Cellular and molecular signatures of cancer stem-like cells in hepatocellular carcinoma / Ain Zubaidah Ayob
Hepatocellular carcinoma (HCC) remains a highly lethal cancer with increasing global incidence and mortality. Cancer stem cells (CSCs), or cancer stem-like cells (CSLCs) have been considered as key drivers of cancer progression, and the enrichment of this subpopulation is associated with aggressi...
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| Format: | Thesis |
| Published: |
2022
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| Online Access: | http://studentsrepo.um.edu.my/14787/5/ain_zubidah.pdf http://studentsrepo.um.edu.my/14787/ |
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| Summary: | Hepatocellular carcinoma (HCC) remains a highly lethal cancer with increasing global
incidence and mortality. Cancer stem cells (CSCs), or cancer stem-like cells (CSLCs)
have been considered as key drivers of cancer progression, and the enrichment of this
subpopulation is associated with aggressive and resistance cancer nature that causes
metastasis and tumour relapse. Better therapeutic strategy specifically targeting CSCs is
required for a more effective cancer treatment; however, the cellular and molecular
features as well as the regulatory mechanisms underlying CSCs remain unclear. This
study aims to investigate the molecular regulatory networks underlying the CSLCs in
HCC through their cellular and molecular signatures. Using in vitro approach as the
strategy to enrich CSLCs, three culture models were established using HepG2 cells
including 1) tumoursphere (3D-HepG2), 2) TGF-β1-induced epithelial-mesenchymal
transition (TβT-HepG2) and 3) cisplatin-induced resistant (CisR-HepG2) model. Using
serum-free media supplemented with 20 ng/ml of bFGF and EGF, 3D-HepG2 which was
established under low-adherent condition exhibited high sphere formation, proliferation,
and self-renewal capacity, with culture for 15 days result in high tumoursphere yield.
TβT-HepG2, established by treatment with 5 ng/ml TGF-β1 for 72 hours at 5000
cells/cm2 seeding density, displayed mesenchymal-like morphology with Vimentin, Snail,
Twist and Zeb1 upregulation corresponding with epithelial-mesenchymal (E-M) hybrid
characteristics. CisR-HepG2 was generated through three cycles of 72-hour cisplatin
treatment at IC50 concentration (6.62 ± 1.103 μM), which acquired resistance evident by
a 3.4-fold increase in IC50 value (22.55 ± 0.33 μM) and upregulation of drug transporters
and anti-apoptotic genes. The enrichment of CSC-like population in these models was
iv
evidenced by the increased fractions of ALDH+ population compared to parental cells,
whereas enhanced CD133 expression was only observed in the 3D-HepG2 model.
Comparatively, these models exhibited common features associated with increased
stemness quiescence, invasion/migration and resistance based on gene expression
analysis, whereas the proliferation and EMT features were differential between them.
Subsequently, integrated miRNA and mRNA analysis was performed to elucidate the key
regulatory networks underlying different CSC-like models. Microarray profiling revealed
22 miRNAs and diverse set of mRNAs were differentially expressed (DE) in the HepG2
derived CSC-like models compared to parental, with exclusive and overlapping
signatures observed between the three models. Seven miRNAs (miR-122-5p, miR-181a-
5p, miR-125a-5p, miR-29a-3p, miR-23a-3p, miR-92a-3p and miR-483-3p) identified to
be highly differentially expressed (>10 fold-change, p<0.05) with miR-19b-3p, miR-23a-
3p and miR-483-3p being commonly expressed by all the three models, suggest that these
CSLC-associated miRNAs might be critical in CSC regulatory networks. Functional
analysis revealed distinct regulatory networks including ‘TNF-signalling pathway’,
‘cAMP signalling’, ‘Rap1 signalling pathway’, ‘Proteoglycans in cancer’, ‘Systemic
lupus erythematosus’ and ‘FoxO signalling pathway’, with ‘Focal adhesion’, ‘p53
signalling’, and ‘Cell cycle’ were identified as common pathways in these CSC-like
models through the regulatory roles of these DE miRNAs. Collectively, this study
revealed distinct yet overlapping miRNA-mRNA signatures and regulatory networks
underlying CSC drives the CSC heterogeneity. The identified molecular regulators and
pathways of CSLCs from this study provide the avenues for future investigations and may
facilitate the development of therapeutic approach for CSC-targeted therapy in HCC
treatment. |
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