Koh, May Zie
(2021)
Profiling of gene and exosomal microRNA as therapy targets and biomarkers of cisplatin resistance in triple negative breast cancer stem cell-like subpopulation.
PhD thesis, University of Nottingham.
Abstract
Breast cancer is the leading cause of death in women worldwide. Among different breast cancer subtypes, triple negative breast cancer (TNBC) which is defined by lack of oestrogen receptor, progesterone receptor and human epidermal growth factor receptors 2 possesses more aggressive behaviour than other breast cancer subtypes. Besides, TNBC is strongly associated with recurrence of metastatic breast cancer. One of the effective drugs used for TNBC treatment is cisplatin that inhibits DNA replication and dissociates p63 and p73, the transcription factors that are co-expressed exclusively in TNBC, thus leading to apoptosis of TNBC. However, the efficacy of cisplatin is compromised over time, resulted in cisplatin resistance and eventually cancer relapse. Cancer stem cells (CSCs) was proposed to contribute to the development of cisplatin resistance in breast cancer.
A short non-coding RNA namely microRNA, is now recognised of being capable to interfere with the onset and progression of cancer tumour via epigenetic regulation of gene expression. Furthermore, microRNAs can be packaged into exosome, forming exosomal-miRNAs that can circulate in various body fluid. As such, screening of the dysregulated profile of highly stable exosomal-miRNAs shows potential role in breast cancer diagnosis, prognosis and therapeutic as the acquirement of exosomal-miRNAs is non-invasive. Therefore, this study aims to investigate the role of CSC-like cells in cisplatin resistance, followed by elucidation of the exosomal-miRNAs profile of cisplatin-treated CSC-like cells for the discovery of potential biomarkers to monitor cisplatin resistance in breast cancer.
TNBC cell line, MDA-MB-231 was used in this study. CSC-like cells were first isolated based on expression of breast CSCs surface markers (CD24-/CD44+) from parental cells and the therapeutic effect of cisplatin on CSC-like cells was compared to that of the parental cells first via phenotypic characterisation. Next, dysregulation of CSC-correlated genes in treated CSC-like cells was identified via PCR array. Followed by that, two platforms which were next generation sequencing (NGS) and Nanostring were used to decipher the exosomal-microRNAs profile in treated CSC-like cells.
Under the same cisplatin treatment condition (56μM of cisplatin for 48 hours), treated CSC-like cells retained higher expression of breast CSCs markers (CD24-/CD44+ and ALDH1A1+), displayed lower G2/M phase cell arrest, sub G0/G1 event and late apoptosis/necrosis as compared to treated parental cells. In comparison to treated parental cells, treated CSC-like cells also demonstrated higher self-renewability. In correlation to its phenotypic characteristics, dysregulation of JAK2, SMO, PTCH1, GSK3β, NOTCH2, DLL1 and WNT1 detected in treated CSC-like cells were associated with JAK/STAT, Hh and angiogenesis signalling pathways and were suggested to contribute to higher drug resistance and cell stemness in treated CSC-like cells as compared to treated parental cells. Not only that, cisplatin treatment induced upregulation of exomiR-196a-5p, exomiR-939-5p and downregulation of exomiR-206 solely in treated CSC-like cells, which may attenuate the cytotoxicity of cisplatin, reflected by lower apoptotic event and higher self-renewability in CSC-like subpopulation in MDA via modulation of NOTCH2, GSK3β and TWIST1. Together, these data showed that CSC-like cells were more tolerant to the cytotoxic effect of cisplatin as compared to parental cells which could be modulated by dysregulated CSC-correlated genes and exosomal-miRNAs in treated CSC-like cells.
In addition, exo-miR-30a-5p, miR-28-3p, miR-126-3p and miR-941 were significantly dysregulated in CSC-like subpopulation detected by both NGS and Nanostring. Therefore, this subset of microRNAs could serve as potential candidates for development of prognostic biomarkers and therapeutic targets of cisplatin-resistant TNBC.
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