The regulation of Wilms' tumour 1 by phospholipid

Lead Research Organisation: University of Bristol
Department Name: Cellular and Molecular Medicine

Abstract

The Wilms' tumour 1 protein, WT1, was first identified in a type of kidney cancer that affects children. WT1 is generally lost in this type of cancer, which suggests that WT1 normally offers protection. An opposite role for WT1 is seen in many adult cancers, including of the lung and breast. Here, WT1 helps to drive formation of the cancer. Thus, WT1 appears to have opposite effects in childhood and adult cancers.

We have discovered an interaction partner of WT1, a protein named BASP1. BASP1 is able to bind to WT1 and change the way that WT1 behaves. BASP1 is responsible for switching the function of WT1 between a protector or a promoter of cancer.

WT1 is a protein that binds to DNA. It binds to the control regions of genes and switches them on. When BASP1 binds to WT1, this causes genes to switch off instead. This is how BASP1 changes the function of WT1 from a cancer-promoter to a cancer-preventer.

We have found that BASP1 needs to bind to a small natural chemical called PIP2. PIP2 is a type of fat that is normally associated with the membrane that surrounds cells. Surprisingly, PIP2 needs to bind to BASP1 when it interacts with WT1 and DNA. Without PIP2, BASP1 does not function correctly with WT1. Our work has demonstrated a new way for switching genes on and off.

This proposal describes experiments to determine how PIP2 functions with BASP1 and WT1 to change the on-off status of genes. There are three specific aims;

1. We will determine how BASP1 and PIP2 cause changes in the ability of WT1 to switch genes on and off. This will involve looking at the way that genes are wrapped into bundles inside the cell. The effect of BASP1 and PIP2 on the bundling of genes will determine how they work with WT1 to switch genes on and off.

2. We will determine how BASP1 binds to PIP2 and how this changes the way that BASP1 binds to WT1. The specific features of BASP1 that are needed for binding to PIP2 will be determined. Insights into how the BASP1-PIP2 partnership changes depending upon the state of the cell will be obtained.

3. WT1 was first identified as a factor that loses function in children's kidney cancer. We will use the information from the first two aims to determine how WT1, BASP1 and PIP2 change the behavior of a cell type that normally develops into a kidney cell. This will involve looking at how they act to make the cells continue to grow in a cancer-like fashion instead of forming specialised kidney cells.

The completion of these studies will provide new insights into the critical roles of WT1 in cancer. They will also open new avenues for therapeutic intervention in cancers that involve WT1 and BASP1.

Technical Summary

WT1 can act as either a tumour suppressor or oncogene in several cancers, but how these activities are controlled is not clear. We identified BASP1 as a critical regulator of WT1. WT1 and BASP1 form a complex that represses transcription of WT1 target genes. Our central hypothesis is that BASP1 acts as a switch to convert WT1 from a transcriptional activator to a transcriptional repressor, and that this mechanism is critical in directing its tumour suppressor and oncogenic activities.

Our recent findings have revealed that the N-terminal myristoylation of BASP1 is required to elicit transcriptional repression at WT1 target genes. Moreover, myristoylated BASP1 binds to nuclear phosphatidylinositol 4, 5-bisphosphate (PIP2), which stimulates the recruitment of HDAC1 to the promoter to mediate transcriptional repression. Our findings have uncovered a novel role for myristoylation in transcription, and a critical function for PIP2 in gene-specific transcriptional repression through the recruitment of histone deacetylase.

This proposal seeks to determine the mechanisms by which the PIP2-BASP1 interaction regulates WT1 function in gene expression and cell fate. These studies will provide new insights into the mechanisms of WT1 action in development, and will also open new avenues for therapy in several major cancers.
The aims are;

1. Determine the role of PIP2 in the modulation of BASP1 contacts with the chromatin remodelling and transcription machinery, and how PIP2 regulates WT1-dependent gene expression.

2. Study how BASP1-PIP2 contact is regulated by the phosphorylation of BASP1 and how this regulates WT1 function.

3. Determine the role of BASP1-PIP2 in the control of cell proliferation and differentiation using a model of kidney podocyte differentiation.

Planned Impact

Who will benefit from this research?

The proposed work is basic science that will increase our understanding of the mechanisms of gene regulation. The work will enhance our understanding of the role of WT1 and BASP1 in kidney development and also several cancer types. The immediate beneficiaries will be academic, biomedical, biotechnology and pharmaceutical industry researchers. Students in the biological sciences will also benefit.


How will they benefit from this research?

The mechanisms involved in the regulation of transcription apply to many areas of biology research. The results of this study are therefore likely to be of significant influence to the work of others in the biology, biomedical and biotechnology research communities. This proposal describes the first evidence that myristoylation plays a role in transcriptional regulation and will therefore stimulate other work in this area. Indeed, it is very likely that other transcription factors will function by a similar mechanism.

Researchers within the pharmaceutical and health-related private sector will benefit in the longer term because the new information derived from this study will help in understanding the roles of WT1 and BASP1 in cancer. Moreover, the regulation of WT1/BASP1 by a small molecule such as PIP2 presents the possibility of modulating WT1/BASP1 function through small molecule therapeutics. Indeed, there are several drugs that are known to modulate PIP2 metabolism that can therefore potentially affect WT1 and BASP1 function. Clinical trials using WT1 peptide vaccines have shown promise in reducing adult leukaemia cell counts and promoting shrinkage of solid tumours including lung cancer and melanoma. While these successes demonstrate the importance of targeting WT1 in the therapy of several types of cancer, their use thus far slows tumour growth but has not yet proven to be curative. The research outlined in this proposal will provide new insights into the regulation of WT1/BASP1 by PIP2. Targeting this pathway will provide a new avenue for therapeutics, and will thus be of direct use to the pharmaceutical industry. The Severnside Alliance for Translational Research (SARTRE) will facilitate in developing discussions with key industrial partners.

The postdoctoral fellow working on this project will gain key skills in research techniques, scientific communication, and supervision. The project involves cutting edge techniques that are at the forefront of current research both in the public and private sector. The postdoctoral fellow will play a central role in the reporting of the work, both by presentation at scientific meetings and in manuscript preparation. The postdoctoral fellow will also have the opportunity to supervise undergraduate and postgraduate students in the laboratory and thus also gain skills in laboratory management and supervision.

The experience obtained by the postdoctoral fellow will be equally applicable to a career in academia, industry, and other science-related endeavour's. Two postdoctoral fellows trained in the applicant's laboratory have gone on to gain independent group leader appointments at academic institutions. Three postdoctoral fellows trained in the applicant's laboratory have gone on to gain permanent research appointments in major pharmaceutical companies. Two postdoctoral fellows from the applicant's laboratory have gone on to fruitful careers in scientific writing. One postdoctoral fellow has gone on to a permanent post in the NHS clinical sector. One postdoctoral fellow gained a position in University research administration involved in industrial collaborations. Another postdoctoral fellow went on to gain an MBA and then to a senior position in marketing in the biotechnology sector. Thus, the skills gained by previous members of the laboratory have gone on to fill diverse roles in the UK economy.

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