Onyido, Emenike K.
(2017)
Mechanisms and roles of FLYWCH1 in colorectal cancer.
PhD thesis, University of Nottingham.
Abstract
Human colorectal cancer (CRC) is the fourth most common cause of cancer-related death in the UK and worldwide. Defects in conserved signalling pathways play key roles in the development of almost all cancers and, in CRCs, over 80% of tumours show hyper-activation of the canonical Wnt signalling pathway. This pathway, through the transcriptional activity of β-catenin (and its binding partner, TCF4), maintains the stem cell compartment of colon and intestinal-crypts as well as cancer-initiating cells. Whilst the role of β-catenin/TCF4 in the development of both normal and neoplastic colon/intestinal tissues is well documented, the molecular basis of these functionally distinct nuclear-transcriptional programs is still under investigation, hence the functional elucidation of nuclear cofactors that interact with nuclear β-catenin contribute to further unravel the mechanisms involved in the β-catenin-mediated nuclear transcription. In addition to LEF/TCFs, interaction of β-catenin with a plethora of other transcriptional co-activators and/or co-repressors remains vital for gene regulation.
To this end, our lab have been dedicated to identifying -catenin/TCF4 interacting partner proteins (CIPs) capable of fine-tuning the Wnt-level in CRC cells. Among other CIPs, my proposed project was focused on FLYWCH1, a totally novel protein with a FLYWCH/Zn-finger DNA-binding domain, called “FLYWCH”. Previous data in our lab demonstrated that FLYWCH1 preferentially binds the nuclear/ un-phosphorylated--catenin whilst -catenin is still bound to TCF4 (Muhammed et al., submitted). Muhammed et al., found that FLYWCH1 is able to modulate transcription of many -catenin target genes including the stem cell marker (Lgr5) and genes that are associated with migration and invasion of CRC cells. They also showed that FLYWCH1 mRNA expression is restricted to a subpopulation of tumour cells in both human CRCs and ApcMin model mouse for intestinal cancer via in-situ hybridization (ISH). However, prior to these almost nothing was known about the FLYWCH1.
In my research project it was proposed, to build on these advances in FLYWCH1，Wnt and CRC, and to undertake a cell & molecular research program on the role of the FLYWCH1-transcription regulator in potential suppression of colon cancer via direct regulation of microRNAs. However, commercially available FLYWCH1-antibodies worked endogenously only for immunocytochemistry/immunofluorescent (IF), but not for immunohisto-chemistry (IHC) and Western blotting analysis.
Here we provide evidences via FLYWCH1 overexpression and shRNA knockdown in cultured fibroblast (TIG119) and CRC cell lines that FLYWCH1 possess tumour suppressor functions mainly by;
i) Inhibition of cell migration via modulating actin cytoskeleton re-modelling and stress fibre formation and by targeting E-cadherin suppressor ZEB1.
ii) Inhibiting cancer stemness in-vitro (colonosphere assays), by modulating the Wnt/β-catenin signalling pathway and possibly in a GSK-3β dependent manner.
iii) The localization of FLYWCH1 speckled nuclear with splicing factor SC-35 foci, a potential mechanism involved in regulation of miRNA expression.
iv) Positively regulating the expression of let-7 miRNA expression via modulating the LIN28A and LIN28B subcellular distribution.
We also showed that FLYWCH1 expression is correlated positively with let-7 miRNA expression in primary colorectal cancer samples and matched metastases from patients. While we are currently striving for obtaining substantial knowledge about FLYWCH1 function in-vivo and mechanistic insight into the regulatory circuitry of FLYWCH1/miRNAs, collectively, our data suggest that FLYWCH1 possesses tumour suppressor activity and may exert its influence on cancer cells homeostasis through miRNA regulation.
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