| Author: | Liu, Xian |
| Title: | Serine-347 phosphorylated Musashi-1 stimulates cell cycle progression, cytoskeletal remodeling, and tumorigenicity of glioblastoma multiforme |
| Advisors: | Lin, Liang-ting (HTI) Law, Ka Wai Helen (HTI) |
| Degree: | Ph.D. |
| Year: | 2024 |
| Subject: | RNA-protein interactions Glioblastoma multiforme Brain -- Tumors Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Health Technology and Informatics |
| Pages: | xxxii, 220 pages : color illustrations |
| Language: | English |
| Abstract: | Glioblastoma (GBM) is a highly aggressive primary brain cancer known for its insensitivity to anti-growth signals and propensity for relapse. GBM cells exhibit uncontrolled proliferation, often leading to their exit from the cell cycle and entry into a dormant state called G1. The recurrence of GBM has been linked to enhanced invasion and tumor regeneration with cytoskeletal reorganization. The RNA-binding protein, Musashi-1 (MSI1), is renowned for its heightened expression and oncogenic characteristics in diverse cancer types, including GBM. However, the precise mechanisms by which MSI1 influences GBM’s resistance to growth-inhibiting signals and relapse remain largely elusive. Targeting key regulators that govern these fundamental hallmarks of cancer may offer a promising therapeutic approach for GBM. My inquiry commenced by scrutinizing the role of the relatively unexplored C-terminal domain of MSI1 in GBM. Three human GBM cell lines, including U87-MG, 05MG, and S1R1, were used in the in vitro experiments. Using Phos-Tag and customized antibody, I identified serine-347 of MSI1 as a novel phosphorylation site. Moreover, the global bioinformatics screenings using MSI1 overexpressed cells unveiled a strong correlation between MSI1 and cell cycle regulation in GBM. The transcriptome analysis between wild-type MSI1 and its loss-of-phosphorylation mutation (MSI1-S347A) form revealed a robust association between cytoskeletal-related pathways and the positive regulation of cancer cell growth as a result of phosphorylated MSI1 (pMSI1(S347)). pMSI1(S347), instead of lacking S347 phosphorylation, was revealed to predominantly facilitate DNA replication using flow cytometry analysis with cell synchronization. An RNase footprinting assay demonstrated that pMSI1(S347) translationally suppressed p21 expression. Furthermore, the attenuation of elevated p21 levels in S347A cells resulted in the abrogation of the G1 phase arrest. Therefore, pMSI1(S347) promotes the cell cycle progression in GBM cells by suppressing the expression of p21. A noticeable decrease in migration and invasion was observed in cells lacking S347 phosphorylation using wound healing and Transwell assay. Cells without S347 phosphorylation had reduced F-actin formation and impaired cytoskeletal polymerization in immunofluorescent images. Furthermore, pMSI1(S347) significantly enhanced GBM initiation in vitro and in vivo, validated by colony formation assays, 3D cell culture, and orthotopic implantation in a mouse GBM model. The expression of CD133 (Prominin-1), a well-established marker of tumor-initiating cells, was concurrently upregulated along with pMSI1(S347), leading to increased clonogenicity, as evidenced by confocal microscopy. Moreover, quantitative reverse transcription PCR analysis revealed an upregulation of a few let-7 microRNA members, including let-7b, e, and i, in the absence of MSI1 phosphorylation. Suppression of let-7i expression in S347A cells restored the oncogenic features, including cytoskeletal remodeling, spheroid growth, and CD133 accumulation. Thus, S347 phosphorylation stimulates cytoskeletal reorganization and tumorigenicity of GBM through downregulating let-7 expression. This study elucidates a crucial correlation between the GBM malignancy and a novel oncogenic post-translational modification at MSI1-S347. The phosphorylation of MSI1-S347 enhances the cell cycle progression through p21 signaling and augments tumorigenicity via the let-7 pathway in GBM. Further investigation into the identification of potential mediators, such as inhibitors targeting the kinase responsible for phosphorylating MSI1-S347 or antagonistic agents reducing S347 phosphorylation, hold promise for offering GBM patients a more favorable prognosis. |
| Rights: | All rights reserved |
| Access: | open access |
Copyright Undertaking
As a bona fide Library user, I declare that:
- I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
- I will use the Database for the purpose of my research or private study only and not for circulation or further reproduction or any other purpose.
- I agree to indemnify and hold the University harmless from and against any loss, damage, cost, liability or expenses arising from copyright infringement or unauthorized usage.
By downloading any item(s) listed above, you acknowledge that you have read and understood the copyright undertaking as stated above, and agree to be bound by all of its terms.
Please use this identifier to cite or link to this item:
https://theses.lib.polyu.edu.hk/handle/200/12816