TRIBBLES1 (TRIB1) Pseudokinase Induces Treatment Resistance in GBM by Promoting Survival of Tumor Cells through the Akt Pathway

Abstract

<h3>Purpose/Objective(s)</h3> Glioblastoma (GBM; WHO grade IV) is the most aggressive form of glioma with a dismal 5-year survival rate. The current therapies are largely ineffective and therapy resistance is commonly encountered. Therefore, there is an urgent need to identify druggable therapeutic targets in GBM. In this study we identified TRIB1, a Ser/Thr pseudokinase that functions as a scaffold to initiate Ubiquitin Proteasome System-mediated degradation of target proteins in the cell. It has also been shown to activate the MAPK and Akt signaling cascades in various cell types. Reports also suggest that TRIB1 contributes to chemotherapy resistance in various cancers. Therefore, we evaluated the role of TRIB1 in GBM cell proliferation/ survival and its contribution to therapy resistance. <h3>Materials/Methods</h3> We utilized patient-centered reverse translational approach to identify novel therapeutic targets. To this end, TRIB1 was identified by statistical association (Cox regression analysis) of the patient-derived mRNA profiling data publicly available from TCGA GBM cohort. TRIB1 was functionally validated <i>in vitro</i> by generating stable overexpression cell lines by antibiotic selection. TRIB1 was conditionally knocked down by doxycycline induction. Co-immunoprecipitation was performed to evaluate protein-protein interactions. Protein levels were detected by western blotting. Site-directed mutagenesis was performed to mutate specific amino acid. Patient derived primary cell lines overexpressing the wild type and mutant (W337A) Trib1 transgenes were injected intracranially to create tumors in nude mice. Changes in tumor volume and overall survival (OS) were determined. <h3>Results</h3> The mRNA profiling of TCGA GBM cohort revealed that increased <i>TRIB1</i> gene expression was associated with worse OS of GBM patients (HR=1.3 (1.0-1.5); P=0.019). Mice bearing TRIB1 transgene overexpressing tumors had highest tumor volume and lowest OS whereas mice bearing TRIB1-W337A tumors had the highest OS and lower tumor volume when measured. We also observed that overexpression of TRIB1 caused a decrease in apoptosis of primary GBM cell lines after RT and TMZ treatments <i>in vitro</i>. Overexpression of TRIB1 also increased the phosphorylation/activation of Akt in patient derived primary cell lines. Akt activation was similarly decreased after TRIB1 knockdown. TRIB1 bound directly to Akt in these cells. Following PI3K inhibition, Akt phosphorylation was detected to a greater level in TRIB1 overexpressing cells. <h3>Conclusion</h3> Our data suggest that heightened expression of TRIB1 in GBM cells contributes to RT/TMZ resistance by activating the prosurvival Akt signaling cascades. Therefore, targeting TRIB1 mediated Akt activation could provide a therapeutic benefit over targeting Akt directly, which may reduce Akt's oncogenic signal without risking the toxicity associated with available Akt- specific inhibitors.