Cellular and molecular signatures of cancer stem-like cells in hepatocellular carcinoma / Ain Zubaidah Ayob

Ain Zubaidah, Ayob (2022) Cellular and molecular signatures of cancer stem-like cells in hepatocellular carcinoma / Ain Zubaidah Ayob. PhD thesis, Universiti Malaya.

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Abstract

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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