- Title
- Molecular characterization of quiescent cancer cells
- Creator
- La, Ting
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2019
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Relapse following initial remission, especially metastasis, is currently a major cause in cancer-related death and occurs in virtually every types of anti-cancer therapy. The tumour recurrence is mainly due to the wake-up of the residual cancer cells, which are remaining quiescent during remission after conventional treatment. Quiescent cancer cells are inherently resistant to cell death and refractory to therapeutic drugs. Specifically targeting quiescent cancer cells by uncover the characteristics would be a promising way to eradicate cancer completely. Nevertheless, the mechanisms responsible for the resistance of quiescent cancer cells to cell death remain largely undefined. This is primarily due to the lack of understanding of their biological characteristics as a consequence of technical hurdles in the isolation and analysis of viable quiescent cells. Quiescent cells characteristically express high levels of the cyclin-dependent kinase (CDK) inhibitor p27. This thesis includes two systems of isolation quiescent cancer cells. First, a model system encompassing an mVenus-tagged p27 mutant lacking the CDK-binding domain (mVenus-p27K⁻) together with a mCherry-tagged truncated mutant of chromatin licensing and DNA replication factor 1 (hCDT1) [mCherry-hCDT1(30/120)] that is exclusively expressed in cells in G0 and G1 phases was employed to isolate putative mVenus-p27K-high/mCherry-hCDT1(30/120)+ mouse melanoma cells. By using this system, we identified a p53-responsive microRNA network that promotes cellular quiescence in both mouse and human melanoma cells. Building on this, we took advantage that quiescent cells are also characteristically negative for the proliferation marker Ki67 to develop a CRISPR/Cas9-based system, in which a green fluorescent protein (EGFP) gene is fused with endogenous CDKN1B, the gene encoding p27, and a red fluorescent protein (mCherry) gene, with endogenous MKI67, the gene encoding Ki67 in the genome of human melanoma cells. By using this system, we have successfully isolated viable p27high/Ki67⁻ human melanoma cells using FACS that were verified to be authentic quiescent cells. This system provides us with an exceptional tool for further characterization of the biological properties of quiescent melanoma cells. By analysing the transcripts and protein profiles in the isolated quiescent and cycling cancer cells, we found that in comparison with cycling cancer cells, the quiescent cancer cells generate energy relying on oxidative phosphorylation (OXPHOS) rather than glycolysis. Of note, by employing OXPHOS inhibitor, quiescent melanoma cells either arrested by serum starvation or by various MAPK inhibitors are dramatically sensitive to apoptosis. Overall, towards targeting quiescent cancer cells by inhibiting OXPHOS may be exploited to overcome cellular quiescence induced cancer recurrence.
- Subject
- quiescence; p53; CRISPR/Cas9; microRNAs; G0; miR-27b-3p; miR-455-3p; cell cycle; tumour dormancy; p27; Ki67
- Identifier
- http://hdl.handle.net/1959.13/1407578
- Identifier
- uon:35744
- Rights
- Copyright 2019 Ting La
- Language
- eng
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 6 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 637 KB | Adobe Acrobat PDF | View Details Download |