Cancer, Development and Adult Tissue Maintenance

 

Summary

Studying the link between normal development and disease, especially cancer. The Wt1 tumor suppressor gene and its role in kidney development has been the starting point for this, and we are studying both molecular and biological roles of the gene. Our work is now being extended to downstream genes, pathways and processes in different developing organs, including kidney, heart and neurons, as well as its potential role as an oncogene in adult cancer. Our current models suggest that whereas in the kidney the role of WT1 appears to be in stimulating cells to differentiate and exist the cell cycle, elsewhere in the developing embryo it could be doing the opposite by stimulating cell proliferation and keeping them in a mofre undifferentiated state.

 

Data from us and others has shown that Wt1 can not just bind DNA but RNA as well, partially depending on the isoform. We are trying to identify physiological RNAs that bind the WT1 protein using RNA pull-down followed by microarray and high-throughput sequencing approaches. As we have shown that some Wt1 isoforms can colocalise with splicing complexes and bind directly to several splicing factors, it is assumed Wt1 might be involved in splicing. No targets for this function are known, but if true, the RNAs that can bind to Wt1 are the prime targets for splicing regulated by Wt1. We are extending this analysis with a whole-genome exon array analysis of material from our Wt1 mouse models. In addition to nuclear functions for Wt1, we have shown the protein actively shuttles between nucleus and cytoplasm, where part of the protein is found in polysomes. We are currently further analysing this cytoplasmic function, also in the context of potential Wt1-binding RNAs. Any interesting RNA targets coming from any of these approaches will be analysed in vivo using mouse models using the systems we have recently developed.

 

WT1 in kidney development

WT1 was originally identified as a tumor suppressor gene responsible for the development of Wilms’ tumors, which are believed to be the result of misdevelopment of the kidney. For this reason we are interested in the role of WT1 in normal kidney development. Previously, in close collaboration with Prof. Jamie Davies we have developed a method for RNAi in kidney organ culture, with which we could provide the first functional evidence that Wt1 is involved in the induction of nephrogenesis, the process that is believed to be disturbed in the development of Wilms’ tumors. We have now developed a conditional knockout model for Wt1 which we are crossing to different Cre expressing lines. We have started to analyse our Wt1 conditional in combination with a Wt1-GFP knock-in allele provided by the lab of Prof. Sugiyama in time-lapse analysis of kidney organ cultures. We are currently accumulating evidence that Wt1 has distinct functions at different stages during kidney development. In the future our aim is to identify the downstream events responsible involved in these different functions.

 

 

Figure 2: Use of RNAi in kidney organ culture to knockdown Wt1 shows the protein is involved in nephrogenesis. An almost complete reduction in nephron numbers is found when Wt1 specific siRNAs are used (B and D) when compared to luciferase control siRNAs (A and C).Red: laminin; green: Wt1. Adapted from Davies et al (2004).

 

 

1. Molecular functions of WT1 (this page)
2. WT1 in kidney development (this page)
3. Wt1 and Wnt4
4. Wt1 outside the Kidney
5. WT1 in MET and EMT
6. Wt1 in Cancer
7. Publications
8. Rosa26 - DEST Vector Information

 

Purpose

The research in our lab focuses around the link between normal development and disease, in particular cancer. Historically the starting point of our work has been the WT1 tumor suppressor gene, but our research is now being extended to several other genes and processes WT1 is linked to. We are now using the developing kidney, heart and neurons as well as in vitro differentiated ES cells as paradigms to study the opposite functions of WT1 in different systems. In all these projects we are trying to understand how the derailing of normal development can lead to disease.

As the WT1 gene encodes many different isoforms with in some cases proven different functions, in vitro overexpression studies will only give limited information. In many cases we are using therefore different mouse models which enable us to look at functions of endogenous Wt1. The last few years have seen a big investment in setting up new in vivo systems which are now starting to provide exiting new data. Although in vivo experiments are more informative for identifying physiological relevant functions for WT1, in vitro experiments are easier to study the more detailed molecular and biochemical functions. For this reason our lab is trying to get the best of both worlds, by taking the material from our mouse models back to the dish, either as ES cells, in kidney organ cultures or as new cell lines generated via crosses with the ‘Immortomouse’ allele. In this way we are generating a system where we can continuous analyse the physiological relevance of our in vitro finding by going back to the complementary mouse models.

Approach, Progress and Future Work

Molecular functions of WT1

WT1 is a multi-functional protein. Its best described molecular function is in transcriptional regulation, where in vitro it can function as either a transcriptional activator or repressor. We have recently identified some potential new direct targets for Wt1 which we are trying to confirm in vivo at the moment. We will soon be embarking on whole genome analysis to identify Wt1 downstream targets in different cell and tissue types, both for activation and repression by Wt1.

 

Figure 1: Heterokaryon assay using mouse M15 (Wt1-positive) and human HeLa (WT1-negative) shows Wt1 can shuttle between nucleus and cytoplasm. Adapted from Niksic et al (2004).