Oncogenic Activities of Tribbles1 (TRIB1) Pseudokinase Overexpressed in GBM are Mediated by Protein-Protein Interactions

Abstract

Glioblastoma (GBM) is the most aggressive form of glioma with a low 5-year survival rate. The current treatments are inadequate and crippled by therapy resistance. Therefore, there is an unmet need to identify druggable therapeutic targets in GBM. In this study we identified TRIB1, a Ser/Thr pseudokinase that acts as a scaffold to initiate Ubiquitin Proteasome System-mediated degradation of its substrates. We and others have found that TRIB1 activates the canonical MAPK and Akt signaling cascades. Previous reports also suggest that TRIB1 contributes to chemotherapy resistance in various cancers. Therefore, we evaluated oncogenic roles of TRIB1 in GBM cells and its contribution to therapy resistance. Patient-centered reverse translational approach was utilized to identify novel therapeutic targets. To this end, TRIB1 was identified by statistical association (Cox regression analysis) of the patient-derived gene expression profiling data publicly available from TCGA GBM cohort. TRIB1 was functionally validated in vitro by generating stable overexpression cell lines (patient-derived) by antibiotic selection. Conditional knockdown of TRIB1 was achieved by doxycycline induction. Protein-protein interactions were evaluated by co-immunoprecipitation. Protein levels were detected by western blotting. Changes in tumor volume and overall survival (OS) were calculated. The mRNA profiling of TCGA GBM cohort revealed that increased TRIB1 gene expression was associated with worse OS of GBM patients [HR = 1.3 (1.0-1.5); P = 0.019]. The same analyses in our institutional cohort revealed a similar association. Mice bearing TRIB1 transgene overexpressing tumors had the increased tumor volume and shorter OS compared to empty vector control at the end of experiment. Overexpression of TRIB1 increased the phosphorylation/activation of ERK and Akt in patient-derived primary cell lines. Akt but not ERK activation was decreased after TRIB1 knockdown. TRIB1 bound directly to ERK and Akt in these cells. TRIB1 also formed a complex with p53, COP1 and HDAC1 in patient-derived primary cell lines. This protein-protein interaction was independent of TP53 mutation status. Our data suggest that TRIB1 overexpressed in GBM executes various oncogenic functions through interaction with different proteins. Activating ERK signaling, can induce cell proliferation. Similarly, by activating Akt it can cause prosurvival effects. Finally, by associating with HDAC1 and COP1, TRIB1 can modulate p53 function. All these protein-protein interactions ultimately contribute to chemoradiotherapy resistance in GBM cells. We are currently developing small molecule inhibitors targeting the above-mentioned interactions of TRIB1 to overcome therapeutic resistance.