Functional Study of EYA1 in Glioma Formation and Progression

Glioma is the most common intracranial tumor, and it constitutes more than half of brain tumors. It is also the leading cause of cancer death worldwide. Infiltrative growth of glioma cells makes it difficult to remove whole tumor and results in inadequate therapeutic response and high recurrence. Understanding the functional implications and interplay of molecular changes and elucidating the molecular drivers underlying glioma formation and progression are among the priority of glioma research.

EYA1 is a protein with dual functions. It serves as a transcription cofactor and a phosphatase. EYA1 regulates cell proliferation, survival, and tissue specification, and it is required for normal cranial facial morphogenesis as well as embryonic development of inner ear, kidney, and lung. The reactivation of EYA1 is also involved in numerous tumor formation and development processes. For example, EYA1 has been reported to control gliomagenesis through transcriptionally regulating MYC to maintain glioblastoma stem cell stemness and survival. However, the function of EYA1 in non-stem-like glioma cells is still unclear.

To date, γH2AX, c-Myc, and Notch1 have been identified to be directly dephosphorylated by EYA1 in different biological contexts. However, the substrates specifically related to glioma remains to be recognized. Also, EYA1-centered interactome and targetome have not been uncovered in glioma. The relationship between the EYA1 expression and glioma prognosis is not well understood.

In this study, we firstly systematically analyzed EYA1 expression patterns in different tumors and normal tissues and found that EYA1 served as a novel marker in glioma. EYA1 was explicitly upregulated in oligodendroglioma tumors with IDH mutation and 1p/19q codeletion, and EYA1 expression was positively correlated with patient survival. Our patient tissue array data also suggested that EYA1 expression was elevated in glioma samples and correlated with glioma grades. We further investigated the function of EYA1 in glioma cell growth in vitro and in vivo, and we found that EYA1 suppression inhibited glioma cell proliferation, clonogenicity, tumorsphere formation, and tumor formation, but extended the survival time of mice bearing glioma.

Mechanistically, we uncovered EYA1-regulated interactome, targetome, and phosphoproteome through affinity-mass spectrometry, RNA sequencing, and phosphoproteomics techniques and systematically determined the downstream targets of EYA1 in glioma cells. We found that EYA1 mainly interacted with factors related to cell cycle regulation and DNA damage repair. Transcriptome data also suggested that EYA1 inhibition downregulated the expression of genes related to cell cycle progression, including CCND1, CDKN1A, CDKN1B, and E2F3. Our experimental data also indicated that EYA1 suppression made cell cycle arrest at the G1 phase. Finally, our phosphoproteome results suggested that BCL2L12, a well-known oncogene in glioma, was a potential substrate of EYA1. Moreover, we validated that EYA1 dephosphorylated BCL2L12 at Thr33 to stabilize it by preventing its ubiquitination, which enhances BCL2L12-mediated cell growth and apoptosis resistance in glioma.

In summary, we systematically studied the role of EYA1 in regulating cell growth during glioma formation and progression in this thesis. Our study may provide a new therapeutic target for glioma.

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