FLYWCH1, a novel transcription regulator with potential tumour suppressor activity

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 More recent unpublished in-situ hybridisation (ISH) data from our lab reported the high Flywch1 expressing population of normal and adjacent tumour-free crypt-based cells (2-6 cells),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

FLYWCH1, a Multi-Functional Zinc Finger Protein Contributes to the DNA Repair Pathway

Over recent years, several Cys2-His2 (C2H2) domain-containing proteins have emerged as critical players in repairing DNA-double strand breaks. Human FLYWCH1 is a newly characterised nuclear transcription factor with (C2H2)-type zinc-finger DNA-binding domains. Yet, our knowledge about FLYWCH1 is still in its infancy. This study explores the expression, role and regulation of FLYWCH1 in the context of DNA damage and repair. We provide evidence suggesting a potential contribution of FLYWCH1 in facilitating the recruitment of DNA-damage response proteins (DDRPs). We found that FLYWCH1 colocalises with γH2AX in normal fibroblasts and colorectal cancer (CRC) cell lines. Importantly, our results showed that enforced expression of FLYWCH1 induces the expression of γH2AX, ATM and P53 proteins. Using an ATM-knockout (ATMKO) model, we indicated that FLYWCH1 mediates the phosphorylation of H2AX (Ser139) independently to ATM expression. On the other hand, the induction of DNA damage using UV-light induces the endogenous expression of FLYWCH1. Conversely, cisplatin treatment reduces the endogenous level of FLYWCH1 in CRC cell lines. Together, our findings uncover a novel FLYWCH1/H2AX phosphorylation axis in steady-state conditions and during the induction of the DNA-damage response (DDR). Although the role of FLYWCH1 within the DDR machinery remains largely uncharacterised and poorly understood, we here report for the first-time findings that implicate FLYWCH1 as a potential participant in the DNA damage response signaling pathways

FLYWCH1, a novel suppressor of nuclear b-catenin, regulates migration and morphology in colorectal cancer

© 2018 American Association for Cancer Research. Wnt/b-catenin signaling plays a critical role during development of both normal and malignant colorectal cancer tissues. Phosphorylation of b-catenin protein alters its trafficking and function. Such conventional allosteric regulation usually involves a highly specialized set of molecular interactions, which may specifically turn on a particular cell phenotype. This study identifies a novel transcription modulator with an FLYWCH/Zn-finger DNA-binding domain, called "FLYWCH1." Using a modified yeast-2-hybrid based Ras-Recruitment system, it is demonstrated that FLYWCH1 directly binds to unphosphorylated (nuclear) b-catenin efficiently suppressing the transcriptional activity of Wnt/ b-catenin signaling that cannot be rescued by TCF4. FLYWCH1 rearranges the transcriptional activity of b-catenin/TCF4 to selectively block the expression of specific downstream genes associated with colorectal cancer cell migration and morphology, including ZEB1, EPHA4, and E-cadherin. Accordingly, overexpression of FLYWCH1 reduces cell motility and increases cell attachment. The expression of FLYWCH1 negatively correlates with the expression level of ZEB1 and EPHA4 in normal versus primary and metastatic colorectal cancer tissues in patients. Thus, FLYWCH1 antagonizes b-catenin/TCF4 signaling during cell polarity/migration in colorectal cancer. Implications: This study uncovers a new molecular mechanism by which FLYWCH1 with a possible tumor suppressive role represses b-catenin-induced ZEB1 and increases cadherin-mediated cell attachment preventing colorectal cancer metastasis

Morphological alterations of cultured human colorectal matched tumour and healthy organoids.

Organoids have extensive applications in many fields ranging from modelling human development and disease, personalised medicine, drug screening, etc. Moreover, in the last few years, several studies have evaluated the capacity of organoids as transplantation sources for therapeutic approaches and regenerative medicine. Nevertheless, depending on the origin of the cells and anatomical complications, an organoid transplant may make tissue regeneration difficult. However, some essential aspects of organoids including the morphological alterations and the growth pattern of the matched tumour and their healthy derived organoids have received less attention. Therefore, the current work focused on culturing matched healthy and tumour organoids from the same patient with colorectal cancer (CRC) and assessed their timed growth and structural differences on a daily basis. The healthy organoids underwent proliferation and branching morphogenesis, while the tumour organoids did not follow the same pattern, and the majority of them developed cystic structures instead. However, the number and size of tumour organoids were different from one patient to another. The differential morphological changes of the healthy versus human colonic tumour organoids likely linked to distinct molecular and cellular events during each day. Thus, while their specific structural features provide valuable in vitro models to study various aspects of human intestinal/colon tissue homeostasis and CRC which avoid or replace the use of animals in research, this model may also hold a great promise for the transplantation and regenerative medicine applications

FLYWCH1, a novel transcription regulator with potential tumour suppressor activity

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

Increased FLYWCH1 Expression is Negatively Correlated with Wnt/β-catenin Target Gene Expression in Acute Myeloid Leukemia Cells

Acute myeloid leukaemia (AML) is a heterogeneous clonal malignancy of hematopoietic progenitor cells. The Wnt pathway and its downstream targets are tightly regulated by β-catenin. We recently discovered a new protein, FLYWCH1, which can directly bind nuclear β-catenin. Herein, we studied the FLYWCH1/β-catenin pathway in AML cells using qRT-PCR, Western blot, and immunofluorescence assays. In addition, the stemness activity and cell cycle were analysed by the colony-forming unit (CFU) using methylcellulose-based and Propidium iodide/flow cytometry assays. We found that FLYWCH1 mRNA and protein were differentially expressed in the AML cell lines. C-Myc, cyclin D1, and c-Jun expression decreased in the presence of higher FLYWCH1 expression, and vice versa. There appeared to be the loss of FLYWCH1 expression in dividing cells. The sub-G0 phase was prolonged and shortened in the low and high FLYWCH1 expression cell lines, respectively. The G0/G1 arrest correlated with FLYWCH1-expression, and these cell lines also formed colonies, whereas the low FLYWCH1 expression cell lines could not. Thus, FLYWCH1 functions as a negative regulator of the Wnt/β-catenin pathway in AML