Introduction: Glioblastoma stem cells (GSC) comprise of a subpopulation of tumor cells that possess the unique ability to self-renew and facilitate tumor growth. We employed in vivo and in vitro phage display biopanning strategies against GSCs to isolate novel targets that may play a role in the maintenance of GSCs.
Methods: In vivo and in vitro phage display biopanning strategies were applied to GSCs with a random library consisting of 7 amino acid length peptides. In vivo biopanning was performed using a combination of negative and positive selection to isolate peptides with specific affinity for binding GSCs grown in culture. In vivo biopanning was performed against GSCs intracranially xenografted into immunodeficient mice. In silico analysis of binding peptides resulted in potential targets specific for GSCs.
Results: One consensus peptide sequence was isolated in both the in vivo and in vitro biopanning strategies. Protein BLAST analysis revealed a match with Eyes Absent protein (Eya1), a transcriptional co-activator involved in cell proliferation, as well as DNA damage repair. We demonstrated that Eya1 is preferentially expressed in GSCs and co-localizes with known GSC markers. Inhibition of Eya1 expression through ribonucleic acid interference inhibited GSC formation as evidenced by decrease in cell viability, tumorsphere formation, and in vivo tumorigenesis. Eya1 was also found to be present in the tumor vasculature, corresponding with endothelial cell markers. Inhibition of EYA1 expression resulted in hindrance of tumor derived endothelial cell formation, indicating a potential role in tumor angiogenesis. Cellular migration and invasion were also decreased with Inhibition of Eya1 expression. Eya1 was found to co-localize with and regulate c-Myc, suggesting Eya1 may interact with c-Myc to induce GSC proliferation.
Conclusions: Phage display biopanning against GSCs isolated Eya1, which regulates c-Myc to play a crucial role in the proliferation and migration of glioblastoma stem cells.
Patient Care: Isolate potential therapeutics targets for the treatment of glioblastoma
Learning Objectives: 1) understand the cancer stem cell hypothesis as it pertains to glioblastoma
2) understand phage display screening techniques and its application to explore novel targets in brain tumors.
3) Understand the role that EYA1 may play in glioblastoma stem cell maintenance and proliferation through regulation of oncogene activity.