2668 - TRIB1 Modulates p53 Pathway in GBM Cells via HDAC1 in Response to Radiation Treatment

Purpose/Objective(s): Glioblastoma (GBM, WHO Grade 4) is the most aggressive form of brain tumor with a low 5-year survival rate of 12 to 15 months. The transcription factor P53 that functions as a tumor suppressor is regulated through protein-protein interactions as well as post-translational modifications. According to TCGA, the p53 pathway is found deregulated in 84% of GBM cases. It is interesting to note that TP53 mutations found in 21-54% of different molecular subtypes of GBM are not correlated with patient survival. However, aberrant regulation of the p53 pathway is implicated in tumor progression and treatment response of these tumors. In that vein, we have previously shown that the pseudokinase TRIB1 could modulate p53 functions by associating with HDAC1 (a histone deacetylase) and COP1 (an E3 ubiquitin ligase) in GBM cells. Previous studies in non-small cell lung carcinoma have shown that TRIB1 reduces p53 activity via HDAC1-mediated deacetylation. In this study, we have explored the biochemical mechanisms of TRIB1- and HDAC1-mediated modulation of p53 in irradiated p53-wild type primary GBM cells.

Materials/Methods: Patient derived xenograft (PDX) cell line stably overexpressing TRIB1 was created by lentiviral transduction followed by antibiotic selection. A radiation (RT) dose of 3Gy was administered by the X-Rad cabinet system in 2-3 minutes. Protein lysate was collected at indicated time points. Immunoprecipitation was performed utilizing Pierce™ Anti-DYKDDDDK Magnetic Agarose beads. Protein levels were detected by western blotting.

Results: Here we found that basal HDAC1 levels were higher in GBM cell lines compared to normal human astrocytes indicating deregulation of p53 in these cell lines. After RT, the acetylated p53 level was increased in both TRIB1-overexpressing and EV (empty vector) cells after 4 hours but decreased at 24 hours in TRIB1-overexpressing cells suggesting deactivation of p53. HDAC1 protein levels were similarly increased after RT in both cell types, but to a higher extent in TRIB1-overexpressing cells. These data suggest that deactivation of p53 at 24h was mediated by HDAC1-induced deacetylation. P53 protein was induced to higher levels in EV cells after RT than TRIB1-overexpressing cells. In contrast, the MDM2 protein level was robustly increased in TRIB1-overexpressing cells compared to EV cells after RT at longer time points pointing towards increased p53 degradation by ubiquitination in these cells. Exogenous TRIB1 protein levels were also increased after RT. Immunoprecipitation with FLAG-TRIB1 revealed that TRIB1 formed a complex with HDAC1, MDM2 and p53 (three key components of p53 pathway) after RT whereas in control cells MDM2 was barely detectable.

Conclusion: TRIB1 may modulate the p53 pathway through HDAC1-mediated deacetylation and/or MDM2-driven ubiquitination of p53 protein. Further studies are underway for a deeper understanding of the role of TRIB1 in the p53 pathway in GBM.