Almozyan, Sheema
(2021)
FLYWCH1, a novel transcription regulator with potential tumour suppressor activity.
PhD thesis, University of Nottingham.
Abstract
Colorectal cancer (CRC) remains the world's third most deadly cancer. The molecular mechanisms underlying cancer development are multifactorial; however, hyperactivation of the Wnt pathway is the most common and primary molecular driver of this disease. Therefore, a better characterisation of the molecular mechanisms underlying the differences of Wnt/β-catenin signalling pathway action in normal versus cancer cells is warranted. Identifying specific protein(s) regulating Wnt/β-catenin differentially in healthy versus cancer cells are particularly of clinical interest.
Previous work in Dr Nateri's lab used a modified yeast two-hybrid Ras-Recruitment System (RRS) and identified several new proteins that bind β-catenin protein. One of the β-catenin interacting proteins was FLYWCH1, a previously uncharacterised protein product of the human FLYWCH1 gene. Hitherto, only a few published studies investigated the interplay of physiological and molecular mechanisms of FLYWCH1. For example, FLYWCH1-mediated transcriptional regulation was particularly crucial for the cardiovascular system. Another study reported that in humans, FLYWCH1 mutation variants might be deleterious and associated with familial mitral valve prolapse (MVP) in humans.
Furthermore, the roles of FLYWCH transcription factors (known as FLH-1 and FLH- 2) regulating microRNAs (miRNAs) were previously reported in C. elegans. Therefore, the FLYWCH1 protein likely plays an essential role in regulating gene expression. Our lab has also shown that overexpression of FLYWCH1 antagonises β-catenin/TCF4 signalling during cell polarity/migration in cultured CRC cells and influences the AML cells proliferation.
However, we are beginning to provide insights into the molecular mechanisms through which the FLYWCH proteins function. More recent unpublished in-situ hybridisation (ISH) data from our lab reported the high Flywch1 expressing population of normal and adjacent tumour-free crypt-based cells (2-6 cells), to be the stem cells and transiently amplifying cells. In contrast, Flywch1 expression was not detected in differentiated epithelial cells in the villi and highly down-regulated at the crypt-based cells of a tumour in ApcMin mouse.
Considering the FLYWCH1 expression data, we hypothesised that FLYWCH1 might play a critical role in regulating Wnt/β-catenin mediated colon/intestinal development and tumour formation activities. Therefore, this project was aimed to explore the biological significance, cellular and molecular mechanism(s) of FLYWCH1 via several in vitro approaches through gain and loss of function analysis of FLYWCH1 in cultured human skin fibroblast cells (TIG119), CRC cell lines, 3D- organoids models, and eventually clinically orientated CRC TMA analyses.
As a result, herein, Chapter 3 demonstrated that Flywch1 crypt-expression is crucial for maintaining the growth and proliferation of the intestinal crypt using murine intestinal organoids. Loss of Flywch1 accelerates the proliferation (Ki-67+ cells) of normal intestinal organoids via increasing the transcription of Lgr5/Olmf4 intestinal stem cell markers. In CRC patient-derived tumour organoids (PDOs), over-expressing human FLYWCH1 protein significantly reduces PDOs sizes and growth by regulating a subset of Wnt target genes involved in cancer invasion, stemness and EMT. Furthermore, we showed that loss of FLYWCH1 in SW620 cell lines conferred more stemness activity in vitro by increasing the colonosphere forming efficiency. Our data provided proof of concept regarding the role of FLYWCH1 in regulating Wnt-mediated biological responses in CRC. Additionally, the data suggested that an over-activation of Wnt signalling decreases FLYWCH1 expression, affecting its stability and cellular localisation. More importantly, our immunohistochemistry-based analysis revealed a significant reduction in FLYWCH1 protein expression in patient's tumour tissues compared to normal samples. Further analyses indicated a significant correlation between low FLYWCH1 expression, cytoplasmic localisation, CRC staging and overall survival. The data indicate that a low expression of FLYWCH1 could predict a poor prognosis in CRCs. In Chapter 4, the data demonstrated for the first time that FLYWCH1 could play as a novel participant in DNA-damage/ repair pathways, co-localising with γH2AX and overexpressing FLYWCH1 induces the expression of γH2AX protein (Almozyan S., et al. 2021).
Altogether, these findings suggest that deregulation of FLYWCH1-WNT signalling and/or FLYWCH1-γH2AX axis via DNA repair pathway could be a significant modulator of tumorigenesis in CRC. However, future studies integrating omics data with in vivo models will allow us to understand the flow of our current developed data that underlies intestinal tissue homeostasis and cancer.
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