Targets Identification of the DNA Polymerase Alpha Inhibitor ST1926 in Glioblastoma: A Proteomic Approach

Abstract

Abstract Background: Glioblastoma multiforme (GBM) is one of the most aggressive forms of malignant brain tumors. Despite the available therapies, the median survival rate of GBM patients does not exceed two years. Tumor heterogeneity and the restrictive nature of the blood-brain barrier are major obstacles detected in GBM management. Therefore, there is an eminent need to develop more efficient treatments. Natural and synthetic retinoids have been investigated in several cancer types and showed promising antitumor effects. Synthetic retinoids outweigh their natural counterparts offering improved selectivity to overcome the non-specificity and toxicity of the latter. The atypical adamantyl retinoid ST1926 induces apoptosis and growth inhibition in different cancer types independent of retinoid signaling pathway. We have demonstrated that ST1926 is an inhibitor of the catalytic subunit of DNA polymerase alpha (POLA1) which is involved in the initiation of DNA synthesis in eukaryotic cells. We have also previously shown that in GBM cells, ST1926, at sub-micromolar concentrations, decreased cell viability, induced apoptosis, and reduced POLA1 protein levels. Thus, ST1926 could be a suitable drug to treat GBM. Aims: Our aim is to identify new molecular targets and novel molecular function, biological processes and signaling pathways related to ST1926 mode of action in GBM via utilizing an advanced proteomic approach and bioinformatics platform that enable pathway enrichment analysis. Data from this work would provide better understanding of the underlying mechanisms of ST1926 in GBM therapy. Methods: LC-MS/MS-based shot gun proteomic analysis was performed on three human GBM cell lines with different p53 status: U87MG (wild-type p53), U251 (mutated p53), and U118 (mutated p53) treated with ST1926. Bioinformatics pathway enrichment analysis was performed via Pathway Studio (v 10.01) to interrogate molecular targets and signaling pathways that are modulated by ST1926 in GBM cells. In silico analysis was performed to investigate the association of differentially regulated genes/proteins upon ST1926 treatment on the U251 cell line with tumorigenesis by assessing mRNA and protein levels between normal brain and GBM tissues. Western blotting was conducted to validate some of the proteomic hits. Results: Analysis of the proteomic data led to the identification of numerous targets of ST1926 in GBM cells. A total of 197, 71, and 167 proteins were altered upon ST1926 treatment of U251, U118, and U87 respectively. DNA topoisomerase 2-alpha, Collagen 3 alpha-1(VI) chain, sequestosome-1/p62, and Tubulin alpha-4A were among the common target proteins by ST1926 observed in the three cell lines. We confirmed by Western blot the downregulation of Collagen alpha-1(VI) chain and the upregulation of sequestosome-1/p62 and Tubulin alpha-4A chain protein levels upon ST1926 treatment. Bioinformatic analysis revealed pathways that are modulated by ST1926 related to cell growth, cell cycle, cell death, apoptosis, neoplasia, malignant transformation, DNA replication, DNA damage, and oxidative stress. Several of these ST1926 target proteins were demonstrated to be differentially modulated in normal human brain versus GBM tissues. Conclusion: ST1926 may play a promising role in GBM treatment. This underscores the importance of exploring the different pathways and target proteins that are involved in its mechanism of action.