A PP2A Regulatory Nexus Modulating Hippo Signalling and Growth

The growth of tissues during development and adult life is the result of a fine balancing act between cell proliferation, differentiation and death. Understanding the mechanisms that regulate tissue growth is one of the key unanswered questions in biology. It is increasingly clear that growth is tightly connected with tissue structure and that, in pathological conditions, tissue structure is severely affected, leading to tissue abnormalities.

The Hippo pathway is one of the most important signalling cascades controlling tissue growth, and it responds to alterations in tissue architecture. This signalling cascade includes several steps, some of which are mediated by proteins whose function can be modified by the attachment of different chemical and biochemical entities, in a process termed post-translational modification. Phosphorylation is one such post-translational process in the cell, which consists of the attachment of a phosphate group to other proteins. This is a reversible process that involves a complex machinery of proteins that attach or remove the modification from other proteins. Phosphorylation changes the properties of proteins by affecting their stability, localisation in the cell or function.

We have previously found that the phosphatase PP2A blocks the activity of the Hippo pathway as part of a large protein complex. Recently, when studying in detail how tissue structure controls Hippo signalling, we discovered that the PP2A phosphatase has an additional role in the regulation of tissue growth and, in certain conditions, it can activate rather than block Hippo signalling. However, it is still unknown what are the signals and mechanisms that control whether PP2A activates or blocks the Hippo pathway.

Using the fruit fly Drosophila as a model organism, we will investigate how tissue growth is regulated by the phosphatase PP2A. The fruit fly is an extremely flexible and highly relevant biological tool that has been widely used in genetic studies due to its ease of manipulation and fast life cycle. Many of the genes known to be important for human development were first identified and characterised as a result of fruit fly genetic studies. Due to the remarkable conservation of genes between flies and mammals, findings from fruit fly studies have been translated to advances in our understanding of important biological processes in humans. Thus, our work will also examine the function of genes identified in Drosophila in human tissue culture cells to precisely study the molecular details of the process.

This project will reveal how the phosphatase PP2A controls tissue growth and what are the signals that regulate its activity and switch its function from a growth-promoting to a growth-suppressive protein, and vice versa. We will study this crucial question both in normal developmental conditions and when tissues respond to changes in their architecture. We will use a combination of genetic and biochemical approaches to uncover the function of the genes involved by switching them off and determining whether cells are impaired in their ability to proliferate and give rise to tissues of the correct size.

We will elucidate how the activity of the phosphatase PP2A is regulated, what are the signals that control its activity, and how it can be exploited. Numerous genes involved in tissue growth are mutated in human disease, leading to developmental abnormalities and cancer. Therefore, identification of genes involved in the regulation of tissue growth may be potentially relevant for future therapeutic interventions. In addition, as we will study the role of our genes of interest in the context of the response to tissue damage, our project will also be very relevant for future studies into tissue regeneration and tissue engineering.

Tissue growth regulation is essential for development and homeostasis, ensuring tissue regeneration is transient and preventing tumourigenesis. The growth-suppressive Hippo pathway monitors cell-cell communication and epithelial tissue organisation by integrating multiple signals. However, the mechanisms controlling tissue growth have been incompletely defined.

We have previously shown that the phosphatase PP2A blocks Hippo signalling via the STRIPAK complex. However, we have recently found that PP2A prevents phosphorylation and degradation of Expanded, a protein that links growth control to the polarity protein Crumbs and activates Hippo signalling. Our aim is to elucidate how PP2A controls Hippo signalling, how its activity is regulated downstream of multiple signals with antagonistic effects on tissue growth, and to identify what controls switching of PP2A function from promoting to suppressing tissue growth. For this, we will address the following aims:

Aim 1: Identify and characterise the Hippo pathway-activating PP2A complex. We will uncover the molecular apparatus assembled by PP2A to sustain the function of the Hippo pathway protein Expanded.

Aim 2: Characterise the signals regulating the growth-suppressing PP2A complex. Hippo signalling is regulated by several upstream signals and proteins and we will define how these modulate the function of the PP2A complex that regulates Expanded.

Aim 3: Study the interplay between the STRIPAK and the growth-suppressing PP2A complex. We will address how the activity of PP2A is regulated in the different complexes and how these are co-regulated to ensure cells respond appropriately to signals controlling tissue growth.

This project will uncover novel mechanisms controlling growth and will provide an in-depth characterisation of in vivo phosphorylation-mediated signalling events. Our findings will be crucial for researchers from such diverse fields as tissue growth, regeneration and cancer biology.

Uncovering how tissue growth is regulated is crucial for our understanding of how tissues grow to attain their correct final size, what signals control this sizing mechanism and how this process remains error-free. Our work focuses on the Hippo tumour suppressor pathway, which has recently emerged as a crucial network in cancer and tissue damage repair. Our work will enhance our knowledge of tissue growth regulation and will have a wide-ranging impact in several fields, including developmental biology, tissue engineering and cancer biology. Thus, our project has the potential to benefit a wide range of academic and non-academic beneficiaries.

Our findings will benefit the wider scientific community by acting as a catalyst for research in other fields. Results will be disseminated to the scientific community and any relevant data or novel reagents will be communicated to the appropriate repositories (e.g. Flybase, ProteomeXchange), thereby allowing their prompt release, which will open new avenues of research in basic biology and applied research. The research community will be targeted by publication in international peer-reviewed high impact journals (2-3 publications predicted) and presentation in national and international meetings (monthly or annually, depending on their scope).

As our work has potential connections to human health, we will publicise our research to the public, highlighting the role of basic research as a cornerstone for applied research and the use of model organisms to replace, refine and reduce the number of animals in scientific research. Data will be communicated via the Queen Mary and Barts Cancer Institute public engagement program, which includes laboratory tours (bimonthly), open days, special seminars or activities (annually). With the Centre of the Cell and the Centre for Public Engagement, we will design activities for local and national beneficiaries. We also plan to apply for external funding to produce short videos and animations that explain our work in a simple way. With the BCI Digital Media and Communications Officer and QMUL press office, publications will be advertised to the general public by press releases and on the QMUL and BCI websites and social media presence.

Due to its potential links to human health and disease, our work will be instrumental for the development of new pharmacological inhibitors and drugs targeting the Hippo pathway machinery, which may be used in future therapies for developmental abnormalities, in the context of tissue regeneration or cancer. We will work alongside QM Innovation and current BCI pharmaceutical company partners to develop a strategy to test or design targeted drugs or inhibitors for future therapeutic use. In future, upon identification of promising therapeutic targets, we will work in close association with Profs Peter Schmid and Thomas Powles, from the Experimental Cancer Medicine Centre in BCI. Profs Schmid and Powles are world experts in the development and testing of cancer treatments and biomarkers of response in early stage clinical trials. Novel targets can be rapidly tested in a disease setting in numerous samples from clinical trials available from the Schmid and Powles groups to support the possible translational nature of our work, thus ensuring our results are meaningful to public health care.

In the long-term, our work will be instrumental for the development and training of a new generation of researchers. The support system currently in place in BCI nurtures, promotes and enhances scientific and personal development of all researchers, and scientific independence is fostered through a mentoring scheme involving junior and senior PIs. Hiring researchers from other national and international institutions will significantly add to the skills portfolio of BCI and foster collaborations. Finally, regular workshops are organised to showcase future scientific and non-academic careers.