NPC cells with or without ionizing radiation. g GMPS expression in immune-purified protein by anti-HA antibody from NPC cells with or without TRIM21 LOF. h GMPS expression in immune-purified protein by anti-MYC antibody from NPC cells with or without SERPINB5 LOF. i GMPS expression in NPC cells with TRIM21 or SERPINB5 overexpression. j Immunofluorescence staining of overexpressed GMPS and ubiquitin in HONE1 cells with or without ionizing radiation. The localization of GMPS and was evaluated in condition of SERPINB5 or TRIM21 LOF. For the co-immunoprecipitation assay, MG132 was added into the cell medium before cell harvesting to avoid GMPS degradation. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

SERPINB5 prevents X-ray radiation–induced NPC cell apoptosis. a Flow cytometry analysis of Annexin V and PI staining in HONE1 cells with TRIM21 GOF or LOF after X-ray radiation. Annexin⁺PI⁻ cells were evaluated and quantified. b, c Quantification of apoptotic HONE1 b and 5-8F c cells. d Absorbance intensity of SERPINB5 GOF (top) and LOF (bottom) tumor cells and their counterpart controls in mice (n = 5 for each group). The tumors were evaluated 2 and 3 weeks, respectively, after implantation, and the mice received radiotherapy (2 Gy daily and a total of 12 Gy) after 2 weeks. e, f Absorbance intensity analysis of the tumors in mice. g GFP expression in HONE1 cells with SERPINB5-V2A-GFP overexpression or TRIM21 knockout. h Flow cytometry analysis of GFP⁺ cell percentages in HONE1 and 5-8F cells. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

SERPINB5 expression increases in radioresistant NPC patients. a, b Immunohistochemistry staining of SERPINB5 and GMPS in radiosensitive a and radioresistant b patients. Based on the staining intensity, the images are divided into three grades from weakest to strongest (from 1 to 3, respectively)