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.