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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.

SUBMITTER: Zhang P

PROVIDER: S-EPMC6995195 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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

Zhang Panpan P Li Xiaomin X He Qiuping Q Zhang Lulu L Song Keqing K Yang Xiaojing X He Qingmei Q Wang Yaqin Y Hong Xiaohong X Ma Jun J Liu Na N

Journal of biomedical science 20200131 1

<h4>Background</h4>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.<h4>Methods</h4>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 downstrea ...[more]

PMID: 32005234

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Project description:Nasopharyngeal carcinoma (NPC) has a particularly high prevalence in southern China, southeastern Asia and northern Africa. Radiation resistance remains a serious obstacle to successful treatment in NPC. This study aimed to explore the metabolic feature of radiation-resistant NPC cells and identify new molecular-targeted agents to improve the therapeutic effects of radiotherapy in NPC. Methods: Radiation-responsive and radiation-resistant NPC cells were used as the model system in vitro and in vivo. Metabolomics approach was used to illustrate the global metabolic changes. 13C isotopomer tracing experiment and Seahorse XF analysis were undertaken to determine the activity of fatty acid oxidation (FAO). qRT-PCR was performed to evaluate the expression of essential FAO genes including CPT1A. NPC tumor tissue microarray was used to investigate the prognostic role of CPT1A. Either RNA interference or pharmacological blockade by Etomoxir were used to inhibit CPT1A. Radiation resistance was evaluated by colony formation assay. Mitochondrial membrane potential, apoptosis and neutral lipid content were measured by flow cytometry analysis using JC-1, Annexin V and LipidTOX Red probe respectively. Molecular markers of mitochondrial apoptosis were detected by western blot. Xenografts were treated with Etomoxir, radiation, or a combination of Etomoxir and radiation. Mitochondrial apoptosis and lipid droplets content of tumor tissues were detected by cleaved caspase 9 and Oil Red O staining respectively. Liquid chromatography coupled with tandem mass spectrometry approach was used to identify CPT1A-binding proteins. The interaction of CPT1A and Rab14 were detected by immunoprecipitation, immunofluorescence and in situ proximity ligation analysis. Fragment docking and direct coupling combined computational protein-protein interaction prediction method were used to predict the binding interface. Fatty acid trafficking was measured by pulse-chase assay using BODIPY C16 and MitoTracker Red probe. Results: FAO was active in radiation-resistant NPC cells, and the rate-limiting enzyme of FAO, carnitine palmitoyl transferase 1 A (CPT1A), was consistently up-regulated in these cells. The protein level of CPT1A was significantly associated with poor overall survival of NPC patients following radiotherapy. Inhibition of CPT1A re-sensitized NPC cells to radiation therapy by activating mitochondrial apoptosis both in vitro and in vivo. In addition, we identified Rab14 as a novel CPT1A binding protein. The CPT1A-Rab14 interaction facilitated fatty acid trafficking from lipid droplets to mitochondria, which decreased radiation-induced lipid accumulation and maximized ATP production. Knockdown of Rab14 attenuated CPT1A-mediated fatty acid trafficking and radiation resistance. Conclusion: An active FAO is a vital signature of NPC radiation resistance. Targeting CPT1A could be a beneficial regimen to improve the therapeutic effects of radiotherapy in NPC patients. Importantly, the CPT1A-Rab14 interaction plays roles in CPT1A-mediated radiation resistance by facilitating fatty acid trafficking. This interaction could be an attractive interface for the discovery of novel CPT1A inhibitors.

Dataset Information

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

Publications

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

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