Vincan Lab ~ Projects

Wnt/FZD signalling pathways in cancer and disease

Recent insights from diverse fields of basic and clinical research reveal that the biological processes that govern embryonic development and organogenesis are also commonly involved in the pathologies that arise in that organ or tissue in the adult. This striking parallel between embryonic development and pathology is exemplified by Wnt signalling in the intestinal tract. Wnt signalling is critical throughout embryonic development of the mammalian gut. Moreover, competent Wnt signalling is essential for the homeostatic control of the adult intestinal epithelium. On the other hand, aberrant Wnt signalling in the adult intestine leads to cancer and other pathologies. This critical role of the Wnt pathway in gut development and homeostasis is conserved through evolution, emphasizing the importance of this pathway in this organ. Our studies strongly implicate Frizzled7 (FZD7) in transmitting this critical Wnt signal. We are interested in defining the role of FZD7 in gut tissues, and will employ molecular techniques (qRT-PCR, Western blot, confocal immunofluorescence microscopy, immunohistochemistry) and molecular tools (FZD7 promoter reporter plasmid, expression plasmid) and in vivo (conditional knock-out and knock-in mice) and in vitro (organoid and monolayer colon cancer cell lines) models systems.

The Cancer Biology Lab collaborates with Prof. Hans Clevers and Dr Nick Barker (Hubrecht Laboratory) on the mouse models, and Prof. Thomas Brabletz (University of Freiburg) on colorectal cancer morphogenesis and metastasis.

Specific questions we are addressing:

1. Is FZD7 necessary during intestinal development?

We will specifically delete FZD7 in the intestinal epithelium during mouse gut development and study the consequences.

2. Is FZD7 necessary for adult crypt homeostasis?

We will induce FZD7 deletion in the intestinal epithelium of adult mice to study the immediate consequences of FZD7 loss on tissue homeostasis.

3. How is FZD7 expression regulated in colon cancer cells?

To gain insight into the cellular pathways that regulate FZD7 expression, we will use a FZD7 reporter assay system where the expression of a reporter gene in under the control of the native FZD7 promoter.

4. Which Wnt ligand activates FZD7 in gut tissues?

Our expression studies implicate at least two Wnt genes in FZD7-mediated colorectal cancer morphogenesis. We will use molecular tools to investigate which Wnt acts in concert with FZD7.

These projects are suitable to and can be adapted for PhD or Honours projects. 

Figure Legend:

Some of the techniques and model systems used in the Cancer Biology Laboratory.

Carcinoma tissue sections: (A) Shows FZD7 protein (brown) detected in a colon cancer tissue section by immunohistochemistry (IH). FZD7 is expressed by moderate to well-differentiated cancer cells.

3D colorectal cancer morphogenesis model: LIM1863-Mph cells model many aspects of colon cancer morphogenesis – the cells spontaneously anchor to tissue culture plastic to form 2D monolayer patches. Over several days, the monolayer cells reorganize cell-cell contacts to assemble 3D spheres (organoids). The cells lift off the tissue culture plastic as the spheres are assembled (B) and the mature organoids float free. The solid and open arrows in (B) indicate maturing organoids and the nucleus of a monolayer cell, respectively (scanning electron microscopy). (C) A cross-section of the organoid spheres (by transmission electron microscopy) shows highly polarised epithelial cells with basal nuclei (N) and abundant apical microvilli extending into the central lumen (L).

Molecular tools: Expression vectors (conventional and retroviral) and short hairpin (sh)RNA retroviral plasmids are employed to increase or deplete gene expression, respectively. (D) Shows FZD7 protein (green) in 293T cells transfected with a FZD7 expression plasmid (detected by confocal immunofluorescence microscopy).